A Dissertation Entitled SGEF Forms a Complex with Scribble and Dlg1

A dissertation

entitled

SGEF forms a complex with Scribble and Dlg1 and regulates epithelial junctions and

contractility

Sahezeel S. Awadia

Submitted to the Graduate Faculty as partial fulfillment of the requirements for the

Doctor of Philosophy Degree in

Biological Sciences ______

Dr. Rafael Garcia-Mata, Committee Chair

______

Dr. Ann Miller, Committee Member

______

Dr. Tomer Avidor-Reiss, Committee Member

______

Dr. Deborah Chadee, Committee Member

______

Dr. Katherine Eisenmann, Committee Member

Dr Cyndee Gruden, Dean College of Graduate Studies

The University of Toledo May 2019

An Abstract of

SGEF forms a complex with Scribble and Dlg1 and regulates epithelial junctions and

contractility

Sahezeel S. Awadia

Submitted to the Graduate Faculty as partial fulfillment of the requirements for the Doctor of Philosophy Degree in Biological Sciences

The University of Toledo

May 2019

The establishment of epithelial cell polarity is essential for growth, differentiation and morphogenesis. Conversely, loss of cell polarity is a hallmark of many disease states, including cancer. The concerted action of three conserved protein complexes – PAR,

Crumbs and Scribble – controls the establishment of cell polarity. In addition, the Rho- family of small GTPases, which regulate actin dynamics, plays a key role in the development of polarity. How polarity complexes regulate Rho GTPase function remains unknown.

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The members of the Scribble polarity complex Scribble, Lgl and Dlg1 are known to interact genetically but the direct interaction between Scribble and Dlg1 is not known completely. Here, we show that SGEF interacts with Scribble, a member of the Scribble polarity complex. A novel internal PDZ binding motif in SGEF interacts with PDZ1 of

Scribble. We also obtained a crystal structure of PDZ1 of Scribble in complex with the peptide comprising internal PDZ binding motif of SGEF. An unstructured 30 amino acid motif at the N-terminus of SGEF interacts with GUK (Guanylate Kinase) domain located at C-terminus of Dlg1. SGEF interacts with Scribble and Dlg1 through different regions in

SGEF. This allows simultaneous binding of both Scribble and Dlg1 to SGEF forming a ternary complex of Scribble-SGEF-Dlg1 in epithelial cells.

The Scribble polarity complex has been implicated in regulation of junctional architecture in epithelial cells. Our finding shows that SGEF localizes to apical junctions of Xenopus embryo and lateral junctions of MDCK (Madin Darby Canine Kidney) epithelial cells in Scribble dependent fashion. Scribble and Dlg1 are known to be involved in Adherens Junctions (AJ) establishment and maintenance. Here we show that knockdown of SGEF in MDCK cells leads to disruption of AJ characterized by almost a complete loss of E-cadherin, diffused localization of E-cadherin binding partners -catenin, -catenin and p120-catenin at lateral cortex. Disruption of lateral junctions leads to a collapse of lateral wall of epithelial cells leading to flattening of epithelial cells silenced for SGEF.

We also show that silencing of SGEF leads to straightening of otherwise curvilinear tight junctions stained by ZO-1 (Zonula Occludens). The loss of curvilinear phenotype is accompanied by localization of myosin to apical actin belt forming beautifully organized actomyosin arrays. Apical actomyosin arrays in SGEF KD cells leads to increased

iv contractility, increased tension, increased permeability and straightening of apical tight junctions. Our results suggested that the scaffolding activity of SGEF to form a ternary complex with Scribble and Dlg1 is essential for the apical actomyosin contractility and proper architecture of the tight junctions whereas the exchange activity of SGEF is required for the E-cadherin based adherens junctions architecture in epithelial cells.

MDCK cells when embedded in a 3D matrix like matrigel form characteristic 3D structures called cysts that are highly polarized and have a single open (fluid filled) lumen.

