University of Nebraska Medical Center DigitalCommons@UNMC

Theses & Dissertations Graduate Studies

Summer 8-19-2016

The Mechanism of Tubular Recycling Endosome Biogenesis

Shuwei Xie University of Nebraska Medical Center

Follow this and additional works at: https://digitalcommons.unmc.edu/etd

Part of the Cell Biology Commons

Recommended Citation Xie, Shuwei, "The Mechanism of Tubular Recycling Endosome Biogenesis" (2016). Theses & Dissertations. 124. https://digitalcommons.unmc.edu/etd/124

This Dissertation is brought to you for free and open access by the Graduate Studies at DigitalCommons@UNMC. It has been accepted for inclusion in Theses & Dissertations by an authorized administrator of DigitalCommons@UNMC. For more information, please contact [email protected]. I

The mechanism of Tubular recycling endosome biogenesis

By

Shuwei Xie

A DISSERTATION

Presented to the Faculty of The University of Nebraska Graduate College In Partial Fulfillment of the Requirements For the Degree of Doctor of Philosophy

Department of Biochemistry & Molecular Biology

Under the Supervision of Professor Steve Caplan

University of Nebraska Medical Center Omaha, Nebraska

June, 2016

Supervisory Committee:

Paul Sorgen, Ph.D. Parmender Mehta, Ph.D.

Anna Dunaevsky, Ph.D. II

TITLE

The mechanism of Tubular recycling endosome biogenesis

BY

Shuwei Xie

APPROVED DATE

Steve Caplan, Ph.D. August 12th 2015

Paul Sorgen, Ph.D. August 12th 2015

Parmender Mehta, Ph.D. August 12th 2015

Anna Dunaevsky, Ph.D. August 12th 2015

SUPERVISORY COMMITTEE

GRADUATE COLLEGE UNIVERSITY OF NEBRASKA

III

The mechanism of Tubular recycling endosome biogenesis Shuwei Xie, Ph.D.

University of Nebraska Medical Center, 2015

Supervisor: Steve Caplan, Ph.D.

Endocytic trafficking is a critical process for cellular homeostasis, and multiple ailments that include cardiovascular disease and cancer are related to the dysregulation of endocytic transport. As vesicles and target membranes are key to endocytic transport, lipids are essential for the regulation of endocytic trafficking pathways. We have shown that the tubular recycling endosomes (TRE) are essential for the regulation of endocytic recycling pathways. However, the mechanisms by which TRE are biosynthesized and carry out their functions remain unsolved. Studies from our lab have shown that phosphatidic acid (PA) recruits Molecule Interacting with Casl-Like protein 1 (MICAL-L1) as well as Syndapin2, and subsequently Eps15 homology domain containing protein

EHD1 to the membrane of TREs as a complex, and this complex is essential for the biogenesis of TRE and efficient recycling of internalized receptors back to the plasma membrane. However, the involvement of PA in endocytic trafficking has not been well characterized. Diacylglycerol kinase (DGK) α is one of ten DGK isoforms that converts diacylglycerol (DAG) to PA. We showed that depletion of DGKα, a kinase devoid of a clathrin-dependent adaptor protein complex 2 binding site, results in an impaired biogenesis of TRE. As a consequence, we observed a delay in MHC I recycling to the plasma membrane. On the other hand, the rate of MHC I internalization remained unaffected. By Co-immunoprecipitation assay, we showed that DGKα forms a complex with the TRE hub protein, MICAL-L1. Given that MICAL-L1 and the F-BAR-containing membrane-tubulating protein Syndapin2 associate selectively with PA, whose generation IV is majorly mediated by DGKα, we propose a positive feedback loop in which DGKα generates PA to drive its own recruitment to TRE via its interaction with MICAL-L1. Our data support a novel role for the involvement of DGKα in TRE biogenesis and MHC I recycling.

While the molecular mechanism of MICAL-L1 decorated TRE biogenesis is further revealed by our studies of the involvement of DGKα, major questions with regards to their function in endocytic recycling still remained unsolved, such as what cargos travel through these tubular membrane structures, where is the destination of these cargos, etc. We showed that TRE preferentially traffic cargos internalized via clathrin-independent endocytosis (CIE), and may originate from the sorting endosomes (SE). Since MICAL-L1

TRE is a major component of the endocytic recycling compartment (ERC), the understanding of the composition and cargo distribution within the ERC would further add to our knowledge of TRE functions. We used 3D Structured Illumination Microscopy, dual- channel and 3D direct Stochastic Optical Reconstruction Microscopy (dSTORM) to obtain new information about ERC morphology and cargo segregation. For the first time, we discovered that cargo internalized either via clathrin-mediated endocytosis (CME) or CIE remains segregated in the ERC, likely on distinct carriers. This suggests that no further sorting occurs upon cargo exit from SE. Moreover, 3D dSTORM data support a model in which some, but not all ERC vesicles are tethered by contiguous ‘membrane bridges’.

These findings support a significantly altered model for endocytic recycling in mammalian cells, in which cargos are sorted in the peripheral endosomes and carried by MICAL-L1 decorated TRE to the ERC, while segregation is maintained at the ERC.

V

TABLE OF CONTENTS

TITLE PAGE...... I ABSTRACT...... III TABLE OF CONTENTS...... V TABLE OF FIGURES...... VIII LIST OF ABBREVIATIONS...... XI ACKNOWLEDGEMENTS...... XVIII

CHAPTER I ...... 1 INTRODUCTION ...... 1 1. ENDOCYTIC TRAFFICKING ...... 2 1.1 Overview ...... 2 1.2 Various routes of internalization into the cell ...... 5 1.3 Sorting of endocytic cargo at sorting/early endosomes (SE/EE) ...... 10 1.4 Different routes of endocytic recycling ...... 14 1.5 The significance of TRE in endocytic recycling ...... 17 2. REGULATORS OF ENDOCYTIC TRAFFICKING ...... 19 2.1 Overview ...... 19 2.2 Rab proteins and their effectors ...... 20 2.3 SNARE proteins ...... 25 2.4 C-terminal EH domain containing proteins (EHDs) and their interaction partners ...... 26 3. MEMBRANE MODELING IN ENDOCYTIC TRAFFICKING ...... 32 3.1 Role of lipid components in endocytic trafficking ...... 32 3.2 Membrane curvature and the biogenesis of tubular recycling endosomes ...... 44 3.3 Membrane vesiculation ...... 48 4. THE ROLE OF DIACYLGLYCEROL KINASE IN ENDOCYTIC TRAFFICKING. 50 4.1 Overview ...... 50 4.2 The role of DGK on exocytosis ...... 56 4.3 DGK mediates MVB formation and secretion ...... 57 4.4 The role of DGK on secretion from the Golgi apparatus ...... 58 VI

4.5 The role of DGK on endocytic recycling ...... 60 4.6 The role of DGK on endocytosis ...... 61 4.7 Summary and conclusion ...... 64 CHAPTER II ...... 65 5 MATERIALS AND METHODS ...... 66 5.1 Cell lines ...... 66 5.2 DNA Constructs, Transfection and siRNA Treatment ...... 66 5.3 Antibodies and Reagents ...... 67 5.4 Immunoblotting ...... 69 5.5 Flow cytometry analysis ...... 69 5.7 Co-immunoprecipitation assay ...... 72 5.8 Pulse-chase recycling assay ...... 72 5.9 Immunofluorescence staining ...... 76 5.10 Confocal microscopy imaging ...... 77 5.11 Post imaging analysis ...... 79 5.12 Statistical analysis ...... 80 5.13 Live cell imaging ...... 80 5.14 Structured Illumination Microscopy (SIM) imaging and data processing . 81 5.15 Direct Stochastic Reconstruction Microscopy (dSTORM) imaging and data processing ...... 81 5.16 Tubular endosome regrowth assay...... 86 5.17 Membrane fractionation...... 86 5.18 Duolink proximity assay ...... 86 CHAPTER III ...... 88 Diacylglycerol Kinase α Regulates Tubular Recycling Endosome Biogenesis and Major Histocompatibility Complex Class I Recycling ...... 88 6 ABSTRACT...... 89 7 INTRODUCTION ...... 90 8 RESULTS ...... 92 8.1 The rate of MHC I internalization is not regulated by DGKα ...... 92 8.2 DGKα controls the half-life of MHC I ...... 96 8.3 DGKα-depletion interferes with MHC I trafficking to the recycling compartment and delays its recycling ...... 99 8.4 DGKα is required for the biogenesis of tubular recycling endosomes 103 VII

8.5 DGKα forms a complex with MICAL-L1...... 108 8.6 The biogenesis of TRE requires the activity of DGKα ...... 111 9 DISCUSSION ...... 115 CHAPTER IV ...... 118 The endocytic recycling compartment maintains cargo segregation acquired upon exit from the sorting endosome ...... 118 10 ABSTRACT ...... 119 11 INTRODUCTION...... 120 12 RESULTS ...... 123 12.1 The ERC is a compartment that is composed of individual endosomal membranes with potential underlying connections...... 123 12.2 Cargo internalized either via CME or CIE remains segregated after being trafficked to the ERC ...... 128 12.3 Rab11a regulates recycling of CME but not CIE cargos...... 137 12.4 TRE are distinct from Rab11 recycling endosomes, which originate from a subset of SE containing Rabenosyn-5, and likely transport cargo from SE to the ERC ...... 147 12.5 TRE selectively transport CIE cargos CD59 from the SE to the ERC ... 152 13 DISCUSSION ...... 163 13.1 Composition of the ERC ...... 163 13.2 The selectivity of TRE ...... 165 13.3 The significance of TRE in endocytic trafficking ...... 167 CHAPTER V ...... 170 Summary and Future Directions ...... 170 14 Summary ...... 171 15 Future directions ...... 174 15.1 The potential role of DGKα on the tubular recycling endosomes ...... 174 15.2 The mechanisms of cargo sorting in the recycling pathways ...... 175 15.3 The role of ERC in endocytic recycling ...... 176 REFERENCES ...... 177

VIII

Table of figures

Chapter I

Figure 1.1 Schematic diagram depicting the endocytic pathways.

Figure 1.2 Pathways of endocytosis and endocytic recycling.

Figure 2 Regulation of endocytic transport by EHD proteins, Rabs and their effectors.

Figure 3 Model for the biogenesis of tubular recycling endosomes.

Figure 4.1 Pathways involved in the metabolism of DAG and PA described in this review.

Figure 4.2 Schematic diagram illustrating the domain architecture of diacylglycerol kinase isoforms.

Chapter II

Figure 5.1 Schematic workflow for quantitatively measuring recycling of Tf and its receptor or MHC I by flow cytometry.

Figure 5.2 Schematic illustration of a humidity chamber.

Chapter III

Figure 8. 1 The rate of MHC I internalization is not regulated by DGKα.

Figure 8. 2 DGKα controls the half-life of MHC I. IX

Figure 8.3 DGKα-depletion interferes with MHC I trafficking to the recycling compartment and delays its recycling.

Figure 8.4 DGKα is required for the biogenesis of tubular recycling endosomes.

Figure 8.5 DGKα forms a complex with MICAL-L1.

Figure 8.6 The biogenesis of tubular recycling endosomes requires the activity of

DGKα.

Figure 12.1 The 3D SIM characterization of Rab11a recycling endosomes in the ERC.

Figure 12.2 Linkage between recycling endosomes in the ERC via “membrane bridges” as observed by SIM and 3D dSTORM imaging.

Figure 12.3 The CIE cargo CD59 is absent from perinuclear Rab11a recycling endosomes in the ERC.

Figure 12. 4 Segregation of CME and CIE cargoes in the ERC.

Figure. 12.5 dSTORM visualization of CD59 and Transferrin in the cell periphery.

Figure. 12.6 dSTORM visualization of CD59 and transferrin in the perinuclear area.

Figure 12.7. Myosin Vb tail over-expression selectively traps CME cargos in the perinuclear area.

Figure. 12.8 Myosin Vb tail overexpression selectively traps clathrin-dependent cargos but not TRE regulators in the perinuclear area.

Figure 12.9 Rab11a regulates the recycling of CME but not CIE cargo.

Figure 12.10 SIM and dSTORM visualization of Rab11a and MICAL-L1-labeled TRE. X

Figure.12.11 dSTORM visualization of Rab11a and MICAL-L1-labeled recycling endosomes.

Figure 12.12 MICAL-L1-decorated TRE are derived from SE.

Figure. 12.13 Rabenosyn-5 partially colocalizes with MICAL-L1 to TRE in untreated cells.

Figure. 12.14 MICAL-L1-decorated TRE do not originate from the PM

Figure 12.15 TRE selectively transport cargos from SE.

Figure.12.16 MICAL-L1 displays limited co-localization with transferrin even after a longer internalization

Figure 12.17 TRE transport CD59 into the recycling compartment.

Figure 12.18 Proposed model depicting the recycling of CME and CIE.

XI

List of abbreviation

[DE]XXX[LI] Dileucine-based sorting motif

µm micrometer

AAA+ ATPases associated with diverse cellular activities

ACAP1 Arf6 GTPase-activating protein 1

ADP Adenosine-5’-diphosphate

AMPA 2-amino-3-(3-hydroxy-5-methyl-isoxazol-4-yl)propanoic acid

AP Adaptor protein

AP-2 Adaptor protein-2

ARF ADP-ribosylation factor

ARH Autosomal recessive hypercholesterolemia

ATP Adenosine-5’-triphosphate

ATPase ATP hydrolysis enzyme

BAR Bin/amphiphysin/Rvs

BSA Bovine serum albumin

CCP Clathrin-coated pits

CCV Clathrin-coated vesicles

CDC42 Cell division cycle 42

CDE Clathrin-dependent endocytosis cDNA Complementary DNA

CIE clathrin-independent endocytosis

CLIC Clathrin-independent carrier

CME Clathrin-mediated endocytosis

COP Coat protein complex XII

CRMP2 Collapsing response mediator protein-2

CTxB Cholera toxin B

DAG Diacylglycerol

DGK Diacylglycerol kinase

DMEM Dulbecco’s Modified Eagle Medium

DNA Deoxyribonucleic acid dSTORM Direct Stochastic Optical Reconstructed Micorscopy

EDTA Ethylenediaminetetraacetic acid

EE Early endosomes

EEA1 Early endosomal antigen-1

EGF Epidermal growth factor

EGFR epidermal growth factor receptor

EH Eps15 homology

EHD Eps 15 homology domain-containing

Eps15 Epidermal growth factor substrate 15

ER Endoplasmic reticulum

ERC endocytic recycling compartment

ESCRT Endosomal sorting complex required for transport

F-BAR FER/Cip4 homology-Bin/Amphiphysin/Rvs

FBS Fetal bovine serum

GAP GTPase-activating protein

GDF GDI displacement factor

GDI GDP-dissociation inhibitor

GDP Guanosine-5’-diphosphate

GEEC GPI-anchored protein-enriched endosomal compartment XIII

GEF Guanine nucleotide exchange factor

GFP Green fluorescent protein

GLUT4 Glucose transporter 4

GPCR G protein-coupled receptor

GPI Glycosylphosphatidylinositol

GPI-AP Glycosylphosphatidylinositol-anchored protein

GRAF1 GTPase Regulator Associated with Focal adhesion kinase-1

GSL glycosphingolipids

GST Glutathione S-transferase

GTP Guanosine-5’-triphosphate

GTPase GTP hydrolysis enzyme h Hour

HA Hemagglutinin

HEPES 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid

HOPS Homotypic fusion and vacuole protein sorting complex

HRP Horseradish peroxidase

Hrs Hepatocyte growth factor-regulated tyrosine kinase substrate

Hsp70 70 kilodalton heat shock protein

IF immunofluorescence kD kiloDalton

LAMP1 Lysosomal-associated membrane protein 1

LDL Low-density lipoprotein

LDLR Low-density lipoprotein receptor

LE Late endosome

LPA Lysophosphatidic acid XIV

LPAAT LPA acyltransferase

LPAT Lysophospholipid acyltransferase

MDCK Madin-Darby canine kidney cells

MHC I Major Histocompatibility Complex Class I

MICAL-L1 Molecule Interacting with CasL-Like 1 min minute

MTOC Microtubule organizing center

MVB Multi-vesicular bodies

NADP+ Nicotinamide adenine dinucleotide phosphate

NADPH Reduced NADP+

N-BAR N-terminal helix and Bin/Amphiphysin/Rvs nm nanometer

NMR Nuclear magnetic resonance

NPF Asparagine-proline-phenylalanine motif

PA Phosphatidic acid

PAP PA phosphatase

PBS Phosphate-buffered saline

PBST Phosphate-buffered saline with Tween

PC phosphatidylcholine

PCR Polymerase chain reaction

PDGF Platelet-derived growth factor

PE Phosphatidylethanolamine

PH Pleckstrin homology

PH domain Pleckstrin homology domain

PI phosphatidylinositol XV

PI(3,5)P2 Phosphatidylinositol 3,5-bisphosphate

PI(4,5)P2 Phosphatidylinositol 4,5-bisphosphate

PI3K Phosphatidylinositol 3-kinase

PI3P Phosphatidylinositol 3-phosphate

PI4P Phosphatidylinositol 4-phosphate

PIKfyve FYVE finger-containing phosphoinositide kinase

PIP Phosphatidylinositol phosphate

PIP5K phosphatidylinositol 4-phosphate 5-kinase

PKC Protein kinase C

PLA Phospholipase A

PLD Phospholipase D

PM plasma membrane

PRD Proline-rich domains

PS Phosphatidylserine

PtdIns phosphatidylinositol

PtdIns(3,5)P2 Phosphatidylinositol 3,5-bisphosphate

PtdIns(4,5)P2 Phosphatidylinositol 4,5-bisphosphate

PX Phox domain

Rab Ras-associated binding proteins

Rab Ras-like in rat brain

Rab11-FIP Rab11 family-interacting protein

RE Recycling endosomes

RhoA Ras homology gene family, member A

Rme-1 Receptor-mediated endocytosis protien1

RNA Ribonucleic acid XVI

RNAi RNA interference

ROI Region of interest

ROS Reactive oxygen species

RPE Retinal pigment epithelium cells s Second

SDS-PAGE Sodium dodecyl sulfate-polyacrylamide gel electrophoresis

SE Sorting endosome

SH3 Src homology 3 domain

SIM Structured Illumination Microscopy siRNA Small interfering RNA

SNARE soluble N-ethylmaleimide-sensitive factor attachment proteins receptors

SNX Sorting nexin

STAM 2 signal transducing adaptor molecule 2

SV40 Simian vacuolating virus 40

Tf Transferrin

TfR Transferrin receptor

TGN Trans-Golgi network

TRE Tubular recycling endosomes

Tris Tris(hydroxymethyl)aminomethane

TX-100 Triton X-100

UIM Ubiquitin-interacting motif

Vps vacuolar protein sorting

WASP Wiskott-Aldrich syndrome protein

WB Western blot

WGA Wheat Germ Agglutinin XVII

Y2H Yeast two-hybrid

YXXΦ Tyrosine-based sorting motif

XVIII

Acknowledgements

Firstly, I would like to thank God for all the precious giving and guiding me through the PhD program.

Secondly, I am in much debt to my mentors, Dr. Steve Caplan and Dr. Naava

Naslavsky. If it weren’t for them, there is now way I could survive the PhD training. Their tolerance, patience, support and encouragement were always with me since the first day

I joined the lab. It is really my fortune to have them as my mentors. I owe also very much to present and past lab members for their friendship and generous help: Dr. Bishuang Cai,

Dr. Sai Srinivas Panapakkam Giridharan, Dr. Jing Zhang, Dr. Laura Simone, Dawn

Katafiza, Dr. James Reinecke, Linda Kelsey, Kriti Bahl and Trey Farmer.

I would also like to thank my dissertation committee members Dr. Paul Sorgen, Dr.

Parmender Mehta, Dr. Anna Dunaevsky for their guidance and support.

Last but not least, I would like to thank my family for their love and affection, especially my husband, who gave me the warmest encouragement and strength, as well as my daughter, Lydia, for her well-behaving.

1

CHAPTER I

INTRODUCTION

2

1. ENDOCYTIC TRAFFICKING

1.1 Overview

The plasma membrane (PM) of mammalian cells is composed of a highly dynamic lipid bilayer that continuously separates the interior of the cell from the environment. The

PM plays an important role in the maintenance of the cell volume, and is essential for communication with other cells and the extracellular environment. Furthermore, in order to respond to extracellular signals, the localization of transmembrane receptors to the PM and the composition of lipids is regulated by endocytic transport, which is a complicated process in which multiple proteins regulate distinct stages of vesiculation, budding, transport and fusion with target organelles (Jovic, Sharma et al. 2010). Dysregulation of endocytic transport is related to various diseases including cancer and heart disease

(Conner and Schmid 2003, Stein, Dong et al. 2003). For example, the internalization of receptor tyrosine kinases is a means to attenuate their signaling pathways (Palfy, Remenyi et al. 2012). Aside from PM homeostasis, cell shape and receptor retrieval, endocytic recycling is also key for a variety of cellular processes including furrow cleavage and cytokinesis (Skop, Bergmann et al. 2001, Fielding, Schonteich et al. 2005, Montagnac,

Echard et al. 2008), cell migration (Caswell and Norman 2008), polarity (Wang, Brown et al. 2000), regulating cell fusion in myoblasts (Doherty, Demonbreun et al. 2008), and controlling α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptors in neurons

(Park, Penick et al. 2004). Understanding the molecular mechanisms by which endocytic transport is regulated will ultimately lend support for clinical applications.

Endocytic transport within the cell is facilitated by an important membrane-bound organelle known as the endosome (Jovic, Sharma et al. 2010). Endosomes are important for the internalization of cargos into the cell (endocytosis), sorting and delivering of cargo 3 molecules to their corresponding destinations, either to degradation or to recycling back to the plasma membrane, which depends upon a series of well-coordinated membrane fission and fusion events (Figure 1.1). A large group of cytosolic proteins assemble onto endosomal membranes to influence their shape, and facilitate the formation of a budding vesicle. These vesicles/carriers are then transported along microtubule tracks to a specific destination where they eventually fuse with a target membrane and release their cargo.

Such proteins often assemble as a complex and can act as an “operational unit.

The endocytic pathway of mammalian cells consists of distinct endosomal membrane compartments: early, late and recycling endosomes (Figure 1.1). Early endosomes (EE), often located in the periphery of the cell, are usually the first station of the endocytic pathway, where sorting events are initiated, determining the subsequent fate of the internalized cargos through the coordinate recruitment of essential regulators and assembly of the sorting machinery. (Jovic, Sharma et al. 2010). The most commonly studied post-sorting destinations for internalized proteins are lysosomes, which are involved in degradation, the trans-Golgi network (TGN) or recycling endosomes involved in traffic back to the PM. In the presence of sorting signals for degradation, early endosomes mature into late endosomes that subsequently fuse with lysosomes for degradation. On the other hand, the recycling process is extremely vital in that it brings back most of the internalized lipids and receptors to the PM, preserving the capacity of surface membrane and reactivity to extracellular stimuli. In this case, cargos are sorted through extensive tubulation of the early endosomes membranes forming recycling endosomes with a high ratio of membrane surface to luminal volume (Maxfield and

McGraw 2004). These tubular membranous structures are termed tubular recycling endosomes (TRE). This thesis aims to discuss the mechanisms by which TRE are

4

Figure 1.1. Schematic diagram depicting the endocytic pathways.

Receptors are internalized either through clathrin-dependent endocytosis or clathrin-independent endocytosis into vesicles that ultimately fuse with early/sorting endosomes. Cargo may be transported to late endosomes and lysosomes for degradation, or it may be recycled back to the plasma membrane directly from SE or indirectly via a perinuclear endocytic recycling compartment (ERC). (image used with permission from (Reineke, Xie et al. 2015))

5 generated, as well as the novel function of TRE as a selective carrier for cargos internalized via clathrin-independent endocytosis (CIE) into the recycling pathway.

1.2 Various routes of internalization into the cell

Endocytosis refers to a cellular process that internalizes a fraction of PM lipids along with its integral protein and extracellular fluid (Conner and Schmid 2003). Based on the size of the endocytic vesicles, there are multiple routes for protein endocytosis into the cell, including but not limited to receptor-mediated endocytosis, pinocytosis, phagocytosis.

While phagocytosis internalizes larger particles such as pathogens and debris in specialized mammalian cells such as macrophages, monocytes and neutrophils (Aderem and Underhill 1999), pinocytosis regulates the uptake of smaller units including fluids and low molecular weight solutes (Conner and Schmid 2003). Pinocytosis is non-specific and non-absorptive.

1.2.1 clathrin-mediated endocytosis

Molecule-specific endocytosis is called receptor-mediated endocytosis (RME), and might be the best studied form of endocytosis. It is characterized by the assembly of clathrin-coated pits (CCP), also referred to as clathrin-mediated endocytosis (CME), which commences when receptors at the PM cluster into CCP, often triggered by ligand binding, and are internalized into membrane-bound vesicles (Figure 1.1). The process of internalization is an active one and relies upon adaptor proteins that recognize amino acid motifs on the cytoplasmic tails of transmembrane receptor (Kirchhausen, Bonifacino et al.

1997, Kirchhausen, Owen et al. 2014). The adaptor protein complex, Adaptor protein-2

(AP-2) consists of large (α and β2), medium (µ2) and small (δ2) subunits, and binds both 6 the cargo and major lipid component of the PM, phosphatidylinositol 4,5-bisphosphate

[PtdIns(4,5)P2] through its μ-subunit and σ-subunit (Collins, McCoy et al. 2002, Kelly,

McCoy et al. 2008). The motifs AP-2 recognized are YXXΦ motifs (where Φ can be F, I,

L, M or V) and [ED]xxxL[LI] acidic dileucine motifs. A classic example of receptor containing the motifs in its cytoplasmic tail for CME is the transferrin receptor (TfR, YXXΦ motif), and the low-density lipoprotein receptors (LDLR, FXNPXY motif). After cargo selection and CCP assembly, the GTPase protein dynamin is recruited to the vesicle neck by BAR (Bin–Amphiphysin–Rvs) domain-containing proteins such as amphiphysin, endophilin and sorting nexin 9 (SNX9). Dynamin then undergoes conformational change via GTP hydrolysis, regulating the scission and budding of CCP (Kosaka and Ikeda 1983,

Wigge, Kohler et al. 1997, Schmidt, Wolde et al. 1999, Bashkirov, Akimov et al. 2008,

Ferguson, Raimondi et al. 2009).

1.2.2 clathrin-independent endocytosis

In addition to CME, transmembrane proteins such as bacterial cholera toxin B subunit (CTxB) (Sandvig and van Deurs 1994), viruses (Madshus, Sandvig et al. 1987), major histocompatibility complex class I (Naslavsky, Weigert et al. 2004), β1-integrin and glycosylphosphatidylinositol-anchored proteins (GPI-APs) (Sabharanjak, Sharma et al.

2002) such as CD59 can also be internalized via deep membrane invaginations originating at the PM that are devoid of clathrin (clathrin-independent endocytosis, CIE; Figure 1.1)

(Massol, Larsen et al. 2005, Sandvig, Torgersen et al. 2008, Sandvig, Pust et al. 2011).

Cargos internalized through this pathway are taken up in structures with various shapes often containing a significant level of cholesterol that is maintained throughout the endocytic pathway (Gagescu, Demaurex et al. 2000, Mayor, Parton et al. 2014), ranging 7 from small vesicles to extended flask-shaped membrane tubules, depending on the protein regulators recruited to the PM at the internalization site.

The first structures identified as the carrier for CIE were caveolae, which are invaginations of the PM forming a flask-shaped morphology (Stan 2002). The major component for the caveolae-mediated endocytosis is caveolin 1 (Singh, Puri et al. 2003).

Caveolar cargos are diverse, ranging from lipids, proteins and lipid-anchored proteins to pathogens. Caveolae-mediated endocytosis also requires the dynamin GTPase for scission and vesicle budding.

A second clathrin-independent pathway requiring dynamin activity is mediated by the small GTPase RhoA (Lamaze, Dujeancourt et al. 2001). This pathway is prominent in immune cells and fibroblasts for the internalization of the β-chain of the interleukin-2 receptor (IL-2R-β). Although the role of RhoA in the generation of clathrin-independent structures has not yet been investigated, it could possibly be required for regulating the actin cytoskeleton dynamics and subsequently affecting this endocytic pathway.

Another important type of CIE involves the uptake of glycosylphosphatidylinositol

(GPI)-anchored proteins (Mayor, Parton et al. 2014). GPI-anchored protein internalization relies on neither clathrin nor caveolae, but rather is found on micro-domains of the PM which contain cholesterol enriched lipid rafts. After internalization, these GPI-anchored proteins can be detected in a population of early endosomal organelles referred to as GPI- enriched early endosomal compartments (GEEC) (Sabharanjak, Sharma et al. 2002). As opposed to CME and caveolae-mediated endocytosis, the GEEC pathway is independent of dynamin activity. Instead, the fission of the clathrin-independent carriers (CLIC) in this poorly characterized pathway is mediated by the GTPase Regulator Associated with Focal adhesion kinase-1 (GRAF1; also known as Oligophrenin-1-Like). GRAF1 is recruited to the membrane tubules derived from the PM via its BAR and Pleckstrin homology (PH) 8 domains (Lundmark, Doherty et al. 2008), and either modifies the activity of small G proteins such as RhoA and Cdc42 (Sabharanjak, Sharma et al. 2002, Jelen, Lachowicz et al. 2009). Previous work from our lab also proposed a model that GRAF1 forms a vesiculation complex comprised of MICAL-L1 and EHD1 on TRE and supports TRE vesiculation (Cai, Caplan et al. 2012, Cai, Xie et al. 2014).

A common characteristic of this dynamin-independent CIE is the involvement of small GTPases, either the Rho family member CDC42 or the Arf family member ARF6.

The CDC42-mediated endosome budding from the PM results in relatively long and wide surface invaginations, compared to small spherical structures generated by the caveolar pathway (Sabharanjak, Sharma et al. 2002), and thus takes up a large volume of fluid phase. The role of CDC42 activation on this CIE pathway is to some extent coupled to the recruitment of actin-polymerization machinery. The other key regulator, ADP-ribosylation factor 6 (Arf6) is a member of an Arf family of GTP-binding proteins. Arf6 stimulates the generation of PIP2 by activating the lipid-modifying enzyme phosphatidylinositol 4- phosphate-5-kinase (PIP5-kinase), and the formation of phosphatidic acid (PA) by activing phospholipase D (PLD). In addition, the changes in membrane lipid composition have a potent role in actin remodeling (Mayor and Pagano 2007). Since both CDC-42 and Arf6 affects the endocytosis of GPI-APs, whether they regulate related or distinct mechanisms of CIE still needs to be determined.

1.2.3 Cargo recognition and segregation for different internalization pathways

Little is known about how cargo is selected for the different endocytic pathways.

For CME, as briefly discussed above, specific adaptor proteins would identify the recognition signal from cytosolic domain of the incoming cargos to initiate the cascades of

CCP formation and fission, thus the CME pathways are highly regulated and the endocytic 9

itineraries of CME cargos are pre-destined. In the cases of different CIE pathways, no such well-defined adaptors have been identified. Furthermore, many cargos internalized via the CIE itinerary are membrane-anchored proteins without a cytoplasmic tail. One prevalent theory with regards to CIE cargo sorting is based on the clustering and association of cargos within micro-domains at the PM.

Regardless of the initial mechanism used to internalize these receptors, most vesicles containing internalized proteins arrive at a common sorting station known as the early/sorting endosome (SE; Figure 1.1) (Naslavsky, Weigert et al. 2003, Jovic, Sharma et al. 2010, Huotari and Helenius 2011), although some receptors such as CD144 and

CD98 bypass the SE (Eyster, Higginson et al. 2009). However, the convergence of cargos at the SE/EE does negate the possibility that the selective lipid/protein marker attached to the cargos prior to endocytosis will be retained in later stages of trafficking and be involved in the post internalization fate of CIE cargos.

With the lack of clear cytoplasmic target signals for CIE cargos, the nanoscale clustering of lipid-tethered proteins such as GPI-APs functions as an intrinsic sorting signal for the CDC42-mediated endocytosis (Sharma, Varma et al. 2004). In a fluorescence resonance energy transfer (FRET)-based assay, it was shown that CtxB unevenly distributed in sub-domains of the PM undergo significant clustering and such clusters led to the formation of non-clathrin-coated invaginations (Parton, Joggerst et al. 1994, Glebov,

Bright et al. 2006). Furthermore, recent studies have shown that glycosphingolipids (GSLs) form clusters in micro-domains of the PM employs the caveolae machinery for internalization (Singh, Liu et al. 2006), while a non-natural GSL analogue that does not form clusters is not taken up in caveolae. Thus far, the basis for this selective internalization is not known, yet as suggested by several studies including the ones described above, it is possible that a “stereochemistry-based sorting” mechanism 10

mediates the preference for different CIE pathways on various sizes of micro-domains and cargo clustering (Madore, Smith et al. 1999, Sharma, Sabharanjak et al. 2002).

In addition to the lipid-based mechanisms, it was reported that ubiquitination of some receptor tyrosine kinases such as epidermal growth factor receptor (EGFR) serves as a protein-based machinery in determining whether the receptor should be internalized via a CME or CIE pathway. Accordingly, ubiquitinated EGFR is shunted from the CME to a cholesterol-sensitive CIE route, by the combined effect of 3 ubiquitin interacting motif

(UIM)-containing proteins: Eps15, epsin and Eps15-related (Eps15R) (Sigismund, Woelk et al. 2005). A recent study on γc-cytokine receptor also revealed the existence of target sequences on its cytoplasmic tail for RhoA- and Arf6-mediated CIE pathways (Birkle, Zeng et al. 2003). Moreover, the integral membrane proteins flotillin-1 and flotillin-2 (also known as reggie-2 and reggie-1) are found with GPI-APs and CtxB in clusters at sites that lack clathrin and caveolin (Stuermer, Lang et al. 2001, Glebov, Bright et al. 2006). Such findings implicate an important role for protein adaptors in cargo selection for CIE pathways; however specific adaptors for these routes remain to be discovered.

1.3 Sorting of endocytic cargo at sorting/early endosomes (SE/EE)

The SE/EE is a compartment where internalized proteins from different endocytosis pathways are converged and constantly undergo sorting. Thus it is not surprising to find it to be a highly dynamic structure usually composed of regions of large vesicles and thin tubular extensions with high propensity to undergo homotypic fusion and fission (Gruenberg, Griffiths et al. 1989, Gruenberg 2001). Cargos sorted to tubular- vesicular regions typically undergo recycling, whereas cargos with post-translational modifications (such as ubiquitination) are often slated for degradation via the late- endosomal/lysosomal (LE, Figure 1.1) pathway (Mellman 1996). The sorting occurred in 11 the SE/EE is well organized and efficiently facilitated by the key proteins and lipid components of SE/EE. Yet the mechanisms by which receptors are trafficked through this organelle have not been fully defined.

Most of the internalized ligands are sorted into LE with an acidic pH (∼5.5), optimal for the degradation pathway. In addition, receptors destined for degradation contain degradative signals, which are usually post-translational modifications (such as ubiquitination) (Figure 1.1). For example, Mono-ubiquitination of one or more lysine residues in the receptor tyrosine kinase epidermal growth factor receptor (EGFR) is sufficient for its sorting to lysosomal degradation (Haglund, Sigismund et al. 2003).

Hepatocyte growth factor-regulated tyrosine kinase substrate (Hrs) containing a ubiquitin interacting motif (UIM) is responsible for the recognition of the mono-ubiquitinated cargos on the SE/EE (Raiborg and Stenmark 2002). In addition, signal transducing adaptor molecule 2 (STAM 2) and Tsg101 subunit of the endosomal sorting complex required for transport (ESCRT-1) form a complex with Hrs for the recognition of ubiquitin signals

(Bache, Brech et al. 2003, Bache, Raiborg et al. 2003). Successful recognition of degradation signals initiates the removal of cargos from recycling membranes into intralumenal vesicles. These vesicles then either mature into LE or undergo invagination and formation of multivesicular bodies (MVBs) (Babst, Katzmann et al. 2002, Hanson and

Cashikar 2012).

Apart from sending cargos to the degradative pathway, the SE/EE compartment also serves as diverging point between the endocytic and biosynthetic routes. Retrograde sorting of cargos starts from receptors clustering in subdomains of SE/EE undergoing retromer-mediated tubulation (Bonifacino and Hurley 2008). The Retromer complex is composed of a sorting nexin (SNX) dimer and the vacuolar protein sorting (Vps) trimer

Vps26-Vps29-Vps35. The SNX dimer contains a PI(3)P-binding Phox domain (PX), which 12 is required for the association with SE/EE membranes, and a curvature-sensing BAR domain, which is necessary for the tubulation of retrograde endosomes (Bonifacino and

Rojas 2006), while the Vps trimer acts as a Rab5 and Rab7 effector (Rojas, van Vlijmen et al. 2008). Active Rab5 on SE/EE recruits a series of Rab5 effectors including a class III phosphatidylinositol 3- phosphate kinase [PtdIns(3)-kinase] that generates phosphatidylinositol 3,5-bisphosphate [PtdIns(3,5)P2] to which the Retromer complex preferentially binds (Rutherford, Traer et al. 2006). To strengthen the binding, Rab5 also recruits a known Rab7 GEF, the VPS C-homotypic fusion and vacuole protein sorting

(HOPS) complex, resulting in Rab7 recruitment and activation (Rink, Ghigo et al. 2005).

Active Rab7 then works together with Rab5 to reinforce the binding of the two subcomplexes to form the retromer cargo-sorting machinery. Our lab has found a

Retromer complex binding partner, EHD1. EHD1 binds to the Rab5 effector Rabankyrin-

5 (Rank-5), and thus interacts with components of the Retromer complex such as Vps26 and Vps35, which retains the integrity of Retromer tubules and facilitates efficient retrograde transport (Gokool, Tattersall et al. 2007). Another EHD family protein member,

EHD3, also regulates the retrograde trafficking and affects Golgi morphology (Zhang,

Naslavsky et al. 2012).

One central function of SE/EE that is of considerable interest is sorting towards the recycling pathway. At least two distinct recycling pathways were described (Maxfield and

McGraw 2004). Receptors are delivered into newly formed extensive tubulation of the EE membranes which subsequently send cargos either directly back to the PM from SE/EE via “fast recycling” (van der Sluijs, Hull et al. 1992, Sheff, Daro et al. 1999), or through a dense compartment composed of vesicular and tubular recycling endosomes called the endocytic recycling compartment (ERC) in a “slow recycling” route. The role of the ERC in endocytic recycling remains unclear, though it has been proposed that the recycling 13 cargos are protected from degradation by concentration into the ERC. Kinetic studies of the recycling rates of a classical recycling receptor, the transferrin receptor (TfR) has demonstrated the existence of both “fast” (t1/2=5 min) and “slow” recycling (t1/2=15-30 min)

(Hopkins 1983) (Figure 1.1). The sorting of cargos into the recycling pathways is exerted through a regulator conversion cascade on the subdomains of the SE/EE where cargos are concentrated. For example, TfR is quickly sorted away from the Rab5-labeled SE/EE subdomain and into a Rab4 subdomain during “fast recycling”. Alternatively, a Rab5-

Rab4-Rab11 conversion occurs on the TfR-containing SE/EE subdomain, facilitating the budding of vesicles carrying TfR to the ERC (Sonnichsen, De Renzis et al. 2000). Other regulatory proteins which exerts their endocytic sorting function by interaction with

Rab4/Rab5 include EEA1, Rabenosyn-5 and Rabptin-5/Rabex-5 complex, which will be discussed in the Section 2 (Vitale, Rybin et al. 1998, de Renzis, Sonnichsen et al. 2002,

Pagano, Crottet et al. 2004, Mattera and Bonifacino 2008). In addition to these well- studied regulators, we have identified EHD1 as an interaction partner of Rabenosyn-5, a bivalent Rab4/Rab5 effector (Naslavsky, Boehm et al. 2004), suggesting that EHD1 exerts its regulatory role on delivering cargos from the SE/EE to the ERC. On the other hand, we have also discovered a novel protein complex EHD1-MICAL-L1-Syndapin2 which resides on and mediates the biogenesis and function of the tubular recycling endosomes (TRE)

(Giridharan, Cai et al. 2013). Depletion of EHD1 results in cargo accumulation at the ERC, indicating its role in mediating the delivery of recycling cargo from the ERC back to the

PM (Naslavsky, Boehm et al. 2004). This complicated effect of EHD1 on the recycling pathways will be addressed in chapter IV of this thesis. It was previously thought that receptors lacking degradation targeting signal would be by default sent to the recycling pathways based on the “geometry-based sorting” mechanism (Gruenberg 2001, Maxfield and McGraw 2004). However, recent studies suggested that Arf6 GTPase-activating protein (GAP), ACAP1, binds to the cytosolic tail of the TfR and serves as a sorting signal 14 for recycling (Dai, Li et al. 2004). In addition to ACAP-1 mediated recycling, alternative recycling sorting motifs also exist that can be transplanted to a non-recycling receptors and induce recycling. For example, phosphorylation of serine 411 of the β2-adrenergic receptor recruits the EBP50 (ezrin-radixin-moesin (ERM)-binding phosphoprotein-50) to regulate recycling of this receptor (Cao, Deacon et al. 1999). Apart from its function as part of a sorting mechanism to the lysosome, Hrs also binds to the cytosolic tail of both the β2-adrenergic and μ-opioid receptors and directs them to a recycling pathway

(Hanyaloglu, McCullagh et al. 2005). In addition to the known sorting machinery, our findings (to be described in chapter IV) suggest a new mode of cargo sorting into different

RE.

1.4 Different routes of endocytic recycling

To maintain the required balance of these molecules at the cell surface, recycling of the internalized molecules is a tightly regulated process. Upon delivery to the SE, internalized receptors can be sorted into one of at least two distinct recycling pathways.

As previously described, recycling pathways have been described as either ‘fast recycling’ or ‘slow recycling,’ depending on whether the recycling cargo is returned to the PM directly from SE, or whether it is first transported to an additional organelle known as the endocytic recycling compartment (ERC), which is often localized near the microtubule organizing center (MTOC) at the perinuclear region of the cell (Grant and Donaldson 2009). Whereas the fast recycling route returns glycosphingolipids to the PM and is regulated by Rab4 (van der Sluijs, Hull et al. 1992, Choudhury, Sharma et al. 2004), many receptors recycle via a

Rab11-mediated pathway that traverses the ERC (Ren, Xu et al. 1998).

In addition to Rab11, a large number of proteins are important regulators for the recycling pathway, including Rab11 effectors FIP2 (Naslavsky, Rahajeng et al. 2006) and 15

FIP5 (Schonteich, Wilson et al. 2008), EHD3, EHD4, arf6 as well as Rab22 (Figure 1.2).

Previous studies revealed that these regulators pose differential effects on the recycling of diverse cargos. For example, some regulatory proteins were identified in the regulation of recycling of CIE cargos, such as Rab22a (Weigert, Yeung et al. 2004), Rab8 (Hattula,

Furuhjelm et al. 2006) , and Arf6 (Radhakrishna and Donaldson 1997, Powelka, Sun et al.

2004). Yet the role of such regulators might not be exclusively on the recycling of cargos from CIE pathway as they also exert regulatory roles on CME cargos such as transferrin

(Hattula, Furuhjelm et al. 2006, Magadan, Barbieri et al. 2006). On the other hand, CME cargos such as Transferrin receptor (TfR) seems to be preferentially mobilized by Myosin

Vb/Rab11a-FIP2/Rab11a complex. Moreover, in HeLa cells, TfR is recycled back to the

PM via recycling endosomes (RE) that are separate from the TRE that carry CIE cargos back to the PM (Radhakrishna and Donaldson 1997, Naslavsky, Weigert et al. 2003,

Naslavsky, Weigert et al. 2004). A growing number of studies have shown that vesicles budding from the PM via different endocytosis pathways may maintain distinct itineraries throughout their intracellular trafficking (Mayor, Parton et al. 2014), and these distinctions potentially lie with the lipid composition of the membranes, which might specifically recruit different sorting machineries that further distinguish the routes for such vesicles. Although the routes for receptor-mediated endocytosis have been intensively studied, little is known about the fates and itinerary of the proteins and lipids after their entry via different internalization mechanisms. Recently, increasing evidence from our work and others has provided support for the notion that cargos from CME and CIE pathways are probably delivered to the ERC through different recycling endosomes (RE) within “slow recycling” despite them entering the ERC, and once the sorting is established, cargos remain in distinct endosomes in the ERC (Figure 12.18).

16

Figure 1.2. Pathways of endocytosis and endocytic recycling.

Itinerary of cargo proteins entering cells by CME (blue cargo) and CIE (red cargo) and subsequent routes of cargo to the EE, ERC and RE is shown. Key regulators of each endocytic pathway are also displayed. Both types of cargo can move from the EE to the ERC by a process mediated by Snx4, dynein, EHD3, Rab10, Rab22a and the Rab11 effectors FIP2, -3 and -5. Rab11 mediates the recycling of both types of cargos, while the recycling of CIE cargos seems to require Rab8-, Rab22a-, or MICAL-L1 decorated TRE, along with the regulation of EHD1/RME-1, Alix and FIP2. This is a composite description of endocytic recycling and all of the components shown here may not be evident in a given cell type. RE = recycling endosome; TRE = tubular recycling endosome; ERC = endocytic recycling compartment.

