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 .....................................................................................