We found that the silencing of SGEF displayed a disorganized cyst phenotype with abnormal lumens formation. Further, we showed that formation of open lumen is regulated by the scaffolding activity of SGEF whereas ability to form a single hollow lumen is regulated by exchange activity of SGEF.

Finally, we found that SGEF overexpression alters junctional architecture and dynamics and promotes apical constriction. Xenopus embryos overexpressing SGEF had increased cell constriction due to localization of myosin to apical region of cells. This increased contractility reduced the apical cell area in SGEF overexpressing embryos.

Taken together, our results suggested that the scaffolding activity of SGEF to form a ternary complex with Scribble and Dlg1 regulates apical actomyosin contractility and TJ properties whereas its catalytic activity to activate RhoG regulates E-cadherin based AJ architecture in epithelial cells.

To my wife, my son and my parents. This would not have been possible without your

unwavering support

ACKNOWLEDGEMENTS

First and foremost, I would like to thank my mentor Dr. Rafael Garcia-Mata for guiding me throughout my PhD journey, be it a scientific or personal, he has been pillar of strength and support for me. I am truly blessed to get an opportunity to work in his lab. It is because of Rafael that I am able to fulfill my dream of becoming a scientist. He has inspired me to become an independent researcher and helped me realize the power of critical reasoning. In addition to this, I am also extremely thankful to my committee members Dr. Ann Miller, Dr. Tomer Avidor-Reiss, Dr. Katherine Eisenmann and Dr.

Deborah Chadee. A special thanks to Dr. Silvia Goicoechea. Finally, I also appreciate the support of the Chair of the Biology Department, Dr. Bruce Bamber.

I am also extremely thankful to Ashtyn Hoover, Kyle Snyder, Torey Arnold, Farah

Huq, Atul Khire, Alan Hammer and for all their help and wonderful memories.

I am extremely grateful to my lovely wife Alisha. Without her support and understanding this thesis and my PhD were unachievable. She has been extremely supportive of me throughout this entire process and has made countless sacrifices to help me get to this point. She always motivated, inspired and most importantly never lost faith in me. Her smiling face always provided me with added fuel to work towards this thesis.

A special thanks to my parents Shamsuddin and Zubeda Awadia and Kusoom

Khoja, Scott and Laurie Hahnlen, my brother Sahil, and sister-in-law Anjum for their unfailing faith and support. A special thanks to kids of our family Ronak, Ijju. Lastly, how can I forget my little angel, my son Aariz. His love and affection always kept me going.

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TABLE OF CONTENTS

ABSTRACT……………………………………………………………………………..iii

ACKNOWLEDGEMENTS ...... vii

TABLE OF CONTENTS ...... viii

LIST OF FIGURES ...... 1

LIST OF ABBREVIATIONS ...... 4

Chapter 1 Introduction ...... 1

1.1 The structure of epithelial tissue and its role in cancer ...... 1 1.1.1 Tight junctions as a barrier in epithelial tissue ...... 2

1.1.2 The adherens junction “sticks” cells together ...... 3

1.1.3 The dynamic actin cytoskeleton regulates apical constriction in epithelial

tissue ...... 5

1.2 Epithelial Cell Polarity ...... 6 1.2.1 The Scribble complex regulates apicobasal cell polarity in epithelial cells ...... 8

1.3 Rho GTPases are key modulators of epithelial polarity and junctional architecture .. 13 1.3.1 RhoG and its function in actin cytoskeleton regulation ...... 14

1.3.3 Structure and function of SGEF, a RhoG exchange factor ...... 15

1.4 PDZ domains interaction with PDZ Binding Domain (PBD) ...... 16 1.5 Model systems to understand junctions in epithelial tissue ...... 17 1.5.1 Fixed MDCK cells for 2D junction analysis ...... 17