17

The ERC is considered a morphologically distinct series of membranes from SE and comprises a series of vesicular and tubular structures that enable sorting and recycling of internalized receptors and lipids back to the plasma membrane (Gruenberg and Maxfield 1995). The composition of the ERC has an important bearing on the differential routes for cargos to recycle back to the PM. However, there remains uncertainty regarding the composition of the ERC, with many proponents of the idea that it is a structure surrounded by a contiguous membrane (Mayor, Presley et al. 1993,

Maxfield and McGraw 2004). Electron microscopy tracking of transferrin seemed to suggest that the ERC is a network with tubular-vesicular endosomes (Marsh, Leopold et al. 1995). Because of the limitation of traditional confocal microscopy in resolving condensed, small endosomal structures (around 100 nm in diameter), whether the ERC is composed of mostly a single contiguous membrane, or whether it is a region where highly dense RE carry different cargos and are clustered together remains a difficult question to resolve. The development of super-resolution microscopy (Structured

Illumination Microscopy, SIM and direct Stochastic Optical Reconstruction Microscopy, dSTORM) provides a powerful tool to address such questions. In Chapter IV of this thesis,

I will provide a more in-depth description of our most updated findings with regards to differential sorting to recycling endosomes distinguishing receptors from the CME and CIE pathways.

1.5 The significance of TRE in endocytic recycling

Tubular endocytic carriers play an important role in mediating endocytic trafficking.

For example, CIE cargos including bacterial toxins (Sandvig and van Deurs 1994, Romer,

Berland et al. 2007), major histocompatibility class I (MHC I) (Massol, Larsen et al. 2005), and the Glycophosphatidylinositol-Anchored protein (GPI-AP), CD59 (Naslavsky, Weigert 18 et al. 2004), are internalized in deep tubular invaginations at the plasma membrane (PM)

(Donaldson, Porat-Shliom et al. 2009, Day, Baetz et al. 2015).

In addition, there is another type of tubular endosomes that function at post- internalization stage, delivering cargos in recycling pathway (Cullen 2008). Such tubular structures are known as tubular recycling endosomes (TRE) (Chi, Harrison et al. 2015).

Tubular intermediates play important roles in sorting and cargo selection (Maxfield and

McGraw 2004) due to their high surface-to-volume ratio. Recycling cargos attached to a tubule-generating membrane can be preferentially segregated from cargos distributed to other subdomains on SE/EE (Maxfield and McGraw 2004). Similar mechanisms are also responsible for the exit of receptors from the ERC to the PM. Recently, we identified

MICAL-L1 as a TRE marker that stably resides on the endosomal membrane and facilitates its tubulation and biogenesis (Sharma, Giridharan et al. 2009). MICAL-L1- decorated TRE bear different lipid components than the tubular invaginations at the PM, and MICAL-L1 does not localize to PM tubules labeled by Wheat Germ Agglutinin (WGA)

(Figure 1.1) (Flesch, Voorhout et al. 1998). TRE are crucial for the recycling of internalized receptors and lipids. Based on the observations that depletion of either MICAL-L1 or EHD1 in HeLa cells resulted in the failure of transferrin and β1-integrin to exit the ERC, TRE are previously considered as carriers for cargos returning to the plasma membrane from the

ERC (Jovic, Naslavsky et al. 2007, Donaldson, Porat-Shliom et al. 2009, Sharma,

Giridharan et al. 2009). Yet there is no solid evidence for the directionality of TRE. Studies described in this thesis (Chapter IV) demonstrated that MICAL-L1-decorated TRE can be generated from Rabenosyn-5-containing SE, indicating that TRE are responsible for the movement of cargo from peripheral SE to the perinuclear ERC (see model in Fig. 12.18). 19

2. REGULATORS OF ENDOCYTIC TRAFFICKING

2.1 Overview

Due to the importance of endocytic trafficking, there are many proteins that regulate the intracellular trafficking of cargos, including Rab GTPases, soluble N- ethylmaleimide-sensitive factor attachment proteins receptors (SNARE) fusion machinery proteins, fission proteins, coat proteins, etc.

Rab GTPase proteins are a family of more than 60 small Ras-related GTP-binding proteins that control endocytic transport steps and localize to endocytic organelles (Pfeffer and Aivazian 2004). When bound to GDP, Rabs are cytosolic and typically inactive, whereas GTP-bound Rabs are active and usually associated with endocytic membranes, which recruit effector proteins that regulate lipid content, membrane fusion/fission, and transport along microtubules or microfilaments (Pfeffer and Aivazian 2004).

Another class of important trafficking regulator is the SNARE family of proteins.

SNARE fusion proteins located on vesicles (v-SNARE) couple with SNARE presented on the target membrane (t-SNARE) and provide the required energy for the fusion of the membrane between the travelling vesicles and targets.

Among other key regulators of endocytic transport, C-terminal Eps15 homology domain (EHD) proteins, a family of four highly homologous membrane-associated

ATPases, regulate membrane tubulation and vesiculation along with their binding partners, and are required for the efficient transport of cargo proteins between endocytic compartments (Naslavsky and Caplan 2011). The members of EHD protein family, EHD1-

4, display high sequence homology, yet they play different roles in the endocytic pathways.

EHD1 and EHD3 are both recruited to endocytic membranes through their interactions with molecule interacting with CasL-like1 (MICAL-L1). MICAL-L1 and the EHDs localize 20 to a unique array of tubular recycling endosomes (TRE) that span a region from the cell periphery to the perinuclear ERC (Cai, Giridharan et al. 2013, Giridharan, Cai et al. 2013).

2.2 Rab proteins and their effectors

Among a wide array of proteins regulate the endocytic trafficking pathway, the small GTP-binding Ras-associated binding (Rab) proteins play a critical role. During one

Rab GTPase cycle, inactive GDP-bound Rabs are initially associated with Guanosine nucleotide dissociation inhibitors (GDIs) and retained in the cytosol. The GDI displacement factor (GDF) release them from GDI molecules and allows their recruitment to the endosome membrane. Upon GTP binding, Rabs can interact with a series of specific protein effectors including adaptors, tethering molecules, kinases, phosphatases, and motor proteins to carry out their regulatory functions on endosome fission, fusion and tethering. The activated Rabs can be inactivated by their specific GTPase-activating proteins (GAPs) through facilitating GTP hydrolysis, which drives Rab molecules back to the cytosol and concludes a Rab cycle (Grosshans, Ortiz et al. 2006). There are over 60

Rab proteins that function in different endocytic pathways, principally regulating endosome functions including vesicle tethering, fusion, budding and motility (Zerial and

McBride 2001, Deneka and van der Sluijs 2002, Pfeffer and Aivazian 2004, Grosshans,

Ortiz et al. 2006).

The Rab proteins recruited to the SE/EE mediate the function of post- internalization sorting, and include Rab4, Rab5, Rab10, Rab11 and Rab22 (van der Sluijs,

Hull et al. 1992, Daro, van der Sluijs et al. 1996, Babbey, Ahktar et al. 2006, Magadan,

Barbieri et al. 2006). Rab5 is the most extensively analyzed Rab of the early endocytic pathway (Gorvel, Chavrier et al. 1991, Bucci, Parton et al. 1992, Barbieri, Roberts et al. 21

1996, Zerial and McBride 2001, Grosshans, Ortiz et al. 2006). It regulates entry of the cargo from the PM to the SE/EE, generation of phosphotidylinositol-3-phosphate (PI3P) lipid which is enriched on EE (Christoforidis, Miaczynska et al. 1999, Murray, Panaretou et al. 2002), homotypic fusion (Gorvel, Chavrier et al. 1991) and the motility of EE on actin and microtubules tracks (Nielsen, Severin et al. 1999, Pal, Severin et al. 2006). In addition, it also functions in activating signaling pathways from EE (Benmerah 2004, Miaczynska,

Christoforidis et al. 2004, Schenck, Goto-Silva et al. 2008). The active or GTP-bound Rab5 is generated at the EE by the action of Rabex-5 which acts as a guanine exchange factor

(GEF) for Rab5 (Horiuchi, Lippe et al. 1997). Active Rab5 is sufficient to recruit effector proteins to the EE where they can carry out their specialized functions in trafficking and sorting (Grosshans, Ortiz et al. 2006). Several key Rab5 effectors include phosphatidylinositol 3-phosphate kinase/hVPS34/p150 (Christoforidis, Miaczynska et al.

1999), Early endosomal antigen-1 (EEA1) (Merithew, Stone et al. 2003), Rabenosyn-5

(Nielsen, Christoforidis et al. 2000) and Adaptor protein containing PH domain (APPL1 and APPL2) (Miaczynska, Christoforidis et al. 2004). The activation of PI3K results in the generation of the most prominent phospholipid, PI3P, on the SE/EE membranes, which I will discuss more thoroughly in section 3.1 (Siddhanta and Shields 1998, Gillooly, Morrow et al. 2000). EEA1 and Rabenosyn-5 are both recruited by binding to PI3P through their

FYVE domain. EEA1 subsequently recruits Syntaxin 13 and Syntaxin 6, which accelerate the fusion of EE with recycling endosomes and the Golgi, respectively (McBride, Rybin et al. 1999, Simonsen, Gaullier et al. 1999). Rabenosyn-5, on the other hand, interacts with human vacuolar protein sorting 45 (hVps45), which then binds to SNARE proteins and mediates vesicular fusion similar to EEA1 (Naslavsky, McKenzie et al. 2009). Rabenosyn-

5 also interacts with members of the C-terminal Eps15 homology domain (EHD)- containing protein family, the EHD1 and EHD3 (which will be discussed in the section 2.4)

(Naslavsky, Boehm et al. 2004), and thus highlighting the significance of mediating 22 receptor recycling from RE back to the PM through these interactions. In addition to Rab5 and its effectors, Rab4 is also localized to EE (van der Sluijs, Hull et al. 1992). Unlike

Rab5, Rab4 regulates the direct exit of recycling cargo containing vesicles from SE/EE to the PM, as well as the sorting of these cargos to the endocytic recycling compartment

(ERC) (van der Sluijs, Hull et al. 1992, Sheff, Daro et al. 1999).

Rab7 is a major player that mediates the maturation of SE/EE to late endosomes, accompanied by the exchange of Rab5 to Rab7 (Peralta, Martin et al. 2010). It is recruited to the EE membrane via homotypic fusion and vacuole protein sorting (HOPS) subunit

Vam6p/Vps39 (Caplan, Hartnell et al. 2001) (Wurmser, Sato et al. 2000). Vps39 interacts with Mon1 which binds to Rab5-GTP, displacing Rabex 5 from the membrane, and together with another protein, Czi1, recruits Rab7. The Mon 1-Ccz 1 complex prevents

Rab5 re-activation as well as promotes Rab7 activation (Nordmann, Cabrera et al. 2010),

EE thus mature into LE. An Rab7 effector, Rab7-interacting lysosomal protein (RILP), interacts with Rab7 on LE and lysosomes (Cantalupo, Alifano et al. 2001). It induces the recruitment of dynein-dynactin motor complexes to Rab7 containing LE, transporting it towards the minus end of microtubules (Jordens, Fernandez-Borja et al. 2001). The HOPS complex interplays with Rab7 as well as SNARES (Stroupe, Hickey et al. 2009), mediating

LE membrane fusion and tethering.

The Rab family proteins that regulate recycling pathways include Rab11 and Rab4, as well as Rab21, Rab22, Rab8, Rab15, and Rab35 (Grosshans, Ortiz et al. 2006, Grant and Donaldson 2009, Hsu and Prekeris 2010). Various Rab effectors also control recycling

(Grosshans, Ortiz et al. 2006), some of which interact with EHD1, another known regulator of endocytic recycling (Naslavsky and Caplan 2011). Rab4, as mentioned above, is most well-known as a regulator for the fast recycling routes as it escorts cargo-containing endosomes back to the PM upon sorting from Rab-5 positive SE/EE. On the other hand, 23 sorting of cargos into Rab4/Rab11 micro-domains of SE/EE leads to their delivery to the

RE (Sonnichsen, De Renzis et al. 2000). Rab11, by binding to its effector, Rab11 family- interacting protein 5 (FIP5) and Rab11-FIP2, recruits important players of the slow recycling that drives transportation from EE to RE respectively, including Kif3B, a component of the kinesin II motor protein (Schonteich, Wilson et al. 2008), myosin Vb

(Roland, Kenworthy et al. 2007), EHD1 and EHD3 (Naslavsky, Rahajeng et al. 2006).

Rab35, like Rab4, also mediates the fast recycling of receptors, such as MHC I (Sato,

Sato et al. 2008, Allaire, Marat et al. 2010). Furthermore, a Rab35 effector, molecule interacting with CasL-like 1 (MICAL-L1), can be localized to the tubular recycling endosomes membrane by Rab35 and ADP-ribosylation factor 6 (Arf6), and serves as a scaffolding proteins connecting various regulators to these TREs for efficient trafficking

(Giridharan, Cai et al. 2012). Apart from these molecules, various types of Rab proteins play different, albeit somewhat interconnected roles in the course of slow recycling. For instance, Rab8 is recruited onto TRE membranes by MICAL-L1 (Huber, Pimplikar et al.

1993) and is involved in the Rab11-Rab8-MyosinVb machinery to recycling cargos from the ERC to the PM (Roland, Kenworthy et al. 2007). It also regulates the transportation from the TGN to the PM in Madin-Darby canine kidney (MDCK) cells (Huber, Pimplikar et al. 1993). Rab22a decorates tubular structures emanating from the ERC, where its activity mediates tubule generation as well as the final fusion of recycling membranes with the surface (Weigert, Yeung et al. 2004). Interestingly, Rab22a seems to preferentially effect the recycling of MHC I, whereas it only modestly affects transferrin trafficking, implicating potential cargo selection by Rab22a within the slow recycling pathways.

Apart from Rab proteins, there is another family of guanine nucleotide-binding proteins that regulate organelle dynamics and membrane trafficking, the ADP-ribosylation factor (ARF) proteins. The Arf proteins are controlled by a similar GTPase cycle to that of 24

Rabs through their specific GEFs and GAPs (Donaldson and Jackson 2000), with the exception of Arf6, whose attachment to membranes seems not affected significantly by its nucleotide status. Among the different categories of ARF proteins, class I (Arf1, Arf2, Arf3)

Arf proteins mediate significant trafficking between ER-to-Golgi (D'Souza-Schorey and

Chavrier 2006), while class III (Arf6) stimulates endocytosis (macropinocytosis) by promoting the formation of membrane invagination, and facilitates the membrane recycling of cargos back to the PM (Naslavsky, Weigert et al. 2003). Arf6 function is more closely associated with membrane lipid modifications and modulation of the actin cytoskeleton, by activation of type I phosphatidylinositol 4-phosphate 5-kinase (PIP5K) for generating

PIP2, a major PM phosphoinositide involved in membrane traffic and actin rearrangements (Czech 2003, Yin and Janmey 2003). Arf6 also activates phospholipase

D (PLD) (Melendez, Harnett et al. 2001, Powner, Hodgkin et al. 2002), an enzyme that hydrolyzes phosphatidic acid (PA), leading to regulated secretion, stimulated membrane ruffling, and other PLD-activity related events (Caumont, Galas et al. 1998, Dana, Eigsti et al. 2000, O'Luanaigh, Pardo et al. 2002). Interestingly, Arf6 regulates the internalization of certain types of cargos, including MHC I, G protein-coupled receptors, E-cadherin and

β1-integrin at the PM (Radhakrishna and Donaldson 1997, Brown, Campbell et al. 2001,

Naslavsky, Weigert et al. 2003, Houndolo, Boulay et al. 2005). Moreover, Arf6 forms a ternary complex with Rab11, Rab11-FIP3 and Rab11-FIP4 (Fielding, Schonteich et al.

2005) and regulates the recycling of β1-integrin (Powelka, Sun et al. 2004). The overexpression of dominant negative Arf6Q67L impairs membrane recycling but not internalization. Arf6 functions with EHD1 to mediate the MHC I-containing TRE (Caplan,

Naslavsky et al. 2002), and controls the localization of Rab8 and MICAL-L1 to tubular membranes (Rahajeng, Giridharan et al. 2012).

25

2.3 SNARE proteins

Soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNARE) are highly conserved membrane proteins with a SNARE motif that mediate fusion events in membrane trafficking pathways (Bennett 1995, Sollner 1995, Fasshauer 2003). The

SNARE motif is a helix-forming unit which contains an evolutionarily conserved domain of

60-70 amino acids with heptad repeats. When the SNARE protein on the vesicle membrane (v-SNARE) interacts with the corresponding SNARE on the target membrane

(t-SNARE), the SNARE motifs associate to form a four-helical bundle with high stability, providing the energy required for membrane fusion (Sutton, Fasshauer et al. 1998, Chen and Scheller 2001). Examples of SNARE proteins involved in SE/EE homotypic fusion includes syntaxin13, Vps 10p tail interactor 1 (vti1a), syntaxin6, and VAMP4 (Brandhorst,

Zwilling et al. 2006, Zwilling, Cypionka et al. 2007), which is specifically recruited onto the

SE/EE membrane by upstream targeting proteins such as the Rab5 effector and tethering protein EEA1 (McBride, Rybin et al. 1999, Simonsen, Gaullier et al. 1999). Treatment with the PtdIns(3)P-kinase inhibitor, Wortmannin, blocks the recruitment of EEA1 to the SE/EE membrane, and thus inhibits the SNARE machinery targeting, as a consequence, endosome fusion is impaired (Patki, Virbasius et al. 1997). After catalyzing homotypic fusion, the SNARE complex is dissociated from the SE/EE membrane facilitated by the

AAA+ (ATPases Associated with diverse cellular Activities) protein N-ethylmaleimide- sensitive factor (NSF) (Mayer, Wickner et al. 1996, Hanson and Whiteheart 2005). SNARE complex dissociation is initiated by binding of 3 molecules of soluble NSF attachment protein (SNAP) with the helical SNARE bundle, followed by recruitment of NSF to the

SNARE complex to dissociate the complex and the four-helical SNARE bundle using the energy derived from the hydrolysis of 3-6 ATP molecules (Sollner 1995).

26

2.4 C-terminal EH domain containing proteins (EHDs) and their interaction partners

Our lab focuses on the four members of the C-terminal Eps15 Homology Domain

(EHD) proteins, EHD1-4 (Figure 2). The mammalian EHD proteins share a high level of sequence identity (approximately 70%-80%), yet have very different functions within the endocytic trafficking pathway.

EHD1 is the best characterized of the four mammalian EHD proteins. It is unique in its localization to tubular-vesicular membranes involved in endocytic recycling (Grant and Caplan 2008, Naslavsky and Caplan 2011). EHD1 regulates the recycling of multiple receptors that are internalized both through clathrin-coated pits and independent of clathrin, including transferrin receptor (TfR) (Lin, Grant et al. 2001), major histocompatibility complex class I proteins (MHC I)(Caplan, Naslavsky et al. 2002), the insulin-regulated GLUT4 transporters (Guilherme, Soriano et al. 2004), the cystic fibrosis transmembrane conductance regulator (Lin, Grant et al. 2001), AMPA type glutamate receptors (Park, Penick et al. 2004), MHC class II molecules (Walseng, Bakke et al. 2008), the hyperpolarization-activated cyclic nucleotide-gated (HCN) ion channel family members HCN1, HCN2 and HCN4 (Hardel, Harmel et al. 2008), G-protein-activated inwardly rectifying potassium channels (Chung, Qian et al. 2009), and the calcium- activated potassium channel KCa2.3 (Gao, Balut et al. 2010), and other channels

(Guilherme, Soriano et al. 2004). β1 integrins are also subject to EHD1 regulation (Jovic,

Naslavsky et al. 2007). The interaction of EHD1 with Rab11-FIP2 (Naslavsky, Rahajeng et al. 2006), and its localization to peripheral endosomes and functional relationship with 27

Figure 2 Regulation of endocytic transport by EHD proteins, Rabs and their effectors.

Internalized receptors converge at the SE/EE and are sorted there into one of at least four pathways. EHD1 and/or EHD3 mediates retrograde transport of some receptors (such as shiga toxin) from SE/EE to the Golgi apparatus. Alternatively, EHD3 facilitates the cargo sorting into the perinuclear ERC in the recycling pathway, while EHD1 plays a key role in both shuttling from SE/EE to the ERC and recycling back to the PM. EHD1/EHD3 interaction partner MICAL-L1 as well as other Rab proteins (Rab5, Rab11, Rab8a and their effectors) also exert a regulatory effect on this pathway. EHD2 locates to the PM and regulates the caveolae-mediated endocytosis. Receptors slated for degradation are sorted to EE subdomains containing EHD4 and transported to late endosomes and lysosomes. (image used with permission from (Naslavsky and Caplan 2011)) 28

Rab35 and connecdenn (Sato, Sato et al. 2008, Allaire, Marat et al. 2010) suggest that

EHD1 also plays a role in the transport of receptors from EE to the ERC. EHD1 is linked to dynein motors via the collapsin response mediator protein-2 (Crmp2) and thus drives the cargo transportation from the SE/EE to ERC (Rahajeng, Giridharan et al. 2010).

Although in vitro experiments show that EHD proteins are capable of inducing lipid tubulation (Daumke, Lundmark et al. 2007, Pant, Sharma et al. 2009), studies in cells seem to indicate an opposing effect of the EHD proteins. Recent studies of the EH domain of EHD1 and the crystal structure of EHD2 have suggested that they serve as dynamin- like ATPases which play an important role in the membrane scission process (Lee, Zhao et al. 2005, Daumke, Lundmark et al. 2007, Jakobsson, Ackermann et al. 2011, Cai,

Caplan et al. 2012). In agreement with this notion, purified EHD1 added to an ATP containing semi-permeablization system promotes the generation of vesicular endosomes

(Cai, Giridharan et al. 2013). Additionally, the nuclear magnetic resonance solution structure of the EHD1 EH domain (Kieken, Jovic et al. 2007) led to the identification of novel interaction partners containing asparagine–proline–phenylalanine (NPF) motifs followed by acidic residues that selectively interact with the positively charged EH domain electrostatic surface area (Henry, Corrigan et al. 2010, Kieken, Sharma et al. 2010).

Based on the binding motif prediction, we have discovered an additional protein,

MICAL-L1, which also decorates these tubular membrane structures. MICAL-L1 directly interacts with the EHD1 EH-domain through one of its asparagine-proline-phenylalanine

(NPF) motifs, and coordinates multiple events that shape the membranes during endocytic transport (Sharma, Giridharan et al. 2009, Giridharan, Cai et al. 2013). MICAL-L1 interacts with many essential membrane regulators to the TRE, including Rab8 (Sharma, Jovic et al. 2009) and proteins that contain Bin–Amphiphysin–Rvs (BAR) domains, which would induce membranes to form highly curved shapes (McMahon and Gallop 2005, 29

Zimmerberg and Kozlov 2006), including the N-BAR protein Amphiphysin/Bin1 (Pant,

Sharma et al. 2009), and the F-BAR protein Syndapin2 (or PACSIN2) (Braun, Pinyol et al.

2005, Giridharan, Cai et al. 2013). MICAL-L1 and Syndapin2 preferentially binds to an essential lipid component of the TRE tubules, PA; accordingly, generation of PA is crucial for the promotion of F-BAR-induced TRE biogenesis (Giridharan, Cai et al. 2013). EHD1 is subsequently recruited to this complex on TRE where fission is facilitated, giving rise to newly formed recycling endosomes containing cargos sorted to and from the ERC.

Along with the growing agreement for EHD1 as a vesiculator of TRE (Jakobsson,

Ackermann et al. 2011), there are other EHD1 interaction partners also involved in the fission process. For example, the GTPase Regulator Associated with Focal adhesion kinase-1 (GRAF1; also known as Oligophrenin-1-Like) interacts with both EHD1 and

MICAL-L1 and serves as a vesiculator (Cai, Xie et al. 2014). Another lipid-modifier enzyme, cPLA2α, directly interacts and cooperates with EHD1, and thus supports the scission process (Cai, Caplan et al. 2012).

It is worthy of mention that, in addition to its role in recycling, EHD1 also mediates the internalization of the low density lipoprotein receptor (LDLR) (Naslavsky, Rahajeng et al. 2007) and the L1/neuron-glia cell adhesion molecule (NgCAM)(Yap, Lasiecka et al.

2010). Furthermore, EHD1 interacts with members of the retrograde complex Vps35-

Vps26-Vps29, stabilizing the SNX-1 tubules, and thus regulates retrograde transport from

SE/EE to the Golgi apparatus (Gokool, Tattersall et al. 2007). Recent studies have also revealed a role for EHD1 on mitosis and cilligenesis (Lu, Insinna et al. 2015, Reinecke,

Katafiasz et al. 2015), and implicated a potential relationship between vesicular trafficking and these cellular events. 30

EHD2 appears to be a unique member of EHD family, displaying only 70% sequence identity with EHD1 (compared to 86% and 74% displayed by EHD3 and EHD4, respectively). The complete EHD2 structure has been solved (Daumke, Lundmark et al.

2007), indicating its role in nucleotide-dependent membrane remodeling in vivo. EHD2 regulates a variety of important functions that include sarcolemmal repair (Marg, Schoewel et al. 2012), myoblast fusion (Doherty, Demonbreun et al. 2008, Posey, Pytel et al. 2011), and control of Rac1 and the actin cytoskeleton (Benjamin, Weidberg et al. 2011, Stoeber,

Stoeck et al. 2012). EHD2 poses a very different regulatory role in endocytic trafficking compared to the other members in the EHD family. It is recruited to the PM by preferentially binding to phosphatidylinositol (4,5)-bisphosphate [PI(4,5)P2] (Simone,

Caplan et al. 2013) and regulates caveolar mobility (Moren, Shah et al. 2012). Previous studies from our lab suggested that the EHD2 NPF phenylalanine residue is crucial for

EHD2 localization to the plasma membrane, whereas the proline residue is essential for

EHD2 dimerization and binding (Bahl, Naslavsky et al. 2015). In addition, EHD2 interacts with EH-domain-binding protein 1 (EHBP1) (Guilherme, Soriano et al. 2004), implicating a functional redundancy with EHD1(George, Ying et al. 2007), as well as a regulatory effect on the internalization of TfR and GlUT4, potentially by linking clathrin-dependent endocytosis to filamentous actin via the EHBP1 CH domain (Guilherme, Soriano et al.

2004).

EHD3 shares the highest degree of homology with EHD1 and forms a hetero- dimerization with EHD1 (Galperin, Benjamin et al. 2002). In the contrary to EHD1, EHD3 does not appear to play a major role in regulating the exit of cargos from the ERC to the

PM, since its absence results in cargos accumulating in the peripheral SE/EE and the failure to reach the ERC (Naslavsky, Rahajeng et al. 2006). There is no precise definition of EHD3’s role on the tubular membrane. On one hand, in vitro experiments demonstrated 31 that EHD3 is required for membrane tubulation (Henmi, Oe et al. 2016), on the other, our most recent findings suggest that EHD3 stabilizes the newly synthesized MICAL-L1- decorated TRE (unpublished data). In some cases, EHD3 displays a certain degree of functional redundancy to EHD1. For example, EHD1 and EHD3 are required for the formation of ciliary vesicles (Lu, Insinna et al. 2015). EHD3 can also cooperate with EHD1 and Rab8a to interact with myosin Vb motors (Roland, Kenworthy et al. 2007). EHD3 is also involved in EE-to-Golgi retrograde trafficking and maintenance of Golgi morphology

(Naslavsky, McKenzie et al. 2009). Interestingly, while EHD1 interacts with Rabankyrin-5

(Zhang, Reiling et al. 2012), which mediates the subcellular localization of the Retromer trimer via binding to Vps26 and Vps35, EHD3 does not bind Rabankyrin-5, despite the fact that they share a high level of amino acid homology; thus the precise rolse of EHD1 and EHD3 in retrograde transport need to be dissected.

EHD4 is primarily localized to SE/EE and has been implicated in the regulation of cargo transport from SE/EE to both the ERC and the lysosomal degradation pathway

(George, Ying et al. 2007, Sharma, Naslavsky et al. 2008). Studies in neuronal cells haves suggested that EHD4 functions upstream to EHD1, regulating the internalization of TrkA and TrkB nerve growth factor receptors and an active fragment of Nogo-A, a highly potent inhibitor of axonal growth (Shao, Akmentin et al. 2002, Valdez, Akmentin et al. 2005, Joset,

Dodd et al. 2010). EHD4 also interacts with EHD1 to control the endocytosis of L1/NgCAM

(Yap, Lasiecka et al. 2010).

32

3. MEMBRANE MODELING IN ENDOCYTIC TRAFFICKING

3.1 Role of lipid components in endocytic trafficking

Membrane lipids are essential for the regulation of endocytic trafficking, by selectively recruiting lipid-binding-domain-containing proteins (Simonsen, Wurmser et al.

2001, Gruenberg 2003). For example, phosphatidylinositol 3-phosphate (PI3P) recruits trafficking-related effectors such as Rab5, EEA1 and Rabenosyn-5 to the early endosomal membrane (Burd and Emr 1998, Gaullier, Simonsen et al. 1998, Patki, Lawe et al. 1998), and phosphatidylinositol 4,5-bisphosphate (PIP2) binds to the pleckstrin-homology (PH) domain of important endocytic regulators such as dynamin2 and GRAF1 (McLaughlin,

Wang et al. 2002). The selective recruitment of proteins to distinct sites is ensured by the localized regulation of the level of lipid components, thus lipid-modifying enzymes might play a central role in endocytic trafficking.

Phospholipids may serve as an essential part of the machinery driving the fusion and/or fission of membranes, based on their shape and geometric features, and therefore may play a role in the recruitment of key endocytic regulators, induction of membrane curvature, budding and generation of vesicles, membrane fusion and tethering, etc. In this section, the endocytic function of some important membrane lipid components will be discussed.

3.1.1 Role of phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2]) on endocytosis

The phosphoinositide PI(4,5)P2 is a major lipid component of the PM and a key regulator of the formation, scission and uncoating of clathrin-coated vesicles (Jost,

Simpson et al. 1998), by specifically recruiting PI(4, 5)P2 binding proteins to the PM such as epsin, clathrin assembly lymphoid myeloid leukemia protein (CALM), and AP180 (Kay, 33

Yamabhai et al. 1999, Ford, Pearse et al. 2001, Itoh, Koshiba et al. 2001). These proteins are important in the initial assembly of the clathrin lattice. AP180 then recruits the clathrin machinery adaptor AP2 onto the PM in cooperation with PI(4,5)P2.

Proteins essential for scission of CCVs also interact with PI(4,5)P2, such as the

GTPase dynamin. Moreover, it is likely that the consumption of PI(4,5)P2 is also crucial for late stages of invagination and scission of CCVs. Indeed, a lipid-modifying enzyme, synaptojanin, which dephosphorylates PI(4,5)P2 into PI4P, is indispensable for the uncoating of CCVs (Cremona, Di Paolo et al. 1999, Harris, Hartwieg et al. 2000).

Recent data indicate that the activation of the class I PI3K and the PI-specific phospholipase C (PI-PLC) is essential for constitutive micropinocytosis. This enzyme cleaves PI(4,5)P2 into diacylglycerol (DAG) and inositol 1,4,5-trisphosphate (Amyere,

Payrastre et al. 2000). Studies using the PI(4,5)P2-binding pleckstrin homology (PH) domain of PLCδ1 as a probe show that there is a local rapid accumulation of PI(4,5)P2 at the phagosomal invagination, which disappears upon recruitment of PLC and local formation of DAG (Botelho, Teruel et al. 2000). PI(4,5)P2 can bind and release actin- binding proteins, leading to free actin ends and actin polymerization (Tolias, Hartwig et al.

2000). PI(4,5)P2 also promotes actin polymerization to form motile comet tails on vesicles enriched in cholesterol-containing rafts (Rozelle, Machesky et al. 2000). The multiple functions of PI(4,5)P2 in endocytosis, phagocytosis, macropinocytosis and vesicle motility imply that specific mechanisms must exist to ensure a localized formation and turnover of this lipid.

34

3.1.2 Role of phosphatidylinositol 3-phosphate (PI3P) on SE/EE function and

autophagy

The SE/EE compartment is enriched in PI3P (Gillooly, Morrow et al. 2000), which is generated by phosphatidylinositol 3-phosphate kinase (PI3K) as an effector of active small GTPase Rab5 (Christoforidis, Miaczynska et al. 1999). This important type of EE phospholipids explicitly enlist FYVE domain-containing PI3P binding proteins such as

EEA1 and Rabenosyn-5 or PX domain containing proteins such as SNX1 (Nielsen,

Christoforidis et al. 2000, Raiborg, Bremnes et al. 2001, Cozier, Carlton et al. 2002). These proteins are responsible for almost all the SE/EE sorting events described in section 1.3, thus the generation of PI3P is essential for SE/EE functions. One of the proteins recruited by PI3P at the SE/EE is a FYVE finger-containing phosphoinositide kinase (PIKFyve), which catalyze the conversion of PI3P to phosphatidylinositol (3,5)-bisphosphate [PI(3,

5)P2] (Cabezas, Pattni et al. 2006), a phosphoinositide that is enriched on late endosomal membranes (Ikonomov, Sbrissa et al. 2003). The level of PI3P and PI(3, 5)P2 on EE determines the lipid environment and can favor homotypic fusion or endosomal fission

(Shisheva 2008). Knockdown of PIKFyve results in a delay in fluid-phase transport to late endosomes and impairs EE-to-Golgi retrograde trafficking (Rutherford, Traer et al. 2006).

This is probably because that PIKFyve is required for maintenance of EE morphology and exit from SE/EE to late endosomes by regulating PI3P and PI(3, 5)P2. Another PI3P consuming enzyme recruited onto SE/EE is the lipid phosphatase MTM1, a member of the myotubularin family of proteins (Blondeau, Laporte et al. 2000, Begley and Dixon 2005,

Cao, Laporte et al. 2007). Similarly, depletion of MTM1 impairs the sorting of EGFR to late endosomes (Cao, Backer et al. 2008). This highlights the importance of maintaining the overall balance of PI3P levels in the cell for proper sorting to occur and suggests that a highly coordinated mechanism between PI3K and phosphatases must exist at the EE. 35

Autophagy is a transport pathway that functions both in yeast and mammalian cells to deliver cytosolic proteins and organelles to the lumen of the vacuole/lysosome for hydrolase-mediated degradation. The autophagy process is characterized by selection and sequestration of cargos in autophagosomes, fusion of autophagosomes and lysosomes, and the subsequent lysosomal degradation of cargos. Vps34 PI3K is shown binding to autophagy-related proteins Vps30/Apg6 and Apg14 (Kihara, Noda et al. 2001), implicating a role for PI3P in autophagy. Interestingly, in mammalian cells it has been demonstrated that the PI3K inhibitor wortmannin impairs autophagy and that the exogenous addition of PI3P increases the rate of autophagy (Petiot, Ogier-Denis et al.

2000). Future work will need to be directed at defining effectors of PI3P in the autophagic pathway.

3.1.3 Role of phosphatidic acid in endocytic trafficking

Phosphatidic acid (PA) is not a prevalent phospholipid on endosome membranes, yet it exerts key endocytic regulatory functions as revealed by recent studies. Generally, there are 3 pathways mediating the generation of PA in mammalian cells: 1) lysophosphatidic acid can be converted to PA by the transfer of an acyl chain by enzymes known as lysophosphatidic acyl transferases (LPAT), and acylglycerol phosphate acyltransferases (AGPAT) (reviewed in (Shindou, Hishikawa et al. 2009)); 2) the removal of the head group of glycerphospholipids such as phosphatidylcholine by phospholipase

D (PLD) (Donaldson 2009); 3) the phosphorylation of diacylglycerol through the action of diacylglycerol kinases (DGK) (reviewed in (Merida, Avila-Flores et al. 2008)).

Among other better-studied phospholipids, the involvement of phosphatidic acid

(PA) has not been well characterized in endocytic transport. Possible mechanisms by 36 which local PA carries out endocytic trafficking regulation include: 1) PA-enriched membranes with higher curvature tend to undergo fission; (Kooijman, Chupin et al. 2003)

2) it serves as a docking site for recruiting specific proteins such as Rab coupling proteins

(RCP) (Rainero, Caswell et al. 2012), Molecules Interacting with CAsL-Like1 (MICAL-L1)

(Giridharan, Cai et al. 2013), and Sorting nexin 27 (SNX27) (Rincon, Santos et al. 2007) to the membrane; 3) it is an intermediate for PI re-synthesis (Shulga, Topham et al. 2011).

The Small cross-sectional area of the phosphomonoester headgroup of phosphatidic acid (PA) relative to the DAG backbone gives pA a cone-shape structure, and suggested that membrane domains enriched with this acidic phospholipids tend to undergo membrane fusion, in conjunction with Ca2+ (Burger 2000). However, the shape of PA varies greatly under different Ca2+ concentrations. Unsaturated PA has a cylindrical, bilayer-preferring structure under normal cytoplasmic conditions (37°C, pH 7.2, 0.5 mM free Mg2+); but at the mildly acidic intra-Golgi conditions (pH 5.9-6.6, 0.3 mM Ca2+), it displays a conical (type-II) shape (Kooijman, Chupin et al. 2003), which is prone to form a highly curved membrane facilitating the fission process.

Recent studies have suggested the involvement of PA in the secretory pathway.

For example, DGK-generated PA is required for the induction of neutrophil exocytosis from azurophilic granules by anti-neutrophil cytoplasmic antibodies (ANCAs), since treatment with DGK inhibitors reduces granule release by inhibiting granule fusion at the plasma membrane (Holden, Savage et al. 2011). In this study, the addition of PA restored the release of myeloperoxidase (MPO) in DGK-inhibited cells, whereas supplementing cells with DAG failed to restore exocytosis. These findings, along with in vivo studies, collectively indicate that ANCA-driven granule exocytosis is mediated by DGK-generated

PA (Williams, Pettitt et al. 2007). Interestingly, only certain PA derived from select pathways is involved in governing this secretory process, since PLD-derived PA does not 37 play a role in this process (Siddiqui and English 1996, Dickey and Faller 2008). Currently, there is incomplete agreement over the involvement of DGK-generated PA in regulating secretion from the Golgi. On one hand, since PA is a direct product of DGK’s activity on

DAG and the up-regulation of DAG at the Golgi does not lead to a concomitant PA level increase, this implies that DGK might not be involved in this mechanism (Sarri, Sicart et al. 2011), or that PA is rapidly transformed into other phospholipids. On the other hand, a study shows that DGK activity on PA production, rather than the consumption of DAG, regulates nascent vesicle secretion from the TGN (Siddhanta and Shields 1998).

Another role implicated for PA in secretion relates to the maintenance of Golgi morphology, which is an organelle found in most eukaryotic cells responsible for the processing, sorting and transporting of proteins and lipids. The formation of Golgi-to- plasma membrane transport carriers is accomplished via budding, elongation, constriction, and finally fission of the Golgi membrane. These events are facilitated by lipid bilayer deformation as well as the concerted efforts of many proteins that act at the various stages of secretion (Bard and Malhotra 2006). Studies suggest that a lipid micro-domain containing interconverting LPA, PA and DAG has the potential to drive membrane fission through changes in membrane deformation at the Golgi apparatus, thus mediating the

Golgi fission (Kooijman, Chupin et al. 2003). Several PA generating proteins have been identified that may induce fission at the Golgi apparatus: CtBP3/BARS and endophilin facilitate the conversion of LPA to PA (Schmidt, Wolde et al. 1999, Weigert, Silletta et al.

1999), PLD mediates the generation of PA (Ktistakis, Brown et al. 1996, Chen, Siddhanta et al. 1997), and protein kinase D (PKD) binds to diacylglycerol (DAG) (Bossard, Bresson et al. 2007).

Recent studies from our lab and others have suggest a novel role of PA in efficient recycling of internalized receptors back to the plasma membrane. Decreasing cellular PA 38 with inhibitors of PLD or DGK leads to delayed TfR recycling (Giridharan, Cai et al. 2013).

DGKα-derived PA binds and recruits the Rab-coupling protein (RCP) to the tips of invasive pseudopods. Since RCP is required for the recycling of α5β1 integrin during cell migration

(Caswell, Chan et al. 2008), DGKα is essential for RCP to drive the recycling of α5β1 integrin (Rainero, Caswell et al. 2012). Another effect of DGK-derived PA in endocytic recycling lies in the regulation of TRE that are decorated by MICAL-L1 and Syndapin2.

MICAL-L1 and Syndapin2 promote the biogenesis of TRE (thus regulating TRE function), and they are both recruited to the TRE membrane through direct interactions with PA

(Giridharan, Cai et al. 2013). DGKα-derived PA mediates the recycling of Major

Histocompatibility Complex Class I (MHC I) without affecting its internalization. In addition, the MICAL-L1-decorated TRE are disrupted upon DGKα-depletion, leading to general defects in endocytic recycling (Xie, Naslavsky et al. 2014). Upon DGKα-knock-down, when PA synthesis is restricted, TRE biogenesis is impaired; however, preventing PA catabolism with the PA phosphatase inhibitor propranolol induces the recovery of TRE, even in the absence of DGKα. This indicates that DGKα regulates endocytic recycling through the level of local PA.

Dynamin is a key regulator of membrane constriction and fission during endocytosis that binds to anionic lipids (including PA) through its Pleckstrin Homology (PH) domain. The presence of PA increases dynamin’s enzymatic activity, and induces its deep penetration into the membrane (Burger, Demel et al. 2000). Moreover, in experiments where liposomes of different lipid components were co-incubated with dynamin, PA- containing liposomes had the most efficient dynamin-coated tubule formation (Takei,

Haucke et al. 1998).

39

3.1.4 Role of diacylglycerol (DAG) in endocytic trafficking

The mechanisms by which DAG regulates membrane trafficking are diverse. It either serves as a second messenger to activate PKC, PKD, and downstream signaling cascades (Simon, Ivanov et al. 1996, Maeda, Beznoussenko et al. 2001, Diaz Anel and

Malhotra 2005); or is involved in the regulation of PI cycling which is essential for the cellular abundance of phosphatidylinositol 4,5-bisphosphate (PIP2) and inositol 1,4,5-

2+ trisphosphate (IP3) (Vicinanza, D'Angelo et al. 2008) , as well as the subsequent Ca influx (Clapham 2007).

One well characterized function of DAG on endocytic trafficking lies in the secretory pathway from the Golgi. For example, upon inhibition of DGK in cells with the inhibitor R59022, impaired DGK activity leads to increased levels of DAG. Simultaneously, the level of PA correspondingly decreases in these cells. In this case, considerable stimulation of acrosomal exocytosis from mammalian sperms is observed (Roldan and

Harrison 1992, Mayorga, Tomes et al. 2007), which is a special type of regulated exocytosis using conserved exocytic mechanisms also found in neuronal, endocrinal and other cells (Mayorga, Tomes et al. 2007). Subsequent studies have identified DAG’s role in stimulating acrosomal exocytosis through PKC and phospholipase D1 (PLD1) activation, promoting the continued production of PIP2 and subsequently, IP3 which is required for the intra-acrosomal calcium efflux during fusion with the plasma membrane of the spermatozoon (See Figure 3 for the metabolism of DAG and related phospholipids).

Furthermore, it was demonstrated that DAG activates Rab3A leading to the assembly of

SNARE complexes and membrane fusion via interaction with Munc-13 (Huang, Yang et al. 2011, Lopez, Pelletan et al. 2012).

Previous studies have revealed that DAG plays a dual role in the generation of transport carriers, and thus mediates the secretion of newly synthesized proteins from the 40

41

Figure 3. Model for the biogenesis of tubular recycling endosomes.

A) Phosphatidic acid (PA) enriched membrane tend to form membrane curvatures recruiting scaffolding proteins; B) MICAL-L1 (via its CC domain) and Syndpin2 (via its F-BAR domain) are recruited to PA-enriched membranes; C) MICAL-L1 and Syndapin2 stabilize each other on the membrane by an interaction between the MICAL-L1 PXXP motifs and the Syndapin2 SH3 domain; D) Syndapin2 through its F-BAR domain facilitates the generation of tubular endosomes; E) Syndapin2 and MICAL-L1 bind to the EH domain of EHD1 via their NPF motifs and recruit EHD1 to TREs, potentially facilitating vesiculation. (image used with permission from (Giridharan, Cai et al. 2013))

42

Golgi. It serves in lipid signaling on the trans-Golgi network (TGN) for the recruitment and activation of essential proteins onto the TGN membrane. Such proteins include protein kinase D (PKD) (Maeda, Beznoussenko et al. 2001), protein kinase Cη (PKCη) (Simon,

Ivanov et al. 1996, Diaz Anel and Malhotra 2005), and the ARF GTPase-activating proteins (ARF GAPs) Gcs1p, Age1p and Age2p (De Matteis, Santini et al. 1993, Poon,

Nothwehr et al. 2001, Benjamin, Poon et al. 2011) (see Figure 4.1 for the downstream effector of DAG). Reducing cellular DAG levels inhibited recruitment and blocked TGN-to- plasma membrane trafficking (Baron and Malhotra 2002). On the other hand, the conical shape of DAG in the outer leaflet provides negative membrane curvature, which is thought to facilitate membrane fission (Shemesh, Luini et al. 2003). Given the importance of DAG levels on TGN-to-plasma membrane transport, the metabolic pathways for the production or consumption of DAG intricately regulate the secretory pathway (Roth 1999, Huijbregts,

Topalof et al. 2000, McMaster 2001). However, the molecular mechanisms mediating

DAG cellular levels during vesicular trafficking under physiological conditions are not well understood.

Studies also show that the level of DAG at the Golgi mediates retrograde transport

(Golgi-to-ER), while anterograde transport (ER-to-Golgi) is insensitive to DAG. PA phosphatase mediated DAG production is required for the formation of COPI vesicles and

Golgi-to-ER transport (Baron and Malhotra 2002, Fernandez-Ulibarri, Vilella et al. 2007,

Asp, Kartberg et al. 2009, Gutierrez-Martinez, Fernandez-Ulibarri et al. 2013).