1.5.2 The MDCK 3D cysts embedded in matrigel to understand junction and polarity

...... 18

viii

1.5.3 Xenopus embryo to understand junctions and polarity in live epithelial

environment ...... 19

Chapter 2 Material and Methods...... 20

Chapter 3 Results and Discussion ...... 36

3.1 RhoG knockdown and rescue with biotin ligase tagged fusion using adenoviral expression system ...... 36 3.2 Novel RhoG interacting membrane receptors and receptor binding proteins identified in Bio-ID screen ...... 38 3.2.1 Membrane receptors identified in Bio-ID screen of RhoG ...... 39

3.2.2 Effectors identified in the Bio-ID screen of RhoG ...... 40

3.3 SGEF interacts with Scribble through a novel PDZ binding motif ...... 41 3.4 The structure of Scribble PDZ1/SGEF-PDZpeptide complex...... 43 3.5 SGEF N-terminal region interacts with the GUK domain of Dlg1 ...... 47 3.6 SGEF forms a ternary complex with Scribble and Dlg1 ...... 51 3.7 SGEF localizes at junctions in epithelial cells ...... 52 3.8 SGEF KD downregulates E-cadherin expression and affects adherens junction architecture ...... 58 3.9 SGEF KD changes tight junction morphology and alters barrier function ...... 63 3.10 SGEF KD stimulates actomyosin contractility...... 65 3.11 SGEF overexpression promotes apical constriction in Xenopus embryos ...... 69 3.12 The nucleotide exchange activity of SGEF is required for junctional maintenance whereas its scaffolding activity is required for apical contractility ...... 73 3.13 SGEF KD does not affect polarity but impairs lumen formation in 3D MDCK cysts ... 77 Chapter 4 Discussion ...... 83

Chapter 5 Conclusions and Future Perspective ...... 93

5.1 Conclusions ...... 93 5.2 Future directions ...... 95 Author Contribution ...... 98

References ...... 100

LIST OF TABLES

2-1 Constructs utilized in this study

2-2 Xenopus microinjection parameters

2-3 Crystallographic Data and Refinement Statistics

3-1 Membrane receptors identified in Bio-ID screen and filtered based on criteria

selected. SC- average Spectral Count of each protein in Mass spectrometry

3-2 RhoG effectors/adaptors identified in Bio-ID screen and filtered based on criteria selected.

SC- average Spectral Count of each protein in Mass spectrometry

LIST OF FIGURES

1-1 The structure of Epithelial Tissue

1-2 The Epithelial cell-cell junctions

1-3 The Epithelial cell polarity complexes

1-4 A Schematic representation of the members of the Scribble polarity

1-5 The Rho GTPase cycle

1-6 A schematic of the structural organization of SGEF

3-1 Exogenously expressed RhoG-BirA is active and biotinylated multiple proteins

3-2 An interactome of novel RhoG interacting membrane receptors and receptor

binding proteins identified in Bio-ID screen.

3-3 SGEF interacts with Scribble PDZ domain through a novel PDZ binding motif

3-4 Mapping the binding domains of SGEF, Scribble and Dlg1 interaction

3-5 SGEF’s N-terminus interacts with Dlg1 GUK domain

3-6 SGEF forms a ternary complex with Scribble and Dlg1

3-7 SGEF localizes at junctions in epithelial cells

3-8 RhoG localization at junctions in MDCK cells

3-9 SGEF regulates adherens junction properties of epithelial cells

3-10 SGEF regulates the establishment of junction in MDCK cells

3-11 SGEF KD regulates tight junction architecture and permeability

3-12 SGEF KD stimulates actomyosin contractility

3-13 SGEF signals through ROCK/myosin pathway to induce contractility in epithelial

cells

3-14 SGEF regulates apical constriction in epithelial cells

3-15 The exchange activity of SGEF is required for junctional maintenance whereas

scaffolding activity of SGEF is required for apical contractility

3-16 SGEF does not affect polarity but regulates lumen formation in 3D MDCK cysts

3-17 Catalytic activity of SGEF is required for the lumen formation in MDCK cyst

grown in matrigel

LIST OF ABBREVIATIONS

2D………………….2 Dimensional 3D………………….3 Dimensional

AJ………………….Adherens Junctions AJC………………...Apical Junctional Complex ASA………………..Accessible Surface Area

BCJ………………...Bicellular Junctions BSA……………...... Bovine Serum Albumin

CD………………....Catalytic Dead Crc………………....Circletail CTRL……………....Control

DAPI………….…....4',6-Diamidino-2-Phenylindole, Dihydrochloride DH………………....Dbl Homology Dlg…………………Discs Large DMEM……………..Dulbecco’s Modified Eagle’s Medium

ECM……………….Extracellular matrix EMT……………….Epithelial to Mesenchymal Transition

F-actin…………...... Filamentous actin

GAP………………..GTPase Activating Proteins GEF………………..Guanine nucleotide Exchange Factor GFP……………...... Green Fluorescent Protein gp 135…………...... glycoprotein 135 GUK……………….Guanylate Kinase GUKH……………..GUK Holder

J/C………………….Junctional to Cytosolic ratio

LAPSD……………..LAP Specific Domains Lgl………………….Lethal Giant Larvae LRR………………...Leucine Rich Repeats

MAGUK…………..Membrane-Associated Guanylate Kinase

MDCK…………….Madin-Darby Canine Kidney MET……………….Mesenchymal to Epithelial Transition NHS……………….Nance-Horan syndrome

OE…………………Overexpression

PAGE…………...... Polyacrylamide Gel Electrophoresis PBD……....…...... PDZ Binding Domain PBS………..……….Phosphate Buffer Solution PDZ………………...PSD95-Disc large-ZO-1 PH………………….Pleckstrin Homology PRR………………...Proline Rich Region

Rho…………...... Ras Homology

SEM……….………Standard Error of the Mean SGEF………...... SH3 containing Guanine nucleotide Exchange Factor SH3……………...... Src Homology-3 ShRNA…………….Short hairpin RNA

TCJ…………...... Tricellular Junctions TEER……………....Transepithelial electrical resistance TJ…………………..Tight Junctions

WT…………...... Wild Type

ZO-1……………….Zonula Occludens-1 ZO-2……………….Zonula Occludens-2 ZO-3……………….Zonula Occludens-3

Chapter 1

Introduction

1.1 The structure of epithelial tissue and its role in cancer

Epithelial cells form a tightly packed sheets of uniformly polarized cells, with an apical membrane contacting the environment, lateral membranes held together by specialized cell-cell junctions, and basal membranes anchored to the extracellular matrix (Rodriguez-

Boulan and Macara, 2014)(Fig 1-1). At the cell-cell interface, junctional complexes forma selectively permeable sheet that separates extracellular and intracellular environment (Fig.

1-1) (Balda et al., 1996; Liang and Weber, 2014) where it is involved in flushing of extracellular debris/microbes through secretion and shedding away of infected cells. Other functions of epithelial tissue include absorption of nutrients and selective transport of molecules across the epithelial monolayer (Caplan et al., 2008; Li et al., 2017; McCaffrey and Macara, 2011).

During embryogenesis epithelial tissue develops into sheets and tubules covering the surface of the body facing outside and lining of the organs. The cohesive structure of the epithelium along with polarized constriction at the apical surface allows epithelial tissues to fold during embryogenesis (e.g. during gastrulation and neural tube closure) (Campanale et al., 2017).

In spite of forming a rigid structure, epithelial tissue is very dynamic undergoing constant turnover of epithelial cells by extrusion and apoptosis. Moreover, certain conditions enable an epithelium to quickly lose or acquire epithelial phenotype, referred to as Epithelial to

Mesenchymal Transition (EMT) or Mesenchymal to Epithelial Transition (MET) (Lim and

Thiery, 2012). Most of the cancers in humans arise from an epithelium through a process

1 similar to EMT (McCaffrey and Macara, 2011; Muthuswamy and Xue, 2012). Therefore, it is of fundamental importance to understand epithelial structure, polarization and its function.