DAG levels mediates the activity of downstream proteins such as PKC, and/or

Munc-13 (Topham and Prescott 2001, Luo, Prescott et al. 2003, Zhong, Hainey et al. 2003,

Crotty, Cai et al. 2006, Merida, Avila-Flores et al. 2008). Classical and novel PKC bind to

DAG (Ohno and Nishizuka 2002, Cai, Crotty et al. 2010), which are major mediators of endocytic recycling. PKCα localizes to transferrin-positive recycling endosomes in the 43

Figure 4.1 Pathways involved in the metabolism of DAG and PA described in this review.

DGK, diacylglycerol kinase. DAG, Diacylglycerol; PA, phosphatidic acid; PC, phosphatidylcholine; PIP2, phosphatidylinositol 4,5-bisphosphate; IP3, inositol 1,4,5- trisphosphate; PLD, phospholipase D; PLC, phospholipase C; PI4P, Phosphatidylinositol 4-phosphate; PI, phosphatidylinositol; PKD, protein kinase D; PKC, protein kinase C; ARF GAP, ARF GTPase-activating protein. Solid arrows indicate the metabolic pathways; hollow arrows indicate the downstream effector activated by DAG or IP3. (Image used with permission from (Xie, Naslavsky et al. 2015))

44 peri-nuclear area, and PKCα stimulation by DAG/ phorbol myristate acetate (PMA) accelerates the recycling of transferrin to the plasma membrane (Becker and Hannun

2003, Idkowiak-Baldys, Becker et al. 2006), while the sorting of LDLR to the lysosome remains unaffected (Idkowiak-Baldys, Becker et al. 2006). Furthermore, perturbation of this DAG gradient through the inhibition of DGK impaired both dynein recruitment and microtubule organizing center (MTOC) polarization (Quann, Merino et al. 2009).

3.2 Membrane curvature and the biogenesis of tubular recycling endosomes

Membrane curvature is mediated by the constitutive physical characteristics of membrane lipid components as well as membrane-remodeling proteins, and is crucial for various cellular events such as membrane trafficking, migration and organelle biogenesis.

At different stages during membrane vesicle generation, transportation and fusion, the different degrees of positive or negative curvature (curve inward towards or outward from cytoplasm, respectively) are key players for the insertion of amphipathic helices and BAR domain containing sensor proteins. Membrane curvature can be generated by multiple mechanisms and in most cases these processes do not work in isolation: 1) changes in membrane composition; 2) influences in integral membrane proteins; 3) cytoskeletal proteins and microtubule motor activity; 4) scaffolding by peripheral membrane proteins

(McMahon and Gallop 2005).

The stereochemical properties of different lipid components greatly influences membrane curvature. Lipid-modifying enzymes are thus important for creating a favorable membrane environment for different endosome dynamics, including vesicle budding, fission and fusion. These enzymes catalyze either the conversion between different lipid types that recruits different trafficking machineries (Brown, Chambers et al. 2003, 45

Kooijman, Chupin et al. 2005, Cai, Caplan et al. 2012, Xie, Naslavsky et al. 2014), or the change of head group size influencing the area occupied by the lipids (Hammond,

Reboiras et al. 1984). For example, lysophosphatidic acid acyl transferase and phospholipase A2 activity mediate the lipid turnover between lysophosphatidic acid (LPA) and phosphatidic acid (PA), favoring opposite membrane curvatures (Farge, Ojcius et al.

1999, Brown, Chambers et al. 2003, Kooijman, Chupin et al. 2005). PI3K-mediated transformation from PI3P into PI(4, 5)P2, is essential for the SE/EE-to-LE switch

(Fernandez-Borja, Wubbolts et al. 1999).

Transmembrane proteins with a conical shape are also capable of inducing membrane curvature. However, only a few integral proteins with defined morphology are found and the role of those proteins on membrane curvature is poorly understood. One example of such a protein is the nicotinic acetylcholine receptor residing at the top of membrane folds at the neuromuscular junction (Unwin 2005). In the presence of attachment proteins, by clustering with each other, acetylcholine receptors pose an enhanced effect on local curvature (Boudin, Doan et al. 2000, Eckler, Kuehn et al. 2005).

This mechanism of inducing high positive curvature by integral receptors might contribute to selective endocytosis which excludes other receptors with different stereochemical shape.

Cytoskeletal dynamics including branching, bundling and treadmilling of actin filaments are tightly related to the regulation of high membrane curvature in many cellular organelles and structures such as filopodia, pseudopodia, phagocytic cups and axonal growth cones (Sheetz 2001, Ledesma and Dotti 2003). The cytoskeleton plays a major role in the maintenance of membrane-shape and tension, during membrane trafficking events, actin rearrangements occur resulting in changes in tension, which would allow higher degree of membrane curvature (Dai, Ting-Beall et al. 1997, Raucher and Sheetz 46

1999, Bettache, Baisamy et al. 2003). Also it is established that actin polymerization in many endocytic invagination events aids the fission process (Shupliakov, Bloom et al.

2002, Yarar, Waterman-Storer et al. 2005).

In addition to transmembrane proteins, several families of membrane scaffolding proteins also induce membrane deformation. For example, the dynamin family proteins bind to inositol lipids and form helical oligomers to sculpt the membrane morphology into a tubular structure (Hinshaw and Schmid 1995, Marks, Stowell et al. 2001), as well as to constrain the neck of invaginations and perform scission. Coat proteins such as clathrin,

COPI and COPII are also well-known membrane benders in conjunction with scaffolding proteins directly recruited by membrane lipids (Nossal 2001, Antonny, Gounon et al. 2003).

Caveolin oligomerizes to form a coat that also induce flask-shaped membrane invaginations (Razani and Lisanti 2001). Furthermore, epsin which binds clathrin, Eps15 and AP2, also induces local curvature and tubulates membranes, possibly by inserting an active amphipathic helix into the inner leaflet of the bilayer depending on the activity of

PI(4,5)P2 (Ford, Mills et al. 2002).

One important class of membrane curvature sensor and bender is the BAR domain containing protein. The BAR domain bears a concave membrane binding region that preferentially binds to curved membrane (Peter, Kent et al. 2004). BAR domains are also frequently found in combination with N-terminal amphipathic helices (known as N-BAR domains). All N-BAR domain-containing proteins including amphiphysin, endophilin,

BRAP, andrin and SNX1 have the ability to form membrane tubules, and thus are involved in multiple trafficking events, like the formation and scission of clathrin-coated vesicles

(amphyphysin) and transportation of mannose-6-phosphate receptors to the TGN (SNX1)

(Takei, Slepnev et al. 1999, Farsad, Ringstad et al. 2001, Razzaq, Robinson et al. 2001,

Carlton, Bujny et al. 2004, Richnau, Fransson et al. 2004). 47

Other than the N-BAR domain, based on the sequence homology and solved structures, domain, BAR-domain containing proteins are categorized into several types: classical BAR, N-BAR, F-BAR, I-BAR, BAR-PH and PX-BAR (Frost, Unger et al. 2009,

Suetsugu 2010). Among these proteins, F-BAR-containing protein Syndapin (also known as PACSIN, Protein kinase C and casein kinase II interacting protein) is an important one which regulates various trafficking events (Kessels and Qualmann 2004, Quan and

Robinson 2013). Our lab has been focused on the mechanisms by which tubular recycling endosomes are generated and carry out their function. Recently we have linked one of the three isoforms of Syndapin, Syndapin2 to TRE biogenesis (Braun, Pinyol et al. 2005,

Giridharan, Cai et al. 2013). Syndapin2 bears a C-terminal Src Homology 3 (SH3) domain

(Kessels and Qualmann 2004) as well as multiple NPF motifs (Braun, Pinyol et al. 2005) which determine its association with various partners: dynamin, synaptojanin, synapsin,

Rac 1 and neural Wiskott-Aldrich syndrome protein (N-WASP), MICAL-L1 and EHD proteins (Kessels and Qualmann 2004, Giridharan, Cai et al. 2013). We have proposed that PA enriched membranes tend to form a curvature matching the concave structure of

Syndapin2, leading to the preferential recruitment of a novel TRE generation complex composed of MICAL-L1, Syndapin2 and EHD1 (See Figure 3 for the proposed model of

Syndapin2’s involvement in biogenesis of TRE). This biogenesis process is vital for cargo sorting into the recycling pathways, in that the depletion of MICAL-L1 and EHD1 results in significant accumulation of cargos in the ERC (Giridharan, Cai et al. 2013). However, once

TREs are generated and cargo has been sorted, for recycling to occur there must be efficient vesiculation of these tubular structures.

48

3.3 Membrane vesiculation

Intracellular trafficking requires the constant formation of carrier vesicles. These vesicles, which bud from the donor membrane, detach and move toward their destination organelle and subsequently fuse with it. The formation of vesicles is one of the most active membrane-shaping processes in the cells, and necessitates fission proteins to help overcome the energy barrier to pinch off the extended membrane tubules (Kooijman,

Chupin et al. 2003, Grimmer, Ying et al. 2005). However, it is noteworthy that there is no discernable barrier between membrane tubulation and membrane scission, since high membrane curvature, often known as the neck, induced by lipid/protein-based mechanisms eventually undergo scission as the last step in vesicle formation (Kooijman,

Chupin et al. 2003, Peter, Kent et al. 2004, McMahon and Gallop 2005). Membrane scission is performed by multiple proteins such as the GTPase dynamin (Lenz, Morlot et al. 2009), EHD proteins (Cai, Katafiasz et al. 2011), GRAF1 (Cai, Xie et al. 2014) and cPLA2α (Cai, Caplan et al. 2012).

The dynamin family of GTPase proteins is among the best-characterized membrane “vesiculator” that mediates the CCVs budding off from the PM. Dynamin consists of a GTPase domain that hydrolyze GTP, a middle domain, a GTPase-activating domain, a pleckstrin homology (PH) domain that anchors the protein to the PIP2 enriched

PM, and a proline-rich domain (PRD) that interacts with SH3 domain containing proteins

(Campelo and Malhotra 2012). Membrane curvature results in dynamin oligomerization, assembling into short collars constricting the neck of budding CCVs in a GTP-dependent manner, upon GTP hydrolysis, dynamin undergoes conformational change that causes vesicle scission (Takei, McPherson et al. 1995, Muhlberg, Warnock et al. 1997, Hinshaw

2000, Praefcke and McMahon 2004). 49

While dynamin is required for budding and scission of CCVs, it is dispensable for the fission of CLIC vesicles in CIE pathway, suggesting an alternative fission machinery for this route (Doherty and Lundmark 2009). Previously, we have demonstrated that EHD1 mediates the vesiculation of endocytosed invaginations containing GPI-AP, CD59 (Cai,

Caplan et al. 2012). The EHD family ATPase proteins shares various common features with dynamin. They are capable of forming ring-like structures in vitro and stimulate nucleotide hydrolysis in response to lipid binding (Daumke, Lundmark et al. 2007).

Ultrastructural analysis on EHD2 has revealed that ATP binding results in oligomerization of EHD proteins into a scissor shape formed by their dimeric G-domain and the helical domain (Daumke, Lundmark et al. 2007). Furthermore, EHD1 promotes the fission of synaptic vesicles in vivo in an ATP-dependent manner (Jakobsson, Ackermann et al.

2011).

EHD proteins are also involved in membrane scission and the generation of recycling vesicles (Jakobsson, Ackermann et al. 2011, Cai, Giridharan et al. 2013,

Giridharan, Cai et al. 2013). Our study indicates that PA is an essential lipid component of RE tubules and that the presence of PA enhances recruitment and the activity of F-BAR domain of Syndapin2, which in cooperation with MICAL-L1, recruits EHD1 to TRE to perform scission, presumably in a similar ATPase dependent manner (See Figure 3 for a proposed model on EHD1’s role on the fission of TRE).

In cooperation with EHD1, we have previously shown that the lipid modifier cytosolic phospholipase A2α (cPLA2α) is involved in the vesiculation of GPI-AP-containing membranes, using endogenous CD59 as a model for GPI-APs. Upon cPLA2α depletion,

CD59-containing endosomes became hypertubular. As a lipid modifier, cPLA2α may act locally to remodel membrane curvature, allowing EHD1 to cleave the curved membranes into vesicles. 50

In a more recent study, we also discovered that GTPase Regulator Associated with Focal adhesion kinase-1 (GRAF1) synergizes with EHD1 to support TRE vesiculation

(Cai, Xie et al. 2014), in addition to its characterized roles in CLIC/GEEC regulation and its functions at the PM (Lundmark, Doherty et al. 2008, Doherty and Lundmark 2009,

Doherty, Ahlund et al. 2011). Interestingly, the BAR domain of GRAF1 is insufficient for the vesiculation process, instead its function is carried out through the GAP domain

(Doherty and Lundmark 2009, Cai, Xie et al. 2014). Via its PH domain, GRAF1 was previously shown to predominantly bind to PIP2 (Lundmark, Doherty et al. 2008, Doherty and Lundmark 2009), which serves as a potential docking site for GRAF1 on tubular and vesicular endosomes. Alternatively, GRAF1 can also localize to the TRE via an indirect interaction with MICAL-L1, which binds selectively to PA-enriched TRE (Jovic, Kieken et al. 2009, Giridharan, Cai et al. 2013). Future studies will be needed to determine whether the combined scission activities of EHD1 and GRAF1 are required in vivo to overcome energy barriers, or whether they are needed to effectively regulate the complex process of recycling from the TRE.

4. THE ROLE OF DIACYLGLYCEROL KINASE IN ENDOCYTIC TRAFFICKING

4.1 Overview

The cytosolic diacylglycerol kinases (DGKs) are a family of kinases that regulate the conversion of diacylglycerol (DAG) to phosphatidic acid (PA) by phosphorylation.

DGKs are widely conserved evolutionarily, and are found in organisms as diverse as bacteria (Badola and Sanders 1997), fungi, Saccharomyces cerevisiae, plants (Gomez-

Merino, Brearley et al. 2004), multi-cellular organisms including Drosophila melanogaster and Caenorhabditis elegans (Harden, Yap et al. 1993, Jose and Koelle 2005), and 51 mammals (Arisz and Munnik 2010). DGK from Escherichia coli share little structural resemblance to the eukaryotic ortholog (Topham and Prescott 1999), although eukaryote

DGKs have a highly conserved catalytic domain. Drosophila melanogaster DGK shares homologous amino acid sequences at the carboxyl-terminal domain with porcine DGK

(Masai, Hosoya et al. 1992). In plants such as Arabidopsis thaliana, DGK has evolved into three phylogenetic clusters. Cluster I, encoded by AtDGK1 and AtDGK2, resembles the mammalian DGKε, while the other two clusters, contain only the conserved kinase domain but lack other accessory motifs, and accordingly are much smaller proteins (Arisz,

Testerink et al. 2009, Arisz and Munnik 2010). Intriguingly, the yeast Saccharomyces cerevisiae DGK does not possess the signature catalytic domain with an ATP-binding domain, but utilizes CTP. It also has a much simpler and less varied amino-terminal regulatory domain than its ATP-dependent homologs (Han, O'Hara et al. 2008). To date, in mammals, ten DGK isoforms have been identified (Sakane, Yamada et al. 1990, Goto and Kondo 1993, Kai, Sakane et al. 1994, Bunting, Tang et al. 1996, Klauck, Xu et al.

1996, Sakane, Imai et al. 1996, Tang, Bunting et al. 1996, Houssa, Schaap et al. 1997,

Ding, Traer et al. 1998, Imai, Kai et al. 2005). All DGK isoforms have a conserved catalytic domain with an ATP-binding site that is required for kinase activity, and cysteine-rich regions that are homologous to the C1A and C1B motifs of protein kinase C (PKC) (Hurley,

Newton et al. 1997).

Beyond the conserved catalytic domains, the regulatory domains of mammalian

DGK isoforms differ greatly, leading to differential localization and regulation of phospholipids in the cells (different subcellular localizations of DGK isoforms are summarized in Table 4). Based on these regulatory domains, DGK isoforms can be divided into five subtypes. Figure 4.2 summarizes the catalytic and diverse regulatory domains in each DGK isoform of each type. In addition to the conserved domains, type I 52

Figure 4.2. Schematic diagram illustrating the domain architecture of diacylglycerol kinase isoforms.

The ten members of the mammalian DGK family are grouped into five types according to their regulatory domains. PH, pleckstrin homology domain; SAM, sterile α motif domain. (Image used with permission from (Xie, Naslavsky et al. 2015)) 53

Localization DGK isoforms Plasma membrane α (Shirai, Segawa et al. 2000, Sanjuan, Jones et al. 2001), δ (Fallman, Lew et al. 1989), β, γ, ζ (Shirai, Segawa et al. 2000, Abramovici and Gee 2007) Endoplasmic reticulum δ (Nagaya, Wada et al. 2002), η, κ Endosome membrane α (Xie, Naslavsky et al. 2014), ζ (Rincon, Santos et al. 2007) Nuclear ζ (Topham, Bunting et al. 1998), ι Multi-Vesicular Body α (Alonso, Mazzeo et al. 2007) Trans-Golgi network α (Shirai, Segawa et al. 2000)

Table 4. Known subcellular localizations of different DGK isoforms. (Image used with permission from (Xie, Naslavsky et al. 2015))

54

DGKs (DGKα, β, and γ) contain a calcium-binding EF hand motif (Yamada, Sakane et al.

1997). Upon activation, DGKα rapidly translocates from the cytosol to the plasma membrane (Sanjuan, Jones et al. 2001). Type II DGKs (DGKδ, η and κ) have a unique pleckstrin homology (PH) domain and a sterile α motif (SAM) domain (Sakane, Imai et al.

1996) that can bind to the endoplasmic reticulum (ER) and affect anterograde transport

(Nagaya, Wada et al. 2002). Type III DGK (DGKε) has a unique substrate specificity for arachidonate-DAG (Shulga, Topham et al. 2011, Jennings, Doshi et al. 2015). This substrate specificity renders DGKε the most important isoform that catalyzes the first step of phosphatidylinositol (PI) re-synthesis. Although there is no evidence supporting a direct role for DGKε on membrane trafficking, it is possible that of the DGK isoforms, only DGKε exerts an additional function mediating membrane trafficking via PI cycling. Type IV DGKs

(DGKζ and ι) have a nuclear localization signal in a MARCKS homologous domain

(Topham, Bunting et al. 1998), four ankyrin repeats, and carboxyl terminal PDZ-binding domains (Hogan, Shepherd et al. 2001). Type V DGK (DGKθ) has three C1 domains, a putative PH domain, and a Ras association (RA) domain. In humans, each subtype of the

DGK family isoforms displays a tissue-specific expression profile. For example, DGKα is mostly detected in brain and immunologic organs, such as spleen and thymus (Sanjuan,

Jones et al. 2001). DGKβ is expressed in neurons in caudate-putamen, the nucleus accumbens, and the olfactory tubercle (Goto and Kondo 1993, Shirai, Kouzuki et al. 2010).

DGKδ is substantially expressed in spleen, ovary and skeletal muscle (Sakane, Imai et al.

1996, Chibalin, Leng et al. 2008, Sakai and Sakane 2012). DGKζ is also highly expressed in spleen and thymus, as well as in heart and pancreas (Goto and Kondo 1996).

Studies using DGK knock-out mouse models have revealed the involvement of various DGK isoforms in different diseases. DGKα or DGKζ-null T cells display defects in immune function (Zhong, Hainey et al. 2003, Zha, Marks et al. 2006). DGKδ 55 haploinsufficiency causes the development of insulin resistance in skeletal muscle and adipose tissue (Chibalin, Leng et al. 2008). In addition, DGKβ/DGKε knock-out mice exhibit brain disorders and behavioral abnormalities (Rodriguez de Turco, Tang et al. 2001,

Kakefuda, Oyagi et al. 2010, Mukherjee, Zeitouni et al. 2013).

Since DGKα was identified in the 1990s, studies on DGK family proteins have primarily focused on their function in regulating signaling pathways. DGK terminates DAG- based signals and accentuates PA signaling, and both DAG and PA serve as important second messengers in cells. The activation of DGK usually involves the translocation of

DGK to a membrane compartment (Besterman, Pollenz et al. 1986). Upon recruitment to the membrane, DGK can be activated by calcium binding to the EF hand motif. DGK activity is initiated or enhanced by some lipid components including the products of phosphatidylinositol-3-kinase (PI3K) such as phosphatidylinositol-3,4,5-trisphosphate

(PIP3) (Cipres, Carrasco et al. 2003), phosphatidylserine, and sphingosine (Topham and

Prescott 1999). It is also activated via Src tyrosine kinase phosphorylation (Cutrupi,

Baldanzi et al. 2000) and serine phosphorylation by protein kinase C (PKC) (Yamaguchi,

Shirai et al. 2006). Some upstream players participating in DGK activation include interleukin2 (IL-2) receptor signaling (Flores, Casaseca et al. 1996), epidermal growth factor receptor (EGFR) (Schaap, van der Wal et al. 1993), T cell antigen receptor signal transduction (Sanjuan, Jones et al. 2001), and hepatocyte growth factor receptor (HGFR)

(Chianale, Rainero et al. 2010).

The effect of DGK on endocytic transport is attributed to its substrate and product of DGK, DAG and PA. DAG and PA respectively play different roles in endocytic events.

These include vesicular trafficking, the secretory pathway regulation, and Golgi apparatus function, whose major function is summarized and discussed in Section 3.1.1 and 3.1.2.

The increasing awareness of the relevance of lipid metabolites such as DAG and PA in 56 membrane trafficking, and understanding the significance of DGK function in regulating the fine balance of DAG-to-PA levels is becoming an increasingly important research goal.

4.2 The role of DGK on exocytosis

By fine-tuning the local lipid component through consuming DAG and generating

PA, DGK plays a role in modifying the membrane curvature tendency to create a favorable environment for membrane fission/fusion, and thus mediates the process of exosome secretion. For instance, upon inhibition of DGK in cells with the inhibitor R59022, the local level of DAG is correspondingly decreased, which stimulates acrosomal exocytosis

(Roldan and Harrison 1992, Mayorga, Tomes et al. 2007). On the other hand, the DGK activity inhibition results in decreased levesl of PA, which reduces neutrophil exocytosis from azurophilic granules by inhibiting granule fusion at the PM (Holden, Savage et al.

2011).

There are some indications that DGK mediates the release of neurotransmitters, possibly through the recruitment of munc-13 (Betz, Ashery et al. 1998). Studies on DGKι knock-out mice show that it is involved in presynaptic glutamate release during 3,5- dihydroxyphenylglycine (DHPG)-induced long-term potentiation (Yang, Seo et al. 2011).

DGK1 knock-out (the homolog of DGKθ) in Caenorhabditis elegans, led to an increase in acetylcholine release (Miller, Emerson et al. 1999), suggesting that DGK negatively regulates synaptic transmission. However, the molecular mechanism underlying this role of DGK has yet to be established.

57

4.3 DGK mediates MVB formation and secretion

MVBs are formed by the inward invagination of the limiting membrane of an endosome, giving rise to intraluminal vesicles (ILVs). Although previously considered a mechanism of cargo sorting for lysosomal degradation, MVBs also fuse with the plasma membrane, secreting their ILVs into the extracellular area (Hanson and Cashikar 2012).

Studies show that DGK is involved in multiple processes related to exosome secretion, including the formation and maturation of Multi-Vesicular Bodies (MVBs) (i.e., the number of MVBs per cell and inward vesiculation of MVBs), the budding and release of exosomes from MVBs, and their fusion with the plasma membrane (Izquierdo, Ruiz-Ruiz et al. 1996,

Alonso, Rodriguez et al. 2005, Alonso, Mazzeo et al. 2007, Quann, Merino et al. 2009). In cytotoxic T lymphocytes, MVBs are responsible for releasing the pro-apoptotic Fas ligand

(FasL) at the immunological synapse (Bossi and Griffiths 1999) with DGKα playing an important regulatory role in this process (Alonso, Rodriguez et al. 2005, Alonso, Mazzeo et al. 2007). Upon receptor stimulation, FasL and DGKα relocate to FasL-containing MVB structures. DGKα is recruited to MVBs and to exosomes, where it plays a dual role;

DGKα kinase activity exerts a negative role in the formation of mature MVBs, as experiments show that treatment with a type I DGK inhibitor, R59949, induces the maturation of CD63-positive/lysobisphosphatidic acid-positive MVBs, and increases the secretion of exosomes (Jiang, Sakane et al. 2000, Alonso, Rodriguez et al. 2005, Alonso,

Mazzeo et al. 2011). In contrast, down regulation of DGKα inhibits polarized exosome secretion, and affects degranulation of MVBs at the immune synapse, while the kinase inhibitor increases polarized secretion of exosomes. These studies imply that

DGKα kinase activity negatively regulates the formation of mature MVBs, while regions outside the kinase domain are required for polarization of MVBs and exosome secretion

(Alonso, Mazzeo et al. 2011). 58

4.4 The role of DGK on secretion from the Golgi apparatus

The role of DGK on the secretory pathway from the Golgi apparatus is mediated through the level of DAG. Previous studies have revealed that DAG plays a dual role in the generation of transport carriers, and thus mediates secretion from the Golgi. It serves in lipid signaling on the trans-Golgi network (TGN) for the recruitment and activation of essential proteins onto the TGN membrane. Such proteins include protein kinase D (PKD)

(Maeda, Beznoussenko et al. 2001), protein kinase Cη (PKCη) (Simon, Ivanov et al. 1996,

Diaz Anel and Malhotra 2005), and the ARF GTPase-activating proteins (ARF GAPs)

Gcs1p, Age1p and Age2p (De Matteis, Santini et al. 1993, Poon, Nothwehr et al. 2001,

Benjamin, Poon et al. 2011) (see Figure 2 for the downstream effector of DAG). Reducing cellular DAG levels inhibited recruitment and blocked TGN-to-plasma membrane trafficking (Baron and Malhotra 2002). On the other hand, the conical shape of DAG in the outer leaflet provides negative membrane curvature, which is thought to facilitate membrane fission (Shemesh, Luini et al. 2003). Given the importance of DAG levels on

TGN-to-plasma membrane transport, the metabolic pathways for the production or consumption of DAG intricately regulate the secretory pathway (Roth 1999, Huijbregts,

Topalof et al. 2000, McMaster 2001). DGK rapidly reduces the level of DAG, resulting in the inhibition of TGN-to-plasma membrane transport, implicating the negative regulation of DGK on Golgi secretory pathways. Earlier studies established that DGKα translocates to the TGN upon receptor stimulation (Shirai, Segawa et al. 2000). Furthermore, research in yeast has revealed that the conversion of DAG to PA by increased DGK expression significantly impairs Golgi function. Sec14p is a phosphatidylinositol (PI)-transfer protein that generates the DAG precursor, phosphatidylcholine (PC). PLD then converts PC into

PA, which is dephosphorylated by phosphatidic acid phosphatase (PAP) into DAG (Figure

4.1). Studies have demonstrated that Sec14p is important in maintaining a favorable lipid 59 environment for TGN-to-plasma membrane trafficking (McGee, Skinner et al. 1994,

Sreenivas, Patton-Vogt et al. 1998, Mousley, Tyeryar et al. 2007), and thus is essential for yeast viability and secretory competence. To date, a large class of loss-of-function mutations in nonessential genes have been identified, the “bypass Sec14p” mutations, that restore cell viability and Golgi secretion with defective Sec14p, indicating that such mutations occur in regulators of TGN transportation downstream of Sec14p. Among the

“bypass Sec14p” mutations, Sac1p deficiency functions by inducing accumulation of phosphatidylinositol 4-phosphate (PI4P), a pro-secretory phospholipid in the Golgi. Indeed, yeast DGK expression compromises the ability of Sac1p deficiency to effect “bypass

Sec14p,” suggesting DGK’s negative role in TGN secretory pathway, possibly through reducing the level of PI4P (Kearns, McGee et al. 1997).

4.5 Anterograde transport is mediated by local PA levels

Although no direct evidence shows that DGK is involved in the regulation of membrane fission, since PA is required for the fission of Golgi structures, it is possible that membrane fission and fusion events may require the DGK-generated PA, potentially implicating DGK as a major player in mediating vesicle trafficking. Among the ten isoforms in the DGK family, only DGKα is localized to the TGN (Alonso, Rodriguez et al. 2005), and a certain portion of cytosolic DGKδ localizes to the ER via its SAM domain (Nagaya, Wada et al. 2002). DGKδ expression blocks the formation of COPII-coated structures in the ER and slows ER-to-Golgi transport of Vesicular Stomatitis Virus G Glycoprotein (VSV-G), indicating that the anterograde transport is inhibited by DGKδ.

On the other hand, COPI structures are unaffected (Nagaya, Wada et al. 2002), suggesting that retrograde transport is independent of DGKδ. Apparently, lipid conversion is not required for anterograde transport, since the overexpression of a DGKδ kinase-dead 60 mutant led to similar inhibition of ER-to-Golgi transport. It is highly possible that the effect of DGK on anterograde transport from the ER to the Golgi is mediated through the production of PA, rather than DAG. Since the level of DAG affects the formation of COPI endosomes and retrograde transport (Golgi-to-ER), but not anterograde transport (ER-to-

Golgi) (Baron and Malhotra 2002, Fernandez-Ulibarri, Vilella et al. 2007).

4.5 The role of DGK on endocytic recycling

DGK’s effect on endocytic recycling is performed through multiple mechanisms.

Firstly, DGK affects the DAG/Ras/ERK signaling pathway, either through regulating the

DAG levels or directly by mediating the activity of downstream proteins such as PKC, and/or Munc-13 (Topham and Prescott 2001, Luo, Prescott et al. 2003, Zhong, Hainey et al. 2003, Crotty, Cai et al. 2006, Merida, Avila-Flores et al. 2008). Either way, PKC serves as an important downstream signaling protein involved in multiple trafficking processes.

Classical and novel protein kinase C (PKC) binds to DAG (Ohno and Nishizuka 2002, Cai,

Crotty et al. 2010). PKCα localizes to transferrin-positive recycling endosomes in the peri- nuclear area, and PKCα stimulation by DAG/PMA accelerates the recycling of transferrin to the plasma membrane (Becker and Hannun 2003, Idkowiak-Baldys, Becker et al. 2006), while the sorting of LDLR to the lysosome is not affected (Idkowiak-Baldys, Becker et al.

2006). Furthermore, perturbation of this DAG gradient through the inhibition of DGKs impaired both dynein recruitment and MTOC polarization (Quann, Merino et al. 2009).

In a recent study, DGKα was linked to the recycling of α5β1 integrin by recruiting a protein required for its recycling during migration, the Rab-coupling protein (RCP) to the tips of invasive pseudopods (Caswell, Chan et al. 2008, Rainero, Caswell et al. 2012). 61

Sorting nexin 27 (SNX27), a member of the SNX family of proteins involved in intracellular sorting and trafficking (Worby and Dixon 2002, Carlton, Bujny et al. 2005), interacts with DGKζ. This interaction is required for the localization of SNX27 to the sorting endosomes in Jurkat T cells. DGKζ-siRNA accelerated the recycling of transferrin receptor from the endocytic recycling compartment, to the plasma membrane in T lymphocytes, which is the opposite of DGKα (Rincon, Santos et al. 2007). Although the reason why

DGKζ–knock-down in T lymphocytes has such a dramatically different impact than DGKζ- knock-down in other cell types remains unknown, it is possible that compensation by other

DGKs and/or other PA-generating pathways in T cells is more robust.

Another effect of DGKα in endocytic recycling lies in the regulation of tubular recycling endosomes (TRE) that are decorated by Molecules Interacting with CAsL-

Like1 (MICAL-L1). MICAL-L1 and Syndapin2 promote the biogenesis of TRE (thus regulating TRE function), and they are both recruited to the TRE membrane through direct interactions with PA (Giridharan, Cai et al. 2013). DGKα-derived PA mediates the recycling of Major Histocompatibility Complex Class I (MHC I) without affecting its internalization. In addition, the MICAL-L1-decorated TRE are disrupted upon DGKα- depletion, leading to general defects in endocytic recycling (Xie, Naslavsky et al. 2014).

When the compromised synthesis of PA upon DGKα-knock-down is mitigated by preventing it catabolism with the PA phosphatase inhibitor propranolol, the loss of TRE is reversed. This indicates that DGKα regulates endocytic recycling through the level of PA.

4.6 The role of DGK on endocytosis

Genome-wide short interfering RNA screening analysis assessing the involvement of different human kinases on endocytosis (Pelkmans, Fava et al. 2005) has predicted 62 that DGKβ negatively influence CIE while DGKγ positively mediates CIE, and DGKδ seems to have a dual effect promoting CIE and inhibiting CME.

By inhibiting type I DGK, total cellular PA levels decreased. Correspondingly, the internalization of epidermal growth factor (EGF) was significantly impaired. However, the uptake of transferrin remained unaffected, suggesting that EGF internalization depends on DGK activity, whereas the uptake of transferrin is independent of the kinase activity.

Moreover, upon inhibition of type I DGK, fewer clathrin-coated pits (CCP) formed, indicating a role for DGK activity in CCP formation (Antonescu, Danuser et al. 2010).

In addition to its kinase activity regulating DAG-PA conversion, DGKs also serve as scaffolding proteins that recruit regulatory proteins required for endocytosis. For example, during clathrin-dependent endocytosis, CCP are formed with the assistance of clathrin and the Adaptor Protein 2 (AP-2) complex, and DGKδ co-localizes with these CCP through its interaction with AP-2 via F369DTFRIL and D746PF sequences in the catalytic domain. Furthermore, the uptake of both transferrin and EGF was significantly reduced in the absence of DGKδ. Importantly, the kinase activity is also required for the endocytic process, as the kinase-dead mutant could not reverse the impaired uptake observed upon

DGKδ knock-down (Kawasaki, Kobayashi et al. 2008). This is probably due to the regulatory effect of PA on CME. Although DAG stimulates the internalization of transferrin in some organisms such as Trypanosomatids brucei, in human cells DAG levels do not influence transferrin endocytosis (Subramanya and Mensa-Wilmot 2010). These studies suggest that DGK mediates clathrin-dependent endocytosis either through kinase activity leading to PA production, or by serving as a scaffold protein that recruits AP-2 for CCP formation.

Phagocytosis and macropinocytosis are processes essential for innate immunity and tissue homeostasis, during which cells such as macrophages ingest particulate 63

(phagocytosis) or soluble (macropinocytosis) pathogens into membrane-bound vacuoles.

The molecular mechanisms mediating phagocytosis include protein tyrosine kinases,

GTP-binding proteins, PKC, actin polymerization and membrane movement (Lennartz

1999). Phosphoinositide metabolism is critical for regulation of the initiation of both processes. A local phagosomal DAG accumulation is observed by biochemical means during particle ingestion (Fallman, Lew et al. 1989). The phosphorylation by DGK is a critical determinant of DAG at the phagocytic sites (Schlam, Bohdanowicz et al. 2013).

DGK inhibition by R59002 or R59949 increases the DAG-positive phagosomes, and enhances reactive oxygen species (ROS) generation by these phagosomes (Ueyama,

Lennartz et al. 2004, Schlam, Bohdanowicz et al. 2013), indicating that DGK terminates the DAG signaling mediating the phagosomal ROS production. Meanwhile, a PM enrichment of PA is detected in phagocytes. Both DGK and PLD are responsible for the

PA production in phagocytic sites (Corrotte, Chasserot-Golaz et al. 2006, Bohdanowicz,

Schlam et al. 2013). Among the ten isoforms of DGK, DGKβ, DGKγ, and DGKζ are found in the PM of macrophages, suggesting that multiple DGK isoforms are involved in the regulation of macropinocytosis. The PM PA abundance parallels membrane ruffling, and accordingly, the DGK inhibitor R59002 treatment depresses both the rate and extent of ruffle formation. Upon DGK inhibition, macropinosome formation and dextran internalization is impaired (Bohdanowicz, Schlam et al. 2013). In addition to regulating the

DAG-PA equilibrium, DGKζ plays a crucial role in phagocytosis and macropinocytosis via the activation of the Rho-GTPase family protein Rac1 (Abramovici and Gee 2007, Okada,

Hozumi et al. 2012).

64

4.7 Summary and conclusion

In addition to the important role DGKs play in cell signaling and immune activity, their effect on endocytic trafficking should not be underestimated. By catalyzing the conversion of DAG to PA, DGKs regulate different stages of endocytic trafficking, including the transport from/to the Golgi apparatus, the formation and secretion of MVBs, endocytosis, and the biosynthesis of recycling endosomes. There are ten mammalian

DGK isoforms, each with different regulatory domains, substrate specificities, and tissue/subcellular distribution, resulting in differential regulation of endocytic trafficking.

The existence of multiple isoforms suggests that there may be different DGKs at distinct subcellular structures, and that there is localized regulation of DAG/PA levels mediating endocytic trafficking. However, lipid metabolism is a dynamic, bi-directional processes, and DGKs may need to cooperate with other lipid modifiers to create and/or maintain an optimal membrane environment for trafficking. It is also possible that DGKs function in a kinase-independent manner. One example is that DGKδ facilitates CME through its binding to AP-2, but not through its kinase activity in regulating DAG or PA levels.

By mediating endocytic trafficking, DGKs control cell morphology, migration, apoptosis, protein biosynthesis, and receptor activation. Although there is no established pathology caused by a malfunctioning DGK isoform, knock-out mouse models reveal significant pathological consequences, including insulin insensitivity, immune function abnormalities and brain disorders, suggesting a potential role for DGKs as therapeutic targets in disease.

65

CHAPTER II

Materials and methods

66

5 MATERIALS AND METHODS

5.1 Cell lines

For the studies described in Chapter III and Chapter IV, HeLa cells from ATCC were grown in DMEM containing 10% FBS, 2 mM glutamine, 100 units/ml penicillin, and

100 units/ml streptomycin.

5.2 DNA Constructs, Transfection and siRNA Treatment

For the studies described in Chapter III, DGKα was cloned from a HeLa cDNA library by PCR amplification with primers (5 ‘-

ACGCGTCGACCATGGCCAAGGAGAGGGGCCTAATAAGC-3’, 5 ‘-

AAGGAAAAAAGCGGCCGCTTAGCTCAAGAAGCCAAAGAAATTGGTG-3’), and cloned into a pHA-CMV vector (Clontech).

Transfection of HeLa cells for 18 h at 37oC was performed using X-tremeGENE 9

(Roche Applied Science) according to the manufacturer’s protocol. siRNA treatment was carried out with Oligofectamine (Invitrogen) for 72 h according to the manufacturer’s protocol, using 0.3 µM oligonucleotide (Dharmacon). Four specific oligonucleotides (On-

Target SMART pool) were directed at human DGKα: 5’-GAGAUAGGGCUCCGAUUAU-

3’, 5’-CAAUCAAGAUCACCCACAA-3’, 5’-CGACCAGUGUGCCAUGAAA-3’, 5’-

ACAGUAGGCUGGAUUCUAG-3’).

For the studies described in Chapter IV, human HA-MICAL-L1 and HA-

Syndapin2 were previously described (Sharma, Giridharan et al. 2009, Giridharan,

Cai et al. 2013). The GFP-MyosinVb tail construct was a gift from Dr. James

R. Goldenring (Vanderbilt University Medical Center, USA) (Lapierre, Kumar et al.

2001). GFP-cellubrevin was kindly provided by Dr. J. Donaldson (NHLBI, National 67

Institutes of Health). Dominant-negative GFP-Rab11 (S25N) was kindly provided by

Dr. Juan S. Bonifacino (NICHD, National Institutes of Health). HeLa cells were transfected for 18 h at 37°C with X-tremeGENE 9 (Roche Applied Science) according to the manufacturer's protocol. siRNA oligonucleotides targeting human Rab11A were synthesized by Dharmacon: sense (5’-GAGUAAUCUCCUGUCUCGA(dT)(dT)-3’).

Rab11a-siRNA treatment was carried out with lipofectamine RNAi-Max (Invitrogen) for 48 h according to the manufacturer's protocol, using 0.3 μM oligonucleotide

(Dharmacon).

5.3 Antibodies and Reagents

The following antibodies were used in studies described in Chapter III: Supernatant from the W6/32 hybridoma, producing antibody against MHC I, was previously described

(Naslavsky, Weigert et al. 2003) and used for immunofluorescence. The mouse monoclonal antibody (HC-10) against MHC I was used for immunoblotting. Other commercial antibodies used were: mouse anti-actin (Novus Biologicals, Inc.), goat anti- mouse horseradish-peroxidase (HRP) (Jackson ImmunoResearch Laboratories, Inc.), donkey anti-rabbit HRP (GE Healthcare), Alexa-568 goat anti-mouse, Alexa-488 goat anti- rabbit, and Alexa-647 goat anti–mouse F(ab)2 (Invitrogen), mouse anti-MICAL-L1

(Abnova), rabbit anti-Rab11 (US Biologicals), rabbit anti-HA (Signalway), rabbit anti-EEA1

(Cell Signaling), rabbit anti-caveolin (Cell Signaling), mouse anti-cytochrome c (BD

Pharmacology), and rat anti-Hsc70 (Stressgene). R59949, propanolol and cycloheximide were purchased from Sigma. 4',6 diamidino-2-phenylindole (DAPI) was obtained from

Invitrogen.

The following antibodies were used in studies described in Chapter IV: Mouse monoclonal MEM-43 ascites antibody against CD59 was kindly provided by Dr. V. Horejsi 68

(Academy of Sciences of the Czech Republic, Prague, Czech Republic), and used previously (Cai, Katafiasz et al. 2011, Cai, Caplan et al. 2012, Cai, Xie et al. 2014). Other commercial antibodies used were: mouse anti- human β1-integrin (CD29) (AbD Serotec,

Inc.), rabbit anti-Low Density Lipoprotein Receptor (LDLR) (Progene, Inc.), mouse anti- human TfR (Zymed), mouse antiMICAL-L1 (Novus Biologicals, Inc.), mouse anti-actin

(Novus Biologicals, Inc.), rabbit antiRabenosyn-5 (Novus Biologicals, Inc.), rabbit anti-

Rab11a (US Biologicals), rabbit anti-HA (Signalway), goat anti-mouse horseradish peroxidase (HRP) (Jackson ImmunoResearch Laboratories, Inc.), donkey anti-rabbit HRP

(GE Healthcare), Alexa-568 conjugated goat antimouse, Alexa-488 conjugated goat anti- rabbit. Secondary antibodies used in dSTORM imaging were: Alexa-647 goat anti-mouse

F(ab)2 (Invitrogen), Atto488 conjugated anti-rabbit (sigma), Atto488 conjugated anti- mouse (sigma), Alexa-647 goat anti-rabbit (Life Technologies). AntiCD59 antibody was conjugated to Alexa-555 with APEXTM -Alexa-Fluor 555 antibody-labeling kit (Life

Technologies) according to the manufacturer’s instructions.

In studies described in Chapter III, to inhibit DGKα or phosphatidic acid phosphatase (PAP) activity, inhibitor treatments were performed as described earlier

(Jovanovic, Brown et al. 2006, Giridharan, Cai et al. 2013). Briefly, 100 µM (w/v) of DGKα inhibitor, R59949, was applied for 1 h at 37oC. 100 µM (w/v) of the PAP inhibitor, propranolol, was incubated for 30 min. at 37oC.

Reagents used in studies described in Chapter IV include: Alexa-568 conjugated

Transferrin, Alexa-633 conjugated Transferrin, Alexa-647-conjugated-Transferrin, and

Wheat Germ Agglutinin were from Life Technologies, Inc. CAY inhibitor cocktail

(CAY10593, CAY10594) was from Cayman Chemical Company, Inc. 20% Glucose solution, Glucose Oxidase from Aspergillus Niger, Catalase from bovine liver, Cysteamine 69

Hydrochloride (MEA), and 100 nm diameter gold nanoparticles were all purchased from

Sigma Aldrich.

5.4 Immunoblotting

For immunoblotting experiments described in both Chapter III and Chapter IV,

HeLa cells were harvested and lysed on ice for 30 min in lysis buffer containing 50 mM

Tris, pH 7.4, 150 mM NaCl, 1% Nonidet P-40, 0.5% sodium deoxycholate, and protease inhibitor mixture (Roche Molecular Biochemicals). Total protein levels in the lysate were then determined by BioRad protein assay (Bio-Rad Laboratories) and normalized for equal protein loading on gels. Protein samples were separated on 10% SDS-PAGE, and detected by immunoblotting with appropriate antibodies.

In Chapter III, in order to perform densitometry analysis, 3 independent experiments were quantified by densitometry, and analyzed using Image J software

(http://rsbweb.nih.gov/ij/links.html). Mean values and standard deviation are presented.

5.5 Flow cytometry analysis

For experiments described in Chapter III, HeLa cells were suspended with cell stripper (Mediatech Inc.) and then washed with phosphate-buffered saline (PBS) 3 times.

To measure MHC I uptake, the cells were incubated with W6/32 anti-MHCI antibodies

(supernatant) for the indicated time at 37oC. Cells were then subjected to a 1 min. acid- strip (0.5 M NaCl, 0.5% acetic acid, pH 3.0) to remove remaining non-internalized MHC I antibody on the surface. Cells were fixed with 4% (v/v) paraformaldehyde in PBS for 10 min. After fixation, the cells were incubated with Alexa-647 goat anti-mouse F(ab)2 in the immunofluorescence staining buffer containing 0.2% saponin (w/v) and 0.5% BSA (w/v) 70 in PBS for 1 h at room temperature, followed by 3 washes in PBS. Cells were then subjected to flow cytometry analysis. Values from 3 independent experiments were normalized. To measure the total/surface level of MHC I at steady-state, HeLa cells were fixed and stained with W6/32 anti-MHC I antibody and then the Alexa-647 goat anti-mouse

F(ab)2 in the presence or absence of detergent (saponin).