Figure 1-1 The structure of Epithelial Tissue. The epithelial tissue separates internal from external environment by forming a semi-permeable sheet. TJ and AJ at apical region of these cells forms a tight seal. Epithelial cells have distinct apical and basolateral membrane domains where their respective protein components and secretory pathways are segregated. 1.1.1 Tight junctions as a barrier in epithelial tissue

Tight junctions (TJ) are specialized intercellular junctional complexes that allow adhesion between adjacent epithelial cells. TJ form a selectively permeable barrier by making tight contacts with adjacent cells. They serve as a boundary between apical and basolateral membrane domains, thus segregating protein machinery and transport to their respective membrane domains (Buckley and Turner, 2018; Fanning et al., 1999; Liang and Weber,

2014; Martin and Jiang, 2009). The TJs were thought of having just a structural role of

2 forming a barrier (Balda et al., 1996). Recently, other roles of the TJs including regulation of polarity, proliferation, transport and differentiation were explored (Aijaz et al., 2006;

Matter et al., 2005; Mitic and Anderson, 1998; Van Itallie and Anderson, 2004). The TJs, also known as kissing points are formed when transmembrane proteins of a cell make trans contact with transmembrane proteins of the adjacent cells. The occludins and claudins forms the major transmembrane proteins of TJ (Cereijido et al., 1981; Furuse et al., 1998).

At their cytoplasmic tail Occludins and Claudins bind to Zonula Occludens proteins (ZO-

1, ZO-2 and ZO-3) (Anderson et al., 1988; Mitic and Anderson, 1998; Stevenson et al.,

1986). ZO proteins amongst others serve as the adapter proteins that connect TJ to cellular actin cytoskeleton through their actin binding domains (Fig. 1-2) (Itoh et al., 1997). Many of these adapter proteins associated with TJ are PDZ domain containing scaffolds that allow other signaling proteins to be recruited to junctions by forming multimeric junctional complexes (Aijaz et al., 2006).

1.1.2 The adherens junction “sticks” cells together

The AJ forms adhesive contacts between cells that facilitates development of new tissues as well as growth and maintenance of an adult tissue (Gumbiner, 1996). The cadherins in

Epithelial tissue form the core of adherens junction along with cellular catenins (,  and p120-Catenin) (Fig. 1-2) (reviewed in (Aberle et al., 1996)). The cadherins are

Figure 1-2 The Epithelial cell-cell junctions. The apical Tight Junctions (TJ) are the ‘gate keepers’ of epithelial tissue, restricting the entry of molecules across the epithelial sheet. Claudins and occludins are the transmembrane proteins of TJs whereas ZO-1 is the cytoplasmic adapter proteins connecting TJ proteins to actin cytoskeleton of the cell. The AJ (Adherens Junctions) ‘glues’ neighboring epithelial cells together. E-cadherin are the transmembrane protein connected to cytoplasmic Catenins (,  and p120-Catenins) at their cytoplasmic tail. Catenins connect AJ to actin cytoskeleton of the cell. Together, TJ and AJ forms the Apical Junctional Complexes (AJC) of epithelial cells. Ca +2 dependent, homophilic, transmembrane proteins consisting of five cadherin repeats bound together by Ca+2 to form a rigid linear structure (Pokutta et al., 1994). The extracellular domains of cadherin establishes cis interactions with cadherins on the same cell as well as trans interaction with cadherins on adjacent cells to form a zipper like structure (Aberle et al., 1996). At the cytosolic face cadherins are connected to the actin cytoskeleton of the cells through α and β-catenins. β-catenin binds to the distal cytoplasmic domain of E-cadherin and in turn α-catenin interacts with β-catenin (Fig. 1-2) (Aberle et

4 al., 1994; Ozawa and Kemler, 1992).-catenin links AJ to the actin cytoskeleton of epithelial cells through actin binding proteins like Vinculin, -actinin and ZO-1(Itoh et al.,

1997; Knudsen et al., 1995; Watabe-Uchida et al., 1998). p120-catenin also interacts at the proximal region of the cytoplasmic domain of E-cadherin (Reynolds et al., 1994;

Shibamoto et al., 1995; Yap et al., 1998). Cadherin-catenin complexes thus allow formation of a functional multiprotein hub at the AJ that connects actin cytoskeleton of epithelial cells providing structural as well as signaling capabilities to epithelial cells.