To carry out flow cytometry analysis of fluorescently labeled Tf as described in

Chapter III and IV, the following steps were followed:

HeLa cells were passaged and plated on a 100-mm dish 24 h prior to the experiment. To have sufficient cells for treatment, they were 60 to 80% confluent. Cells were then serum-starved for 1 h, followed by an incubation in 4 mL of Cell Stripper

(Mediatech Inc.) for 1 min on ice. Pipetting up and down was done to detach cells from the bottom of the dish. Cells were collected in 15 mL Falcon tubes and pelleted by centrifugation at 1,300 rpm at for 5 min. The supernatant was carefully aspirated without disrupting the cell pellet and the cells were washed three times in 1 mL of cold PBS, in between each wash the cells were pelleted by centrifugation at 1,300 rpm at 4 oC for 5 min. After the third PBS wash, cells were re-suspended in 1 mL of PBS and divided into three aliquots (about 330 mL) in 1.5 mL Eppendorf tubes. Tube A was labeled as the Tf pulse treatment (see later), Tube B served as the Tf chase treatment, and Tube C served as the pre-uptake control, which accounts for auto-fluorescence artifacts and background noise.

For Tube C, the cells were fixed (PBS in formaldehyde; final concentration 3.7%) for 10 min at room temperature. For tube A and B, Tf-488 was diluted to a concentration of 25 m g/mL in DMEM (containing 0.1% BSA). Cells were then re-suspended in 150 m L

o of Tf-488 solution and rotated in a CO2-regulated incubator at 37 C for 15 min (pulse), after which they were washed three times with 1 mL of cold PBS, and pelleted by 71 centrifugation at 1300 rpm at 4 oC for 5 min after each wash. Pellets from Tube A were re- suspended in 3.7% paraformaldehyde in PBS and fixed for 10 min at room temperature.

At the meanwhile, pellets from Tube B were re-suspended in 1 mL of complete DMEM

(containing fetal bovine serum) and rotated at 37 oC for 45 min (chase) before wash and fixation as described for Tube A.

Cells were transferred from Tubes A, B, and C into flow cytometry-compatible tubes and 488 fluorescence was measured by flow cytometry. Median fluorescence intensity (MFI, arbitrary units) of different samples was assessed by flow cytometry. Tube

C was used to “gate” (set the fluorescence threshold) for Tubes A and B. The MFI of Tube

A equals the total amount of internalized Tf while Tube B represents the amount of Tf remaining in the cells after the chase (non-recycled). The recycling rate was calculated by the following equation (Equation (1)):

(MFI of chase sample (Tube B)) 푅푒푐푦푐푙푖푛푔 푟푎푡푒 = 1 − ( × 100%) (1) (MFI of pulse−only sample (Tube A))

5.6 Cycloheximide treatment and MHC I degradation

Cells grown on 35 mm dishes were treated with 150 µg/ml cycloheximide for 12 h or 24 h. They were then scraped off and lysed for immunoblotting. The amount of remaining MHC I after the indicated times was analyzed by densitometry from 3 independent experiments. The level of MHC I observed prior to cycloheximide treatment

(0 h) was set as 100%.

72

5.7 Co-immunoprecipitation assay

To identify potential interaction partners of MICAL-L1, HeLa cells growing in 100- mm dishes were transfected with either HA-DGKα or HA-MARCKS (negative control) using XtremeGENE 9. Cells were then suspended and washed with pre-warmed PBS, and incubated for 1 h in the presence of 100 µM DMSO (control) or 100 µM R59949.

Inhibitor-treated cells were lysed with lysis buffer containing 50 mM Tris pH 7.4, 150 mM

NaCl, 0.5% Triton X-100 and 1.8 g/l Iodoacetamide on ice for 1 h. For coimmunoprecipitations, lysates were incubated with anti-MICAL-L1 antibody at 4oC overnight. Protein L beads were added to the lysate-antibody mix at 4oC for 4 h. Samples were then washed 3 times with 0.1% Triton X-100. Proteins were eluted from the protein

L beads by boiling in the presence of 4× loading buffer (250 mM Tris, pH 6.8, 8% SDS,

40% glycerol, 5% β-mercaptoethanol, 0.2% bromophenol blue [w/v]) for 10 min. Eluted proteins were then identified by immunoblotting. HA-DGKα or HA-MARCKS were each detected by anti-HA antibody.

5.8 Pulse-chase recycling assay

In experiments described in Chapter III and Chapter IV, quantification of the rate of or recycling of different cargos (such as MHC I/CD59/ β1-integrin, etc.) was performed as followings (See figure 5.1 for the workflow for measuring the rate of internalization or recycling of cargos):

HeLa cells were passaged and plated on 35 mm plates containing three coverslips. Cells were approximately 75% confluent by the time of experiment to help maintain cell viability during the acid stripping of non-internalized antibody. 73

During the assay, anti-MHC I/CD59/ β1-integrin antibody was diluted in complete DMEM in a dilution rate according to manufacturer’s recommendation. Each coverslip was inverted using forceps onto 50 m L of antibody-containing solution in a humidity chamber (see Figure 5.2 for an example of humidity chamber setting) for 30 min at 37 oC (“pulse”). After pulse, coverslips were transferred to a 12-well plate to remove non-internalized antibody by incubating the coverslips in 1 mL of acid- stripping buffer (0.5 M NaCl, 0.5% acetic acid, pH 3.0, pre-warmed to 37 oC) for 1 min.

To achieve consistent stripping, the plates were gently agitated. After acid-strip, coverslips were washed three times with 1 mL pre-warmed PBS. Two of the coverslips, denoted A and B, respectively, were fixed in 3.7% formaldehyde-PBS for

10 min at room temperature (“pulse-only”). The rest of the coverslips (assigned as C) were incubated in 2 mL pre-warmed complete DMEM media in a 12-well plate for 3 h

(chase) at 37 oC and fixed. Alexa-568 goat anti-mouse antibody was diluted in the

Abdil solution (3% BSA and 2% w/v saponin in PBS). Immunofluorescence staining was performed as described in section 5.9 “Immunofluorescence staining”.

For coverslip A, staining was performed in the Abdil solution containing 0.2% w/v saponin, while the staining of B and C was done without this permeabilizing reagent. Coverslips A - C measure the following:

A. the total internalized MHC I/CD59/ β1-integrin after pulse;

B. the basal surface level of MHC I/CD59/ β1-integrin remaining after the pulse and stripping;

C. the level of MHC I/CD59/ β1-integrin that is recycled back to PM after the chase. 74

Figure 5.1 Schematic workflow for quantitatively measuring recycling of Tf and its receptor or MHC I by flow cytometry. (Image used with permission from (Reineke, Xie et al. 2015)) 75

Figure 5.2 Schematic illustration of a humidity chamber.

The moist filter paper attached to the cover of the plate on which the glass coverslips are incubated with the antibody solution ensures that the coverslip and cells do not dry out.

(Image used with permission from (Reineke, Xie et al. 2015))

76

Image acquisition was performed as described in section 5.10 “Confocal microscopy imaging”. The Mean fluorescence intensity (MFI, arbitrary unit) as well as the area of each individual cell was assessed by ImageJ (http://rsbweb.nih.gov/ij/links.html) as described in section 5.11 “post-imaging analysis” . At least 60 cells from different fields of each sample from 3 repeated independent experiments were measured. For each independent experiment, the total fluorescence was calculated using Equation (2):

Total fluorescence = Area X MFI (2)

The rate of MHC I/CD59/ β1-integrin recycling was calculated by the following equation (Equation (3)) and the significance of the different tubes was calculated as described in section 5.12 “Statistical analysis”. The recycling rates measured in each experiment were presented in a bar graph with standard deviation.

푇표푡푎푙 푓푙푢표푟푒푠푐푒푛푐푒 표푓 퐶−푇표푡푎푙 푓푙푢표푟푒푠푐푒푛푐푒 표푓 퐵 Recycling rate = 푋 100 % (3) 푇표푡푎푙 푓푙푢표푟푒푠푒푛푐푒 표푓 퐴

5.9 Immunofluorescence staining

At each time point described in section 5.8, forceps were used to quickly but carefully transfer each coverslip cell side up into a single well of a 12-well plate that already contains 1 mL of PBS. Cells were then fixed in 3.7% formaldehyde in PBS for 10 min at room temperature. After fixation, the coverslips were washed three times with PBS, gently removing the fluid by aspiration, and pipetting in 1-2 mL PBS. Before staining, the cells were blocked and permeabilized in Abdil solution (3%BSA and 0.2% w/v saponin in PBS) for 30 min. at room temperature. The appropriate antibodies (such as markers for endosomes) were diluted in Abdil solution according to manufacturer’s brochure. The cells were placed cell-side down onto a drop of primary antibody containing solution inside a humidity chamber (see figure 5.3 for an illustration of a humidity chamber) to save on 77 reagents, and incubated for 1 h at room temperature or overnight at 4°C. Then the coverslips were carefully transferred back to a 12-well dish and wash 3 times with PBS.

The appropriate Alexa Fluor-conjugated secondary antibody was diluted in Abdil solution.

The coverslips were again placed onto antibody containing solution drops and incubated for 1 h at room temperature. DAPI were counter-stained to label the nuclei following the manufacturer’s recommendations. Eventually, cells were washed 3 times in PBS in a 12- well dish and rinsed once with ddH2O, and then mounted cell-side down onto a drop of

FluormountG (about 10 - 20 m L) that is placed on microscope slides. Allow coverslips to air dry for about 1 h at room temperature. Coverslips with sealed with clear nail polish and visualize by confocal or epifluorescence microscopy as described in section 5.10

“Confocal microscopy Imaging”.

5.10 Confocal microscopy imaging

For most of the immune-stained cells described in Chapter III and IV, except for

Superresolution images, the imaging process was performed with an inverted Zeiss LSM5

Pascal confocal microscope. Before imaging, the coverslips were prepared as described in section 5.9 “immunofluorescence staining”. A small drop of oil was placed on the processed coverslip. The eyepiece with a wide-excitation mercury bulb and appropriate filters was used to visualize the cells and select appropriate views. To maximize the objectivity and repeatability of this method, Cell fields was selected randomly, and the fields imaged contained cells that were evenly spread out without growing on top of each other.

To image the internalized cargo, the confocal mode was selected with appropriate laser and filter combinations to scan. A 543 nm laser was used to activate Alexa Fluor 568, in combination with a long-pass 560 nm filter (collects all light above 560 nm). For Alexa 78

Fluor 488, a 488 nm laser was selected for excitation, with a 505–530 nm band pass filter

(collects all light in between 505 nm and 530 nm). For DAPI, a 405 nm laser was used, with a 420–480 nm band pass filter (collects all light in between 420 nm and 480 nm).

During the focusing and calibration stage, fast scans with the lowest laser intensity possible were used to prevent photobleaching. The intensity gain was set so that the pixels are slightly oversaturated, ensuring that the signal is of sufficient intensity when obtaining averaged and higher resolution final images. Pinhole was set at one airy unit which correlates with 0.4 - 0.7 µm thick slices. Blue, green and red fluorescence were acquired sequentially to reduce the possibility of cross-activation and/or bleed-through artifacts.

Images were obtained with pixel dimensions of 1024 X 1024.For some images taken in experiments described in Chapter III and Chapter IV, z-sections of 0.5 µm interval for 6 slices were performed. The gain level and all other parameters were recorded and kept at identical settings throughout the imaging process to ensure that comparisons are valid.

After acquisition, images were saved and exported as 16-bit tiffs, and images with z- sections were processed first to make maximal projection XY images before saving. Post imaging processing were carried out as described in section 5.11 “Post imaging analysis”.

For experiments described in Chapter IV, Structured Illumination Microscopy (SIM) was done with a Zeiss Elyra PS.1 Microscope (Carl Zeiss) by using a 63× objective with numerical aperture of 1.4. Microscopy parameters settings were further described in section 5.14 “Structured Illumination Microscopy (SIM) imaging and data processing”.

For dSTORM sample preparation, cells plated on glass bottom Mattek dishes were fixed and stained with primary antibody in staining buffer, washed and then incubated with secondary antibody. The stained cells were fixed again for 10 min in 4% (v/v) paraformaldehyde, and incubated in pre-sonicated 100 nm diameter gold nanoparticles for 20 min in room temperature before dSTORM acquisition. Imaging procedure in detail 79 was described in section 5.15 “Direct Stochastic Reconstruction Microscopy (dSTORM)

Imaging and Data Processing”.

5.11 Post imaging analysis

With the confocal microscopy images acquired as described in section 5.10

“Confocal microscopy imaging”, post imaging processing was carried out for either data presentation, the measurement of the co-localization degree, or analysis of the amount of intracellular/surface pool of receptors for the calculation of internalization/recycling rate.

For data presentation, In general, 16-bit tiff file was imported into ImageJ

(http://rsbweb.nih.gov/ij/links.html) and processed to 300 dpi with dimensions of 1024 X

1024. Scale bar was added accordingly. Images with Z-sections were processed to maximal projection XY images using Z project tool. Minimal adjustment was made to contrast/brightness of the images, and any changes were maintained throughout all the images acquired for a legitimate comparison. To reveal the changes of fluorescence intensity within a cell, a straight line was drawn over the area of interest using the Straight tool, and the signal intensity of pixels along the line was achieved using Plot Profile tool.

To measure the degree of co-localization, the two channels to be compared was split by the Split Channels tool. Pearson’s coefficient as well as Mander’s coefficient was assessed with ImageJ open source plugin “Just Another Colocalisation Plugin (JACoP)”

(http://rsb.info.nih.gov/ij/plugins/track/jacop.html). The numbers from at least 60 cells from

3 independent confocal microscopy experiments, or 12 cells from 3 independent SIM experiments were averaged and plotted into a bar graph with standard deviations.

In order to measure the amount of intracellular/surface receptors, for each image, the outline of a single cell was circled with the Freehand selections tool. In order to 80 preserve data integrity and repeatability, only cells that are intact, flat and preferably spread out were selected so that the cell edge was easily recognized DAPI staining of the cell nuclei was performed to better visualize the cells. The MFI (arbitrary unit) and area for each selected cell was assessed by the Measure Tool, which was then used to calculate the total fluorescence of the receptors within a cell using equation 2 described in section

5.9 “Pulse-chase recycling assay”.

5.12 Statistical analysis

For experiments described in Chapter III and IV, quantifications on immunoblots densitometry, confocal microscopy images intensity, as well as the degree of colocalization were done by 3 independent experiments. Data sets were averaged and presented on bar graphs with standard deviations, and the significance of difference was analyzed by Student’s one-tailed paired t-test analysis. Data were considered significantly different for p-values less than 0.05. The calculated significance shown in each graph were categorized by the number of stars. * = p < 0.05, ** = p < 0.0025,

***=p<0.0005, **** = p < 0.0001.

5.13 Live cell imaging

HeLa cells on 35 mm glass bottom tissue culture dish (World Precision Instruments,

Inc.) were transiently transfected with GFP-MICAL-L1 for 18 h, and incubated with PLD inhibitors for 30 min at 37°C. The cells were then washed 3 times and incubated in fresh complete medium for 10 min at 37°C, followed by a 10 min-pulse of anti-CD59 antibody conjugated with Alexa-555. Then the cells were washed and imaged in pre-warmed

Ringer’s solution (152 mM NaCl, 25 mM HEPES, pH 7.4, 5.6 mM glucose, 4.8 mM KCl, 81

1.3 mM CaCl2, 1.2 mM KH2PO4, 1.2 mM MgSO4) covered by a thin layer of mineral oil.

Confocal microscopy imaging was done with a Zeiss LSM 5 Pascal confocal microscope

(Carl Zeiss) using a 63× objective with a numerical aperture of 1.4 and appropriate filters.

Images in each channel were obtained at 512 x 512 pixels with a large pinhole (210). The imaging was performed every 15 s, and the images taken at 45 s, 1 min 45 s, 2 min 45 s,

3 min 45 s, and 4 min 45 s were presented.

5.14 Structured Illumination Microscopy (SIM) imaging and data processing

SIM images were collected with a Zeiss ELYRA PS.1 illumination system (Carl

Zeiss) using a 63X oil objective lens with a numerical aperture of 1.4. Three lasers were used in the image acquisition: 642 nm, 568 nm, and 488 nm. Three orientation angles of the excitation grid were acquired for each Z plane, with Z spacing of 110 nm between planes. SIM processing was performed with the SIM module of the Zen BLACK software

(Carl Zeiss). The processed SIM images were then exported in TIF format. Selected cell regions were cropped in ImageJ, and rendered into a three-dimensional reconstruction with a 180-frame rotation series along the Y-axis of the processed SIM images, and exported as AVI videos.

5.15 Direct Stochastic Reconstruction Microscopy (dSTORM) imaging and data

processing

Direct Stochastic Reconstruction Microscopy (dSTORM) was performed on a

Zeiss Elyra PS.1 microscope with a 100X oil-immersion objective with 1.46 numerical aperture. For the imaging of Alexa-647 and Atto-488, 642, 488 and 405 nm laser lines were used. Cell samples were kept in imaging buffer (50 mM Tris, 10 mM NaCl, 10% 82

Glucose, pH 8.0) containing 100 mM Cysteamine Hydrochloride (MEA), 0.56 mg/mL

Glucose Oxidase and 34 µg/mL catalase, and imaged sequentially to detect Alexa-647 and then Atto-488. Images in each channel were obtained at 256 x 256 pixels. The imaging acquisition was performed at 33 Hz. 10,000-25,000 frames were obtained for each channel. 3D dSTORM was performed as described above except using a 63 X oil- immersion objective with 1.4 numerical aperture, with a 3D-PALM slider. 40,000 frames were obtained for 3D dSTORM.

Acquired data were processed using the ImageJ plugin, ThunderSTORM (Ovesny,

Krizek et al. 2014). The parameters used in the processing are listed in the table below. The parameters used in the processing are listed in the table below. Lateral drift was corrected using the ThunderSTORM correlation algorithm and pixels displaying uncertainty (precision) above 60 nm were filtered out before reconstruction to normalized

Gaussian images with 10 nm pixel size. Regions of Interest (ROI) were selected, and the number of pixels (n) within the region of interest (ROI) and area (A, nm2) of ROI were calculated. The Nyquist resolution (r) of the ROI was calculated as: The precision for each pixel within the ROI were plotted on the frequency distribution graph with 30 bins.

To carry out dual channel alignment, at least 3 fiducial gold nanoparticles fluorescing at both corresponding green and red wavelengths were used for calibration.

The lateral (X and Y) distances for same particles in the two wavelengths were measured by ImageJ, and the reconstructed image was adjusted accordingly.

Prior to 3D dSTORM processing, a cylindrical lens calibration was performed.

Fiducial gold nanoparticles were plated on glass bottom Mattek dishes, and were imaged with 3D dSTORM conditions with a Z stage step of 10 nm and 4000 nm Z range limit. The image series was analyzed by Thunderstorm 3D calibration using parameters listed below.

The calibration file was saved for 3D analysis. 3D dSTORM processing was carried out 83 using ThunderSTORM with parameters listed below. The processed data were drift corrected and filtered as described for 2D dSTORM, and rendered into Normalized

Gaussian images with 40 Z slices of 25 nm thickness. Single-slice images were presented.

To validate the image processing, the data collected were reconstructed into normalized Gaussian images with 200 nm pixel size using ThunderSTORM as described above. The images were then convolved by a Gaussian Blur filter with the mean point spread function (PSF).

Co-localization was determined by ImageJ. Histogram images of both channels with 10 nm pixel size were generated respectively, and then aligned as described above.

The pixels from each image were converted to “0” (no pixel intensity) or “1” (pixel intensity from 1-255). Colocalization was defined as a pixel with values of “1” in each channel. Total

2-channel represents the number of pixels, and total co-localized is the number of those pixels that have values of 1 in each channel. The degree of co-localization was determined by the ratio of total co-localized to total 2 channel.

Histograms representing the calculated uncertainty for each channel were plotted on frequency distribution graphs with 30 bins, the mode of the plotted curve was calculated as the precision for the image.

Parameters used in the ThunderSTORM analysis

Pixel size (nm) 200

Photoelectron per A/D count 7.0

Base level [A/D count] 100.0

EM gain 250.0

Filter Wavelet filter (B-Spline)

B-Spline order 3 84

B-Spline scale 2.0

Approximate localization of Molecules

Method Local Maximum

Peak intensity threshold Std(Wave.F1)

Connectivity 8-neighborhood

Sub-pixel localization of molecules

Method PSF: integrated Gaussian

Fitting radius [px] 3

Fitting Least squares

Initial sigma [px] 1.6

Parameters used in the 3D dSTORM calibratoin

Pixel size (nm) 320

Photoelectron per A/D count 7.0

Base level [A/D count] 100.0

EM gain 250.0

Filter Wavelet filter (B-Spline)

B-Spline order 3

B-Spline scale 2.0

Approximate localization of Molecules

Method Local Maximum

Peak intensity threshold 2*Std(Wave.F1)

Connectivity 8-neighborhood

Sub-pixel localization of molecules

Method Elliptic Gaussian w/angle 85

Fitting radius [px] 3

Fitting method Least squares

Initial sigma [px] 1.6

3D defocus model ThnderSTORM

Z stage step [nm] 10.0

Z range limit [nm] 4000.0

Parameters used in the 3D dSTORM analysis

Pixel size (nm) 320

Photoelectron per A/D count 7.0

Base level [A/D count] 100.0

EM gain 250.0

Filter Wavelet filter (B-Spline)

B-Spline order 3

B-Spline scale 2.0

Approximate localization of Molecules

Method Local Maximum

Peak intensity threshold 2*Std(Wave.F1)

Connectivity 8-neighborhood

Sub-pixel localization of molecules

Method PSF: Elliptical Gaussian (3D astigmatism)

Fitting radius [px] 3

Fitting method Least squares

Initial sigma [px] 1.6 86

5.16 Tubular endosome regrowth assay

HeLa cells were incubated with 100 nM (w/v) CAY inhibitor cocktail

(CAY10593 + CAY10594) for 30 min at 37°C, and the inhibitor was then washed away with complete medium. The cells were incubated in fresh medium for 20 min or 1 h before fixation with 4% (v/v) paraformaldehyde. The coverslips were stained for

MICAL-L1 to observe the loss and regrowth of TRE by confocal microscopy.

5.17 Membrane fractionation

The isolation of membrane and cytosol fractions of HeLa cells was performed as previously described (Sharma, Naslavsky et al. 2008). Cells grown on 100 mm dishes were suspended and homogenized on ice in homogenization buffer containing 25 mM

HEPES, 100 mM NaCl, 1 mM EDTA pH 7.4, and protease inhibitor cocktail. Cell debris was removed by 1,000 g centrifugation at 4oC for 10 min. To isolate the membrane and cytosolic fractions, the supernatant was subjected to ultracentrifugation (108,000 g) at 4oC for 1 h. The isolated membranes were dissolved in urea buffer (70 mM Tris-HCl pH 6.8, 8 mM urea, 10 mM n-ethlmalemide, 10 mM iodoacetamide, 2.5% SDS and 0.1 mM DTT) at

37oC for 15 min. Immunoblotting was used to detect protein in the fractions. 10% of the cytosolic fraction was loaded onto the gel. Cytochrome c served as specific marker for the cytosol fraction, and caveolin as a membrane fraction marker.

5.18 Duolink proximity assay

For the Duolink assay (Olink Bioscience), the manufacturer’s protocol was followed to visualize two proteins separated by a distance of less than 40 nm (Soderberg, Gullberg et al. 2006). HeLa cells grown on coverslips were transfected with GFP-MARCKS

(negative control) or GFP-DGKα. After fixation, cells were double- stained with mouse 87 anti-MICAL-L1 and rabbit anti-HA antibody at room temperature for 1 h, followed by the incubation with PLA probe oligonucleotides-conjugated secondary antibody at 37oC for 1 h. Ligase was then added at 37oC for an additional 30 min. Polymerase was added for

100 min. at 37oC for amplification, along with red fluorochrome-labeled primer

(complementary to the amplified oligonucleotides). In this case, red dots indicating individual proximity events were counted and plotted from to 3 independent experiments.

88

CHAPTER III

Diacylglycerol Kinase α Regulates Tubular

Recycling Endosome Biogenesis and Major

Histocompatibility Complex Class I Recycling

Data from the following chapter has been published in J.Biol. Chem., 2014

89

6 ABSTRACT

Major histocompatibility complex class I (MHC I) presents intracellular-derived peptides to cytotoxic T lymphocytes and its subcellular itinerary is important in regulating the immune response. Yet the mode by which MHC I is internalized is poorly studied, since it lacks the typical motifs known to mediate clathrin-dependent endocytosis.

Furthermore, a number of diacylglycerol kinase (DGK) isoforms have been implicated in clathrin-dependent internalization, yet the role of DGK on clathrin-independent endocytosis that uptakes MHC I remained unsolved.

In this chapter, we show that depletion of an isoform of diacylglycerol kinase

(DGKα), a kinase devoid of a clathrin-dependent adaptor protein complex 2 binding site, caused a delay in MHC I recycling to the plasma membrane without affecting the rate of

MHC I internalization. We demonstrate that DGKα knock-down impairs MHC I degradation, resulting in intracellular and surface MHC I accumulation. Since DGKα is required for the generation of phosphatidic acid (PA) which recruits a tubular recycling endosome (TRE) biogenesis machinery, we hypothesize that DGKα mediates the endocytic trafficking of

MHC I via the regulation of TREs generation. By Co-immunoprecipitation assay, we show that DGKα forms a complex with the TRE hub protein, MICAL-L1. Given that MICAL-L1 and the F-BAR-containing membrane-tubulating protein Syndapin2 associate selectively with PA, we propose a positive feedback loop in which DGKα generates PA to drive its own recruitment to TRE via its interaction with MICAL-L1. Our data support a novel role for the involvement of DGKα in TRE biogenesis and MHC I recycling.

90

7 INTRODUCTION

Major histocompatibility complex class I (MHC I) molecules are an essential component of the cellular immune response to a variety of intracellular pathogens and tumors. MHC I molecules are ubiquitously expressed in the surface of most cell types, carrying out their function of foreign peptide antigens presentation to cytotoxic T lymphocytes, resulting in an immune response and the killing of the infected or tumor cell

(reviewed in (Anton and Yewdell 2014)). Accordingly, expression of functional peptide- loaded MHC I on the plasma membrane depends upon the rate of MHC I synthesis, assembly and peptide binding, and the rate at which MHC I molecules are internalized from the plasma membrane.

The classic and best-characterized mode by which receptors are internalized from the plasma membrane is through clathrin-coated pits (Kirchhausen, Owen et al. 2014), via the specific recognition signal that exist on the cytoplasmic tail of cargos by adaptor proteins. However, some plasma membrane proteins, including MHC I, are devoid of such conventional clathrin-dependent endocytosis signals, and is uptake within deep invaginations that is not coated by clathrin, along a pathway regulated in part by the small

GTP-binding protein ADP-ribosylation factor 6 (Arf6) (Donaldson, Porat-Shliom et al.

2009). Although an increasing number of advances has been made in deciphering the internalization and endocytic trafficking of clathrin-independent cargos (Mayor, Parton et al. 2014), our understanding of these pathways nonetheless lags behind the more extensive knowledge of clathrin-dependent internalization.

One important specific adaptor protein for the clathrin-dependent endocytosis machinery is the adaptor protein complex 2 (AP-2), which binds to the cytoplasmic tails of transmembrane receptors, serving as a focal point for the recruitment of clathrin and a wealth of endocytic accessory molecules (Mettlen, Stoeber et al. 2009) including (but not 91 limited to) epsin (Tebar, Sorkina et al. 1996), intersectin (Yamabhai, Hoffman et al. 1998,

Adams, Thorn et al. 2000), Eps15 (Tebar, Sorkina et al. 1996), endophilin (Gad, Ringstad et al. 2000) and the vesiculation protein dynamin (Hinshaw and Schmid 1995). However,

MHC I and other clathrin-independent cargo are devoid of conventional clathrin- dependent endocytosis signals, and do not interact with classical endocytic accessory proteins. Accordingly, attempts are being made to define specific accessory molecules involved in clathrin-independent endocytosis.

Through a genome-wide RNA interference (RNAi) screening for kinases that impact endocytosis (Pelkmans, Fava et al. 2005), one novel protein, diacylglycerol kinase

δ (DGKδ) has been added to the list of clathrin-dependent endocytic regulators. The family of 10 mammalian DGKs catalyze the conversion of diacylglycerol (DAG) to phosphatidic acid (PA) (Merida, Avila-Flores et al. 2008). It was recently demonstrated that DGKδ localizes to clathrin-coated pits, and its depletion led to impaired internalization of transferrin, a well-characterized clathrin-dependent cargo (Kawasaki, Kobayashi et al.

2008). Indeed, DGKδ interacts with the AP-2μ ear domain through a sequence in its catalytic region, and disruption of DGKδ binding to AP-2 the internalization of clathrin- dependent cargo such as epidermal growth factor (Kawasaki, Kobayashi et al. 2008).

DGKα is another member of the type I DGK family that does not bear an AP-2 recognition motif. While many of the DGK proteins have not been studied extensively, there is growing evidence that PA impacts receptor internalization (Antonescu, Danuser et al. 2010) as well as their recycling back to the PM. Indeed, the depletion of local PA level by treatment of phospholipase D (PLD) inhibitor cocktail results in the delayed recycling of transferrin (Giridharan, Cai et al. 2013). PA has also been implicated in the recruitment of proteins such as Molecules Interacting with CAsL-Like1 (MICAL-L1) and

Syndapin2 to endocytic membranes to promote tubular recycling endosome biogenesis 92 and recycling events (Giridharan, Cai et al. 2013). Furthermore, recent findings suggest that DGKα is essential to support β5γ1 integrin recycling and invasive migration (Rainero,

Caswell et al. 2012). Since AP-2-binding sequence is absent from DGKα (similar to that of DGKδ (Kawasaki, Kobayashi et al. 2008), and because integrin trafficking is at least in part mediated through clathrin-independent pathways (Brown, Rozelle et al. 2001, Jovic,

Naslavsky et al. 2007), we hypothesized that DGKα plays a role in the endocytic trafficking of clathrin-independent cargo, such as MHC I.

In this study, we show that DGKα-depletion leads to an increased level of total as well as surface MHC I, resulting from a delayed degradation, while it is dispensable for the internalization of MHC I. On the other hand, MHC I recycling to the plasma membrane was impaired in the absence of DGKα. Our findings support a role for DGKα in the regulation of MHC I trafficking, consistent with its function in the conversion of DAG to PA, a step necessary for the generation of MICAL-L1-containing tubular recycling endosomes

(TRE).

8 RESULTS

8.1 The rate of MHC I internalization is not regulated by DGKα

MHC I is expressed on, and constantly transported to and from the cell surface, carrying an intracellular-digested peptide, which is presented to cytotoxic T cells, as part of the mechanism of the immune system surveillance. As a consequence, regulation of

MHC I trafficking to and from the plasma membrane is a basic requirement for an efficient immune response.

DGKδ is a novel kinase that comprises part of the clathrin-mediated internalization machinery which mediate the internalization of classic clathrin-dependent receptor, the 93 transferrin receptor (Kawasaki, Kobayashi et al. 2008). Since the endocytosis of MHC I is independent of clathrin, and DGKδ poses modest effects on its internalization, we therefore investigated whether a related member of the DGK family, DGKα regulates its endocytic itinerary.

Firstly, we took advantage of a “pulse-chase” assay to measure the internalization rate of MHC I. HeLa cells were incubated with W6-32 anti-MHC I antibodies at 37oC for a short period as a “pulse” process. Then the non-internalized antibody was removed by a brief acid-strip, and the cells were immediately fixed by 4% paraformaldehyde. After fixation, internalized MHC I bound to the antibody was visualized with a secondary antibody. Depletion of DGKα with siRNA treatment (Fig. 8.1A) did not alter levels of internalized MHC I at 10, 20 and 30 min as detected by flow cytometry (Fig. 8.1B), thus

DGKα does not impact the rate of MHC I internalization.

The next stage for MHC I-containing vesicles after internalization is the sorting endosomes (Naslavsky, Weigert et al. 2003). To examine whether internalized MHC I properly reached sorting endosomes marked by the early endosomal autoantigen 1

(EEA1), without the regulation of DGKα, we treated HeLa cells with either Mock or DGKα siRNA, and then allowed the internalization of anti-MHC I antibody for 15 min. at 37°C before being acid-stripped and fixed-permeabilized. Previous experiments indicated that within first 15 min. of internalization, MHC I is present at sorting endosomes (Naslavsky,

Weigert et al. 2003). The internalized MHC I and EEA1 was co-stained with different dyes and visualized by confocal microscopy with appropriate laser settings (Fig. 8.1C-H).

Images were quantified by Image J with two different statistical tests (Pearson’s and

Manders’ coefficients, Fig. 8.1I-J correspondingly). Both analyses indicate that the loss of

DGKα did not alter the normal influx of MHC I into sorting endosomes.

94

95

Figure 8. 1: The rate of MHC I internalization is not regulated by DGKα.

A) HeLa cells were treated with siRNA against DGKα for 72 h and then transfected with HA-DGKα for 18 h before cell lysis. HA-DGKα expression was measured by immunoblotting with anti-HA. Hsc70 was used as a loading control. B) The graph depicts flow cytometry analysis of 3 experiments measuring the rate of MHC I internalization. Cells were incubated with W6/32 anti-MHC I for 10, 20, and 30 min. at 37oC, followed by a brief acid-strip to remove non-internalized antibody. After fixation and permeabilization they were stained with Alexa-647 goat anti-mouse F(ab)2. In parallel, the steady-state surface level of MHC I was determined for each treatment by staining with anti-MHC I followed by secondary antibody, both in the absence of saponin. To normalize for different levels of surface MHC I between the treatments (see Fig. 8.2A), the internalization rate was calculated as the ratio between internalized MHC I / steady-state surface MHC I. C-J) Mock and DGKα-depleted HeLa cells were incubated with W6/32 mouse anti-MHCI antibody for 15 min. at 37oC, stripped and fixed-permeabilized. Internalized MHC I was detected with Alexa-568 goat anti-mouse. Early endosomes were stained with rabbit anti- EEA1 and Alexa-488 goat anti-rabbit DAPI designates the nuclei. Orange arrows exemplify co-localization between MHC I and EEA1, as measured using Image J (I-J) and statistically analyzed with Pearson’s coefficient (I) and Manders’ coefficient (J). Standard deviation is shown. Bar, 10 µm. (Images used with permission from (Xie, Naslavsky et al. 2014))

96

We have also shown that a longer internalization (30 min.) resulted in a decreased degree of co-localization between MHC I and EEA1 in both mock and DGKα-siRNA treated cells (data not shown), suggesting that the exit of MHC I-containing vesicles from early endosomes was similarly unaffected by DGKα-depletion. Taken together, our data suggest that MHC I internalization and its access to sorting endosomes are not regulated by DGKα.

8.2 DGKα controls the half-life of MHC I

The ability of MHC I to present antigens to T lymphocytes directly depends upon its level at the plasma membrane. Having seen that DGKα does not participate in MHC I internalization, we then turned to inquire whether DGKα affects MHC I partitioning at the plasma membrane by knocking down DGKα and measuring the level of cell-surface residing MHC I. To our surprise, the MHC I level on the cell surface was dramatically up- regulated in the absence of DGKα, as shown by immunofluorescence staining of non- permeabilized cells (Fig. 8.2A right panel) compared to Mock-treated cells (Fig. 8.2A left panel). Quantification of the fluorescence intensity by Image J from 3 independent experiments (Fig. 8.2B) revealed nearly a 2-fold increase in the amount of MHC I residing on the cell surface of DGKα-depleted cells. Under similar condition, flow cytometry analysis on non-permeablized cells confirmed increased levels of MHC I on the plasma membrane upon DGKα knock down, although to a lesser extent than that measured by immunofluorescence (Fig. 8.2C, 25% increase).

97

98

Figure 8. 2 DGKα controls the half-life of MHC I.

A-C) The surface level of MHC I for mock and DGKα-knock-down cells was measured by immunofluorescence staining (A, and quantified by Image J in B) and by flow cytometry (C). Briefly, Mock and DGKα knock-down cells were fixed and stained with anti-MHC I and secondary antibody in the absence of permeabilizing detergent. D-F) The total cellular MHC I protein from Mock and DGKα-depleted cells was measured by flow cytometry (D) with anti MHC I and secondary antibody in the presence of saponin, and by immunoblotting (E; Hsc70 serves as a loading control). F) The graph depicts densitometry analysis of 3 independent experiments as in E. G) 150 µg/ml cycloheximide was applied to HeLa cells for 0, 12 and 24 h prior to lysis. Immunoblotting of total cellular MHC I protein was done with anti-MHCI antibody. H) Densitometry analysis of 3 independent experiments as in G is shown with standard deviation. * = p < 0.05, ** = p < 0.0025, **** = p < 0.0001. Bar, 10 µm. (Images used with permission from (Xie, Naslavsky et al. 2014))

99

Apart from the elevated surface- MHC I in Mock- vs. DGKα-depleted cells, we also measured the total cellular MHC I level. Indeed, we observed a similar overall increase in the total MHC I level, demonstrated by both flow cytometry measurements (in Fig. 8.2D) of cells permeabilized with saponin, as well as an immunoblotting analysis. Both experiments revealed that siRNA knock-down of DGKα caused a similar elevation in total

MHC I to that observed on the cell surface (~33%). (Fig. 8. 2E; Hsc70 serves as a loading control). Fig. 8.2F depicts densitometric quantification of 3 separate immunoblotting experiments, as done in Fig. 8.2E).

We hypothesized that the increased total/surface MHC I level in the absence of

DGKα is due to an impaired degradation of MHC I. To test this hypothesis, we assessed the rate of MHC I degradation by following the cellular MHC I level over time upon inhibition of de novo protein synthesis with cycloheximide. Immunoblotting (Fig. 8.2G, upper gel) and densitometry analysis of 3 similar experiments in Fig. 8.2H, showed that ~70% of

MHC I underwent degradation in the first 12 h in Mock-treated cells, with little additional degradation at 24 h. In contrast, in DGKα-depleted HeLa, only ~40% of MHC I was degraded at 12 and 24 h. We conclude, therefore, that there is mis-sorting of MHC I in

DGKα-depleted cells at a post sorting-endosome stage that causes its inefficient degradation. This, in turn, leads to its accumulation both inside the cell and at the plasma membrane.

8.3 DGKα-depletion interferes with MHC I trafficking to the recycling compartment

and delays its recycling

To examine which stages of post-internalization trafficking are regulated by DGKα, we followed the intracellular itinerary of internalized MHC I over time using a “pulse-chase” immunofluorescence-based assay. We incubated cells with W6/32 anti-MHC I antibody 100 for 30 min. at 37oC in the process of “pulse”, followed by a 1 min. acid-strip to remove non- internalized antibody-receptor complexes, and then ‘chased’ in complete media for 30 min.,

1 h, and 2 h at 37oC. Preliminary data suggested that within 1-2 h chase, most internalized

MHC I reaches the perinuclear endocytic recycling compartment (ERC) in HeLa cells

(Caplan, Naslavsky et al. 2002, Naslavsky, Weigert et al. 2003, Naslavsky, Weigert et al.

2004, Donaldson and Williams 2009). Thus we measured the recycling of MHC I at a shorter time point (30 min. pulse and 30 min. chase), as we show here in Fig. 8.3 A and

D. By immunofluorescence staining, MHC I were seen localized to scattered endosomes in both mock- and DGKα siRNA-treated cells, in consistent with the MHC I distribution obtained after the short 15 min. uptake described in Fig. 8.1C-H, and its association with sorting endosomes. At this time point, some MHC I could be observed accumulating in the perinuclear area. It is noteworthy that upon DGKα-depletion (in Fig. 8.3D), more internalized MHC I signal was recorded. Although the internalization rate was not changed, more MHC I molecules were internalized per individual cell potentially because the initial surface MHC I pool was larger in DGKα-deficient cells.

On the other hand, at 1 h and 2 h chase in Mock-treated cells, the MHC I reached a compact and bright perinuclear region (Fig. 8.2B-C; arrows) corresponding to the

Endocytic Recycling Compartment (ERC). For DGKα-depleted cells (Fig. 8.3 E-F), after either a 1 h or 2 h chase, MHC I maintained its scattered distribution pattern in the cell periphery. Only at a later time point (3 h; data not shown), MHC I began to concentrate at the perinuclear area, suggesting a delay in MHC I trafficking to the ERC upon DGKα depletion.

101

102

Figure 8.3 DGKα-depletion interferes with MHC I trafficking to the recycling compartment and delays its recycling.

A-F) Mock and DGKα-depleted HeLa cells were incubated with anti-MHC I antibody for 30 min. at 37oC (pulse), stripped, and chased in complete media for 30 min., 1 h, and 2 h at 37oC, followed by a second strip and fixation-permeabilization. MHC I remaining in the cell was detected with Alexa-568 goat anti-mouse. Arrows point to MHC I concentrated in the ERC. G) Quantification of immunostained MHC I that re-appeared at the PM (underwent recycling). Mock and DGKα-depleted cells were pulsed as in A-F, then chased for 3 h, followed by fixation. The cells were stained with Alexa-568 goat anti-mouse antibody in the absence of detergent. The total MHC I was measured in parallel with fixed cells incubated with anti-MHC I in the presence of detergent. The return of MHC I to the cell surface (following 3 h chase) was calculated as a portion of the total MHC I. 120 cells from 3 independent experiments were analyzed with Image J. Shown is standard deviation. ** = p < 0.0025. Bar, 10 µm. H) Graph depicts flow cytometry analysis of three experiments measuring the rate of transferrin recycling. Mock- and DGKα-depleted HeLa cells were incubated with Transferrin Alexa Fluor® 633 conjugate for 1 h at 37 °C (pulse), and chased in complete medium for 1 h at 37 °C. Cells were then fixed and subjected to flow cytometry. I–J) Total level of β1-Integrin for Mock and DGKα-knock-down cells was measured by immunoblotting. Densitometry analysis of three independent experiments as in A is shown in B with standard deviation. K, surface level of β1-integrin for Mock and DGKα-knock- down cells was measured by flow cytometry with anti-β1-integrin and appropriate secondary antibody staining in the absence of permeabilizing detergent. Statistical analysis was performed with data from three independent flow cytometry experiments. Shown is standard deviation. *, p < 0.05; **, p < 0.0025. (Images used with permission from (Xie, Naslavsky et al. 2014))

103

The delayed delivery of MHC I to the ERC in DGKα-knock-down cells prompted us to hypothesize that MHC I recycling back to the plasma membrane might be similarly delayed. To justify for this hypothesis, we used an immunofluorescence-based assay to measure the re-appearance (recycling) of MHC I at the plasma membrane. HeLa cells were first pulsed with W6/32 anti-MHC I antibodies for 30 min. at 37oC and briefly acid- stripped, followed by a 3 h chase in fresh complete media when MHC I returns back to the plasma membrane. After pulse-chase, the surface MHC I level was monitored with a

W6/32 anti-MHC I antibody, using a secondary anti-mouse antibody in the absence of detergent. Surface anti-MHC I was measured by Image J. To normalize the difference in surface MHC I levels observed at the starting point (see Fig. 8.3.2A-C), a parallel set of coverslips was stained using detergent to measure the total MHC I level. Thus, the amount of recycled MHC I was calculated as a portion of the total pool. In Mock-treated cells (Fig.

8.3G), ~40% of MHC I returned to the cell surface within 3 h chase, while in DGKα knock- down cells, only ~10% returned. This supports the notion that DGKα affects MHC I recycling.

8.4 DGKα is required for the biogenesis of tubular recycling endosomes

In search of a mechanism by which DGKα mediates the recycling of MHC I, we turned to the enzymatic reaction catalyzed by DGKα; the generation of phosphatidic acid

(PA) by phosphorylating diacylglycerol (DAG) (Fig. 8.4E). Having previously described a role for PA in the biogenesis of TRE (Giridharan, Cai et al. 2013), we next questioned whether DGKα is also essential for the biogenesis of TRE, which are required for MHC I recycling.

104

105

Figure 8.4: DGKα is required for the biogenesis of tubular recycling endosomes.

A–D) Morphology of TRE was assessed by immunostaining with the endogenous TRE marker, MICAL-L1. HeLa cells were treated with Mock (A) or DGKα-siRNA (B) for 72 h. C, Mock- or D, DGKα-siRNA-treated cells were also incubated with 100 μm propranolol, a phosphatidic acid phosphatase (PAP) inhibitor, for 30 min at 37 °C (A-B were treated with DMSO). After fixation, cells were stained with anti-MICAL-L1 antibodies and Alexa-568 goat anti-mouse secondary antibody. Yellow boxes depict the insets shown below. D) Image J analysis of A-C; MICAL-L1 area per cell, using 60 cells from 3 independent immunofluorescence staining experiments. E) Image J analysis of A–D; MICAL-L1 area per cell, using 60 cells from three independent immunofluorescence staining experiments. F) Schematic diagram illustrating the function of DGK, PAP, and their corresponding inhibitors. G) HeLa cells were treated as in A–C, and then homogenized. Cytoplasmic and membrane fractions were separated by ultracentrifugation. The entire membrane fraction and 10% of the cytosolic fractions were separated on SDS-PAGE, and MICAL-L1 was detected by immunoblotting. H) Densitometry analysis from 3 independent membrane fractionation experiments (as done in F) is shown as the ratio of membrane-bound MICAL-L1 to cytosolic MICAL-L1. Error bars represent standard deviation. **, p < 0.0025; ***, p < 0.0005; ****, p < 0.0001. Bars, 10 μm. (Images used with permission from (Xie, Naslavsky et al. 2014))

106

To visualize TRE by immunofluorescence we used MICAL-L1, previously described stably recruited to the membrane of tubular recycling endosomes (TRE), and thus can be used as an endogenous marker for these tubular structures (Sharma,

Giridharan et al. 2009). During interphase, MICAL-L1-decorated TRE are typically detected in approximately 50% of HeLa cells and they are depicted in a field of Mock- treated cells in Fig. 8.4A (and inset). On the contrary, when DGKα was depleted, TRE appeared as significantly shorter tubular structures (Fig. 8.4B, see inset). Upon DGKα knock-down, where synthesis of PA is compromised, we sought to preserve PA levels by preventing its catabolism. Propranolol is an inhibitor of phosphatidic acid phosphatase

(PAP), which dephosphorylates PA to generate DAG (illustrated in Fig. 8.4E). Thus,

DGKα-depleted cells were incubated with propranolol for 30 min. at 37oC. As shown in Fig.