1.1.3 The dynamic actin cytoskeleton regulates apical constriction in epithelial tissue

The establishment and maintenance of polarized epithelium requires a characteristic organization of the actin cytoskeleton and its regulators (Eriksson et al., 2009; Etienne-

Manneville, 2010; Harris and Tepass, 2010; Jaworski et al., 2008; Niessen et al., 2011).

The cellular junctional complexes of epithelial cells, including TJ and AJ are anchored to actin cytoskeleton of the cell through actin binding proteins which provides physical strength as well as mechanosensing properties to the epithelium. The actin cytoskeleton associates with a variety of other cellular structures in epithelial cells. One of the most noticeable structure of F-actin is the apical actin belt where bundled actomyosin fibers associate with apical TJ and AJ to regulate apical contractility (Davidson, 2012; Owaribe et al., 1981). The other actin structures include lateral membrane-associated actomyosin cables, (Breckler and Burnside, 1994; Hartman et al., 1989; Maples et al., 1997; Tyska and

Mooseker, 2002), stress fibers at the basolateral plane (Geiger et al., 2001), the parallel actin fibers forming the core of microvilli, short actin filaments associated with Spectrin based cytoskeleton (Bennett and Healy, 2008) and apical actin web associated with non-

5 muscle myosin regulating apical constriction (reviewed in (Coravos et al., 2017; Davidson,

2012; Martin and Goldstein, 2014; Vasquez and Martin, 2016).

The actin cytoskeleton of epithelial tissue is very dynamic, undergoing constant assembly and disassembly allowing epithelial tissue to remodel its shape during its lifetime

(reviewed in (Heer and Martin, 2017; Martin and Goldstein, 2014)). The tissue remodeling results from cell shape change of individual or a group of epithelial cells during development and adulthood (Heisenberg and Bellaiche, 2013; Quintin et al., 2008). Apical constriction of a group of epithelial cells allows bending and turning of the epithelial tissue while maintaining cell-cell junctions (Alvarez and Navascues, 1990; Hardin and Keller,

1988; Lewis, 1947; Sweeton et al., 1991; Wallingford et al., 2013). Apical cell constriction also contributes to important physiological processes including EMT (Anstrom, 1992;

Harrell and Goldstein, 2011; Nance and Priess, 2002; Williams et al., 2012), cell extrusion by apoptosis (Marinari et al., 2012; Slattum et al., 2009; Toyama et al., 2008), and wound healing (Antunes et al., 2013; Davidson et al., 2002). The force required to perform apical constriction is mediated by contraction of apical F-actin bundles by associated non-muscle myosin II motors proteins. Actomyosin contraction generates force on apical junctional complexes that maintains their structure while allowing apical constriction (Kinoshita et al., 2008; Lee and Harland, 2007; Zimmerman et al., 2010). Even though the process of apical constriction is mediated by similar machinery involving actin and myosin in different cell types, the organization and the dynamics of this machinery varies dramatically (Blanchard et al., 2010; Martin and Jiang, 2009; Solon et al., 2009)

1.2 Epithelial Cell Polarity

Epithelial cell polarization is a process in which cells produces distinct apical, lateral and basolateral membrane domains. The establishment of polarity involves asymmetric distribution of protein components to membrane domains within the cell and is critical for the structure and function of epithelial tissue (Campanale et al., 2017; Macara, 2004b;