8.4C, arresting PA catabolism with propranolol, rescued TRE formation, giving rise to elaborate MICAL-L1-decorated tubular structures.

The formation of TRE was assessed by measuring MICAL-L1-containing tubular area per cell by Image J analysis. 3 different experiments (done as in Fig. 8.4A-C) are plotted in Fig. 8.4D (analyzing 60 cells taken from each treatment). We discovered that the average tubular area of MICAL-L1-containing structures was measured to approximately 10 μm2/Mock-treated cell, while DGKα-depleted cells had only 5μm2/cell.

We next sought to examine whether the TRE tubulated area is affected by DGKα via the regulation of phosphatidic acid level. To this aim, we treated cells with propranolol to preserve the pool of phosphatidic acid even in the presence of DGKα siRNA. By analysis of 3 independent experiments, we observe that in the DGKα-depleted cells that received propranolol treatment, TRE area reached as high as 40 μm2/cell, suggesting that: I) other enzymes, such as PLD and LPAT may allow generation of basal levels of PA in the absence of DGKα, and II) PAP inhibition by propranolol effectively caused PA 107 accumulation, possibly due to a rapid in vivo turnover of PA to diacylglycerol by PAP (see discussion).

The requirement of PA for TRE formation was further tested biochemically in Fig.

8.4F. Endogenous membrane-bound MICAL-L1 (representing intact TRE) was isolated by homogenization and ultracentrifugation. As described in the Experimental Procedures, either mock-treated or DGKα-siRNA treated cells (in the presence or absence of propranolol), were homogenized and subjected to ultracentrifugation to separate supernatant containing cytosolic proteins from the pellet containing membrane-bound proteins. The level of endogenous MICAL-L1 in each fraction was detected by immunoblotting; caveolin was used as a membrane-bound control, whereas cytochrome

C was used as a cytosolic protein marker (Fig. 8.4F). Note that the band representing caveolin shifted upward in the membrane fraction compared to total and cytosol fractions, possibly due to the use of urea to solubilize the membrane sample.

As shown in Fig. 8.4F (upper panel), membrane-bound MICAL-L1 in the mock cells decreased substantially upon DGKα-depletion (10% of the cytosolic fraction was loaded).

This is in line with our immunofluorescence staining, which showed diminished and dramatically shortened TRE under these conditions (compare to Fig. 8.4A-B). In contrast, when the DGKα-depleted cells were treated with propranolol (Fig. 8.4F right upper panel), almost all of the MICAL-L1 shifted to the membrane fraction, reflecting the increase of

MICAL-L1-decorated TRE observed in Fig. 8.4C.

Densitometry measurements of 3 fractionation experiments (as in Fig. 8.4F), were used to compare the ratio of MICAL-L1 in the membrane vs. cytosolic fraction (Fig. 8.4G).

As calculated, upon DGKα-depletion, membrane-bound MICAL-L1 comprised less than a half of its normal membrane-association in mock-treated cells. The extent of this reduction is similar to the immunofluorescence-based analysis in Fig. 8.4D (left and middle bars on 108 the graph). Propanolol treatment caused a massive recruitment of MICAL-L1 to membranes, as evidenced in morphologic and biochemical assessments (Fig. 8.4C-D, F-

G). Taken together, DGKα is important for TRE formation and maintenance, possibly by providing a means for local PA elevation in membranes.

8.5 DGKα forms a complex with MICAL-L1.

Having proved that DGKα is indispensable for the recruitment of MICAL-L1 onto tubular membranes, we next investigated whether they interact with each other. Although both proteins partition into cytosolic and membranous fractions, since active DGKα is recruited onto the membranes, we assumed that an interaction between them might occur on membranes. To address their potential association, we performed co- immunoprecipitation and in vivo co-staining. For co-immunoprecipitation, HA-DGKα or

HA-MARCKS-transfected cells (control) were lysed (Fig. 8.5A upper panel), and endogenous MICAL-L1 was pulled-down with specific antibodies. As demonstrated in Fig.

8.5A (lower panel), HA-DGKα, but not HA-MARCKS, was immunoprecipitated by MICAL-

L1. This association could not be detected by a selective yeast two-hybrid assay (data not shown), suggesting an indirect interaction. 109

110

Figure 8.5 DGKα forms a complex with MICAL-L1.

DGKα and MICAL-L1 co-immunoprecipitate. A) HA-MARCKS (negative control) and HA-DGKα-transfected cells were lysed and blotted with anti-HA. B) In the same lysates, endogenous MICAL-L1 was pulled-down with anti-MICAL-L1 and eluted. Eluates were immunoblotted with anti-HA and anti-MICAL-L1. C–H) Micrographs depicted show representative data from proximity ligation assays. Cells grown on coverslips were transfected with GFP-DGKα or HA-MARCKS (negative control). Duolink (proximity ligation) assay was performed using mouse anti-MICAL-L1 and rabbit anti-GFP. Dark dots in C–D indicate proximity (<40 nm) between MICAL-L1 and the HA- tagged protein. E–F, the same cells were also immunostained with anti-HA, to show similar degrees of transfection. G and H, endogenous MICAL-L1 was immunostained to display TRE morphology. I) number of proximity ligation events from 3 independent experiments (as done in C–D) was counted by Image J and plotted with standard deviation. Bar, 10 μm. (Images used with permission from (Xie, Naslavsky et al. 2014))

111

To assess whether DGKα and MICAL-L1 associate in vivo, a proximity ligation assay (Duolink®) was performed. This assay detects in situ proximity (about 40nm) between two proteins (Soderberg, Gullberg et al. 2006) and unlike conventional immunofluorescence methods, such interactions can be detected even when a major portion of the protein is cytoplasmic (cytosolic DGKα). Cells transfected with similar expression levels of HA-DGK α (Fig.8.5, C, E, and G) or HA-MARCKS (negative control:

Fig.8. 5, D, F, and H) were fixed and processed for the proximity assay according to the manufacturer’s protocol, using primary antibodies: mouse anti-MICAL-L1 to detect endogenous MICAL-L1 and rabbit anti-HA. Oligonucleotides conjugated to secondary antibodies underwent ligation if residing about 40nm apart from each other. A specific fluorescent label detected such ligation events (seen as dark dots in Fig.8. 5, C–D, and nuclei are stained with DAPI).

A significant number of proximity events (dots) were observed in HA-DGKα transfected cells (Fig. 8.5E), compared to the HA-MARCKS-transfected negative control

(Fig. 8.5F). Image J quantification of 3 independent proximity assays is summarized in Fig.

8.5G, indicating that DGKα and MICAL-L1 are in close proximity in vivo. Fig. 8.5H-I are controls, showing equal levels of transfection for both HA-tagged proteins, whereas the

MICAL-L1 immunostaining (J-K) demonstrates the efficiency of the DGKα-depletion (with shorter and fewer TRE observed in J).

8.6 The biogenesis of TRE requires the activity of DGKα

We have showed here that DGKα is required for the biogenesis of TRE and that it partially localizes to and associates with MICAL-L1-containing TRE in vivo. We next 112 investigated whether local PA generation was directly required for the association between

MICAL-L1 and DGKα. To this aim, we used the diacylglycerol kinase inhibitor, R59949, to arrest PA production (illustrated in Fig.8.4E). As shown in Fig. 8.6A-B, upon 1 h treatment with R59949, the number and size of MICAL-L1-containing TRE dropped substantially.

Quantification of the mean square area of TRE from 3 independent experiments is summarized in Fig. 8.6C. This loss of TRE is similar to the one observed upon DGKα- siRNA treatment (see Fig. 8.4A-B). Thus, under these conditions of DGKα-depletion and subsequent decrease of MICAL-L1-containing TRE, we now asked whether DGKα and

MICAL-L1 continue to associate. As shown in Fig. 8.6D, although the DGKα inhibition did not alter the MICAL-L1 protein level substantially (upper left panel), co- immunoprecipitation indicated that impaired PA generation resulted in the loss of interaction between MICAL-L1 and DGKα (Fig. 8.6D right panel). Taken together, we propose that the activity of DGKα is required for TRE formation and MHC I recycling.

113

114

Figure 8.6 The biogenesis of tubular recycling endosomes requires the activity of

DGKα.

A and B) cells grown on coverslips were treated with either DMSO or 100 μm R59949 (DGK inhibitor) for 1 h at 37 °C, fixed-permeabilized, and then stained with anti-MICAL- L1. C) Mean square area of TRE-localized endogenous MICAL-L1, taken from three independent experiments (from the experiments depicted in A and B), was measured with Image J and plotted with standard deviation. D) quantification of immunostained MHC I that re-appeared at the PM (underwent recycling). DMSO- and R59949-treated cells were pulsed with anti-MHC I antibody for 30 min at 37 °C, then chased for 3 h, followed by fixation. The cells were stained with Alexa- 568 goat anti-mouse antibody in the absence of detergent. The total MHC I was measured in parallel with fixed cells incubated with anti-MHC I in the presence of detergent. The return of MHC I to the cell surface (following 3 h chase) was calculated as a portion of the total MHC I. 120 cells from three independent experiments were analyzed with Image J. E) Surface level of MHC I for DMSO- and R59949-treated cells was measured by flow cytometry. F) Total MHC I level of DMSO- or R59949-treated cells was measured by immunoblotting with anti-MHC I (HC10). Hsc70 was used as a loading control. G, graph depicts densitometry analysis of three independent experiments as in D. Shown is standard deviation. H) Impaired PA production results in the loss of interaction between MICAL-L1 and DGKα. HA- DGKα transfected cells, growing on 100-mm dish were incubated with either DMSO or 100 μm R59949 for 1 h at 37 °C, and lysed (see input upper panel in H; anti-actin is a loading control). Lysates were co-immunoprecipitated with anti-MICAL-L1 antibodies (lower panel in H), and immunoblotted with anti-HA, and anti MICAL-L1. I) Proposed model depicting the role of DGKα in TRE biogenesis. DGKα is recruited onto membranes through its interaction with a complex containing MICAL-L1. This leads to further generation of PA, and further recruitment of MICAL-L1, which serves as a membrane hub. Its association with the BAR-containing protein, Syndapin2, and the local enrichment of PA culminate in elongation of the membrane into a tubular endosome. Potential “vesiculators” such as EHD1, GRAF1, and cPLA2α are then recruited by MICAL- L1 and form a constriction site, ultimately leading to scission/budding of a transport vesicle. ***, p < 0.0005; ****, p < 0.0001. Bar, 10 μm. (Images used with permission from (Xie, Naslavsky et al. 2014)) 115

9 DISCUSSION

The local generation of PA within the membranes of cellular organelles has been shown with increasing evidence to mediate the trafficking through the endocytic pathways.

For instance, the presence of PA is required for efficient transferrin receptor internalization

(Antonescu, Danuser et al. 2010), as well as for the biogenesis of TRE and the recycling of receptors back to the plasma membrane (Giridharan, Cai et al. 2013). However, till today there is little knowledge about the mechanisms by which the temporal generation of local PA concentrations is regulated.

Three major pathways have been depicted that lead to the generation of PA in mammalian cells. First, an enzyme known as lysophosphatidic acyl transferases (LPAT) catalyzes the conversion of lysophosphatidic acid to PA by the transfer of an acyl chain

(reviewed in (Shindou, Hishikawa et al. 2009)). The most prominent pathway for PA generation might be mediated by Phospholipase D (PLD) removing the head group of glycerphospholipids such as phosphatidylcholine (Donaldson 2009). In addition, the phosphorylation of diacylglycerol can lead to conversion to PA through the action of diacylglycerol kinases (reviewed in (Merida, Avila-Flores et al. 2008)).

Several reasons drove us to investigate the role of DGKα in the trafficking of MHC

I, a receptor that is typically internalized independently of clathrin, in this study. First, among the 10 isoforms of the DGKs family, DGKα is ubiquitously expressed in a variety of cell types, including fibroblasts, T lymphocytes, endothelial cells and various stem cells

(Merida, Avila-Flores et al. 2008). Second, whereas the Adaptor Protein-2 (AP-2) α-ear binding sites mediates the association of type II DGKs (δ, ε and ζ) to the clathrin-mediated endocytosis mechanisms, type I DGKs including DGKα lack these motifs. Based on this sequence difference, we hypothesize that DGKα more likely regulates clathrin- independent cargos. In addition, a recent study demonstrated that DGKα plays a role in 116

α5β1 integrin recycling and thus regulates cell invasiveness, by tethering the Rab11 effector, Rab-coupling protein (RCP), to pseudopodal tips (Rainero, Caswell et al. 2012), implicating a potential role for DGKα on the endocytic recycling routes.

Through the experiments described in the results, we have demonstrated that while DGKα-depletion has little or no impact on the internalization of MHC I, it poses other effects on the intracellular itinerary of this receptor, including recycling and degradation.

Despite that internalization is not affected, surprisingly we observed a ~2-fold increase in the level of MHC I at the cell surface at steady-state. This increase, however, was concurrent with a similar increase in the total cellular level of MHC I that accumulated in the absence of DGKα. Moreover, from previous studies, we have seen that delays in receptor recycling are transient effects, and cells have mechanisms to overcome these delays over time (Naslavsky, Boehm et al. 2004, Naslavsky, Rahajeng et al. 2006). Thus, our observation of increased MHC I at the plasma membrane after 48 h of DGKα-depletion can be attributed to compensatory recycling or the use of alternate recycling pathways.

However, our data showing delayed MHC I degradation in the absence of DGKα suggest that in addition to regulating MHC I recycling, DGKα might also affect MHC I trafficking at an earlier step in the pathway.

The potential role of DGKα on the tubular recycling endosomes should be addressed in future studies. The most striking effect of DGKα knock-down is the delayed

MHC I recycling, leading to an acute postponement in the return of the receptors back to the plasma membrane. Since we have currently demonstrated that TRE biogenesis is required for normal receptor recycling, and PA is an essential lipid needed for the initiation of TRE biogenesis (Giridharan, Cai et al. 2013), we proposed that DGKα activity facilitates

TRE formation and recycling by the conversion of diacylglycerol to PA. Our data designated that the absence of DGKα results in the loss of MICAL-L1-containing TRE, 117 and that this effect could be blocked by treatment with propranolol, an inhibitor of the PA phosphatase enzyme that converts PA to diacylglycerol.

Since DGKα is mostly localized to the cytoplasm, its effective recruitment to membranes is key to its activity in the cell. Our co-immunoprecipitation studies provide evidence that active DGKα forms a complex with MICAL-L1, leading us to speculate that in its capacity as a membrane hub (Rahajeng, Panapakkam Giridharan et al. 2012),

MICAL-L1 recruits DGKα to recycling endosomes. As such, we envision a mechanism in which the generation of PA by DGKα serves as a positive feedback loop for recruitment of more MICAL-L1 and DGKα, spurring additional PA generation that facilitates TRE biogenesis (see working model in Fig. 8.6E). In (A), MICAL-L1 and Syndapin2, which bind with high selectivity to PA (Giridharan, Cai et al. 2013), are recruited onto membranes containing DAG and some PA. (B) DGKα then associates with the membrane, forming a possible complex with MICAL-L1. (C) Local generation of additional membrane- embedded PA further shapes membrane elongation into TRE that contain cargo. (D)

“Vesiculators,” such as EHD1 (Cai, Caplan et al. 2012, Cai, Giridharan et al. 2013),

GRAF1 and the lipid modifier cPLA2 (Cai, Caplan et al. 2012) constrict the TRE by mechanical force and changes in lipid composition, respectively. (E) Scission gives rise to mobile and small recycling endosomes, which are transported along microtubules towards the PM. It is not clear if any of the membrane “sculpting” proteins remain on the highly- curved recycling endosomes.

Although further studies will be required to dissect the function of DGKα in regulating MHC I post-internalization trafficking and prior to recycling, our study highlights a novel role for this enzyme in the generation of TRE and the subsequent regulation of endocytic recycling.

118

CHAPTER IV

The endocytic recycling compartment maintains

cargo segregation acquired upon exit from the

sorting endosome

Data from this chapter was published in Mol.Biol.Cell, 2016

119

10 ABSTRACT

The recycling of receptors occurs either directly (fast recycling), or via an organelle known as the endocytic recycling compartment (ERC). The morphology and function of the ERC is poorly understood and is typically described as a series of tubular-vesicular membrane-bound organelles localized to the peri-nuclear region of the cell. Our understanding of endocytic recycling improved significantly from following internalized receptors such as the transferrin receptor, as they internalize through clathrin-coated pits and reach the recycling compartment. After internalization, transferrin receptor is sorted into recycling endosomes, which are regulated by the small GTPases Rab11. Rab11 is regarded as a major “sorter” of clathrin-derived cargo recycled to the plasma membrane in concert with the motor protein, Myosin Vb. However, the sorting and recycling of clathrin-independent cargos such as Major Histocompatibility Complex Class I (MHC I) and the glycosylphosphatidylinositol-anchored protein, CD59, remains poorly-defined.

Despite evidence that cargos from both CME and CIE pathways are sorted at the early/sorting endosome (SE), whether cargo mixes downstream at the ERC or remains segregated is still an unanswered question. Herein, we use 3D Structured Illumination

Microscopy, dual-channel and 3D direct Stochastic Optical Reconstruction Microscopy

(dSTORM) to obtain new information about ERC morphology and cargo segregation. We show that cargo internalized either via clathrin-mediated endocytosis (CME) or independently of clathrin (CIE) remains segregated in the ERC, likely on distinct carriers.

This suggests that no further sorting occurs upon cargo exit from SE. Moreover, 3D dSTORM data support a model in which some, but not all ERC vesicles are tethered by contiguous ‘membrane bridges.’ Furthermore, TRE preferentially traffic CIE cargo, and may originate from SE membranes. These findings support a significantly altered model 120 for endocytic recycling in mammalian cells, in which sorting occurs in peripheral endosomes and segregation is maintained at the ERC.

11 INTRODUCTION

The plasma membrane (PM) of mammalian cells, a highly dynamic compartment, continuously samples the environment and internalizes receptors and membrane lipids.

In general, there are two major routes for internalization: 1) cargos with specific signals in their cytoplasmic tails, including transferrin receptor (TfR) and low density lipoprotein receptor (LDLR), enter the cell through clathrin-mediated endocytosis (CME)

(Kirchhausen, Owen et al. 2014), and 2) internalized cargos via clathrin-independent endocytosis (CIE) such as CD59 and β1-integrin (Sandvig, Torgersen et al. 2008).

Despite their entry mechanism, after their internalization, endosomes carrying cargos from both CME and CIE tends to fuse with early/sorting endosomes (SE) (Naslavsky, Weigert et al. 2003). In SE, cargos is triaged to either lysosomes for degradation, or recycling pathway that returns cargo to the PM (Jovic, Sharma et al. 2010). Recycling internalized molecules is a tightly regulated process that maintains the homeostasis of these molecules at the cell surface. Aside from PM homeostasis, the key factors for a variety of cellular processes including furrow cleavage and cytokinesis (Skop, Bergmann et al. 2001,

Montagnac, Echard et al. 2008), cell migration (Caswell and Norman 2008) and even polarity (Wang, Brown et al. 2000) also includes also includes cell shape and receptor retrieval, endocytic recycling.

Simply speaking, described as either ‘fast recycling’ or ‘slow recycling,’ recycling pathways depends on whether the recycling cargo is returned directly from SE to the PM, or whether it is initially translocated to another organelle known as the endocytic recycling compartment (ERC), which is predominantly found to localize near the microtubule 121 organizing center (MTOC) at the perinuclear region of the cell (Grant and Donaldson 2009).

While glycosphingolipids is returned to the PM via the fast recycling route that is regulated by Rab4 (van der Sluijs, Hull et al. 1992, Choudhury, Sharma et al. 2004), many receptors recycle via a Rab11a-mediated pathway that traverses the ERC (Ren, Xu et al. 1998).

Regardless of the significance of the ERC, studies focused on its composition, structure and the mode by which this organelle functions are extremely rare to date.

Described as a group of tubular and vesicular membrane-bound structures, ERC often condenses around the MTOC (Maxfield and McGraw 2004), likely to be connected by an elaborate network of tubular cisternae (Hopkins 1983). Although complex cryo-EM and tomography is ultimately required for achieving resolution of the precise three-dimensional nature of the ERC, it is essential to investigate whether cargo internalized via distinct pathways merges at the ERC or segregated in distinct regions or subcompartments within the ERC. This study also has important biological significance; the possibility of different cargos returning to the PM via separate pathways has already been implicated by previous studies (Mayor, Parton et al. 2014), indicating an active mechanism(s) for segregation of cargo post-sorting at the SE.

The identification plethora of membrane-bound tubular recycling endosomes (TRE) leads to a profound understanding of the composition and function of the ERC (Naslavsky and Caplan 2011). TRE are known as tubular endosomes with lengths of up to 10 µm and diameters of up to 200 nm. It has been shown by our group that the mechanism of the biogenesis of these structures includes the in situ generation of phosphatidic acid (PA) on membranes (Xie, Naslavsky et al. 2014), which assists with the recruitment of both

Molecules Interacting with CasL-Like1 (MICAL-L1) and Syndapin2 to the endosomal membrane, facilitating the bending of these structures by the Syndapin2 F-BAR domain to form TRE (Giridharan, Cai et al. 2013). Nevertheless, while the mechanism of TRE 122 biogenesis has been addressed, TRE function remains unclear. For example, while TRE was suggested to play a critical role in regulating the transport of receptors from the perinuclear ERC back to the PM (“outgoing cargo”) (Radhakrishna and Donaldson 1997,

Caplan, Naslavsky et al. 2002, Sharma, Giridharan et al. 2009, Giridharan, Cai et al. 2013), it is not known yet whether cargo was also transported from SE to the ERC (“incoming cargo”) via TRE. Additionally, the relationship between the ‘classical’ Rab11a tubulo- vesicular endosomes (at or near the ERC) and MICAL-L1-containing TRE is also poorly understood. It is worth noting that it is not well understood whether Rab11a endosomes and TRE transport distinct or overlapping cargo.

Taking advantage of super-resolution microscopy, our current study addresses the composition, morphology and structure of the ERC at sub-diffraction resolution. Employing

Structured Illumination Microscopy (SIM) and dual-channel 2D direct Stochastic Optical

Reconstruction Microscopy (dSTORM), as well as 3D dSTORM, we obtained new information about ERC morphology and cargo segregation. This study demonstrated that

CME and CIE cargo remain segregated at the ERC, suggesting that additional sorting may not occur after exit from the SE. In addition, SIM and 3D dSTORM data favors the notion that only part of the vesicles is tethered by contiguous ‘membrane bridges.’ Finally, we show that TRE preferentially facilitate CIE cargo trafficking, and that some TRE originate from SE membranes rather than from the ERC. Overall, our results support a model for endocytic recycling in which sorting occurs in peripheral endosomes while segregation is maintained at the ERC.

123

12 RESULTS

12.1 The ERC is a compartment that is composed of individual endosomal

membranes with potential underlying connections.

Despite considerable advances in understanding internalization and early endocytic events, the nature and the function of the ERC remain incompletely understood.

Although a multitude of studies have addressed the differential internalization requirements for receptors that traffic via CME as compared to CIE, whether these cargos undergo mixing at the ERC or remain segregated throughout their recycling itineraries has yet to be determined.

The ERC is typically considered to be a highly complex series of vesicles and tubules concentrated at the perinuclear area. However, given the lack of cryo-EM tomography or super-resolution data to date, the precise nature of the ERC region remains unknown. To address the nature and organization of the ERC, we applied super-resolution

SIM imaging and compared it with micrographs obtained by confocal microscopy (Fig.

12.1). Whereas ~300 nm resolution by confocal microscopy images depict Rab11a (the

ERC marker selected) in a compact perinuclear region (Fig. 12.1A; yellow arrows), 3D

SIM imaging at ~110 nm resolution suggests that although the Rab11a structures are indeed densely packed, many appear to be single, isolated vesicles, suggesting that the

ERC may not be entirely enveloped by a single contiguous membrane (Fig. 12.1B; and inset). To ensure that membrane-contiguous organelles were not disrupted by our SIM fixation process, we co-stained for Rab11a and the Golgi marker, GM130, and subjected the cells to SIM analysis. As demonstrated in Fig. 12.1C-E, the Golgi retained its typical contiguous ribbon-like structure under these conditions, whereas Rab11a was mostly localized to 124

Figure 12.1 The 3D SIM characterization of Rab11a recycling endosomes in the ERC.

(A, B) Rab11a-labeled recycling endosomes were immunostained with anti-Rab11a and Alexa 488–conjugated anti-rabbit secondary antibody and visualized by confocal microscopy (A) and SIM (B). Yellow arrows point to the dense perinuclear area (A). The dashed rectangle in the SIM micrograph (B) is shown enlarged in the inset. (C–E) HeLa cells were stained with anti-Rab11a and anti-GM130 together with the corresponding secondary antibodies and imaged by SIM. Scale bar, 10 μm. (Images used with permission from (Xie, Bahl et al. 2016))

125 vesicles as expected. These observations support the notion that the ERC may be a compartment that contains a complex combination of linked endosomal membranes and potentially independent structures.

Since Rab11a can segregate to micro-domains of sorting endosomes (Sonnichsen,

De Renzis et al. 2000), we cannot rule out the possibility that ‘membrane bridges’ (with or without Rab11a bound to the membrane) may connect individual membrane-bound vesicles in the recycling compartment. To help address this prospect, we applied 3D SIM imaging to cells that we pulsed and chased with labeled transferrin. We then monitored

ERC-localized TfR and endogenous cellubrevin, a SNARE protein and marker of ERC membranes (Daro, van der Sluijs et al. 1996). As demonstrated in Fig. 12.2A-C, although many TfR-containing endosomes appeared to be on distinct membranes, the 3D SIM imaging allowed us to frequently observe ‘membrane bridges’ marked by cellubrevin

(green) that linked TfR-containing endosomes (Fig. 12.2A-C; see green arrows). In some cases, we detected structures containing both cellubrevin and TfR (Fig. 12.2D; bottom left corner), whereas in other cases TfR- and cellubrevin-containing structures clearly remained distinct from one another (Fig. 12.2D; top half of micrograph). Cellubrevin

‘bridges’ between transferrin-labeled membranes were as long as 400-500 nm in some cases (Fig. 12.2E). To better resolve the potential linkage between apparently distinct endosomal structures within the ERC, we turned to 3D dSTORM imaging. 3D dSTORM displayed a dramatic increase in resolution compared to standard epifluorescence

(compare Fig. 12.2G with 2F). Although the ~60 nm lateral x/y precision (88 nm Nyquiste resolution) obtained with 3D dSTORM (Fig. 12.2H) did not approach the resolution that we typically achieved for 2D dSTORM (Figs. 12. 4 and 12. 7), the 3D imaging allowed us to identify connections of up to 500 nm between Rab11a-containing endosomes at the

ERC (Fig. 12.2 G (1)). On the other hand, other Rab11a-containing clusters of endosomes 126

127

Figure 12.2 Linkage between recycling endosomes in the ERC via “membrane bridges” as observed by SIM and 3D dSTORM imaging.

A–E) HeLa cells on coverslips were transiently transfected with GFP-cellubrevin for 18 h, serum starved for 1 h, and then allowed to internalize Alexa 568–conjugated transferrin for 15 min at 37°C, followed by a 15-min incubation in fresh medium before fixation. The sample was subjected to SIM imaging. F–H) Endogenous Rab11a in the perinuclear area is depicted by epifluorescence microscopy (F) or a reconstructed 3D dSTORM image (G). HeLa cells plated on glass- bottom MatTek dishes were fixed and stained with anti-Rab11a antibody, followed by Alexa 647–conjugated anti-rabbit antibody. The 3D dSTORM was performed by acquiring >9 × 106 localizations during 40,000 frames obtained every 50 ms. The single- molecule localizations were reconstructed into a normalized Gaussian image at 10-nm pixel size using the ThunderSTORM plug-in in ImageJ. Enlarged images of the dashed box areas in G are shown in (1) and (2). A histogram (H) representing the calculated uncertainty was plotted on frequency distribution graphs with 27 bins, and the mode of the distribution was taken as the lateral precision. I) The Nyquist resolution. (Images used with permission from (Xie, Bahl et al. 2016))

128 within the ERC appeared to be independent of one another (Fig. 12.2 G (2)), suggesting the possibility that parts of the ERC maintain an underlying connection, while others do not.

12.2 Cargo internalized either via CME or CIE remains segregated after being

trafficked to the ERC

Given the complexity of the ERC, we next asked whether cargo internalized via

CME and CIE would both localize to the same Rab11-containing regions of this compartment. To address this question, we followed TfR and CD59, a GPI anchored protein which is transported via EHD1- and MICAL-L1-containing TRE (Cai, Katafiasz et al. 2011). As demonstrated by high-resolution SIM imaging, TfR displayed a high level of overlap with Rab11a (Fig. 12.3A-C and insets). To monitor CD59 at the ERC, cells were incubated with anti-CD59 antibody for 30 min. Non-internalized antibody was then stripped from receptors still at the cell surface, and then the internalized CD59-antibody complex was chased for 2 h to allow it to reach the ERC. Although standard confocal microscopy micrographs suggest some potential overlap between CD59 and Rab11a (Fig. 12.3D-F), higher resolution SIM images (Fig. 12.3G-I and insets) depict only limited co-localization.

Based on these experiments showing that TfR, but not CD59, displays considerable overlap with Rab11a at the ERC, we next predicted that cargo internalized either via CME (i.e., TfR), or CIE (i.e., CD59) would remain segregated after being trafficked to the ERC. To test this, we cointernalized transferrin and anti-CD59 antibody for 30 min (pulse) to examine peripheral SE, or for 30 min followed by a 30 min chase into the ERC, and subjected the cells to SIM analysis (Fig. 12.4A-C) or dSTORM (Fig. 12.4D and E; Fig. 12.5 and Fig. 12.6). SIM analysis partially resolved the CD59 and TfR after 129

130

Figure 12.3 The CIE cargo CD59 is absent from perinuclear Rab11a recycling endosomes in the ERC.

A–C) HeLa cells were fixed and stained with anti-TfR and anti-Rab11a antibodies, along with corresponding secondary antibodies. The localization of TfR (A) and Rab11a recycling endosomes (B) was analyzed with SIM. Insets highlight the boxed area with higher magnification. Scale bar, 10 μm. D–F) HeLa cells were incubated with anti-CD59 antibody for 30 min at 37°C, followed by an acid strip. The cells were then incubated in fresh medium for 2 h before fixation. CD59 was visualized with Alexa 568–conjugated anti-mouse antibody (D). Rab11a was visualized with rabbit anti-Rab11a antibody and Alexa 488–conjugated anti-rabbit antibody (E). Cells were imaged by confocal microscopy. Scale bar, 10 μm. G–I) The same sample from D–F was subjected to SIM analysis. The dashed box denotes the perinuclear area. Insets exhibit the boxed area with higher magnification. Scale bars, 10 μm. (Images used with permission from (Xie, Bahl et al. 2016))

131

132

Figure 12. 4 Segregation of CME and CIE cargoes in the ERC.

A–C) HeLa cells were serum starved for 1 h and then incubated with anti-CD59 antibody (A) and Alexa 568–conjugated transferrin for 30 min at 37°C (B) and then acid stripped. The cells were then incubated in fresh medium for 30 min before fixation. CD59 was visualized with Alexa 488–conjugated mouse secondary antibody (A). The cells were subjected to SIM analysis. Dashed boxes denote the perinuclear area where CD59 and TfR were highly concentrated. Insets, magnified images of the boxed areas. Scale bars, 10 μm. D, E) HeLa cells plated on glass-bottom MatTek dishes were serum starved for 1 h, followed by incubation of anti-CD59 antibody, as well as of Alexa 647–conjugated transferrin, for 30 min at 37°C. Then the cells were acid stripped and either fixed (D; periphery) or chased in fresh medium for 30 min before fixation (E; perinuclear area). The plates were then incubated with Atto 488–conjugated anti-mouse antibody. dSTORM was performed by acquiring >1 × 106 localizations during >20,000 frames obtained every 50 ms. The single-molecule localizations were reconstructed into a normalized Gaussian image at 10-nm pixel size using the ThunderSTORM plug-in in ImageJ. Scale bar, 500 nm. (Images used with permission from (Xie, Bahl et al. 2016))

133

134

Figure. 12.5. dSTORM visualization of CD59 and Transferrin in the cell periphery.

A-C The processed images acquired in Figure 12. 4D were convolved to validate the processing. HeLa cells plated on the glass bottom Mattek dish were stained for CD59 and transferrin as described in Figure 12.4D. dSTORM was performed, with a 642 nm wavelength laser for Transferrin, and then a 488 nm wavelength laser for CD59. Approximately 10,000 frames were obtained for each channel. For channel drift alignment, 100 nm-gold bead fiducial markers were placed on the plate prior to the imaging acquisition. Gold beads are depicted by the larger dots in the upper left and bottom right quadrants of the micrographs. The pixel information collected was reconstructed into normalized Gaussian images with 200 nm pixel size using ThunderSTORM in ImageJ (for details of parameters, please refer to Methods). After shift correction, filters were applied so that pixels with uncertainty values larger than 60 nm were filtered out. The images were then convolved by a Gaussian Blur filter with the mean point spread function (PSF). The PSF was 0.179 µm for CD59, and 0.257 µm for Transferrin. D-F The acquired data from dSTORM were processed using ThunderSTORM into pixel information (for details of parameters, please refer to Methods), shift-corrected and filtered as described above, and then reconstructed to normalized Gaussian images with 10 nm pixel size. Yellow boxes represent CD59 (D) and transferrin (E) in the cell periphery. The boxed region is displayed in larger image as Figure 12. 4D. Bar, 10 μm. G The degree of co-localization between CD59 and Transferrin was determined by ImageJ. The histogram images of CD59 and Transferrin with 10 nm pixel size were generated respectively, and then aligned as described above. The pixels from each images were converted to “0” (no pixel intensity) or “1” (pixel intensity from 1-255). Co- localization was defined as a pixel with values of “1” in each channel. Total 2-channel represents the number of pixels, and Total co-localized is the number of those pixels that have values of 1 in each channel. The degree of co-localization was determined by the ratio of “total co-localized” to “total 2 channel”. Results from the entire image as well as the zoomed regions are given. H Histograms representing the calculated uncertainty for each channel were plotted on frequency distribution graphs with 30 bins, and the mode of the distribution was taken as the precision for each channel. I Nyquist resolution for the two boxed region was calculated. The number of pixels (n) within the region of interest (ROI) and the area (A, nm2) of ROI were calculated. The 1 Nyquist resolution (r) can be calculated as below: r=2X . (Images used with permission √푛/퐴 from (Xie, Bahl et al. 2016))

135

136

Figure. 12.6 dSTORM visualization of CD59 and transferrin in the perinuclear area.

A-C) The processed images acquired in Figure 12.4E were convolved to validate the processing. HeLa cells plated on the glass bottom Mattek dish were stained for CD59 and transferrin as described in Figure 12.4E. dSTORM was performed and acquired data were processed as described in Fig. 12.5 and Methods. Approximately 25,000 frames were obtained for each channel. The PSF was 0.265 µm for CD59, and 0.256 µm for MICAL- L1. D-F) The acquired data from dSTORM were processed into normalized Gaussian images with 10 nm pixel size as described in Fig. 12.5 and Methods. Yellow boxes represent CD59 (D) and transferrin (E) in the perinuclear area. The boxed region in D-F is enlarged and presented in Figure 12. 4E. Bar, 10 μm. G) The degree of co-localization between CD59 and transferrin were determined by ImageJ as described in Fig.12.5. Calculations from the entire image and the boxed regions are given. H) Histograms representing the calculated uncertainty for each channel were plotted on frequency distribution graphs with 30 bins, and the mode of the distribution was taken as the precision for each channel. I) Nyquist resolution for the two boxed region was calculated as described in Fig. 12.5. (Images used with permission from (Xie, Bahl et al. 2016))

137 chasing into the dense ERC region (Fig. 12.4A-C; insets). In particular, rotation of the 3D images along the Y-axis shows that CD59- and TfR-containing structures are either mostly distinct, or at least well-segregated. dSTORM imaging at ~10 nm precision and 33-39 nm

Nyquiste resolution further supported the SIM data, showing that CD59 and TfR were mostly segregated within the ERC (Fig. 12.4E and 12.6). dSTORM imaging in the periphery of cells that had taken up both cargos for 30 min also yielded very limited co- localization of TfR and CD59 (Fig. 12.4D and Fig. 12.5), although areas of overlap could be discerned (see arrows). Although we cannot rule out the possibility that these are distinct compartments that have not been clearly resolved in the z-axis, they most likely represent microdomains of peripheral SE displaying ongoing sorting.

12.3 Rab11a regulates recycling of CME but not CIE cargos.

The significant segregation between CME and CIE cargos at the ERC, along with the failure of the latter cargos to localize to Rab11a-containing recycling endosomes (RE) raised the possibility of very limited post-SE sorting, as these two classes of cargos remain separate all throughout their recycling itinerary to the PM. To clarify the role of Rab11a in sorting of distinct cargo classes, we used the Myosin Vb tail as a ‘surrogate’ for Rab11a

(Hales, Vaerman et al. 2002). This truncated motor protein becomes trapped in the ERC, and due to its coupling with Rab11a, cargos that use the Rab11a-Myosin Vb pathway are similarly coalesced in the ERC. As demonstrated, the CME cargos TfR (Fig. 12.7A-C; green arrows) and low-density lipoprotein receptor (LDLR) (Fig. 12.7D-F; blue arrows) were retained in the ERC and highly co-localized with the Myosin Vb tail (quantified in Fig.

12.7M and with profile-scans in Fig. 12.8 A and B). On the other hand, CIE cargo, such 138

139

Figure 12.7. Myosin Vb tail over-expression selectively traps CME cargos in the perinuclear area.

A-C HeLa cells transfected with GFP-Myosin Vb tail were incubated with transferrin for 30 min at 37°C, washed, and then incubated in fresh medium for 2 h. Green arrows point to the perinuclear area where TfR (B) co-localized with the GFP-Myosin Vb tail (A).

D-F HeLa cells transfected with GFP-Myosin Vb tail were incubated with anti-LDLR antibody for 30 min at 37°C, and then acid-stripped and incubated in fresh medium for 2 h. Blue arrows point to the perinuclear area where LDLR (E) co-localized with the GFP- Myosin Vb tail (D).

G-I HeLa cells transfected with GFP-Myosin Vb tail were incubated with CD59 for 30 min at 37°C, and then acid-stripped and incubated in fresh medium for 2 h. Magenta arrows point to the perinuclear area where GFP-Myosin Vb tail (G) is highly compact, while CD59 (H) displayed limited localization to this region.

J-L HeLa cells transfected with GFP-Myosin Vb tail were incubated with β1-integrin for 30 min at 37°C, acid-stripped and incubated in fresh medium for 2 h. Yellow arrows denote the perinuclear area where β1-integrin (B) showed limited co-localization with the GFP- Myosin Vb tail (J). Scale bar, 10 µm.

M The degree of co-localization between GFP-Myosin Vb tail and different cargos was measured using ImageJ with Manders’ overlap coefficients. 120 cells from three independent experiments were measured and subjected to statistical analysis. Error bars represent standard deviation. ***, p<0.0001. (Images used with permission from (Xie, Bahl et al. 2016)) 140

141

Figure. 12.8. Myosin Vb tail overexpression selectively traps clathrin-dependent cargos but not TRE regulators in the perinuclear area.

A-B HeLa cells were transfected with GFP-Myosin Vb tail and treated with transferrin or anti-LDLR antibody as described in Figure 5 A-F. SIM images were taken using a Zeiss Elyra PS.1 microscope and processed by Zen Black. Maximal projections were generated from SIM slices spaced 110 nm apart. The intensity profile along the white arrow for both channels was tracked using RGB Profiler in ImageJ.

C-D HeLa cells were transfected with GFP-Myosin Vb tail and treated with anti-CD59 or anti-β1-integrin antibody as described in Figure 5 G-L. The image acquisition, processing, and intensity profile tracking were performed as described in A-B. Bar, 10 μm.

E-G HeLa cells transfected with GFP-Myosin Vb tail were fixed, and incubated with anti-MICAL-L1 antibody for 1 h at room temperature, followed by appropriate secondary antibody staining. Confocal microscopy images were acquired with a Zeiss LSM5 Pascal microscope as described in the Methods.

H-J HeLa cells transfected with GFP-Myosin Vb tail and HA-Syndapin2 were fixed, and observed by confocal microscopy as described in the Methods. Bar, 10 μm. (Images used with permission from (Xie, Bahl et al. 2016))

142 as CD59 (Fig. 12.7 G-I; red arrows) and β1-integrin (Fig. 12.10 J-L; yellow arrows) displayed very limited localization to the Myosin Vb tail structures (quantified in Fig. 12.7

M and profile-scan in Fig. 12.8 C and D). Moreover, the Myosin Vb tail did not interfere with TRE generation as discerned by MICAL-L1 or Syndapin2 immunostaining (Fig. 12.8

E-J), both of which are required to facilitate TRE biogenesis and endocytic recycling

(Giridharan, Cai et al. 2013), although occasionally MICAL-L1 could also be observed in partial overlap with the Myosin Vb tail. These data support the idea that MICAL-L1 acts independently of Rab11a in controlling endocytic recycling, and that CME and CIE cargos undergo distinct recycling pathways from their internalization and throughout their recycling itineraries.

To further address the seclusion of the clathrin-independent recycling pathway and its lack of reliance on Rab11a, we depleted Rab11a from cells (Fig. 12.9 A) and determined the degree to which TfR recycling is impacted compared to the CIE cargos,

CD59 and β1- integrin. As demonstrated, whereas reduced Rab11a expression severely impaired TfR recycling, no significant impact was measured for CD59 or β1-integrin (Fig.

12.9 B and C). Moreover, overexpression of a dominant-negative Rab11 slowed the rate of TfR recycling without significantly impacting the rate of CD59 recycling (Fig. 12.9 D and

E). Overall, these data are consistent with the notion that recycling CME and CIE cargos are segregated within the ERC, and that Rab11a regulates recycling of CME but not CIE cargos. 143

144

Figure 12.9. Rab11a regulates the recycling of CME but not CIE cargo.

A) HeLa cells were treated with siRNA against Rab11a for 48 h before cell lysis. Rab11a expression was measured by immunoblotting. Actin was used as a loading control. B) Mock- and Rab11a siRNA-treated cells were serum starved for 1 h, and pulsed with Alexa-633 conjugated transferrin for 1 h at 37°C, and then chased in fresh medium for 30 min. The remaining transferrin in the cell was measured by flow cytometry. Statistical analysis was performed with data from three independent flow cytometry assays. Standard deviation is shown. *, p C) Mock- and Rab11a siRNA-treated cells were pulsed with anti- β1-integrin or anti-CD59 antibody for 30 min at 37°C, acid stripped, and chased for 3 h before fixation. The cells were then stained with alexa-568 anti-mouse antibody in the absence of saponin. The total levels of β1-integrin and CD59 were also measured with fixed cells incubated with anti- β1-integrin or anti-CD59 antibody in the presence of saponin. The return of β1- integrin/CD59 to the cell surface after 3 h chase was calculated as a portion of the total protein level. 180 cells from three independent experiments were analyzed with ImageJ. Bars indicate standard deviation. D) HeLa cells transfected with dominant-negative GFP-Rab11 (S25N) were serum starved for 1 h, and pulsed with Alexa-568-conjugated transferrin for 1 h at 37°C, and then chased in fresh medium for 30 min. The samples were imaged by confocal microscopy, and the remaining transferrin in non-transfected and dominant-negative GFP-Rab11-transfected cells was determined by ImageJ intensity measurements. Standard deviation is shown. **, p<0.01. E) HeLa cells transfected with dominant-negative GFP-Rab11 (S25N) were pulsed with anti-CD59 antibody for 30 min at 37°C, acid stripped, and chased for 3 h before fixation. The cells were then stained with alexa-568 anti-mouse antibody in the absence of saponin. The total levels of CD59 were also measured with fixed cells incubated with anti-CD59 antibody in the presence of saponin. The return of CD59 to the cell surface after 3 h chase was calculated as a portion of the total protein level. 120 cells from three independent experiments were analyzed with ImageJ. Standard deviation is shown. (Images used with permission from (Xie, Bahl et al. 2016)) 145

146

Figure 12.10. SIM and dSTORM visualization of Rab11a and MICAL-L1-labeled TRE.

A-C) HeLa cells were stained with anti-Rab11a and anti-MICAL-L1 together with the corresponding secondary antibody, and imaged by SIM. Scale bar, 10 µm.

D) Reconstructed dSTORM image showing Rab11a and MICAL-L1 in the perinuclear area (a-a’’) and in the periphery (b-b’’). HeLa cells plated on glass bottom Mattek dishes were fixed and stained with anti-Rab11a and anti-MICAL-L1 antibodies, followed by Atto-488 conjugated anti-rabbit and Alexa-647 conjugated anti-mouse antibody. dSTORM was performed by acquiring over 1 x 106 localizations during more than 20,000 frames obtained every 50 milliseconds. The single molecule localizations were reconstructed into a normalized Gaussian image at 10 nm pixels using the ThunderSTORM plugin in ImageJ. The panel D shows the entire cell from where these images are cropped (dashed rectangles a and b). (Images used with permission from (Xie, Bahl et al. 2016))

147

12.4 TRE are distinct from Rab11 recycling endosomes, which originate from a

subset of SE containing Rabenosyn-5, and likely transport cargo from SE to

the ERC

TRE containing MICAL-L1 and Rab11a are partially concentrated in the ERC. A major unanswered question is what is the relationship between MICAL-L1-TRE and

Rab11a-RE. Based on the segregation that we observed between CME and CIE cargos, and the select role of Rab11a in regulating the recycling of CME cargos, we hypothesized that Rab11a and MICALL1 may segregate within the ERC. As demonstrated by SIM imaging (Fig. 12.10 A-C), despite the localization of the distal TRE tips to the dense ERC, the TRE show no appreciable overlap with Rab11a. Since SIM is limited to ~100 nm resolution, we turned to study the ERC at higher resolution, using two-channel dSTORM to image cells costained for endogenous Rab11a and MICAL-L1 (Fig. 12.10 D and 12.11).

Two areas of the cell were enlarged in Fig. 12.10 D; “a” represents a TRE in the midst of the dense Rab11a ERC, whereas “b” depicts a TRE from a more peripheral area of the cell. With calculated precision levels of 10 and 13 nm for MICAL-L1 and 18 and 16 nm for

Rab11a in “a” and “b” respectively, and Nyquiste resolution of 31-48 nm (Fig. 12.11), we observed minimal co-localization between Rab11a and MICAL-L1, both at the ERC and in the periphery. These data support our model in which the ERC maintains segregation of resident proteins and cargos.

Previous studies support a role for TRE in regulating cargo transport from the ERC to the PM ((Caplan, Naslavsky et al. 2002) and reviewed in (Maldonado-Baez, Williamson et al. 2013)). Establishing whether TRE might also carry cargo from the SE to the ERC has been a difficult question to address (Fig. 12.12 A). We have previously shown that phospholipase D (PLD) inhibitors (which prevent PA generation) induce the loss of TRE

(Fig. 12.12 B and C and (Giridharan, Cai et al. 2013)). To determine the membrane origin 148

149

Figure.12.11. dSTORM visualization of Rab11a and MICAL-L1-labeled recycling endosomes.

A-C) The processed images acquired in Figure 7D were convolved to validate the processing. HeLa cells plated on the glass bottom Mattek dish were stained for Rab11 and MICAL-L1 as described in Figure 7D. dSTORM was performed and acquired data were processed as described in Fig. 12.5 and Methods. Approximately 25,000 frames were obtained for each channel. The PSF was 0.21 µm for MICAL-L1 and 0.277 µm for Rab11a.

D-F) The acquired data from dSTORM were processed into normalized Gaussian images with 10 nm pixel size as described in Fig. 12.5 and Methods. Yellow rectangles represent Rab11a and MICAL-L1 in the perinuclear area (box a) and periphery (b), respectively. Bar, 10 μm.

G) The degree of co-localization between Rab11a and MICAL-l1 was determined by ImageJ as described in Fig. 12.5. Calculations from the entire image and the boxed regions are given.

H) Nyquist resolution for the two boxed region was calculated as described in Fig. 12.5.

I,J) Histograms representing the calculated uncertainty for each channel were plotted on frequency distribution graphs with 30 bins, and the mode of the distribution was taken as the precision for each channel. (Images used with permission from (Xie, Bahl et al. 2016))

150

151

Figure 12.12. MICAL-L1-decorated TRE are derived from SE.

A) Schematic diagram depicting the ERC and potentially SE as likely sources of TRE membrane. B-E) TRE analysis by confocal microscopy was done on (B) untreated cells, (C) cells incubated with phospholipase D inhibitors for 30 min, or (D) following inhibitor washout and chase in fresh DMEM for 20 min (D) or 1 h (E). Scale bar, 10 µm. F-H) TRE co-localized with the endosomal marker Rabenosyn-5 upon regeneration. HeLa cells treated with PLD inhibitors for 30 min, and then chased in fresh DMEM for 20 min (as in (D)) were co-stained with MICAL-L1 (F) and Rabenosyn-5 (G) antibodies. Arrows depict the nucleation of a MICAL-L1-containing TRE from a Rabenosyn-5-endosome. Scale bars, 10 µm. (Images used with permission from (Xie, Bahl et al. 2016))

152 of TRE, we developed an assay in which washout of the PLD inhibitors led to synchronized de novo TRE generation (“burst”) beginning within 20 min (Fig. 12.12 D) and reaching a peak at 1 h after inhibitor removal (Fig. 12.12 E). Using this assay, we co-stained cells undergoing TRE regeneration 20 min after washout with markers for the ERC and SE. As demonstrated (Fig. 12.12 F-H), the SE marker Rabenosyn-5 could be detected on punctae at the apex of TRE (see inset; arrows), and along TRE membranes, and this colocalization was also observed in untreated cells (Fig. 12.13 A-C and insets). However, other markers for SE such as Rab5 and EEA1 were not observed, suggesting that TRE are derived from a select subset of SE (unpublished observations). Moreover, surface-stained wheat germ agglutinin (WGA) that was internalized for 5 min did not appear in MICAL-L1-containing

TRE, indicating that these TRE are not derived from the PM (Fig. 12.14). In addition, we observed that newly generated TRE localize to the periphery and are typically absent from the ERC. Overall, these data lead us to suggest that some TRE originate from a subset of

SE containing Rabenosyn-5, and likely transport cargo from SE to the ERC.

12.5 TRE selectively transport CIE cargos CD59 from the SE to the ERC

Our next goal was to determine which cargos might potentially be transported along peripheral TRE to the ERC. To address cargo selectivity in TRE, we initially examined the carriers that transport the CIE cargo, CD59. As depicted by SIM micrographs in Fig. 12.15 A-C, CD59 internalized for 9 min clearly localized to MICAL-L1-

TRE. On the other hand, TfR internalized via CME was absent from MICAL-L1-TRE, after either 3 min internalization (Fig. 12.15 D-F) or 9 min internalization (Fig. 12.16 A-C and insets). We next analyzed cargo selectivity on newly-generated TRE derived from

Rabenosyn-5-containing SE. To this aim, cells were treated for 30 min with PLD inhibitors,

153 followed by 20 min washout in complete media, to allow generation of MICAL-L1- containing TRE. In the last 10 min of washout, internalized cargo was monitored. As demonstrated (Fig. 12.15 G-J), internalized CD59 was sorted into newly formed

Rabenosyn-5- and MICAL-L1-containing TRE (quantified in Fig. 12.16 D and E). In contrast, while TfR was detected in partial overlap with Rabenosyn-5 at SE (Fig. 12.15 K-

N; depicted by the yellow arrow), it was entirely absent from the TRE containing both

MICALL1 and Rabenosyn-5 (Fig. 12.15 K-N; quantified in Fig. 12.16 D and E). These data further support the notion that MICAL-L1-TRE preferentially mediate the transport of CIE cargo from SE to the ERC.

To further support or negate the idea that TRE transport CD59 to the ERC from

SE, we carried out live cell imaging experiments to follow CD59 trafficking along newly generated TRE. To this aim, cells were first incubated with PLD inhibitors to disrupt TRE.

The inhibitors were then washed out (leading to acute TRE biogenesis) and anti-CD59 was provided to the cells so that CD59 could be internalized and monitored. As shown in

Fig. 12.17, at the initial time point imaged (45 s), little or no CD59 was observed yet in the

ERC (marked by a dashed, yellow, region of interest), and the TRE monitored had not yet undergone biogenesis and was not visible. At the 1:45 time point, no CD59 had yet reached the ERC (dashed yellow region of interest and blue arrow), but the MICAL-L1- marked TRE began to grow in the cell periphery (red arrow). At 2:45 and 3:45 time points, the TRE continued to grow from the periphery toward and into the ERC region, with some

CD59 accumulating at the ERC. Finally, at the 4:45 time point, the newly-generated TRE, marked by MICAL-L1 and CD59, was extended fully into the ERC (dashed yellow region of interest and blue arrow). Although the signal was weaker than that of MICAL-L1, CD59 partially co-localized with MICAL-L1 on the TRE and reached the ERC. These data strongly support the notion that, in addition to its characterized role in regulating ERC-to- 154

PM trafficking, the source of some TRE membrane derives from peripheral SE, and these

TRE can transport CIE such as CD59 from SE to the ERC. 155

Figure. 12.13. Rabenosyn-5 partially colocalizes with MICAL-L1 to TRE in untreated cells.

A-C) HeLa cells plated on coverslips were fixed, and then stained with anti-MICAL-L1 and anti-Rabenosyn-5 together with the corresponding secondary antibody, and imaged by SIM. Dashed boxes denote the peripheral cell area, where a higher degree of co- localization between MICAL-L1 and Rabenosyn-5 was observed, and are shown with higer magnification in the inset. Scale bar, 10 μm. (Images used with permission from (Xie, Bahl et al. 2016))

156

157

Figure. 12.14. MICAL-L1-decorated TRE do not originate from the PM

A-C) HeLa cells were incubated with Alexa-488 conjugated WGA for 5 min at 37°C to label the PM (A). After fixation, cells were stained with anti-MICAL-L1 antibody and Alexa-568 conjugated secondary antibody (B) and examined by confocal microscopy. Yellow boxes indicate a representative region of the cell periphery that contains MICAL-L1 and WGA. The inset panels show the boxed area with higher magnification. Scale bar, 10 µm. D-F) HeLa cells were incubated with Alexa-488 conjugated WGA (D) and mouse anti-CD59 antibody (E) for 5 min at 37°C, and subsequently fixed and stained with Alexa-568 conjugated anti-mouse antibody. Yellow boxes focus on the region of the cell where CD59 is internalized and localized beneath the PM. The inset panels show the boxed area with higher magnification. Scale bar, 10 µm. (Images used with permission from (Xie, Bahl et al. 2016))

158

HA-MICAL-L1 CD59 9 min pulse Merge A B C

MICAL-L1 Transferrin 568 3 min pulse Merge D E F

30 min PLD inhibitor + 20 min washout

Cargo 10 min pulse Rabenosyn-5 GFP-MICAL-L1 Merge G H I J

CD59 K L M N

1 µm Transferrin

159

Figure 12.15. TRE selectively transport cargos from SE.

A-F) HeLa cells on coverslips were transiently transfected with HA-MICAL-L1 for 18 h, and incubated with anti-CD59 antibody for 9 min at 37°C (A-C) or pulsed for 3 min with Alexa-568 conjugated Transferrin (D-F). Surface-bound CD59 antibody was removed by acid-strip prior to fixation. Internal CD59 was visualized with Alexa-568 conjugated anti- mouse antibody (B) in the presence of saponin. MICAL-L1 was visualized with Rabbit-anti HA antibody and Alexa- 488 conjugated anti-rabbit antibody (A). Scale bar, 10 µm. G-N) HeLa cells transfected with GFP-MICAL-L1 were treated for 30 min with PLD inhibitors, and then either incubated with mouse anti-CD59 antibody for 10 min at 37°C, followed by staining with Alexa-568 conjugated anti-mouse antibody (G-J), or incubated with Alexa-568 conjugated Transferrin for 10 min at 37°C. Rabenosyn-5 endosomes were detected by rabbit anti-Rabenosyn-5 followed by Alexa-647 conjugated anti-rabbit secondary antibody. All images are maximal projections of ~20 SIM slices separated by 110 nm in the z-axis. Arrows in K-N depict TfR in Rabenosyn-5-containing endosomes that are absent from TRE. Scale bar, 1 µm. The degree of co-localization between different cargos and Rabenosyn-5 or MICAL-L1 is quantified and summarized in Fig. 12.16 D-E. (Images used with permission from (Xie, Bahl et al. 2016))

160

Figure.12.16. MICAL-L1 displays limited co-localization with transferrin even after a longer internalization

A-C) HeLa cells growing on coverslips were serum-starved, incubated with Alexa-568 conjugated transferrin for 9 min at 37 °C, and fixed (B). Endogenous MICAL-L1 was detected with mouse anti-MICAL-L1 antibody and Alexa-488 conjugated anti-mouse antibody with saponin (A). Insets depict the boxed areas in higher magnification. Scale bar, 10 µm. D-E) HeLa cells were treated and imaged by SIM as described in Figure 9G-N. The degree of co-localization between different cargos and Rabenosyn-5 (D) or MICAL-L1 (E) was measured using ImageJ with Manders’ overlap coefficients. 10 cells from three independent experiments were measured and subjected to statistical analysis. Error bars represent standard deviation. ****, p<0.0001. **, p<0.01. (Images used with permission from (Xie, Bahl et al. 2016)) 161

CD59 chase GFP-MICAL-L1 Inset 00:45 00:45 00:45

ERC ERC

01:45 01:45 01:45

pulse pulse

ERC ERC

02:45 02:45 02:45

ERC ERC

03:45 03:45 03:45

30 min PLD inhibitor + 20 min washout + 10 min CD59 + min 10 washout 20 + min inhibitor PLD 30 min ERC ERC

04:45 04:45 04:45

ERC ERC

162

Figure 12.17. TRE transport CD59 into the recycling compartment.

HeLa cells on 35 mm glass bottom plates were transiently transfected with GFP-MICAL- L1 for 18 h, and incubated with PLD inhibitors for 30 min at 37°C. The cells were then washed 3 times and incubated in fresh complete medium for 10 min at 37°C, followed by a 10 min-pulse of Alexa-555 conjugated anti-CD59 antibody. The cells were then washed and imaged in pre-warmed Ringer’s solution. Red arrows indicate the initiation site of a newly generated MICALL1-TRE. Blue arrows point to the growing ERC. Dashed boxes denote the perinuclear area, and are shown with higher magnification in the inset. Scale bars, 10 µm. (Images used with permission from (Xie, Bahl et al. 2016))

163

13 DISCUSSION

13.1 Composition of the ERC

The long-standing paradigm for the ERC holds that it is situated in a perinuclear region of the cell adjacent to the MTOC and comprises a complex series of tubular- vesicular endosomes (Yamashiro, Tycko et al. 1984, Mayor, Presley et al. 1993, Marsh,

Leopold et al. 1995, Maxfield and McGraw 2004). Distinguishing whether the ERC is enveloped mostly by a single contiguous membrane as originally proposed by Hopkins

(Hopkins 1983), or whether it is a region of mostly individual vesicles and tubules has been a difficult question to resolve. Although ultimately techniques that directly label membranes and allow 3D analysis (such as cryo-EM coupled with tomography) will help determine the nature of the ERC more precisely, the inherent fusion-fission dynamics of this organelle may complicate this endeavor. In this study, however, we took advantage of advanced super-resolution and single molecule imaging techniques to address cargo segregation within the ERC, and the physical separation of cargo localized to ERC vesicles and TRE.

Given that a growing body of evidence, including our findings herein, supports the idea that CME and CIE cargo sorting occurs at the SE prior to transport to the ERC, it was rational to hypothesize that the cargos remain segregated to either distinct ERC endosomes or membrane subdomains within the ERC until they are recycled back to the

PM (see model in Fig. 12.18). To address this question and circumvent the limitations of light microscopy in resolving distances below ~300 nm, we used both SIM and dSTORM to re-examine the ERC organization and cargos that recycle via this compartment. By these two super-resolution techniques, with Rab11a and cellubrevin as ERC markers, and following the distribution of internalized TfR and CD59 by “pulse-chase,” we provide evidence suggesting that some of the endosomes at the ERC are linked by ‘membrane 164

Figure 12.18. Proposed model depicting the recycling of CME and CIE.

Cargos internalized through CME and CIE merge in Rabenosyn-5-containing sorting endosomes (SE). Rab11a vesicles (or small labile tubules) are formed to escort CME cargos such as TfR and LDLR to the perinuclear area. MICAL-L1-decorated TRE are also generated from n-5- containing SE, carrying CIE cargos such as CD59, CD98 and β1- integrin to the perinuclear area. The sorted cargos remain segregated in the dense perinuclear ERC. Cargo are then linked to motor proteins along microtubules in either vesicular or tubular recycling endosomes, and transported back to the PM. (Images used with permission from (Xie, Bahl et al. 2016))

165 bridges’ as long as 500 nm. On the other hand, other endosomes appear to be individual structures and have no detectable Rab11a or cellubrevin ‘linking’ them. Although tomography will be needed to ascertain the individual nature of these vesicles, the ERC appears to be comprised of a network of both partially connected and individual vesicles and tubules. While the issue of ERC continuity cannot be resolved from our study, our data is consistent with the notion that the ERC maintains segregation of CME and CIE cargo. Moreover, we demonstrate that whereas CME are transported to the PM via Myosin

Vb/Rab11a-FIP2/Rab11a complexes, CIE cargos are recycled independently of Rab11a and Myosin Vb. Overall, these findings hint at the intriguing possibility that CIE may be coupled directly to microtubules, whereas CME may rely first on a connection with Myosin

Vb and microfilaments before being linked to microtubule-dependent transport to the cell surface.

13.2 The selectivity of TRE

The routes for cargo entry into the cell have been studied intensively, and can be categorized into two very general mechanisms: CME and CIE, depending on whether the clathrin-coated pits are assembled during endocytosis (see model in Fig. 12.18). In comparison to the “canonical” internalization via CME, CIE controls the internalization of distinct cargos and is mediated by a different set of regulatory proteins and lipids (Mayor,

Parton et al. 2014). However, much less is known about the fates and itineraries of these proteins and lipids after their entry via different internalization mechanisms. Some regulatory proteins were identified that regulate the recycling of CIE cargos, such as

Rab22a (Weigert, Yeung et al. 2004), Rab8 (Hattula, Furuhjelm et al. 2006), and Arf6

(Radhakrishna and Donaldson 1997, Powelka, Sun et al. 2004). Although potentially selective for CIE cargo, these regulators may also exert a regulatory role on the recycling 166 of CME cargos such as TfR (Hattula, Furuhjelm et al. 2006, Magadan, Barbieri et al. 2006).

We have determined that the TRE biogenesis proteins, MICAL-L1 and its interaction partner Syndapin2, serve as markers for TRE that selectively transport CIE cargos from

SE into the ERC, in addition to their role in regulating ERC-to-PM trafficking. Moreover,

MICAL-L1 and the CIE cargos remain in segregated endosomal structures throughout their retention in the ERC and as they recycle back to the PM. Our data are consistent with the notion that additional sorting at the ERC is unlikely to occur once cargo has exited the SE. Accordingly, once sorting has been established, cargos remain in distinct endosomes or regions of the ERC and are returned to the PM independently of one another. These findings shed new light on the mechanism of cargo sorting into different

RE.

How are select CME cargo sorted to Rab11a-decorated membranes whereas CIE cargo localize to MICAL-L1-containing TRE at the SE? Potential mechanisms distinguishing CME and CIE cargos at the SE include: 1) Cargos entering through CME often contain signals in their cytoplasmic domains that are recognized by specific regulatory proteins such as ACAP1 (Dai, Li et al. 2004). It is possible that there are regulators for the recognition of CIE cargos that have yet to be identified. 2) The lipid microenvironment that escorts CME and CIE cargos from the PM to SE differs. Vesicles derived from CME typically contain phosphatidylinositol-(4,5)-bisphosphate (PIP2) from the PM (Antonescu, Aguet et al. 2011). On the other hand, generation of tubular invaginations bearing CIE cargos is often from cholesterol-enriched domains (Donaldson,

Porat-Shliom et al. 2009). Additionally, MICAL-L1 is recruited onto the membrane of TRE by PA (Giridharan, Cai et al. 2013). The differential lipid microenvironments of recycling endosomes likely also account for the longer and wider tubular structures observed in

TRE. 167

13.3 The significance of TRE in endocytic trafficking

Tubular endocytic carriers play an important role in mediating transport within the endocytic pathways (Chi, Harrison et al. 2015). Recently, we identified MICAL-L1 as a

TRE marker that stably resides on the endosomal membrane and facilitates its tubulation and biogenesis (Sharma, Giridharan et al. 2009). MICAL-L1-decorated TRE bear different lipid components than the tubular invaginations at the PM, and MICAL-L1 does not localize to PM tubules labeled by WGA (Figure 12.5) (Flesch, Voorhout et al. 1998). Indeed, TRE are crucial for the recycling of internalized receptors and lipids. Since depletion of either

MICAL-L1 or EHD1 resulted in the failure of TfR and β1-integrin to exit the ERC, TRE have been primarily considered as carriers of cargos returning to the PM from the ERC

(Jovic, Naslavsky et al. 2007, Donaldson, Porat-Shliom et al. 2009, Sharma, Giridharan et al. 2009). Despite this evidence, the directionality and transport itinerary of TRE has not been firmly established, and whether TRE might also support SE-to-ERC trafficking has remained an open question until now.

Our study demonstrates that MICAL-L1-decorated TRE can be generated from

Rabenosyn-5containing SE, suggesting that TRE are responsible for the movement of cargo from peripheral SE to the perinuclear ERC (see model in Fig. 12.18). Furthermore,

MICAL-L1 recruits EHD1 to the TRE membrane, which mediates the fission of tubules and supports vesicular transport (Naslavsky and Caplan 2011). Our findings indicate that cargo-containing TRE can originate from SE, and that EHD1 likely cleaves TRE to generate vesicular endosomes that ultimately bring in cargos to the ERC. This notion is reinforced by the imaging of TRE either by SIM or dSTORM (Fig. 12.10), or by live imaging

(Fig. 12.17). In these images, MICAL-1 localizes to a contiguous tubular membrane in the cell periphery, while it resides on more vesicular structures in the perinuclear area. 168

As noted, the effect of either MICAL-L1- or EHD1-depletion leads to cargo accumulation in the ERC. These phenotypic observations fit a model in which TRE facilitate the exit of cargo from the ERC, rather than the transport of cargo into the ERC.

This raises the question as to why cargo appears to be delayed at the ERC rather than the SE? One possibility is that TRE might bidirectionally transport cargos both to and out of the ERC. In this scenario, we hypothesize that cargo at the SE has various compensatory or alternate pathways to reach the ERC (in the absence of EHD1 or MICAL-

L1), but fewer alternate pathways to exit the ERC. Thus, the absence of MICAL-L1/EHD1 may induce non-physiological transport of cargo to the ERC via alternate pathways, but then cause the cargo to remain trapped within the ERC. Indeed, impaired function of

Rab11, which appears to be involved in sorting at the SE (Sonnichsen, De Renzis et al.

2000) as well as ERC-to-PM trafficking, also causes accumulation of cargo at the ERC

(Ren, Xu et al. 1998). It has been proposed that EHD proteins mediate membrane scission upon ATP hydrolysis (Daumke, Lundmark et al. 2007). In this scenario, EHD1-depletion would impair TRE vesiculation, and we speculate that this process might also be essential for exit of recycling cargo from the ERC, once cargo has arrived from the SE.

Another potential reason for cargo accumulation at the ERC (as opposed to the peripheral SE) upon EHD1- or MICAL-L1-depletion might result from the coalescence of entire SE populations into the ERC region. A recent study from our lab suggests that

MICAL-L1 is linked to dynein-mediated trafficking via the collapsin response mediator protein 2 (CRMP2), and that MICAL-L1- or CRMP2-depletion led to more rapid cargo transport to the ERC (Rahajeng, Giridharan et al. 2010). We cannot rule out the possibility that intact peripheral SE migrate and cluster to the ERC region; if this is the case, then one explanation for delayed cargo recycling at the ERC upon EHD1- or MICAL-L1- depletion could be due to failure of the cargo to exit intact SE that have already migrated 169 and coalesced into the ERC region. It is of interest that recycling endosomes are tightly connected with the appendages of the mother centriole, since they are both found in the perinuclear region (Hehnly, Chen et al. 2012). Accordingly, we hypothesized that MICAL-

L1 and EHD1 might serve as a link between RE and centrosomes (Reinecke, Katafiasz et al. 2015). MICAL-L1/EHD1 may have a non-selective effect on RE attachment to the microtubules for transportation to the PM, and thus contribute to the regulation of endocytic recycling from the ERC.

Our studies alter the current understanding of the role that TRE play in endocytic recycling. Since we view receptor sorting as less likely to occur in the ERC, we speculate that the transport of endosomes to this central area of the cell may serve to focus RE at a

“staging ground” where RE (from both CME and CIE) are linked to microtubule tracks and/or the actin cytoskeleton by select motor proteins (see model in Fig. 12.18).

Accordingly, the involvement of Myosin Vb in CME trafficking hints at a possible requirement for both microfilament- and microtubule dependent transport mechanisms, whereas we predict that CIE might rely more exclusively on microtubules. Although future studies will be needed to address this idea, we speculate that the ERC region provides a concentrated platform on which vesicles/tubules, motor proteins and the cytoskeleton may be coupled to facilitate efficient transport to the PM.

170

CHAPTER V Summary and Future Directions

171

14 Summary

Endocytic trafficking is a crucial process required for cellular homeostasis, and multiple ailments that include cardiovascular disease and cancer are related to the dysregulation of endocytic transport. Our laboratory’s major focus has been to comprehensively address the underlying mechanisms controlling the internalization, recycling and membrane transport of various receptors. Among the many known regulatory proteins of these pathways, our lab has identified and studied the Molecule

Interacting with CasL Like-1 (MICAL-L1). We have shown that this molecule labels a type of tubular membrane structures termed as the tubular recycling endosomes, carrying cargos such as MHC I and CD59 back to the plasma membrane (Giridharan, Cai et al.

2013). Further, MICAL-L1 acts as a membrane hub that recruits the regulatory machinery onto the TRE, including Syndapin2, eps15 homology domain containing protein EHD1 and

EHD3, Rab8, Rab35, Arf6, etc (Rahajeng, Giridharan et al. 2012, Giridharan, Cai et al.

2013). We have also demonstrated that MICAL-L1 binds with high selectivity to phosphatidic acid (PA), indicating that PA serves as a docking site for MICAL-L1 on membranes, and thus is essential for efficient recycling of internalized receptors.

However, little is known about the potential role of PA in endocytic transport. Our long-term goal is to determine the role that different lipid components, with emphasis on

PA, play in the regulation of endocytic transport. Ultimately, these findings will help to elucidate the molecular basis of endocytic trafficking from the standpoint of lipids, and provide novel strategies for the treatment of endocytic trafficking-related diseases.

PA is generated by 3 pathways: 1) by phospholipase D activity on glycerophospholipids; 2) by diacylglycerol kinase (DGK) activity on diacylglycerol (DAG); or 3) by lyphosphatidic acid acyl transferase activity (LPAT). Among these pathways we found DGK activity especially intriguing, since several DGK isoforms regulate crucial 172 cellular functions; for example, DGKα mediates α5β1 integrin recycling and thus plays a role in cell motility. The underlying mechanism of DGK function, however, remains entirely unresolved. DGKα is one of ten DGK isoforms that convert DAG to PA. It is a better candidate to study in our system than the other 9 DGK isoforms because: (1) DGKα is ubiquitously expressed in human cells and highly expressed in HeLa; (2) DGKα mediates multiple cellular functions, including α5β1 integrin recycling and cell motility, cell proliferation, immune responses, etc.; (3) Little is known about its mode of cellular function.

Evidence supporting a role for DGKα in endocytic trafficking is provided by my data described in Chapter III, using siRNA-based knock-down assays. In the absence of DGKα where synthesis of PA is compromised, I found that MICAL-L1-decorated TRE were disrupted. However, when PA level is preserved by preventing its catabolism by propranolol, an inhibitor of phosphatidic acid phosphatase (PAP), TRE formation is rescued. Furthermore, loss of DGKα caused the failure of Major Histocompatibility

Complex I (MHCI) on the plasma membrane (PM) to reach the endocytic recycling compartment (ERC) following internalization, while their uptake and reach to the sorting endosomes marked by early endosomal autoantigen 1 (EEA1) are not affected, suggesting that DGKα is required for the recycling pathway without altering the normal influx of cargos into sorting endosomes. We have also shown that DGKα controls the half- life of MHC I. In cohesion with this finding, DGKα knock-down significantly impairs the lysosomal degradation of MHC I, possibly by retaining it in the cell, allowing for less rounds of internalization and recycling when a partition of these molecules are sorted to the lysosome for degradation, However, further researches are required for the precise mechanism. The delayed delivery of MHC I to the ERC in DGKα-knock-down cells is further confirmed by our find that MHC I recycling back to the plasma membrane is similarly postponed. 173

By both proximity ligation assay (Duolink®) and co-immunoprecipitation, we were able to show that DGKα and MICAL-L1 associate in vivo in an activity-dependent manner, since R59949, the diacylglycerol kinase inhibitor disrupts the interaction between DGKα and MICAL-L1.

Our research highlights a novel role for DGKα, as a lipid modifying enzyme, in the generation of TRE and its subsequent regulatory role in endocytic recycling. Based on these observations, we proposed a positive feedback model for the biogenesis of TRE, in that DGKα is recruited onto the membrane of MICAL-L1 decorated TRE, which facilitates the generation of PA, and as a subsequence is required for the membrane docking of

MICAL-L1. In this project, we have also established a protocol to quantitatively measure the rate of endocytic recycling, by either immunofluorescence staining, or flow cytometry

(Reineke, Xie et al. 2015).

Inspired by our observation that DGKα preferentially mediates the recycling of

MHC I, a cargo internalized via the clathrin-independent pathway (CIE), instead of

Transferrin Receptor which is uptake via the clathrin-mediated endocytosis (CME), we next sought to investigate the selectivity of TRE. Surprisingly, while very limited amount of TfR are seen on the MICAL-L1 decorated TRE, incoming CD59 are carried by TRE to the REC, implicating that these TRE selectively sort CIE cargos to the recycling pathway.

On the contrary, we have shown that the recycling of CME cargos such as transferrin and

LDLR is mediated through the Rab11-Myosin Vb machinery, while CIE cargos including

MHC I, CD59 and CD98 are independent of such mechanism. In agreement with this notion, higher resolution techniques (SIM and dSTORM) have revealed that Transferrin and CD59 reside in different subtypes of recycling endosomes.

Our study demonstrated that MICAL-L1-decorated TRE are generated from

Rabenosyn-5-containing SE, suggesting that TRE are primarily responsible for the 174 movement of cargo from peripheral SE to the perinuclear ERC. These data in combination suggest that cargos are sorted into different recycling endosomes from sorting endosomes.

Additionally, by super-resolution techniques, using Rab11 as a marker, and following the distribution of internalized transferrin and CD59 by “pulse-chase”, our data strongly supported the idea that the ERC is a dense region with distinct RE, rather than a unified organelle with a contiguous membrane where cargos are actively sorted. Since our studies show that the cargos are sorted before they enter the ERC, it is rational to hypothesize that they remain in distinct endosomes until they are recycled back to the plasma membrane, and that the ERC is a region where densely focused RE carrying different cargos are clustered. These finds provided new evidence on the mechanism of cargo sorting into different recycling endosomes, and alter the current understanding of the role that the ERC plays in endocytic recycling.

15 Future directions

15.1 The potential role of DGKα on the tubular recycling endosomes

The most dramatic effect of DGKα knock-down is the delayed recycling of MHC I, resulting in an acute postponement in the return of the receptors back to the plasma membrane. We have related the role of DGKα on recycling to its regulation of PA, which is needed for the initiation of TRE biogenesis (Giridharan, Cai et al. 2013). Based on our novel data, we envision a mechanism in which the generation of PA by DGKα serves as a positive feedback loop for recruitment of more MICAL-L1 and DGKα, stimulating additional PA generation that facilitates TRE biogenesis. These findings shed new lights on the role of lipid conversion on the TRE regulation. 175

However, important questions with regards to the role of DGKα on the tubular recycling endosomes remain unsolved, including 1) the molecular mechanisms by which

DGKα is recruited to membranes and activated in the cell; 2) the temporal relationship of the recruitment of different TRE regulation components DGKα, MICAL-L1, Syndapin2,

EHD1, EHD3, etc; 3) how does DGKα collaborate with other lipid modifiers such as cPLA2

(Cai, Caplan et al. 2012) to reconcile changes in local lipid composition of the TRE; 4) the substrate specificity of DGKα; 5) how is the activity of DGKα mediated, and whether they remain on the highly-curved recycling endosomes. Similarly, since multiple enzymes mediate the production and consumption of PA, the role of other lipid modifiers on TRE biogenesis are intriguing future directions.

15.2 The mechanisms of cargo sorting in the recycling pathways

The routes for cargo internalization have been studied intensively (Mayor, Parton et al. 2014), yet much less is known about the fates and itinerary of the proteins and lipids after their entry via different internalization mechanisms. In addition to some known regulatory proteins mediating the recycling of CIE cargos, such as Rab22a (Weigert,

Yeung et al. 2004), Rab8 (Hattula, Furuhjelm et al. 2006), and Arf6 (Radhakrishna and

Donaldson 1997, Powelka, Sun et al. 2004), we have identified that MICAL-L1 labeled

TRE selectively transport CIE cargos from SE into the ERC. Further studies are needed to solve the mechanisms for post-internalization sorting at the SE. Potential mechanisms distinguishing different cargos lies in: 1) different sorting motifs in the cytoplasmic domains of cargos such as ACAP1 (Dai, Li et al. 2004); 2) the lipid composition of membranes that accompany cargos from the PM; 3) Cargos clustering and their role on the stereo-chemical nature of cargo-containing invaginations. 176

15.3 The role of ERC in endocytic recycling

Our data strongly supported the idea that the ERC is a dense region with distinct

RE, where sorted cargos remain segregated until returning back to the PM. This finding helps to solve the long-standing paradigm for the composition of the ERC, however, new questions arise as to the role of ERC in the recycling pathway. Based on the subcellular distribution of ERC in the peri-nuclear region adjacent to the MTOC, we proposed that the

ERC serves as a “staging ground”, where cargos destined for recycling are delivered to be protected from lysosomal/proteasomal degradation. Further studies are necessary to prove this hypothesis. Also we are interested in the degree of cargo segregation and whether homotypic fusion and exchange of cargos occur within the ERC.

177

REFERENCES

Abramovici, H. and S. H. Gee (2007). "Morphological changes and spatial regulation of diacylglycerol kinase- zeta, syntrophins, and Rac1 during myoblast fusion." Cell Motil Cytoskeleton 64(7): 549-567.

Adams, A., J. M. Thorn, M. Yamabhai, B. K. Kay and J. P. O'Bryan (2000). "Intersectin, an adaptor protein involved in clathrin-mediated endocytosis, activates mitogenic signaling pathways." J Biol Chem 275(35):

27414-27420.

Aderem, A. and D. M. Underhill (1999). "Mechanisms of phagocytosis in macrophages." Annu Rev Immunol

17: 593-623.

Allaire, P. D., A. L. Marat, C. Dall'Armi, G. Di Paolo, P. S. McPherson and B. Ritter (2010). "The Connecdenn

DENN domain: a GEF for Rab35 mediating cargo-specific exit from early endosomes." Mol Cell 37(3): 370-

382.

Alonso, R., C. Mazzeo, I. Merida and M. Izquierdo (2007). "A new role of diacylglycerol kinase alpha on the secretion of lethal exosomes bearing Fas ligand during activation-induced cell death of T lymphocytes."

Biochimie 89(2): 213-221.

Alonso, R., C. Mazzeo, M. C. Rodriguez, M. Marsh, A. Fraile-Ramos, V. Calvo, A. Avila-Flores, I. Merida and

M. Izquierdo (2011). "Diacylglycerol kinase alpha regulates the formation and polarisation of mature multivesicular bodies involved in the secretion of Fas ligand-containing exosomes in T lymphocytes." Cell

Death Differ 18(7): 1161-1173.

Alonso, R., M. C. Rodriguez, J. Pindado, E. Merino, I. Merida and M. Izquierdo (2005). "Diacylglycerol kinase alpha regulates the secretion of lethal exosomes bearing Fas ligand during activation-induced cell death of T lymphocytes." J Biol Chem 280(31): 28439-28450.

Amyere, M., B. Payrastre, U. Krause, P. Van Der Smissen, A. Veithen and P. J. Courtoy (2000). "Constitutive macropinocytosis in oncogene-transformed fibroblasts depends on sequential permanent activation of phosphoinositide 3-kinase and phospholipase C." Mol Biol Cell 11(10): 3453-3467.

Anton, L. C. and J. W. Yewdell (2014). "Translating DRiPs: MHC class I immunosurveillance of pathogens and tumors." Journal of leukocyte biology 95(4): 551-562.

Antonescu, C. N., F. Aguet, G. Danuser and S. L. Schmid (2011). "Phosphatidylinositol-(4,5)-bisphosphate regulates clathrin-coated pit initiation, stabilization, and size." Molecular Biology of the Cell 22(14): 2588-2600.

Antonescu, C. N., G. Danuser and S. L. Schmid (2010). "Phosphatidic acid plays a regulatory role in clathrin- mediated endocytosis." Molecular Biology of the Cell 21(16): 2944-2952. 178

Antonescu, C. N., G. Danuser and S. L. Schmid (2010). "Phosphatidic acid plays a regulatory role in clathrin- mediated endocytosis." Mol Biol Cell 21(16): 2944-2952.

Antonny, B., P. Gounon, R. Schekman and L. Orci (2003). "Self-assembly of minimal COPII cages." EMBO

Rep 4(4): 419-424.

Arisz, S. A. and T. Munnik (2010). Diacylglycerol kinase. Lipid signaling in plants, Springer: 107-114.

Arisz, S. A., C. Testerink and T. Munnik (2009). "Plant PA signaling via diacylglycerol kinase." Biochim Biophys

Acta 1791(9): 869-875.

Asp, L., F. Kartberg, J. Fernandez-Rodriguez, M. Smedh, M. Elsner, F. Laporte, M. Barcena, K. A. Jansen, J.

A. Valentijn, A. J. Koster, J. J. Bergeron and T. Nilsson (2009). "Early stages of Golgi vesicle and tubule formation require diacylglycerol." Mol Biol Cell 20(3): 780-790.

Babbey, C. M., N. Ahktar, E. Wang, C. C. Chen, B. D. Grant and K. W. Dunn (2006). "Rab10 regulates membrane transport through early endosomes of polarized Madin-Darby canine kidney cells." Mol Biol Cell

17(7): 3156-3175.

Babst, M., D. J. Katzmann, E. J. Estepa-Sabal, T. Meerloo and S. D. Emr (2002). "Escrt-III: an endosome- associated heterooligomeric protein complex required for mvb sorting." Dev Cell 3(2): 271-282.

Bache, K. G., A. Brech, A. Mehlum and H. Stenmark (2003). "Hrs regulates multivesicular body formation via

ESCRT recruitment to endosomes." J Cell Biol 162(3): 435-442.

Bache, K. G., C. Raiborg, A. Mehlum and H. Stenmark (2003). "STAM and Hrs are subunits of a multivalent ubiquitin-binding complex on early endosomes." J Biol Chem 278(14): 12513-12521.

Badola, P. and C. R. Sanders, 2nd (1997). "Escherichia coli diacylglycerol kinase is an evolutionarily optimized membrane enzyme and catalyzes direct phosphoryl transfer." J Biol Chem 272(39): 24176-24182.

Bahl, K., N. Naslavsky and S. Caplan (2015). "Role of the EHD2 unstructured loop in dimerization, protein binding and subcellular localization." PLoS One 10(4): e0123710.

Barbieri, M. A., R. L. Roberts, A. Mukhopadhyay and P. D. Stahl (1996). "Rab5 regulates the dynamics of early endosome fusion." Biocell 20(3): 331-338.

Bard, F. and V. Malhotra (2006). "The formation of TGN-to-plasma-membrane transport carriers." Annu Rev

Cell Dev Biol 22: 439-455.

Baron, C. L. and V. Malhotra (2002). "Role of diacylglycerol in PKD recruitment to the TGN and protein transport to the plasma membrane." Science 295(5553): 325-328. 179

Bashkirov, P. V., S. A. Akimov, A. I. Evseev, S. L. Schmid, J. Zimmerberg and V. A. Frolov (2008). "GTPase cycle of dynamin is coupled to membrane squeeze and release, leading to spontaneous fission." Cell 135(7):

1276-1286.

Becker, K. P. and Y. A. Hannun (2003). "cPKC-dependent sequestration of membrane-recycling components in a subset of recycling endosomes." J Biol Chem 278(52): 52747-52754.

Begley, M. J. and J. E. Dixon (2005). "The structure and regulation of myotubularin phosphatases." Curr Opin

Struct Biol 15(6): 614-620.

Benjamin, J. J., P. P. Poon, S. M. Lewis, A. Auger, T. A. Wong, R. A. Singer and G. C. Johnston (2011). "The yeast Arf GTPase-activating protein Age1 is regulated by phospholipase D for post-Golgi vesicular transport."

J Biol Chem 286(7): 5187-5196.

Benjamin, S., H. Weidberg, D. Rapaport, O. Pekar, M. Nudelman, D. Segal, K. Hirschberg, S. Katzav, M.

Ehrlich and M. Horowitz (2011). "EHD2 mediates trafficking from the plasma membrane by modulating Rac1 activity." Biochem J 439(3): 433-442.

Benmerah, A. (2004). "Endocytosis: signaling from endocytic membranes to the nucleus." Curr Biol 14(8):

R314-316.

Bennett, M. K. (1995). "SNAREs and the specificity of transport vesicle targeting." Curr Opin Cell Biol 7(4):

581-586.

Besterman, J. M., R. S. Pollenz, E. L. Booker, Jr. and P. Cuatrecasas (1986). "Diacylglycerol-induced translocation of diacylglycerol kinase: use of affinity-purified enzyme in a reconstitution system." Proc Natl

Acad Sci U S A 83(24): 9378-9382.

Bettache, N., L. Baisamy, S. Baghdiguian, B. Payrastre, P. Mangeat and A. Bienvenue (2003). "Mechanical constraint imposed on plasma membrane through transverse phospholipid imbalance induces reversible actin polymerization via phosphoinositide 3-kinase activation." J Cell Sci 116(Pt 11): 2277-2284.

Betz, A., U. Ashery, M. Rickmann, I. Augustin, E. Neher, T. C. Sudhof, J. Rettig and N. Brose (1998). "Munc13-

1 is a presynaptic phorbol ester receptor that enhances neurotransmitter release." Neuron 21(1): 123-136.

Birkle, S., G. Zeng, L. Gao, R. K. Yu and J. Aubry (2003). "Role of tumor-associated gangliosides in cancer progression." Biochimie 85(3-4): 455-463.

Blondeau, F., J. Laporte, S. Bodin, G. Superti-Furga, B. Payrastre and J. L. Mandel (2000). "Myotubularin, a phosphatase deficient in myotubular myopathy, acts on phosphatidylinositol 3-kinase and phosphatidylinositol

3-phosphate pathway." Hum Mol Genet 9(15): 2223-2229. 180

Bohdanowicz, M., D. Schlam, M. Hermansson, D. Rizzuti, G. D. Fairn, T. Ueyama, P. Somerharju, G. Du and

S. Grinstein (2013). "Phosphatidic acid is required for the constitutive ruffling and macropinocytosis of phagocytes." Mol Biol Cell 24(11): 1700-1712, S1701-1707.

Bonifacino, J. S. and J. H. Hurley (2008). "Retromer." Curr Opin Cell Biol 20(4): 427-436.

Bonifacino, J. S. and R. Rojas (2006). "Retrograde transport from endosomes to the trans-Golgi network." Nat

Rev Mol Cell Biol 7(8): 568-579.

Bossard, C., D. Bresson, R. S. Polishchuk and V. Malhotra (2007). "Dimeric PKD regulates membrane fission to form transport carriers at the TGN." J Cell Biol 179(6): 1123-1131.

Bossi, G. and G. M. Griffiths (1999). "Degranulation plays an essential part in regulating cell surface expression of Fas ligand in T cells and natural killer cells." Nat Med 5(1): 90-96.

Botelho, R. J., M. Teruel, R. Dierckman, R. Anderson, A. Wells, J. D. York, T. Meyer and S. Grinstein (2000).

"Localized biphasic changes in phosphatidylinositol-4,5-bisphosphate at sites of phagocytosis." J Cell Biol

151(7): 1353-1368.

Boudin, H., A. Doan, J. Xia, R. Shigemoto, R. L. Huganir, P. Worley and A. M. Craig (2000). "Presynaptic clustering of mGluR7a requires the PICK1 PDZ domain binding site." Neuron 28(2): 485-497.

Brandhorst, D., D. Zwilling, S. O. Rizzoli, U. Lippert, T. Lang and R. Jahn (2006). "Homotypic fusion of early endosomes: SNAREs do not determine fusion specificity." Proc Natl Acad Sci U S A 103(8): 2701-2706.

Braun, A., R. Pinyol, R. Dahlhaus, D. Koch, P. Fonarev, B. D. Grant, M. M. Kessels and B. Qualmann (2005).

"EHD proteins associate with syndapin I and II and such interactions play a crucial role in endosomal recycling." Mol Biol Cell 16(8): 3642-3658.

Brown, F. D., A. L. Rozelle, H. L. Yin, T. Balla and J. G. Donaldson (2001). "Phosphatidylinositol 4,5- bisphosphate and Arf6-regulated membrane traffic." J Cell Biol 154(5): 1007-1017.

Brown, S. E., R. D. Campbell and C. M. Sanderson (2001). "Novel NG36/G9a gene products encoded within the human and mouse MHC class III regions." Mamm Genome 12(12): 916-924.

Brown, W. J., K. Chambers and A. Doody (2003). "Phospholipase A2 (PLA2) enzymes in membrane trafficking: mediators of membrane shape and function." Traffic 4(4): 214-221.

Bucci, C., R. G. Parton, I. H. Mather, H. Stunnenberg, K. Simons, B. Hoflack and M. Zerial (1992). "The small

GTPase rab5 functions as a regulatory factor in the early endocytic pathway." Cell 70(5): 715-728.

Bunting, M., W. Tang, G. A. Zimmerman, T. M. McIntyre and S. M. Prescott (1996). "Molecular cloning and characterization of a novel human diacylglycerol kinase zeta." J Biol Chem 271(17): 10230-10236. 181

Burd, C. G. and S. D. Emr (1998). "Phosphatidylinositol(3)-phosphate signaling mediated by specific binding to RING FYVE domains." Mol Cell 2(1): 157-162.