Rodriguez-Boulan and Macara, 2014). The establishment of cell polarity is orchestrated by the concerted actions of three highly conserved protein complexes; PAR, Crumbs and

Scribble (Fig. 1-3) (Bilder et al., 2003). Together, these polarity complexes control the response of cells to both intracellular and extracellular cues in order to establish cellular asymmetry signals, which ultimately result in the reorganization of the cytoskeleton, and membrane trafficking (Rodriguez-Boulan and Macara, 2014). Both PAR and Crumbs are localized at the apical surface, whereas Scribble is basolateral (Bulgakova and Knust, 2009;

Humbert et al., 2006). The localization of the polarity complexes is mutually exclusive, with PAR and Crumbs restricting the Scribble complex from targeting to the apical membrane, and Scribble excluding PAR and Crumbs from the basolateral membrane

(Bilder et al., 2003; Tanentzapf and Tepass, 2003). The epithelial cell shape and directional cellular transport has been attributed to the establishment of apico-basal cell polarity. Loss of apico-basal polarity is an early hallmark of carcinomas, the cancers that arise from epithelial tissues, which comprise 85% of all human cancers (Schock and Perrimon, 2002).

The polarity complexes contain proteins which act as scaffolds to recruit other proteins,

7 including Rho GTPases, to build spatially distinct signaling complexes (reviewed in (Mack and Georgiou, 2014)).

1.2.1

The

Scribble

Figure 1-3 The epithelial cell polarity complexes. Epithelial cell polarity is initiated and maintained by three cell polarity complexes, namely the Crumbs, Par, and Scribble complexes. The Crumbs complex consists of Crumbs-3, PALS1, and PATJ; the Par complex consists of PAR3, PAR6, and aPKC; and the Scribble complex is composed of Scribble, Dlg, and Lgl. The Crumbs and PAR complexes localize to the apical region of the cell and work together to antagonize the function of the Scribble complex at the basolateral region of the cell

8 complex regulates apicobasal cell polarity in epithelial cells

The Scribble complex is one of the three polarity complexes required to maintain cell polarity and junctional architecture. The Scribble complex is highly conserved from

Drosophila to mammals, and has been primarily associated with the regulation of apicobasal polarity, but also plays a role in cell proliferation, cell migration, planar-cell polarity and as a tumor suppressor (Elsum et al., 2012). Originally identified in Drosophila, the Scribble complex comprises three proteins: Scribble, Dlg (Discs Large) and Lgl (Lethal

Giant Larvae) (Bilder and Perrimon, 2000; Gateff and Schneiderman, 1974; Mechler et al.,

1985; Woods and Bryant, 1991). Mutations in each of these proteins result in loss of apicobasal polarity and uncontrolled proliferation, suggesting that Scribble, Dlg and Lgl function in a common pathway (Bilder and Perrimon, 2000). Surprisingly, little evidence is available regarding the molecular mechanisms that control the function of the Scribble complex. Most information to date originates from genetic studies in flies, or loss of function experiments in mammals (Elsum et al., 2012). In Drosophila, mutation in either of Scribble, Dlg or Lgl leads to imaginal disc overgrowth, disruption of epithelial structure and ability to differentiate, formation of solid tumors until the death of larvae (Bilder and

Perrimon, 2000; Gateff, 1978; Murphy, 1974; Stewart et al., 1972). We know that the members of the Scribble complex work as a functional module, where the function of each protein in the complex depends on the function of the others. However, we know very little about how the proteins in the Scribble complex - Scribble, Dlg and Lgl - interact with each other, either physically or functionally, or which downstream signaling pathways are regulated by the Scribble complex.