Burger, K. N. (2000). "Greasing membrane fusion and fission machineries." Traffic 1(8): 605-613.

Burger, K. N., R. A. Demel, S. L. Schmid and B. de Kruijff (2000). "Dynamin is membrane-active: lipid insertion is induced by phosphoinositides and phosphatidic acid." Biochemistry 39(40): 12485-12493.

Cabezas, A., K. Pattni and H. Stenmark (2006). "Cloning and subcellular localization of a human phosphatidylinositol 3-phosphate 5-kinase, PIKfyve/Fab1." Gene 371(1): 34-41.

Cai, B., S. Caplan and N. Naslavsky (2012). "cPLA2alpha and EHD1 interact and regulate the vesiculation of cholesterol-rich GPI-anchored protein-containing endosomes." Molecular Biology of the Cell.

Cai, B., S. Caplan and N. Naslavsky (2012). "cPLA2alpha and EHD1 interact and regulate the vesiculation of cholesterol-rich, GPI-anchored, protein-containing endosomes." Mol Biol Cell 23(10): 1874-1888.

Cai, B., S. S. Giridharan, J. Zhang, S. Saxena, K. Bahl, J. A. Schmidt, P. L. Sorgen, W. Guo, N. Naslavsky and S. Caplan (2013). "Differential roles of C-terminal Eps15 homology domain proteins as vesiculators and tubulators of recycling endosomes." J Biol Chem 288(42): 30172-30180.

Cai, B., S. S. Giridharan, J. Zhang, S. Saxena, K. Bahl, J. A. Schmidt, P. L. Sorgen, W. Guo, N. Naslavsky and S. Caplan (2013). "Differential roles of C-terminal Eps15 homology domain proteins as vesiculators and tubulators of recycling endosomes." The Journal of biological chemistry 288(42): 30172-30180.

Cai, B., D. Katafiasz, V. Horejsi and N. Naslavsky (2011). "Pre-sorting endosomal transport of the GPI- anchored protein, CD59, is regulated by EHD1." Traffic 12(1): 102-120.

Cai, B., S. Xie, S. Caplan and N. Naslavsky (2014). "GRAF1 forms a complex with MICAL-L1 and EHD1 to cooperate in tubular recycling endosome vesiculation." Front Cell Dev Biol 2: 22.

Cai, B., S. Xie, F. Liu, L. C. Simone, S. Caplan, X. Qin and N. Naslavsky (2014). "Rapid degradation of the complement regulator, CD59, by a novel inhibitor." The Journal of biological chemistry 289(17): 12109-12125.

Cai, J., T. M. Crotty, E. Reichert, K. L. Carraway, 3rd, D. M. Stafforini and M. K. Topham (2010). "Diacylglycerol kinase delta and protein kinase C(alpha) modulate epidermal growth factor receptor abundance and degradation through ubiquitin-specific protease 8." J Biol Chem 285(10): 6952-6959.

Campelo, F. and V. Malhotra (2012). "Membrane fission: the biogenesis of transport carriers." Annu Rev

Biochem 81: 407-427.

Cantalupo, G., P. Alifano, V. Roberti, C. B. Bruni and C. Bucci (2001). "Rab-interacting lysosomal protein

(RILP): the Rab7 effector required for transport to lysosomes." EMBO J 20(4): 683-693. 182

Cao, C., J. M. Backer, J. Laporte, E. J. Bedrick and A. Wandinger-Ness (2008). "Sequential actions of myotubularin lipid phosphatases regulate endosomal PI(3)P and growth factor receptor trafficking." Mol Biol

Cell 19(8): 3334-3346.

Cao, C., J. Laporte, J. M. Backer, A. Wandinger-Ness and M. P. Stein (2007). "Myotubularin lipid phosphatase binds the hVPS15/hVPS34 lipid kinase complex on endosomes." Traffic 8(8): 1052-1067.

Cao, T. T., H. W. Deacon, D. Reczek, A. Bretscher and M. von Zastrow (1999). "A kinase-regulated PDZ- domain interaction controls endocytic sorting of the beta2-adrenergic receptor." Nature 401(6750): 286-290.

Caplan, S., L. M. Hartnell, R. C. Aguilar, N. Naslavsky and J. S. Bonifacino (2001). "Human Vam6p promotes lysosome clustering and fusion in vivo." J Cell Biol 154(1): 109-122.

Caplan, S., N. Naslavsky, L. M. Hartnell, R. Lodge, R. S. Polishchuk, J. G. Donaldson and J. S. Bonifacino

(2002). "A tubular EHD1-containing compartment involved in the recycling of major histocompatibility complex class I molecules to the plasma membrane." EMBO J 21(11): 2557-2567.

Carlton, J., M. Bujny, B. J. Peter, V. M. Oorschot, A. Rutherford, H. Mellor, J. Klumperman, H. T. McMahon and P. J. Cullen (2004). "Sorting nexin-1 mediates tubular endosome-to-TGN transport through coincidence sensing of high- curvature membranes and 3-phosphoinositides." Curr Biol 14(20): 1791-1800.

Carlton, J., M. Bujny, A. Rutherford and P. Cullen (2005). "Sorting nexins--unifying trends and new perspectives." Traffic 6(2): 75-82.

Caswell, P. and J. Norman (2008). "Endocytic transport of integrins during cell migration and invasion." Trends in cell biology 18(6): 257-263.

Caswell, P. T., M. Chan, A. J. Lindsay, M. W. McCaffrey, D. Boettiger and J. C. Norman (2008). "Rab-coupling protein coordinates recycling of alpha5beta1 integrin and EGFR1 to promote cell migration in 3D microenvironments." J Cell Biol 183(1): 143-155.

Caumont, A. S., M. C. Galas, N. Vitale, D. Aunis and M. F. Bader (1998). "Regulated exocytosis in chromaffin cells. Translocation of ARF6 stimulates a plasma membrane-associated phospholipase D." J Biol Chem

273(3): 1373-1379.

Chen, Y. A. and R. H. Scheller (2001). "SNARE-mediated membrane fusion." Nat Rev Mol Cell Biol 2(2): 98-

106.

Chen, Y. G., A. Siddhanta, C. D. Austin, S. M. Hammond, T. C. Sung, M. A. Frohman, A. J. Morris and D.

Shields (1997). "Phospholipase D stimulates release of nascent secretory vesicles from the trans-Golgi network." J Cell Biol 138(3): 495-504. 183

Chi, R. J., M. S. Harrison and C. G. Burd (2015). "Biogenesis of endosome-derived transport carriers." Cell

Mol Life Sci.

Chianale, F., E. Rainero, C. Cianflone, V. Bettio, A. Pighini, P. E. Porporato, N. Filigheddu, G. Serini, F.

Sinigaglia, G. Baldanzi and A. Graziani (2010). "Diacylglycerol kinase alpha mediates HGF-induced Rac activation and membrane ruffling by regulating atypical PKC and RhoGDI." Proc Natl Acad Sci U S A 107(9):

4182-4187.

Chibalin, A. V., Y. Leng, E. Vieira, A. Krook, M. Bjornholm, Y. C. Long, O. Kotova, Z. Zhong, F. Sakane, T.

Steiler, C. Nylen, J. Wang, M. Laakso, M. K. Topham, M. Gilbert, H. Wallberg-Henriksson and J. R. Zierath

(2008). "Downregulation of diacylglycerol kinase delta contributes to hyperglycemia-induced insulin resistance." Cell 132(3): 375-386.

Choudhury, A., D. K. Sharma, D. L. Marks and R. E. Pagano (2004). "Elevated endosomal cholesterol levels in Niemann-Pick cells inhibit rab4 and perturb membrane recycling." Mol Biol Cell 15(10): 4500-4511.

Christoforidis, S., M. Miaczynska, K. Ashman, M. Wilm, L. Zhao, S. C. Yip, M. D. Waterfield, J. M. Backer and

M. Zerial (1999). "Phosphatidylinositol-3-OH kinases are Rab5 effectors." Nat Cell Biol 1(4): 249-252.

Chung, H. J., X. Qian, M. Ehlers, Y. N. Jan and L. Y. Jan (2009). "Neuronal activity regulates phosphorylation- dependent surface delivery of G protein-activated inwardly rectifying potassium channels." Proc Natl Acad Sci

U S A 106(2): 629-634.

Cipres, A., S. Carrasco, E. Merino, E. Diaz, U. M. Krishna, J. R. Falck, A. C. Martinez and I. Merida (2003).

"Regulation of diacylglycerol kinase alpha by phosphoinositide 3-kinase lipid products." J Biol Chem 278(37):

35629-35635.

Clapham, D. E. (2007). "Calcium signaling." Cell 131(6): 1047-1058.

Collins, B. M., A. J. McCoy, H. M. Kent, P. R. Evans and D. J. Owen (2002). "Molecular architecture and functional model of the endocytic AP2 complex." Cell 109(4): 523-535.

Conner, S. D. and S. L. Schmid (2003). "Regulated portals of entry into the cell." Nature 422(6927): 37-44.

Corrotte, M., S. Chasserot-Golaz, P. Huang, G. Du, N. T. Ktistakis, M. A. Frohman, N. Vitale, M. F. Bader and

N. J. Grant (2006). "Dynamics and function of phospholipase D and phosphatidic acid during phagocytosis."

Traffic 7(3): 365-377.

Cozier, G. E., J. Carlton, A. H. McGregor, P. A. Gleeson, R. D. Teasdale, H. Mellor and P. J. Cullen (2002).

"The phox homology (PX) domain-dependent, 3-phosphoinositide-mediated association of sorting nexin-1 with an early sorting endosomal compartment is required for its ability to regulate epidermal growth factor receptor degradation." J Biol Chem 277(50): 48730-48736. 184

Cremona, O., G. Di Paolo, M. R. Wenk, A. Luthi, W. T. Kim, K. Takei, L. Daniell, Y. Nemoto, S. B. Shears, R.

A. Flavell, D. A. McCormick and P. De Camilli (1999). "Essential role of phosphoinositide metabolism in synaptic vesicle recycling." Cell 99(2): 179-188.

Crotty, T., J. Cai, F. Sakane, A. Taketomi, S. M. Prescott and M. K. Topham (2006). "Diacylglycerol kinase delta regulates protein kinase C and epidermal growth factor receptor signaling." Proc Natl Acad Sci U S A

103(42): 15485-15490.

Cullen, P. J. (2008). "Endosomal sorting and signalling: an emerging role for sorting nexins." Nat Rev Mol Cell

Biol 9(7): 574-582.

Cutrupi, S., G. Baldanzi, D. Gramaglia, A. Maffe, D. Schaap, E. Giraudo, W. van Blitterswijk, F. Bussolino, P.

M. Comoglio and A. Graziani (2000). "Src-mediated activation of alpha-diacylglycerol kinase is required for hepatocyte growth factor-induced cell motility." EMBO J 19(17): 4614-4622.

Czech, M. P. (2003). "Dynamics of phosphoinositides in membrane retrieval and insertion." Annu Rev Physiol

65: 791-815.

D'Souza-Schorey, C. and P. Chavrier (2006). "ARF proteins: roles in membrane traffic and beyond." Nat Rev

Mol Cell Biol 7(5): 347-358.

Dai, J., J. Li, E. Bos, M. Porcionatto, R. T. Premont, S. Bourgoin, P. J. Peters and V. W. Hsu (2004). "ACAP1 promotes endocytic recycling by recognizing recycling sorting signals." Dev Cell 7(5): 771-776.

Dai, J., H. P. Ting-Beall and M. P. Sheetz (1997). "The secretion-coupled endocytosis correlates with membrane tension changes in RBL 2H3 cells." J Gen Physiol 110(1): 1-10.

Dana, R. R., C. Eigsti, K. L. Holmes and T. L. Leto (2000). "A regulatory role for ADP-ribosylation factor 6

(ARF6) in activation of the phagocyte NADPH oxidase." J Biol Chem 275(42): 32566-32571.

Daro, E., P. van der Sluijs, T. Galli and I. Mellman (1996). "Rab4 and cellubrevin define different early endosome populations on the pathway of transferrin receptor recycling." Proc Natl Acad Sci U S A 93(18):

9559-9564.

Daumke, O., R. Lundmark, Y. Vallis, S. Martens, P. J. Butler and H. T. McMahon (2007). "Architectural and mechanistic insights into an EHD ATPase involved in membrane remodelling." Nature 449(7164): 923-927.

Day, C. A., N. W. Baetz, C. A. Copeland, L. J. Kraft, B. Han, A. Tiwari, K. R. Drake, H. De Luca, D. J.

Chinnapen, M. W. Davidson, R. K. Holmes, M. G. Jobling, T. A. Schroer, W. I. Lencer and A. K. Kenworthy

(2015). "Microtubule motors power plasma membrane tubulation in clathrin-independent endocytosis." Traffic

16(6): 572-590. 185

De Matteis, M. A., G. Santini, R. A. Kahn, G. Di Tullio and A. Luini (1993). "Receptor and protein kinase C- mediated regulation of ARF binding to the Golgi complex." Nature 364(6440): 818-821. de Renzis, S., B. Sonnichsen and M. Zerial (2002). "Divalent Rab effectors regulate the sub-compartmental organization and sorting of early endosomes." Nat Cell Biol 4(2): 124-133.

Deneka, M. and P. van der Sluijs (2002). "'Rab'ing up endosomal membrane transport." Nat Cell Biol 4(2):

E33-35.

Diaz Anel, A. M. and V. Malhotra (2005). "PKCeta is required for beta1gamma2/beta3gamma2- and PKD- mediated transport to the cell surface and the organization of the Golgi apparatus." J Cell Biol 169(1): 83-91.

Dickey, A. and R. Faller (2008). "Examining the contributions of lipid shape and headgroup charge on bilayer behavior." Biophys J 95(6): 2636-2646.

Ding, L., E. Traer, T. M. McIntyre, G. A. Zimmerman and S. M. Prescott (1998). "The cloning and characterization of a novel human diacylglycerol kinase, DGKiota." J Biol Chem 273(49): 32746-32752.

Doherty, G. J., M. K. Ahlund, M. T. Howes, B. Moren, R. G. Parton, H. T. McMahon and R. Lundmark (2011).

"The endocytic protein GRAF1 is directed to cell-matrix adhesion sites and regulates cell spreading." Mol Biol

Cell 22(22): 4380-4389.

Doherty, G. J. and R. Lundmark (2009). "GRAF1-dependent endocytosis." Biochem Soc Trans 37(Pt 5): 1061-

1065.

Doherty, K. R., A. R. Demonbreun, G. Q. Wallace, A. Cave, A. D. Posey, K. Heretis, P. Pytel and E. M. McNally

(2008). "The endocytic recycling protein EHD2 interacts with myoferlin to regulate myoblast fusion." J Biol

Chem 283(29): 20252-20260.

Donaldson, J. G. (2009). "Phospholipase D in endocytosis and endosomal recycling pathways." Biochimica et biophysica acta 1791(9): 845-849.

Donaldson, J. G. and C. L. Jackson (2000). "Regulators and effectors of the ARF GTPases." Curr Opin Cell

Biol 12(4): 475-482.

Donaldson, J. G., N. Porat-Shliom and L. A. Cohen (2009). "Clathrin-independent endocytosis: a unique platform for cell signaling and PM remodeling." Cell Signal 21(1): 1-6.

Donaldson, J. G., N. Porat-Shliom and L. A. Cohen (2009). "Clathrin-independent endocytosis: a unique platform for cell signaling and PM remodeling." Cellular signalling 21(1): 1-6.

Donaldson, J. G. and D. B. Williams (2009). "Intracellular assembly and trafficking of MHC class I molecules."

Traffic 10(12): 1745-1752. 186

Eckler, S. A., R. Kuehn and M. Gautam (2005). "Deletion of N-terminal rapsyn domains disrupts clustering and has dominant negative effects on clustering of full-length rapsyn." Neuroscience 131(3): 661-670.

Eyster, C. A., J. D. Higginson, R. Huebner, N. Porat-Shliom, R. Weigert, W. W. Wu, R. F. Shen and J. G.

Donaldson (2009). "Discovery of new cargo proteins that enter cells through clathrin-independent endocytosis." Traffic 10(5): 590-599.

Fallman, M., D. P. Lew, O. Stendahl and T. Andersson (1989). "Receptor-mediated phagocytosis in human neutrophils is associated with increased formation of inositol phosphates and diacylglycerol. Elevation in cytosolic free calcium and formation of inositol phosphates can be dissociated from accumulation of diacylglycerol." J Clin Invest 84(3): 886-891.

Farge, E., D. M. Ojcius, A. Subtil and A. Dautry-Varsat (1999). "Enhancement of endocytosis due to aminophospholipid transport across the plasma membrane of living cells." Am J Physiol 276(3 Pt 1): C725-

733.

Farsad, K., N. Ringstad, K. Takei, S. R. Floyd, K. Rose and P. De Camilli (2001). "Generation of high curvature membranes mediated by direct endophilin bilayer interactions." J Cell Biol 155(2): 193-200.

Fasshauer, D. (2003). "Structural insights into the SNARE mechanism." Biochim Biophys Acta 1641(2-3): 87-

97.

Ferguson, S. M., A. Raimondi, S. Paradise, H. Shen, K. Mesaki, A. Ferguson, O. Destaing, G. Ko, J. Takasaki,

O. Cremona, O. T. E and P. De Camilli (2009). "Coordinated actions of actin and BAR proteins upstream of dynamin at endocytic clathrin-coated pits." Dev Cell 17(6): 811-822.

Fernandez-Borja, M., R. Wubbolts, J. Calafat, H. Janssen, N. Divecha, S. Dusseljee and J. Neefjes (1999).

"Multivesicular body morphogenesis requires phosphatidyl-inositol 3-kinase activity." Curr Biol 9(1): 55-58.

Fernandez-Ulibarri, I., M. Vilella, F. Lazaro-Dieguez, E. Sarri, S. E. Martinez, N. Jimenez, E. Claro, I. Merida,

K. N. Burger and G. Egea (2007). "Diacylglycerol is required for the formation of COPI vesicles in the Golgi- to-ER transport pathway." Mol Biol Cell 18(9): 3250-3263.

Fielding, A. B., E. Schonteich, J. Matheson, G. Wilson, X. Yu, G. R. Hickson, S. Srivastava, S. A. Baldwin, R.

Prekeris and G. W. Gould (2005). "Rab11-FIP3 and FIP4 interact with Arf6 and the exocyst to control membrane traffic in cytokinesis." EMBO J 24(19): 3389-3399.

Flesch, F. M., W. F. Voorhout, B. Colenbrander, L. M. van Golde and B. M. Gadella (1998). "Use of lectins to characterize plasma membrane preparations from boar spermatozoa: a novel technique for monitoring membrane purity and quantity." Biol Reprod 59(6): 1530-1539. 187

Flores, I., T. Casaseca, A. C. Martinez, H. Kanoh and I. Merida (1996). "Phosphatidic acid generation through interleukin 2 (IL-2)-induced alpha-diacylglycerol kinase activation is an essential step in IL-2-mediated lymphocyte proliferation." J Biol Chem 271(17): 10334-10340.

Ford, M. G., I. G. Mills, B. J. Peter, Y. Vallis, G. J. Praefcke, P. R. Evans and H. T. McMahon (2002). "Curvature of clathrin-coated pits driven by epsin." Nature 419(6905): 361-366.

Ford, M. G., B. M. Pearse, M. K. Higgins, Y. Vallis, D. J. Owen, A. Gibson, C. R. Hopkins, P. R. Evans and H.

T. McMahon (2001). "Simultaneous binding of PtdIns(4,5)P2 and clathrin by AP180 in the nucleation of clathrin lattices on membranes." Science 291(5506): 1051-1055.

Frost, A., V. M. Unger and P. De Camilli (2009). "The BAR domain superfamily: membrane-molding macromolecules." Cell 137(2): 191-196.

Gad, H., N. Ringstad, P. Low, O. Kjaerulff, J. Gustafsson, M. Wenk, G. Di Paolo, Y. Nemoto, J. Crun, M. H.

Ellisman, P. De Camilli, O. Shupliakov and L. Brodin (2000). "Fission and uncoating of synaptic clathrin-coated vesicles are perturbed by disruption of interactions with the SH3 domain of endophilin." Neuron 27(2): 301-

312.

Gagescu, R., N. Demaurex, R. G. Parton, W. Hunziker, L. A. Huber and J. Gruenberg (2000). "The recycling endosome of Madin-Darby canine kidney cells is a mildly acidic compartment rich in raft components." Mol

Biol Cell 11(8): 2775-2791.

Galperin, E., S. Benjamin, D. Rapaport, R. Rotem-Yehudar, S. Tolchinsky and M. Horowitz (2002). "EHD3: a protein that resides in recycling tubular and vesicular membrane structures and interacts with EHD1." Traffic

3(8): 575-589.

Gao, Y., C. M. Balut, M. A. Bailey, G. Patino-Lopez, S. Shaw and D. C. Devor (2010). "Recycling of the Ca2+- activated K+ channel, KCa2.3, is dependent upon RME-1, Rab35/EPI64C, and an N-terminal domain." J Biol

Chem 285(23): 17938-17953.

Gaullier, J. M., A. Simonsen, A. D'Arrigo, B. Bremnes, H. Stenmark and R. Aasland (1998). "FYVE fingers bind PtdIns(3)P." Nature 394(6692): 432-433.

George, M., G. Ying, M. A. Rainey, A. Solomon, P. T. Parikh, Q. Gao, V. Band and H. Band (2007). "Shared as well as distinct roles of EHD proteins revealed by biochemical and functional comparisons in mammalian cells and C. elegans." BMC Cell Biol 8: 3.

Gillooly, D. J., I. C. Morrow, M. Lindsay, R. Gould, N. J. Bryant, J. M. Gaullier, R. G. Parton and H. Stenmark

(2000). "Localization of phosphatidylinositol 3-phosphate in yeast and mammalian cells." EMBO J 19(17):

4577-4588. 188

Giridharan, S. S., B. Cai, N. Naslavsky and S. Caplan (2012). "Trafficking cascades mediated by Rab35 and its membrane hub effector, MICAL-L1." Commun Integr Biol 5(4): 384-387.

Giridharan, S. S., B. Cai, N. Vitale, N. Naslavsky and S. Caplan (2013). "Cooperation of MICAL-L1, syndapin2, and phosphatidic acid in tubular recycling endosome biogenesis." Molecular Biology of the Cell 24(11): 1776-

1790, S1771-1715.

Giridharan, S. S., B. Cai, N. Vitale, N. Naslavsky and S. Caplan (2013). "Cooperation of MICAL-L1, syndapin2, and phosphatidic acid in tubular recycling endosome biogenesis." Mol Biol Cell 24(11): 1776-1790, S1771-

1715.

Glebov, O. O., N. A. Bright and B. J. Nichols (2006). "Flotillin-1 defines a clathrin-independent endocytic pathway in mammalian cells." Nat Cell Biol 8(1): 46-54.

Gokool, S., D. Tattersall and M. N. Seaman (2007). "EHD1 interacts with retromer to stabilize SNX1 tubules and facilitate endosome-to-Golgi retrieval." Traffic 8(12): 1873-1886.

Gomez-Merino, F. C., C. A. Brearley, M. Ornatowska, M. E. Abdel-Haliem, M. I. Zanor and B. Mueller-Roeber

(2004). "AtDGK2, a novel diacylglycerol kinase from Arabidopsis thaliana, phosphorylates 1-stearoyl-2- arachidonoyl-sn-glycerol and 1,2-dioleoyl-sn-glycerol and exhibits cold-inducible gene expression." J Biol

Chem 279(9): 8230-8241.

Gorvel, J. P., P. Chavrier, M. Zerial and J. Gruenberg (1991). "rab5 controls early endosome fusion in vitro."

Cell 64(5): 915-925.

Goto, K. and H. Kondo (1993). "Molecular cloning and expression of a 90-kDa diacylglycerol kinase that predominantly localizes in neurons." Proc Natl Acad Sci U S A 90(16): 7598-7602.

Goto, K. and H. Kondo (1996). "A 104-kDa diacylglycerol kinase containing ankyrin-like repeats localizes in the cell nucleus." Proc Natl Acad Sci U S A 93(20): 11196-11201.

Grant, B. D. and S. Caplan (2008). "Mechanisms of EHD/RME-1 protein function in endocytic transport."

Traffic 9(12): 2043-2052.

Grant, B. D. and J. G. Donaldson (2009). "Pathways and mechanisms of endocytic recycling." Nature reviews.

Molecular cell biology 10(9): 597-608.

Grant, B. D. and J. G. Donaldson (2009). "Pathways and mechanisms of endocytic recycling." Nat Rev Mol

Cell Biol 10(9): 597-608.

Grimmer, S., M. Ying, S. Walchli, B. van Deurs and K. Sandvig (2005). "Golgi vesiculation induced by cholesterol occurs by a dynamin- and cPLA2-dependent mechanism." Traffic 6(2): 144-156. 189

Grosshans, B. L., D. Ortiz and P. Novick (2006). "Rabs and their effectors: achieving specificity in membrane traffic." Proc Natl Acad Sci U S A 103(32): 11821-11827.

Gruenberg, J. (2001). "The endocytic pathway: a mosaic of domains." Nat Rev Mol Cell Biol 2(10): 721-730.

Gruenberg, J. (2003). "Lipids in endocytic membrane transport and sorting." Curr Opin Cell Biol 15(4): 382-

388.

Gruenberg, J., G. Griffiths and K. E. Howell (1989). "Characterization of the early endosome and putative endocytic carrier vesicles in vivo and with an assay of vesicle fusion in vitro." J Cell Biol 108(4): 1301-1316.

Gruenberg, J. and F. R. Maxfield (1995). "Membrane transport in the endocytic pathway." Curr Opin Cell Biol

7(4): 552-563.

Guilherme, A., N. A. Soriano, S. Bose, J. Holik, A. Bose, D. P. Pomerleau, P. Furcinitti, J. Leszyk, S. Corvera and M. P. Czech (2004). "EHD2 and the Novel EH Domain Binding Protein EHBP1 Couple Endocytosis to the

Actin Cytoskeleton." J Biol Chem 279(11): 10593-10605.

Guilherme, A., N. A. Soriano, P. S. Furcinitti and M. P. Czech (2004). "Role of EHD1 and EHBP1 in perinuclear sorting and insulin-regulated GLUT4 recycling in 3T3-L1 adipocytes." J Biol Chem 279(38): 40062-40075.

Gutierrez-Martinez, E., I. Fernandez-Ulibarri, F. Lazaro-Dieguez, L. Johannes, S. Pyne, E. Sarri and G. Egea

(2013). "Lipid phosphate phosphatase 3 participates in transport carrier formation and protein trafficking in the early secretory pathway." J Cell Sci 126(Pt 12): 2641-2655.

Haglund, K., S. Sigismund, S. Polo, I. Szymkiewicz, P. P. Di Fiore and I. Dikic (2003). "Multiple monoubiquitination of RTKs is sufficient for their endocytosis and degradation." Nat Cell Biol 5(5): 461-466.

Hales, C. M., J. P. Vaerman and J. R. Goldenring (2002). "Rab11 family interacting protein 2 associates with

Myosin Vb and regulates plasma membrane recycling." J Biol Chem 277(52): 50415-50421.

Hammond, K., M. D. Reboiras, I. G. Lyle and M. N. Jones (1984). "Characterisation of phosphatidylcholine/phosphatidylinositol sonicated vesicles. Effects of phospholipid composition on vesicle size." Biochim Biophys Acta 774(1): 19-25.

Han, G. S., L. O'Hara, S. Siniossoglou and G. M. Carman (2008). "Characterization of the yeast DGK1- encoded CTP-dependent diacylglycerol kinase." J Biol Chem 283(29): 20443-20453.

Hanson, P. I. and A. Cashikar (2012). "Multivesicular body morphogenesis." Annu Rev Cell Dev Biol 28: 337-

362.

Hanson, P. I. and S. W. Whiteheart (2005). "AAA+ proteins: have engine, will work." Nat Rev Mol Cell Biol

6(7): 519-529. 190

Hanyaloglu, A. C., E. McCullagh and M. von Zastrow (2005). "Essential role of Hrs in a recycling mechanism mediating functional resensitization of cell signaling." EMBO J 24(13): 2265-2283.

Hardel, N., N. Harmel, G. Zolles, B. Fakler and N. Klocker (2008). "Recycling endosomes supply cardiac pacemaker channels for regulated surface expression." Cardiovasc Res 79(1): 52-60.

Harden, N., S. F. Yap, M. A. Chiam and L. Lim (1993). "A Drosophila gene encoding a protein with similarity to diacylglycerol kinase is expressed in specific neurons." Biochem J 289 ( Pt 2): 439-444.

Harris, T. W., E. Hartwieg, H. R. Horvitz and E. M. Jorgensen (2000). "Mutations in synaptojanin disrupt synaptic vesicle recycling." J Cell Biol 150(3): 589-600.

Hattula, K., J. Furuhjelm, J. Tikkanen, K. Tanhuanpaa, P. Laakkonen and J. Peranen (2006). "Characterization of the Rab8-specific membrane traffic route linked to protrusion formation." J Cell Sci 119(Pt 23): 4866-4877.

Hehnly, H., C. T. Chen, C. M. Powers, H. L. Liu and S. Doxsey (2012). "The centrosome regulates the Rab11- dependent recycling endosome pathway at appendages of the mother centriole." Curr Biol 22(20): 1944-1950.

Henmi, Y., N. Oe, N. Kono, T. Taguchi, K. Takei and K. Tanabe (2016). "Phosphatidic acid induces EHD3- containing membrane tubulation and is required for receptor recycling." Exp Cell Res 342(1): 1-10.

Henry, G. D., D. J. Corrigan, J. V. Dineen and J. D. Baleja (2010). "Charge effects in the selection of NPF motifs by the EH domain of EHD1." Biochemistry 49(16): 3381-3392.

Hinshaw, J. E. (2000). "Dynamin and its role in membrane fission." Annu Rev Cell Dev Biol 16: 483-519.

Hinshaw, J. E. and S. L. Schmid (1995). "Dynamin self-assembles into rings suggesting a mechanism for coated vesicle budding." Nature 374(6518): 190-192.

Hogan, A., L. Shepherd, J. Chabot, S. Quenneville, S. M. Prescott, M. K. Topham and S. H. Gee (2001).

"Interaction of gamma 1-syntrophin with diacylglycerol kinase-zeta. Regulation of nuclear localization by PDZ interactions." J Biol Chem 276(28): 26526-26533.

Holden, N. J., C. O. Savage, S. P. Young, M. J. Wakelam, L. Harper and J. M. Williams (2011). "A dual role for diacylglycerol kinase generated phosphatidic acid in autoantibody-induced neutrophil exocytosis." Mol Med

17(11-12): 1242-1252.

Hopkins, C. R. (1983). "Intracellular routing of transferrin and transferrin receptors in epidermoid carcinoma

A431 cells." Cell 35(1): 321-330.

Horiuchi, H., R. Lippe, H. M. McBride, M. Rubino, P. Woodman, H. Stenmark, V. Rybin, M. Wilm, K. Ashman,

M. Mann and M. Zerial (1997). "A novel Rab5 GDP/GTP exchange factor complexed to Rabaptin-5 links nucleotide exchange to effector recruitment and function." Cell 90(6): 1149-1159. 191

Houndolo, T., P. L. Boulay and A. Claing (2005). "G protein-coupled receptor endocytosis in ADP-ribosylation factor 6-depleted cells." J Biol Chem 280(7): 5598-5604.

Houssa, B., D. Schaap, J. van der Wal, K. Goto, H. Kondo, A. Yamakawa, M. Shibata, T. Takenawa and W.

J. van Blitterswijk (1997). "Cloning of a novel human diacylglycerol kinase (DGKtheta) containing three cysteine-rich domains, a proline-rich region, and a pleckstrin homology domain with an overlapping Ras- associating domain." J Biol Chem 272(16): 10422-10428.

Hsu, V. W. and R. Prekeris (2010). "Transport at the recycling endosome." Curr Opin Cell Biol 22(4): 528-534.

Huang, C. C., D. M. Yang, C. C. Lin and L. S. Kao (2011). "Involvement of Rab3A in vesicle priming during exocytosis: interaction with Munc13-1 and Munc18-1." Traffic 12(10): 1356-1370.

Huber, L. A., S. Pimplikar, R. G. Parton, H. Virta, M. Zerial and K. Simons (1993). "Rab8, a small GTPase involved in vesicular traffic between the TGN and the basolateral plasma membrane." J Cell Biol 123(1): 35-

45.

Huijbregts, R. P., L. Topalof and V. A. Bankaitis (2000). "Lipid metabolism and regulation of membrane trafficking." Traffic 1(3): 195-202.

Huotari, J. and A. Helenius (2011). "Endosome maturation." EMBO J 30(17): 3481-3500.

Hurley, J. H., A. C. Newton, P. J. Parker, P. M. Blumberg and Y. Nishizuka (1997). "Taxonomy and function of C1 protein kinase C homology domains." Protein Sci 6(2): 477-480.

Idkowiak-Baldys, J., K. P. Becker, K. Kitatani and Y. A. Hannun (2006). "Dynamic sequestration of the recycling compartment by classical protein kinase C." J Biol Chem 281(31): 22321-22331.

Ikonomov, O. C., D. Sbrissa, M. Foti, J. L. Carpentier and A. Shisheva (2003). "PIKfyve controls fluid phase endocytosis but not recycling/degradation of endocytosed receptors or sorting of procathepsin D by regulating multivesicular body morphogenesis." Mol Biol Cell 14(11): 4581-4591.

Imai, S., M. Kai, S. Yasuda, H. Kanoh and F. Sakane (2005). "Identification and characterization of a novel human type II diacylglycerol kinase, DGK kappa." J Biol Chem 280(48): 39870-39881.

Itoh, T., S. Koshiba, T. Kigawa, A. Kikuchi, S. Yokoyama and T. Takenawa (2001). "Role of the ENTH domain in phosphatidylinositol-4,5-bisphosphate binding and endocytosis." Science 291(5506): 1047-1051.

Izquierdo, M., M. C. Ruiz-Ruiz and A. Lopez-Rivas (1996). "Stimulation of phosphatidylinositol turnover is a key event for Fas-dependent, activation-induced apoptosis in human T lymphocytes." J Immunol 157(1): 21-

28.

Jakobsson, J., F. Ackermann, F. Andersson, D. Larhammar, P. Low and L. Brodin (2011). "Regulation of synaptic vesicle budding and dynamin function by an EHD ATPase." J Neurosci 31(39): 13972-13980. 192

Jelen, F., P. Lachowicz, W. Apostoluk, A. Mateja, Z. S. Derewenda and J. Otlewski (2009). "Dissecting the thermodynamics of GAP-RhoA interactions." J Struct Biol 165(1): 10-18.

Jennings, W., S. Doshi, K. D'Souza and R. M. Epand (2015). "Molecular properties of diacylglycerol kinase- epsilon in relation to function." Chem Phys Lipids.

Jiang, Y., F. Sakane, H. Kanoh and J. P. Walsh (2000). "Selectivity of the diacylglycerol kinase inhibitor 3-[2-

(4-[bis-(4-fluorophenyl)methylene]-1-piperidinyl)ethyl]-2, 3-dihydro-2-thioxo-4(1H)quinazolinone (R59949) among diacylglycerol kinase subtypes." Biochem Pharmacol 59(7): 763-772.

Jordens, I., M. Fernandez-Borja, M. Marsman, S. Dusseljee, L. Janssen, J. Calafat, H. Janssen, R. Wubbolts and J. Neefjes (2001). "The Rab7 effector protein RILP controls lysosomal transport by inducing the recruitment of dynein-dynactin motors." Curr Biol 11(21): 1680-1685.

Jose, A. M. and M. R. Koelle (2005). "Domains, amino acid residues, and new isoforms of Caenorhabditis elegans diacylglycerol kinase 1 (DGK-1) important for terminating diacylglycerol signaling in vivo." J Biol Chem

280(4): 2730-2736.

Joset, A., D. A. Dodd, S. Halegoua and M. E. Schwab (2010). "Pincher-generated Nogo-A endosomes mediate growth cone collapse and retrograde signaling." J Cell Biol 188(2): 271-285.

Jost, M., F. Simpson, J. M. Kavran, M. A. Lemmon and S. L. Schmid (1998). "Phosphatidylinositol-4,5- bisphosphate is required for endocytic coated vesicle formation." Curr Biol 8(25): 1399-1402.

Jovanovic, O. A., F. D. Brown and J. G. Donaldson (2006). "An effector domain mutant of Arf6 implicates phospholipase D in endosomal membrane recycling." Mol Biol Cell 17(1): 327-335.

Jovic, M., F. Kieken, N. Naslavsky, P. L. Sorgen and S. Caplan (2009). "Eps15 homology domain 1-associated tubules contain phosphatidylinositol-4-phosphate and phosphatidylinositol-(4,5)-bisphosphate and are required for efficient recycling." Mol Biol Cell 20(11): 2731-2743.

Jovic, M., N. Naslavsky, D. Rapaport, M. Horowitz and S. Caplan (2007). "EHD1 regulates beta1 integrin endosomal transport: effects on focal adhesions, cell spreading and migration." J Cell Sci 120(Pt 5): 802-814.

Jovic, M., M. Sharma, J. Rahajeng and S. Caplan (2010). "The early endosome: a busy sorting station for proteins at the crossroads." Histol Histopathol 25(1): 99-112.

Jovic, M., M. Sharma, J. Rahajeng and S. Caplan (2010). "The early endosome: a busy sorting station for proteins at the crossroads." Histology and histopathology 25(1): 99-112.

Kai, M., F. Sakane, S. Imai, I. Wada and H. Kanoh (1994). "Molecular cloning of a diacylglycerol kinase isozyme predominantly expressed in human retina with a truncated and inactive enzyme expression in most other human cells." J Biol Chem 269(28): 18492-18498. 193

Kakefuda, K., A. Oyagi, M. Ishisaka, K. Tsuruma, M. Shimazawa, K. Yokota, Y. Shirai, K. Horie, N. Saito, J.

Takeda and H. Hara (2010). "Diacylglycerol kinase beta knockout mice exhibit lithium-sensitive behavioral abnormalities." PLoS One 5(10): e13447.

Kawasaki, T., T. Kobayashi, T. Ueyama, Y. Shirai and N. Saito (2008). "Regulation of clathrin-dependent endocytosis by diacylglycerol kinase delta: importance of kinase activity and binding to AP2alpha." The

Biochemical journal 409(2): 471-479.

Kawasaki, T., T. Kobayashi, T. Ueyama, Y. Shirai and N. Saito (2008). "Regulation of clathrin-dependent endocytosis by diacylglycerol kinase delta: importance of kinase activity and binding to AP2alpha." Biochem

J 409(2): 471-479.

Kay, B. K., M. Yamabhai, B. Wendland and S. D. Emr (1999). "Identification of a novel domain shared by putative components of the endocytic and cytoskeletal machinery." Protein Sci 8(2): 435-438.

Kearns, B. G., T. P. McGee, P. Mayinger, A. Gedvilaite, S. E. Phillips, S. Kagiwada and V. A. Bankaitis (1997).

"Essential role for diacylglycerol in protein transport from the yeast Golgi complex." Nature 387(6628): 101-

105.

Kelly, B. T., A. J. McCoy, K. Spate, S. E. Miller, P. R. Evans, S. Honing and D. J. Owen (2008). "A structural explanation for the binding of endocytic dileucine motifs by the AP2 complex." Nature 456(7224): 976-979.

Kessels, M. M. and B. Qualmann (2004). "The syndapin protein family: linking membrane trafficking with the cytoskeleton." J Cell Sci 117(Pt 15): 3077-3086.

Kieken, F., M. Jovic, N. Naslavsky, S. Caplan and P. L. Sorgen (2007). "EH domain of EHD1." J Biomol NMR

39(4): 323-329.

Kieken, F., M. Sharma, M. Jovic, S. S. Giridharan, N. Naslavsky, S. Caplan and P. L. Sorgen (2010).

"Mechanism for the selective interaction of C-terminal Eps15 homology domain proteins with specific Asn-

Pro-Phe-containing partners." J Biol Chem 285(12): 8687-8694.

Kihara, A., T. Noda, N. Ishihara and Y. Ohsumi (2001). "Two distinct Vps34 phosphatidylinositol 3-kinase complexes function in autophagy and carboxypeptidase Y sorting in Saccharomyces cerevisiae." J Cell Biol

152(3): 519-530.

Kirchhausen, T., J. S. Bonifacino and H. Riezman (1997). "Linking cargo to vesicle formation: receptor tail interactions with coat proteins." Curr Opin Cell Biol 9(4): 488-495.

Kirchhausen, T., D. Owen and S. C. Harrison (2014). "Molecular structure, function, and dynamics of clathrin- mediated membrane traffic." Cold Spring Harb Perspect Biol 6(5): a016725. 194

Klauck, T. M., X. Xu, B. Mousseau and S. Jaken (1996). "Cloning and characterization of a glucocorticoid- induced diacylglycerol kinase." J Biol Chem 271(33): 19781-19788.

Kooijman, E. E., V. Chupin, B. de Kruijff and K. N. Burger (2003). "Modulation of membrane curvature by phosphatidic acid and lysophosphatidic acid." Traffic 4(3): 162-174.

Kooijman, E. E., V. Chupin, N. L. Fuller, M. M. Kozlov, B. de Kruijff, K. N. Burger and P. R. Rand (2005).

"Spontaneous curvature of phosphatidic acid and lysophosphatidic acid." Biochemistry 44(6): 2097-2102.

Kosaka, T. and K. Ikeda (1983). "Reversible blockage of membrane retrieval and endocytosis in the garland cell of the temperature-sensitive mutant of Drosophila melanogaster, shibirets1." J Cell Biol 97(2): 499-507.

Ktistakis, N. T., H. A. Brown, M. G. Waters, P. C. Sternweis and M. G. Roth (1996). "Evidence that phospholipase D mediates ADP ribosylation factor-dependent formation of Golgi coated vesicles." J Cell Biol

134(2): 295-306.

Lamaze, C., A. Dujeancourt, T. Baba, C. G. Lo, A. Benmerah and A. Dautry-Varsat (2001). "Interleukin 2 receptors and detergent-resistant membrane domains define a clathrin-independent endocytic pathway." Mol

Cell 7(3): 661-671.

Lapierre, L. A., R. Kumar, C. M. Hales, J. Navarre, S. G. Bhartur, J. O. Burnette, D. W. Provance, Jr., J. A.

Mercer, M. Bahler and J. R. Goldenring (2001). "Myosin vb is associated with plasma membrane recycling systems." Mol Biol Cell 12(6): 1843-1857.

Ledesma, M. D. and C. G. Dotti (2003). "Membrane and cytoskeleton dynamics during axonal elongation and stabilization." Int Rev Cytol 227: 183-219.

Lee, D. W., X. Zhao, S. Scarselletta, P. J. Schweinsberg, E. Eisenberg, B. D. Grant and L. E. Greene (2005).

"ATP binding regulates oligomerization and endosome association of RME-1 family proteins." J Biol Chem

280(17): 17213-17220.

Lennartz, M. R. (1999). "Phospholipases and phagocytosis: the role of phospholipid-derived second messengers in phagocytosis." Int J Biochem Cell Biol 31(3-4): 415-430.

Lenz, M., S. Morlot and A. Roux (2009). "Mechanical requirements for membrane fission: common facts from various examples." FEBS Lett 583(23): 3839-3846.

Lin, S. X., B. Grant, D. Hirsh and F. R. Maxfield (2001). "Rme-1 regulates the distribution and function of the endocytic recycling compartment in mammalian cells." Nat Cell Biol 3(6): 567-572.

Lopez, C. I., L. E. Pelletan, L. Suhaiman, G. A. De Blas, N. Vitale, L. S. Mayorga and S. A. Belmonte (2012).

"Diacylglycerol stimulates acrosomal exocytosis by feeding into a PKC- and PLD1-dependent positive loop that continuously supplies phosphatidylinositol 4,5-bisphosphate." Biochim Biophys Acta 1821(9): 1186-1199. 195

Lu, Q., C. Insinna, C. Ott, J. Stauffer, P. A. Pintado, J. Rahajeng, U. Baxa, V. Walia, A. Cuenca, Y. S. Hwang,

I. O. Daar, S. Lopes, J. Lippincott-Schwartz, P. K. Jackson, S. Caplan and C. J. Westlake (2015). "Early steps in primary cilium assembly require EHD1/EHD3-dependent ciliary vesicle formation." Nat Cell Biol 17(3): 228-

240.

Lundmark, R., G. J. Doherty, M. T. Howes, K. Cortese, Y. Vallis, R. G. Parton and H. T. McMahon (2008).

"The GTPase-activating protein GRAF1 regulates the CLIC/GEEC endocytic pathway." Curr Biol 18(22):

1802-1808.

Luo, B., S. M. Prescott and M. K. Topham (2003). "Association of diacylglycerol kinase zeta with protein kinase

C alpha: spatial regulation of diacylglycerol signaling." J Cell Biol 160(6): 929-937.

Madore, N., K. L. Smith, C. H. Graham, A. Jen, K. Brady, S. Hall and R. Morris (1999). "Functionally different

GPI proteins are organized in different domains on the neuronal surface." EMBO J 18(24): 6917-6926.

Madshus, I. H., K. Sandvig, S. Olsnes and B. van Deurs (1987). "Effect of reduced endocytosis induced by hypotonic shock and potassium depletion on the infection of Hep 2 cells by picornaviruses." J Cell Physiol

131(1): 14-22.

Maeda, Y., G. V. Beznoussenko, J. Van Lint, A. A. Mironov and V. Malhotra (2001). "Recruitment of protein kinase D to the trans-Golgi network via the first cysteine-rich domain." EMBO J 20(21): 5982-5990.

Magadan, J. G., M. A. Barbieri, R. Mesa, P. D. Stahl and L. S. Mayorga (2006). "Rab22a regulates the sorting of transferrin to recycling endosomes." Mol Cell Biol 26(7): 2595-2614.

Maldonado-Baez, L., C. Williamson and J. G. Donaldson (2013). "Clathrin-independent endocytosis: a cargo- centric view." Exp Cell Res 319(18): 2759-2769.

Marg, A., V. Schoewel, T. Timmel, A. Schulze, C. Shah, O. Daumke and S. Spuler (2012). "Sarcolemmal repair is a slow process and includes EHD2." Traffic 13(9): 1286-1294.

Marks, B., M. H. Stowell, Y. Vallis, I. G. Mills, A. Gibson, C. R. Hopkins and H. T. McMahon (2001). "GTPase activity of dynamin and resulting conformation change are essential for endocytosis." Nature 410(6825): 231-

235.

Marsh, E. W., P. L. Leopold, N. L. Jones and F. R. Maxfield (1995). "Oligomerized transferrin receptors are selectively retained by a lumenal sorting signal in a long-lived endocytic recycling compartment." J Cell Biol

129(6): 1509-1522.

Marsh, E. W., P. L. Leopold, N. L. Jones and F. R. Maxfield (1995). "Oligomerized transferrin receptors are selectively retained by a lumenal sorting signal in a long-lived endocytic recycling compartment." The Journal of cell biology 129(6): 1509-1522. 196

Masai, I., T. Hosoya, S. Kojima and Y. Hotta (1992). "Molecular cloning of a Drosophila diacylglycerol kinase gene that is expressed in the nervous system and muscle." Proc Natl Acad Sci U S A 89(13): 6030-6034.

Massol, R. H., J. E. Larsen and T. Kirchhausen (2005). "Possible role of deep tubular invaginations of the plasma membrane in MHC-I trafficking." Exp Cell Res 306(1): 142-149.

Mattera, R. and J. S. Bonifacino (2008). "Ubiquitin binding and conjugation regulate the recruitment of Rabex-

5 to early endosomes." EMBO J 27(19): 2484-2494.

Maxfield, F. R. and T. E. McGraw (2004). "Endocytic recycling." Nat Rev Mol Cell Biol 5(2): 121-132.

Mayer, A., W. Wickner and A. Haas (1996). "Sec18p (NSF)-driven release of Sec17p (alpha-SNAP) can precede docking and fusion of yeast vacuoles." Cell 85(1): 83-94.

Mayor, S. and R. E. Pagano (2007). "Pathways of clathrin-independent endocytosis." Nat Rev Mol Cell Biol

8(8): 603-612.

Mayor, S., R. G. Parton and J. G. Donaldson (2014). "Clathrin-independent pathways of endocytosis." Cold

Spring Harb Perspect Biol 6(6).

Mayor, S., J. F. Presley and F. R. Maxfield (1993). "Sorting of membrane components from endosomes and subsequent recycling to the cell surface occurs by a bulk flow process." J Cell Biol 121(6): 1257-1269.

Mayorga, L. S., C. N. Tomes and S. A. Belmonte (2007). "Acrosomal exocytosis, a special type of regulated secretion." IUBMB Life 59(4-5): 286-292.

McBride, H. M., V. Rybin, C. Murphy, A. Giner, R. Teasdale and M. Zerial (1999). "Oligomeric complexes link

Rab5 effectors with NSF and drive membrane fusion via interactions between EEA1 and syntaxin 13." Cell

98(3): 377-386.

McGee, T. P., H. B. Skinner, E. A. Whitters, S. A. Henry and V. A. Bankaitis (1994). "A phosphatidylinositol transfer protein controls the phosphatidylcholine content of yeast Golgi membranes." J Cell Biol 124(3): 273-

287.

McLaughlin, S., J. Wang, A. Gambhir and D. Murray (2002). "PIP(2) and proteins: interactions, organization, and information flow." Annu Rev Biophys Biomol Struct 31: 151-175.

McMahon, H. T. and J. L. Gallop (2005). "Membrane curvature and mechanisms of dynamic cell membrane remodelling." Nature 438(7068): 590-596.

McMaster, C. R. (2001). "Lipid metabolism and vesicle trafficking: more than just greasing the transport machinery." Biochem Cell Biol 79(6): 681-692.

Melendez, A. J., M. M. Harnett and J. M. Allen (2001). "Crosstalk between ARF6 and protein kinase Calpha in Fc(gamma)RI-mediated activation of phospholipase D1." Curr Biol 11(11): 869-874. 197

Mellman, I. (1996). "Membranes and sorting." Curr Opin Cell Biol 8(4): 497-498.

Merida, I., A. Avila-Flores and E. Merino (2008). "Diacylglycerol kinases: at the hub of cell signalling." Biochem

J 409(1): 1-18.

Merida, I., A. Avila-Flores and E. Merino (2008). "Diacylglycerol kinases: at the hub of cell signalling." The

Biochemical journal 409(1): 1-18.

Merithew, E., C. Stone, S. Eathiraj and D. G. Lambright (2003). "Determinants of Rab5 interaction with the N terminus of early endosome antigen 1." J Biol Chem 278(10): 8494-8500.

Mettlen, M., M. Stoeber, D. Loerke, C. N. Antonescu, G. Danuser and S. L. Schmid (2009). "Endocytic accessory proteins are functionally distinguished by their differential effects on the maturation of clathrin- coated pits." Molecular Biology of the Cell 20(14): 3251-3260.

Miaczynska, M., S. Christoforidis, A. Giner, A. Shevchenko, S. Uttenweiler-Joseph, B. Habermann, M. Wilm,

R. G. Parton and M. Zerial (2004). "APPL proteins link Rab5 to nuclear signal transduction via an endosomal compartment." Cell 116(3): 445-456.

Miller, K. G., M. D. Emerson and J. B. Rand (1999). "Goalpha and diacylglycerol kinase negatively regulate the Gqalpha pathway in C. elegans." Neuron 24(2): 323-333.

Montagnac, G., A. Echard and P. Chavrier (2008). "Endocytic traffic in animal cell cytokinesis." Current opinion in cell biology 20(4): 454-461.

Moren, B., C. Shah, M. T. Howes, N. L. Schieber, H. T. McMahon, R. G. Parton, O. Daumke and R. Lundmark

(2012). "EHD2 regulates caveolar dynamics via ATP-driven targeting and oligomerization." Mol Biol Cell 23(7):

1316-1329.

Mousley, C. J., K. R. Tyeryar, P. Vincent-Pope and V. A. Bankaitis (2007). "The Sec14-superfamily and the regulatory interface between phospholipid metabolism and membrane trafficking." Biochim Biophys Acta

1771(6): 727-736.

Muhlberg, A. B., D. E. Warnock and S. L. Schmid (1997). "Domain structure and intramolecular regulation of dynamin GTPase." EMBO J 16(22): 6676-6683.

Mukherjee, S., S. Zeitouni, C. F. Cavarsan and L. A. Shapiro (2013). "Increased seizure susceptibility in mice

30 days after fluid percussion injury." Front Neurol 4: 28.

Murray, J. T., C. Panaretou, H. Stenmark, M. Miaczynska and J. M. Backer (2002). "Role of Rab5 in the recruitment of hVps34/p150 to the early endosome." Traffic 3(6): 416-427.

Nagaya, H., I. Wada, Y. J. Jia and H. Kanoh (2002). "Diacylglycerol kinase delta suppresses ER-to-Golgi traffic via its SAM and PH domains." Mol Biol Cell 13(1): 302-316. 198

Naslavsky, N., M. Boehm, P. S. Backlund, Jr. and S. Caplan (2004). "Rabenosyn-5 and EHD1 interact and sequentially regulate protein recycling to the plasma membrane." Mol Biol Cell 15(5): 2410-2422.

Naslavsky, N. and S. Caplan (2011). "EHD proteins: key conductors of endocytic transport." Trends Cell Biol

21(2): 122-131.

Naslavsky, N., J. McKenzie, N. Altan-Bonnet, D. Sheff and S. Caplan (2009). "EHD3 regulates early- endosome-to-Golgi transport and preserves Golgi morphology." J Cell Sci 122(Pt 3): 389-400.

Naslavsky, N., J. Rahajeng, D. Rapaport, M. Horowitz and S. Caplan (2007). "EHD1 regulates cholesterol homeostasis and lipid droplet storage." Biochem Biophys Res Commun 357(3): 792-799.

Naslavsky, N., J. Rahajeng, M. Sharma, M. Jovic and S. Caplan (2006). "Interactions between EHD proteins and Rab11-FIP2: a role for EHD3 in early endosomal transport." Mol Biol Cell 17(1): 163-177.

Naslavsky, N., R. Weigert and J. G. Donaldson (2003). "Convergence of non-clathrin- and clathrin-derived endosomes involves Arf6 inactivation and changes in phosphoinositides." Mol Biol Cell 14(2): 417-431.

Naslavsky, N., R. Weigert and J. G. Donaldson (2004). "Characterization of a nonclathrin endocytic pathway: membrane cargo and lipid requirements." Mol Biol Cell 15(8): 3542-3552.

Nielsen, E., S. Christoforidis, S. Uttenweiler-Joseph, M. Miaczynska, F. Dewitte, M. Wilm, B. Hoflack and M.

Zerial (2000). "Rabenosyn-5, a novel Rab5 effector, is complexed with hVPS45 and recruited to endosomes through a FYVE finger domain." J Cell Biol 151(3): 601-612.

Nielsen, E., F. Severin, J. M. Backer, A. A. Hyman and M. Zerial (1999). "Rab5 regulates motility of early endosomes on microtubules." Nat Cell Biol 1(6): 376-382.

Nordmann, M., M. Cabrera, A. Perz, C. Brocker, C. Ostrowicz, S. Engelbrecht-Vandre and C. Ungermann

(2010). "The Mon1-Ccz1 complex is the GEF of the late endosomal Rab7 homolog Ypt7." Curr Biol 20(18):

1654-1659.

Nossal, R. (2001). "Energetics of clathrin basket assembly." Traffic 2(2): 138-147.

O'Luanaigh, N., R. Pardo, A. Fensome, V. Allen-Baume, D. Jones, M. R. Holt and S. Cockcroft (2002).

"Continual production of phosphatidic acid by phospholipase D is essential for antigen-stimulated membrane ruffling in cultured mast cells." Mol Biol Cell 13(10): 3730-3746.

Ohno, S. and Y. Nishizuka (2002). "Protein kinase C isotypes and their specific functions: prologue." J

Biochem 132(4): 509-511.

Okada, M., Y. Hozumi, K. Iwazaki, K. Misaki, M. Yanagida, Y. Araki, T. Watanabe, H. Yagisawa, M. K. Topham,

K. Kaibuchi and K. Goto (2012). "DGKzeta is involved in LPS-activated phagocytosis through IQGAP1/Rac1 pathway." Biochem Biophys Res Commun 420(2): 479-484. 199

Ovesny, M., P. Krizek, J. Borkovec, Z. Svindrych and G. M. Hagen (2014). "ThunderSTORM: a comprehensive

ImageJ plug-in for PALM and STORM data analysis and super-resolution imaging." Bioinformatics 30(16):

2389-2390.

Pagano, A., P. Crottet, C. Prescianotto-Baschong and M. Spiess (2004). "In vitro formation of recycling vesicles from endosomes requires adaptor protein-1/clathrin and is regulated by rab4 and the connector rabaptin-5." Mol Biol Cell 15(11): 4990-5000.

Pal, A., F. Severin, B. Lommer, A. Shevchenko and M. Zerial (2006). "Huntingtin-HAP40 complex is a novel

Rab5 effector that regulates early endosome motility and is up-regulated in Huntington's disease." J Cell Biol

172(4): 605-618.

Palfy, M., A. Remenyi and T. Korcsmaros (2012). "Endosomal crosstalk: meeting points for signaling pathways." Trends Cell Biol 22(9): 447-456.

Pant, S., M. Sharma, K. Patel, S. Caplan, C. M. Carr and B. D. Grant (2009). "AMPH-1/Amphiphysin/Bin1 functions with RME-1/Ehd1 in endocytic recycling." Nat Cell Biol 11(12): 1399-1410.

Park, M., E. C. Penick, J. G. Edwards, J. A. Kauer and M. D. Ehlers (2004). "Recycling endosomes supply

AMPA receptors for LTP." Science 305(5692): 1972-1975.

Parton, R. G., B. Joggerst and K. Simons (1994). "Regulated internalization of caveolae." J Cell Biol 127(5):

1199-1215.

Patki, V., D. C. Lawe, S. Corvera, J. V. Virbasius and A. Chawla (1998). "A functional PtdIns(3)P-binding motif." Nature 394(6692): 433-434.

Patki, V., J. Virbasius, W. S. Lane, B. H. Toh, H. S. Shpetner and S. Corvera (1997). "Identification of an early endosomal protein regulated by phosphatidylinositol 3-kinase." Proc Natl Acad Sci U S A 94(14): 7326-7330.

Pelkmans, L., E. Fava, H. Grabner, M. Hannus, B. Habermann, E. Krausz and M. Zerial (2005). "Genome- wide analysis of human kinases in clathrin- and caveolae/raft-mediated endocytosis." Nature 436(7047): 78-

86.

Peralta, E. R., B. C. Martin and A. L. Edinger (2010). "Differential effects of TBC1D15 and mammalian Vps39 on Rab7 activation state, lysosomal morphology, and growth factor dependence." J Biol Chem 285(22):

16814-16821.

Peter, B. J., H. M. Kent, I. G. Mills, Y. Vallis, P. J. Butler, P. R. Evans and H. T. McMahon (2004). "BAR domains as sensors of membrane curvature: the amphiphysin BAR structure." Science 303(5657): 495-499. 200

Petiot, A., E. Ogier-Denis, E. F. Blommaart, A. J. Meijer and P. Codogno (2000). "Distinct classes of phosphatidylinositol 3'-kinases are involved in signaling pathways that control macroautophagy in HT-29 cells." J Biol Chem 275(2): 992-998.

Pfeffer, S. and D. Aivazian (2004). "Targeting Rab GTPases to distinct membrane compartments." Nat Rev

Mol Cell Biol 5(11): 886-896.

Poon, P. P., S. F. Nothwehr, R. A. Singer and G. C. Johnston (2001). "The Gcs1 and Age2 ArfGAP proteins provide overlapping essential function for transport from the yeast trans-Golgi network." J Cell Biol 155(7):

1239-1250.

Posey, A. D., Jr., P. Pytel, K. Gardikiotes, A. R. Demonbreun, M. Rainey, M. George, H. Band and E. M.

McNally (2011). "Endocytic recycling proteins EHD1 and EHD2 interact with fer-1-like-5 (Fer1L5) and mediate myoblast fusion." J Biol Chem 286(9): 7379-7388.

Powelka, A. M., J. Sun, J. Li, M. Gao, L. M. Shaw, A. Sonnenberg and V. W. Hsu (2004). "Stimulation- dependent recycling of integrin beta1 regulated by ARF6 and Rab11." Traffic 5(1): 20-36.

Powner, D. J., M. N. Hodgkin and M. J. Wakelam (2002). "Antigen-stimulated activation of phospholipase D1b by Rac1, ARF6, and PKCalpha in RBL-2H3 cells." Mol Biol Cell 13(4): 1252-1262.

Praefcke, G. J. and H. T. McMahon (2004). "The dynamin superfamily: universal membrane tubulation and fission molecules?" Nat Rev Mol Cell Biol 5(2): 133-147.

Quan, A. and P. J. Robinson (2013). "Syndapin--a membrane remodelling and endocytic F-BAR protein."

FEBS J 280(21): 5198-5212.

Quann, E. J., E. Merino, T. Furuta and M. Huse (2009). "Localized diacylglycerol drives the polarization of the microtubule-organizing center in T cells." Nat Immunol 10(6): 627-635.

Radhakrishna, H. and J. G. Donaldson (1997). "ADP-ribosylation factor 6 regulates a novel plasma membrane recycling pathway." J Cell Biol 139(1): 49-61.

Rahajeng, J., S. S. Giridharan, B. Cai, N. Naslavsky and S. Caplan (2012). "MICAL-L1 is a tubular endosomal membrane hub that connects Rab35 and Arf6 with Rab8a." Traffic 13(1): 82-93.

Rahajeng, J., S. S. Giridharan, N. Naslavsky and S. Caplan (2010). "Collapsin response mediator protein-2

(Crmp2) regulates trafficking by linking endocytic regulatory proteins to dynein motors." J Biol Chem 285(42):

31918-31922.

Rahajeng, J., S. S. Panapakkam Giridharan, B. Cai, N. Naslavsky and S. Caplan (2012). "MICAL-L1 is a tubular endosomal membrane hub that connects Rab35 and Arf6 with Rab8a." Traffic 13(1): 82-93. 201

Raiborg, C., B. Bremnes, A. Mehlum, D. J. Gillooly, A. D'Arrigo, E. Stang and H. Stenmark (2001). "FYVE and coiled-coil domains determine the specific localisation of Hrs to early endosomes." J Cell Sci 114(Pt 12): 2255-

2263.

Raiborg, C. and H. Stenmark (2002). "Hrs and endocytic sorting of ubiquitinated membrane proteins." Cell

Struct Funct 27(6): 403-408.

Rainero, E., P. T. Caswell, P. A. Muller, J. Grindlay, M. W. McCaffrey, Q. Zhang, M. J. Wakelam, K. H.

Vousden, A. Graziani and J. C. Norman (2012). "Diacylglycerol kinase alpha controls RCP-dependent integrin trafficking to promote invasive migration." The Journal of cell biology 196(2): 277-295.

Rainero, E., P. T. Caswell, P. A. Muller, J. Grindlay, M. W. McCaffrey, Q. Zhang, M. J. Wakelam, K. H.

Vousden, A. Graziani and J. C. Norman (2012). "Diacylglycerol kinase alpha controls RCP-dependent integrin trafficking to promote invasive migration." J Cell Biol 196(2): 277-295.

Raucher, D. and M. P. Sheetz (1999). "Membrane expansion increases endocytosis rate during mitosis." J

Cell Biol 144(3): 497-506.

Razani, B. and M. P. Lisanti (2001). "Caveolins and caveolae: molecular and functional relationships." Exp

Cell Res 271(1): 36-44.

Razzaq, A., I. M. Robinson, H. T. McMahon, J. N. Skepper, Y. Su, A. C. Zelhof, A. P. Jackson, N. J. Gay and

C. J. O'Kane (2001). "Amphiphysin is necessary for organization of the excitation-contraction coupling machinery of muscles, but not for synaptic vesicle endocytosis in Drosophila." Genes Dev 15(22): 2967-2979.

Reinecke, J. B., D. Katafiasz, N. Naslavsky and S. Caplan (2015). "Novel functions for the endocytic regulatory proteins MICAL-L1 and EHD1 in mitosis." Traffic 16(1): 48-67.

Reineke, J. B., S. Xie, N. Naslavsky and S. Caplan (2015). "Qualitative and quantitative analysis of endocytic recycling." Methods Cell Biol 130: 139-155.

Ren, M., G. Xu, J. Zeng, C. De Lemos-Chiarandini, M. Adesnik and D. D. Sabatini (1998). "Hydrolysis of GTP on rab11 is required for the direct delivery of transferrin from the pericentriolar recycling compartment to the cell surface but not from sorting endosomes." Proc Natl Acad Sci U S A 95(11): 6187-6192.

Richnau, N., A. Fransson, K. Farsad and P. Aspenstrom (2004). "RICH-1 has a BIN/Amphiphysin/Rvsp domain responsible for binding to membrane lipids and tubulation of liposomes." Biochem Biophys Res

Commun 320(3): 1034-1042.

Rincon, E., T. Santos, A. Avila-Flores, J. P. Albar, V. Lalioti, C. Lei, W. Hong and I. Merida (2007). "Proteomics identification of sorting nexin 27 as a diacylglycerol kinase zeta-associated protein: new diacylglycerol kinase roles in endocytic recycling." Mol Cell Proteomics 6(6): 1073-1087. 202

Rink, J., E. Ghigo, Y. Kalaidzidis and M. Zerial (2005). "Rab conversion as a mechanism of progression from early to late endosomes." Cell 122(5): 735-749.

Rodriguez de Turco, E. B., W. Tang, M. K. Topham, F. Sakane, V. L. Marcheselli, C. Chen, A. Taketomi, S.

M. Prescott and N. G. Bazan (2001). "Diacylglycerol kinase epsilon regulates seizure susceptibility and long- term potentiation through arachidonoyl- inositol lipid signaling." Proc Natl Acad Sci U S A 98(8): 4740-4745.

Rojas, R., T. van Vlijmen, G. A. Mardones, Y. Prabhu, A. L. Rojas, S. Mohammed, A. J. Heck, G. Raposo, P. van der Sluijs and J. S. Bonifacino (2008). "Regulation of retromer recruitment to endosomes by sequential action of Rab5 and Rab7." J Cell Biol 183(3): 513-526.

Roland, J. T., A. K. Kenworthy, J. Peranen, S. Caplan and J. R. Goldenring (2007). "Myosin Vb interacts with

Rab8a on a tubular network containing EHD1 and EHD3." Mol Biol Cell 18(8): 2828-2837.

Roldan, E. R. and R. A. Harrison (1992). "The role of diacylglycerol in the exocytosis of the sperm acrosome.

Studies using diacylglycerol lipase and diacylglycerol kinase inhibitors and exogenous diacylglycerols."

Biochem J 281 ( Pt 3): 767-773.

Romer, W., L. Berland, V. Chambon, K. Gaus, B. Windschiegl, D. Tenza, M. R. Aly, V. Fraisier, J. C. Florent,

D. Perrais, C. Lamaze, G. Raposo, C. Steinem, P. Sens, P. Bassereau and L. Johannes (2007). "Shiga toxin induces tubular membrane invaginations for its uptake into cells." Nature 450(7170): 670-675.

Roth, M. G. (1999). "Lipid regulators of membrane traffic through the Golgi complex." Trends Cell Biol 9(5):

174-179.

Rozelle, A. L., L. M. Machesky, M. Yamamoto, M. H. Driessens, R. H. Insall, M. G. Roth, K. Luby-Phelps, G.

Marriott, A. Hall and H. L. Yin (2000). "Phosphatidylinositol 4,5-bisphosphate induces actin-based movement of raft-enriched vesicles through WASP-Arp2/3." Curr Biol 10(6): 311-320.

Rutherford, A. C., C. Traer, T. Wassmer, K. Pattni, M. V. Bujny, J. G. Carlton, H. Stenmark and P. J. Cullen

(2006). "The mammalian phosphatidylinositol 3-phosphate 5-kinase (PIKfyve) regulates endosome-to-TGN retrograde transport." J Cell Sci 119(Pt 19): 3944-3957.

Sabharanjak, S., P. Sharma, R. G. Parton and S. Mayor (2002). "GPI-anchored proteins are delivered to recycling endosomes via a distinct cdc42-regulated, clathrin-independent pinocytic pathway." Dev Cell 2(4):

411-423.

Sakai, H. and F. Sakane (2012). "Recent progress on type II diacylglycerol kinases: the physiological functions of diacylglycerol kinase delta, eta and kappa and their involvement in disease." J Biochem 152(5): 397-406. 203

Sakane, F., S. Imai, M. Kai, I. Wada and H. Kanoh (1996). "Molecular cloning of a novel diacylglycerol kinase isozyme with a pleckstrin homology domain and a C-terminal tail similar to those of the EPH family of protein- tyrosine kinases." J Biol Chem 271(14): 8394-8401.

Sakane, F., K. Yamada, H. Kanoh, C. Yokoyama and T. Tanabe (1990). "Porcine diacylglycerol kinase sequence has zinc finger and E-F hand motifs." Nature 344(6264): 345-348.

Sandvig, K., S. Pust, T. Skotland and B. van Deurs (2011). "Clathrin-independent endocytosis: mechanisms and function." Curr Opin Cell Biol 23(4): 413-420.

Sandvig, K., M. L. Torgersen, H. A. Raa and B. van Deurs (2008). "Clathrin-independent endocytosis: from nonexisting to an extreme degree of complexity." Histochem Cell Biol 129(3): 267-276.

Sandvig, K. and B. van Deurs (1994). "Endocytosis and intracellular sorting of ricin and Shiga toxin." FEBS

Lett 346(1): 99-102.

Sanjuan, M. A., D. R. Jones, M. Izquierdo and I. Merida (2001). "Role of diacylglycerol kinase alpha in the attenuation of receptor signaling." J Cell Biol 153(1): 207-220.

Sarri, E., A. Sicart, F. Lazaro-Dieguez and G. Egea (2011). "Phospholipid synthesis participates in the regulation of diacylglycerol required for membrane trafficking at the Golgi complex." J Biol Chem 286(32):

28632-28643.

Sato, M., K. Sato, W. Liou, S. Pant, A. Harada and B. D. Grant (2008). "Regulation of endocytic recycling by

C. elegans Rab35 and its regulator RME-4, a coated-pit protein." EMBO J 27(8): 1183-1196.

Schaap, D., J. van der Wal, W. J. van Blitterswijk, R. L. van der Bend and H. L. Ploegh (1993). "Diacylglycerol kinase is phosphorylated in vivo upon stimulation of the epidermal growth factor receptor and serine/threonine kinases, including protein kinase C-epsilon." Biochem J 289 ( Pt 3): 875-881.

Schenck, A., L. Goto-Silva, C. Collinet, M. Rhinn, A. Giner, B. Habermann, M. Brand and M. Zerial (2008).

"The endosomal protein Appl1 mediates Akt substrate specificity and cell survival in vertebrate development."

Cell 133(3): 486-497.

Schlam, D., M. Bohdanowicz, A. Chatgilialoglu, B. E. Steinberg, T. Ueyama, G. Du, S. Grinstein and G. D.

Fairn (2013). "Diacylglycerol kinases terminate diacylglycerol signaling during the respiratory burst leading to heterogeneous phagosomal NADPH oxidase activation." J Biol Chem 288(32): 23090-23104.

Schmidt, A., M. Wolde, C. Thiele, W. Fest, H. Kratzin, A. V. Podtelejnikov, W. Witke, W. B. Huttner and H. D.

Soling (1999). "Endophilin I mediates synaptic vesicle formation by transfer of arachidonate to lysophosphatidic acid." Nature 401(6749): 133-141. 204

Schonteich, E., G. M. Wilson, J. Burden, C. R. Hopkins, K. Anderson, J. R. Goldenring and R. Prekeris (2008).

"The Rip11/Rab11-FIP5 and kinesin II complex regulates endocytic protein recycling." J Cell Sci 121(Pt 22):

3824-3833.

Shao, Y., W. Akmentin, J. J. Toledo-Aral, J. Rosenbaum, G. Valdez, J. B. Cabot, B. S. Hilbush and S.

Halegoua (2002). "Pincher, a pinocytic chaperone for nerve growth factor/TrkA signaling endosomes." J Cell

Biol 157(4): 679-691.

Sharma, M., S. S. Giridharan, J. Rahajeng, N. Naslavsky and S. Caplan (2009). "MICAL-L1 links EHD1 to tubular recycling endosomes and regulates receptor recycling." Mol Biol Cell 20(24): 5181-5194.

Sharma, M., M. Jovic, F. Kieken, N. Naslavsky, P. Sorgen and S. Caplan (2009). "A model for the role of

EHD1-containing membrane tubules in endocytic recycling." Commun Integr Biol 2(5): 431-433.

Sharma, M., N. Naslavsky and S. Caplan (2008). "A role for EHD4 in the regulation of early endosomal transport." Traffic 9(6): 995-1018.

Sharma, P., S. Sabharanjak and S. Mayor (2002). "Endocytosis of lipid rafts: an identity crisis." Semin Cell

Dev Biol 13(3): 205-214.

Sharma, P., R. Varma, R. C. Sarasij, Ira, K. Gousset, G. Krishnamoorthy, M. Rao and S. Mayor (2004).

"Nanoscale organization of multiple GPI-anchored proteins in living cell membranes." Cell 116(4): 577-589.

Sheetz, M. P. (2001). "Cell control by membrane-cytoskeleton adhesion." Nat Rev Mol Cell Biol 2(5): 392-396.

Sheff, D. R., E. A. Daro, M. Hull and I. Mellman (1999). "The receptor recycling pathway contains two distinct populations of early endosomes with different sorting functions." J Cell Biol 145(1): 123-139.

Shemesh, T., A. Luini, V. Malhotra, K. N. Burger and M. M. Kozlov (2003). "Prefission constriction of Golgi tubular carriers driven by local lipid metabolism: a theoretical model." Biophys J 85(6): 3813-3827.

Shindou, H., D. Hishikawa, T. Harayama, K. Yuki and T. Shimizu (2009). "Recent progress on acyl CoA: lysophospholipid acyltransferase research." Journal of lipid research 50 Suppl: S46-51.

Shirai, Y., T. Kouzuki, K. Kakefuda, S. Moriguchi, A. Oyagi, K. Horie, S. Y. Morita, M. Shimazawa, K. Fukunaga,

J. Takeda, N. Saito and H. Hara (2010). "Essential role of neuron-enriched diacylglycerol kinase (DGK),

DGKbeta in neurite spine formation, contributing to cognitive function." PLoS One 5(7): e11602.

Shirai, Y., S. Segawa, M. Kuriyama, K. Goto, N. Sakai and N. Saito (2000). "Subtype-specific translocation of diacylglycerol kinase alpha and gamma and its correlation with protein kinase C." J Biol Chem 275(32): 24760-

24766.

Shisheva, A. (2008). "PIKfyve: Partners, significance, debates and paradoxes." Cell Biol Int 32(6): 591-604. 205

Shulga, Y. V., M. K. Topham and R. M. Epand (2011). "Substrate specificity of diacylglycerol kinase-epsilon and the phosphatidylinositol cycle." FEBS Lett 585(24): 4025-4028.

Shupliakov, O., O. Bloom, J. S. Gustafsson, O. Kjaerulff, P. Low, N. Tomilin, V. A. Pieribone, P. Greengard and L. Brodin (2002). "Impaired recycling of synaptic vesicles after acute perturbation of the presynaptic actin cytoskeleton." Proc Natl Acad Sci U S A 99(22): 14476-14481.

Siddhanta, A. and D. Shields (1998). "Secretory vesicle budding from the trans-Golgi network is mediated by phosphatidic acid levels." J Biol Chem 273(29): 17995-17998.

Siddiqui, R. A. and D. English (1996). "Phosphatidic acid binding to human neutrophils: effects on tyrosine kinase-regulated intracellular Ca2+ mobilization." Cell Signal 8(5): 349-354.

Sigismund, S., T. Woelk, C. Puri, E. Maspero, C. Tacchetti, P. Transidico, P. P. Di Fiore and S. Polo (2005).

"Clathrin-independent endocytosis of ubiquitinated cargos." Proc Natl Acad Sci U S A 102(8): 2760-2765.

Simon, J. P., I. E. Ivanov, M. Adesnik and D. D. Sabatini (1996). "The production of post-Golgi vesicles requires a protein kinase C-like molecule, but not its phosphorylating activity." J Cell Biol 135(2): 355-370.

Simone, L. C., S. Caplan and N. Naslavsky (2013). "Role of phosphatidylinositol 4,5-bisphosphate in regulating EHD2 plasma membrane localization." PLoS One 8(9): e74519.

Simonsen, A., J. M. Gaullier, A. D'Arrigo and H. Stenmark (1999). "The Rab5 effector EEA1 interacts directly with syntaxin-6." J Biol Chem 274(41): 28857-28860.

Simonsen, A., A. E. Wurmser, S. D. Emr and H. Stenmark (2001). "The role of phosphoinositides in membrane transport." Curr Opin Cell Biol 13(4): 485-492.

Singh, R. D., Y. Liu, C. L. Wheatley, E. L. Holicky, A. Makino, D. L. Marks, T. Kobayashi, G. Subramaniam, R.

Bittman and R. E. Pagano (2006). "Caveolar endocytosis and microdomain association of a glycosphingolipid analog is dependent on its sphingosine stereochemistry." J Biol Chem 281(41): 30660-30668.

Singh, R. D., V. Puri, J. T. Valiyaveettil, D. L. Marks, R. Bittman and R. E. Pagano (2003). "Selective caveolin-

1-dependent endocytosis of glycosphingolipids." Mol Biol Cell 14(8): 3254-3265.

Skop, A. R., D. Bergmann, W. A. Mohler and J. G. White (2001). "Completion of cytokinesis in C. elegans requires a brefeldin A-sensitive membrane accumulation at the cleavage furrow apex." Current biology : CB

11(10): 735-746.

Soderberg, O., M. Gullberg, M. Jarvius, K. Ridderstrale, K. J. Leuchowius, J. Jarvius, K. Wester, P. Hydbring,

F. Bahram, L. G. Larsson and U. Landegren (2006). "Direct observation of individual endogenous protein complexes in situ by proximity ligation." Nat Methods 3(12): 995-1000.

Sollner, T. (1995). "SNAREs and targeted membrane fusion." FEBS Lett 369(1): 80-83. 206

Sonnichsen, B., S. De Renzis, E. Nielsen, J. Rietdorf and M. Zerial (2000). "Distinct membrane domains on endosomes in the recycling pathway visualized by multicolor imaging of Rab4, Rab5, and Rab11." J Cell Biol

149(4): 901-914.

Sreenivas, A., J. L. Patton-Vogt, V. Bruno, P. Griac and S. A. Henry (1998). "A role for phospholipase D (Pld1p) in growth, secretion, and regulation of membrane lipid synthesis in yeast." J Biol Chem 273(27): 16635-16638.

Stan, R. V. (2002). "Structure and function of endothelial caveolae." Microsc Res Tech 57(5): 350-364.

Stein, M. P., J. Dong and A. Wandinger-Ness (2003). "Rab proteins and endocytic trafficking: potential targets for therapeutic intervention." Adv Drug Deliv Rev 55(11): 1421-1437.

Stoeber, M., I. K. Stoeck, C. Hanni, C. K. Bleck, G. Balistreri and A. Helenius (2012). "Oligomers of the ATPase

EHD2 confine caveolae to the plasma membrane through association with actin." EMBO J 31(10): 2350-2364.

Stroupe, C., C. M. Hickey, J. Mima, A. S. Burfeind and W. Wickner (2009). "Minimal membrane docking requirements revealed by reconstitution of Rab GTPase-dependent membrane fusion from purified components." Proc Natl Acad Sci U S A 106(42): 17626-17633.

Stuermer, C. A., D. M. Lang, F. Kirsch, M. Wiechers, S. O. Deininger and H. Plattner (2001).

"Glycosylphosphatidyl inositol-anchored proteins and fyn kinase assemble in noncaveolar plasma membrane microdomains defined by reggie-1 and -2." Mol Biol Cell 12(10): 3031-3045.

Subramanya, S. and K. Mensa-Wilmot (2010). "Diacylglycerol-stimulated endocytosis of transferrin in trypanosomatids is dependent on tyrosine kinase activity." PLoS One 5(1): e8538.

Suetsugu, S. (2010). "The proposed functions of membrane curvatures mediated by the BAR domain superfamily proteins." J Biochem 148(1): 1-12.

Sutton, R. B., D. Fasshauer, R. Jahn and A. T. Brunger (1998). "Crystal structure of a SNARE complex involved in synaptic exocytosis at 2.4 A resolution." Nature 395(6700): 347-353.

Takei, K., V. Haucke, V. Slepnev, K. Farsad, M. Salazar, H. Chen and P. De Camilli (1998). "Generation of coated intermediates of clathrin-mediated endocytosis on protein-free liposomes." Cell 94(1): 131-141.

Takei, K., P. S. McPherson, S. L. Schmid and P. De Camilli (1995). "Tubular membrane invaginations coated by dynamin rings are induced by GTP-gamma S in nerve terminals." Nature 374(6518): 186-190.

Takei, K., V. I. Slepnev, V. Haucke and P. De Camilli (1999). "Functional partnership between amphiphysin and dynamin in clathrin-mediated endocytosis." Nat Cell Biol 1(1): 33-39.

Tang, W., M. Bunting, G. A. Zimmerman, T. M. McIntyre and S. M. Prescott (1996). "Molecular cloning of a novel human diacylglycerol kinase highly selective for arachidonate-containing substrates." J Biol Chem

271(17): 10237-10241. 207

Tebar, F., T. Sorkina, A. Sorkin, M. Ericsson and T. Kirchhausen (1996). "Eps15 is a component of clathrin- coated pits and vesicles and is located at the rim of coated pits." J Biol Chem 271(46): 28727-28730.

Tolias, K. F., J. H. Hartwig, H. Ishihara, Y. Shibasaki, L. C. Cantley and C. L. Carpenter (2000). "Type Ialpha phosphatidylinositol-4-phosphate 5-kinase mediates Rac-dependent actin assembly." Curr Biol 10(3): 153-

156.

Topham, M. K., M. Bunting, G. A. Zimmerman, T. M. McIntyre, P. J. Blackshear and S. M. Prescott (1998).

"Protein kinase C regulates the nuclear localization of diacylglycerol kinase-zeta." Nature 394(6694): 697-700.

Topham, M. K. and S. M. Prescott (1999). "Mammalian diacylglycerol kinases, a family of lipid kinases with signaling functions." J Biol Chem 274(17): 11447-11450.

Topham, M. K. and S. M. Prescott (2001). "Diacylglycerol kinase zeta regulates Ras activation by a novel mechanism." J Cell Biol 152(6): 1135-1143.

Ueyama, T., M. R. Lennartz, Y. Noda, T. Kobayashi, Y. Shirai, K. Rikitake, T. Yamasaki, S. Hayashi, N. Sakai,

H. Seguchi, M. Sawada, H. Sumimoto and N. Saito (2004). "Superoxide production at phagosomal cup/phagosome through beta I protein kinase C during Fc gamma R-mediated phagocytosis in microglia." J

Immunol 173(7): 4582-4589.

Unwin, N. (2005). "Refined structure of the nicotinic acetylcholine receptor at 4A resolution." J Mol Biol 346(4):

967-989.

Valdez, G., W. Akmentin, P. Philippidou, R. Kuruvilla, D. D. Ginty and S. Halegoua (2005). "Pincher-mediated macroendocytosis underlies retrograde signaling by neurotrophin receptors." J Neurosci 25(21): 5236-5247. van der Sluijs, P., M. Hull, P. Webster, P. Male, B. Goud and I. Mellman (1992). "The small GTP-binding protein rab4 controls an early sorting event on the endocytic pathway." Cell 70(5): 729-740.

Vicinanza, M., G. D'Angelo, A. Di Campli and M. A. De Matteis (2008). "Function and dysfunction of the PI system in membrane trafficking." EMBO J 27(19): 2457-2470.

Vitale, G., V. Rybin, S. Christoforidis, P. Thornqvist, M. McCaffrey, H. Stenmark and M. Zerial (1998). "Distinct

Rab-binding domains mediate the interaction of Rabaptin-5 with GTP-bound Rab4 and Rab5." EMBO J 17(7):

1941-1951.

Walseng, E., O. Bakke and P. A. Roche (2008). "Major histocompatibility complex class II-peptide complexes internalize using a clathrin- and dynamin-independent endocytosis pathway." J Biol Chem 283(21): 14717-

14727. 208

Wang, E., P. S. Brown, B. Aroeti, S. J. Chapin, K. E. Mostov and K. W. Dunn (2000). "Apical and basolateral endocytic pathways of MDCK cells meet in acidic common endosomes distinct from a nearly-neutral apical recycling endosome." Traffic 1(6): 480-493.

Weigert, R., M. G. Silletta, S. Spano, G. Turacchio, C. Cericola, A. Colanzi, S. Senatore, R. Mancini, E. V.

Polishchuk, M. Salmona, F. Facchiano, K. N. Burger, A. Mironov, A. Luini and D. Corda (1999). "CtBP/BARS induces fission of Golgi membranes by acylating lysophosphatidic acid." Nature 402(6760): 429-433.

Weigert, R., A. C. Yeung, J. Li and J. G. Donaldson (2004). "Rab22a regulates the recycling of membrane proteins internalized independently of clathrin." Mol Biol Cell 15(8): 3758-3770.

Wigge, P., K. Kohler, Y. Vallis, C. A. Doyle, D. Owen, S. P. Hunt and H. T. McMahon (1997). "Amphiphysin heterodimers: potential role in clathrin-mediated endocytosis." Mol Biol Cell 8(10): 2003-2015.

Williams, J. M., T. R. Pettitt, W. Powell, J. Grove, C. O. Savage and M. J. Wakelam (2007). "Antineutrophil cytoplasm antibody-stimulated neutrophil adhesion depends on diacylglycerol kinase-catalyzed phosphatidic acid formation." J Am Soc Nephrol 18(4): 1112-1120.

Worby, C. A. and J. E. Dixon (2002). "Sorting out the cellular functions of sorting nexins." Nat Rev Mol Cell

Biol 3(12): 919-931.

Wurmser, A. E., T. K. Sato and S. D. Emr (2000). "New component of the vacuolar class C-Vps complex couples nucleotide exchange on the Ypt7 GTPase to SNARE-dependent docking and fusion." J Cell Biol

151(3): 551-562.

Xie, S., K. Bahl, J. B. Reinecke, G. R. Hammond, N. Naslavsky and S. Caplan (2016). "The endocytic recycling compartment maintains cargo segregation acquired upon exit from the sorting endosome." Mol Biol Cell 27(1):

108-126.

Xie, S., N. Naslavsky and S. Caplan (2014). "Diacylglycerol Kinase alpha Regulates Tubular Recycling

Endosome Biogenesis and Major Histocompatibility Complex Class I Recycling." J Biol Chem 289(46): 31914-

31926.

Xie, S., N. Naslavsky and S. Caplan (2014). "Diacylglycerol kinase alpha regulates tubular recycling endosome biogenesis and major histocompatibility complex class I recycling." The Journal of biological chemistry.

Xie, S., N. Naslavsky and S. Caplan (2015). "Diacylglycerol kinases in membrane trafficking." Cellular

Logistics 5(2): e1078431. 209

Yamabhai, M., N. G. Hoffman, N. L. Hardison, P. S. McPherson, L. Castagnoli, G. Cesareni and B. K. Kay

(1998). "Intersectin, a novel adaptor protein with two Eps15 homology and five Src homology 3 domains." J

Biol Chem 273(47): 31401-31407.

Yamada, K., F. Sakane, N. Matsushima and H. Kanoh (1997). "EF-hand motifs of alpha, beta and gamma isoforms of diacylglycerol kinase bind calcium with different affinities and conformational changes." Biochem

J 321 ( Pt 1): 59-64.

Yamaguchi, Y., Y. Shirai, T. Matsubara, K. Sanse, M. Kuriyama, N. Oshiro, K. Yoshino, K. Yonezawa, Y. Ono and N. Saito (2006). "Phosphorylation and up-regulation of diacylglycerol kinase gamma via its interaction with protein kinase C gamma." J Biol Chem 281(42): 31627-31637.

Yamashiro, D. J., B. Tycko, S. R. Fluss and F. R. Maxfield (1984). "Segregation of transferrin to a mildly acidic

(pH 6.5) para-Golgi compartment in the recycling pathway." Cell 37(3): 789-800.

Yang, J., J. Seo, R. Nair, S. Han, S. Jang, K. Kim, K. Han, S. K. Paik, J. Choi, S. Lee, Y. C. Bae, M. K. Topham,

S. M. Prescott, J. S. Rhee, S. Y. Choi and E. Kim (2011). "DGKiota regulates presynaptic release during mGluR-dependent LTD." EMBO J 30(1): 165-180.

Yap, C. C., Z. M. Lasiecka, S. Caplan and B. Winckler (2010). "Alterations of EHD1/EHD4 protein levels interfere with L1/NgCAM endocytosis in neurons and disrupt axonal targeting." J Neurosci 30(19): 6646-6657.

Yarar, D., C. M. Waterman-Storer and S. L. Schmid (2005). "A dynamic actin cytoskeleton functions at multiple stages of clathrin-mediated endocytosis." Mol Biol Cell 16(2): 964-975.

Yin, H. L. and P. A. Janmey (2003). "Phosphoinositide regulation of the actin cytoskeleton." Annu Rev Physiol

65: 761-789.

Zerial, M. and H. McBride (2001). "Rab proteins as membrane organizers." Nat Rev Mol Cell Biol 2(2): 107-

117.

Zha, Y., R. Marks, A. W. Ho, A. C. Peterson, S. Janardhan, I. Brown, K. Praveen, S. Stang, J. C. Stone and

T. F. Gajewski (2006). "T cell anergy is reversed by active Ras and is regulated by diacylglycerol kinase- alpha." Nat Immunol 7(11): 1166-1173.

Zhang, J., N. Naslavsky and S. Caplan (2012). "EHDs meet the retromer: Complex regulation of retrograde transport." Cell Logist 2(3): 161-165.

Zhang, J., C. Reiling, J. B. Reinecke, I. Prislan, L. A. Marky, P. L. Sorgen, N. Naslavsky and S. Caplan (2012).

"Rabankyrin-5 interacts with EHD1 and Vps26 to regulate endocytic trafficking and retromer function." Traffic

13(5): 745-757. 210

Zhong, X. P., E. A. Hainey, B. A. Olenchock, M. S. Jordan, J. S. Maltzman, K. E. Nichols, H. Shen and G. A.

Koretzky (2003). "Enhanced T cell responses due to diacylglycerol kinase zeta deficiency." Nat Immunol 4(9):

882-890.

Zimmerberg, J. and M. M. Kozlov (2006). "How proteins produce cellular membrane curvature." Nat Rev Mol

Cell Biol 7(1): 9-19.

Zwilling, D., A. Cypionka, W. H. Pohl, D. Fasshauer, P. J. Walla, M. C. Wahl and R. Jahn (2007). "Early endosomal SNAREs form a structurally conserved SNARE complex and fuse liposomes with multiple topologies." EMBO J 26(1): 9-18.