1.2.1.1 Scribble

Scribble was identified as a critical regulator of development and morphogenesis regulating epithelial cell structure and proliferation in Drosophila (Bilder et al., 2000). In Drosophila,

Scribble localizes at the boundary of apical and basolateral septate junction whereas in mammalian epithelial cells its localization is throughout the basal and lateral membrane domains (Ivanov et al., 2010; Mathew et al., 2002). During development, Scribble localizes to membrane before septate junction formation and thus might predetermine the site of SJ formation (Zeitler et al., 2004). The structure of Scribble consists of 16 LRR (Leucine Rich

Repeats) at its N-terminus followed by LAPSD domain (LAP Specific Domains) (Fig.1-

4). Both LRR and LAPSD domains allow Scribble targeting to cell membrane in epithelial cells (Legouis et al., 2003). Scribble also contains 4 PDZ repeats at its C-terminus which contribute to its scaffolding activity. Immunofluorescence analysis of Scribble mutant flies demonstrated mislocalization of apical proteins and AJ components to basolateral domain whereas localization of basolateral proteins was unchanged (Bilder and Perrimon, 2000)

1.2.1.2 Dlg

Dlg was identified first in Drosophila where mutation in the dlg gene showed large imaginal discs fused with each other (Gateff, 1978; Murphy, 1974; Stewart et al., 1972).

Dlg was identified as a member of the membrane-associated guanylate kinase (MAGUK) family, that localizes to cell junctions in epithelial cells. Dlg function in maintaining architecture of junctions as well as apicobasal cell polarity (Woods and Bryant, 1989;

Woods and Bryant, 1991; Woods et al., 1996). In mammals, five orthologues of Drosophila

Dlg have been identified(Dlg1-Dlg5) and Dlg1 is the most ubiquitous of all its orthologues

(Lue et al., 1994). The structure of Dlg consist of a L27 motif and 3 PDZ domains at its N-

Figure 1-4 A Schematic representation of the members of the Scribble polarity complex. Scribble is a member of LAP family consisting of 16 LRR repeats and four PDZ domains. Dlg belongs to the MAGUK family and contains three PDZ domains, an SH3 domain and a GUK domain. Lgl contains multiple WD-40 repeats. Members of Scribble polarity complex are highly conserved from fly to worms to mammals. terminus followed by a HOOK, a SH3 and a GUK domain at its C-terminus (Fig. 1-4). Dlg utilizes PDZ-2 and HOOK domain for its targeting to junctions (Hough et al., 1997;

Perrimon, 1988; Woods and Bryant, 1991). In mammalian cells Dlg1 has been shown to co-localize with E-cadherin in intestinal and renal epithelial cells. Interestingly, a knockdown of Dlg1 alters E-cadherin based AJ integrity (Laprise et al., 2004). Overall,

Dlg1 is an important regulator of AJ and TJ integrity and polarity in mammalian epithelial cells, although exact mechanism of action for Dlg1 is not known completely.

1.2.1.3 Lgl

The Drosophila lgl (lethal giant larvae) tumor suppressor gene was the first member of the

Scribble polarity complex to be identified (Hadorn, 1937; Scharrer and Hadorn, 1938). In

Drosophila, the mutation in the lgl gene caused enlarged imaginal discs and uncontrolled

proliferation, loss in epithelial structure and ability to differentiate (Hadorn, 1937; Mechler

et al., 1985; Scharrer and Hadorn, 1938; Stewart et al., 1972). The structure of Lgl consists

of multiple WD-40 repeats that are important for protein-protein interaction (Fig. 1-4). Lgl

utilizes its WD-40 repeats for its interaction with Scribble(Kallay et al., 2006). Lgl has also

been shown to interact with non-muscle myosin IIB at cell-cell junctions where it is

regulated by phosphorylation through polarity protein Par3 (Kalmes et al., 1996; Strand et

al., 1994a). Lgl has also been shown to associate with cytoskeleton and thus plays a role in

modulating cellular architecture in epithelial cells (Strand et al., 1994b). Overall, Lgl is an

Figure 1-5 The Rho GTPase cycle. Rho GTPases cycle between an inactive GDP- bound form and an active GTP-bound form. The Rho GTPases are activated by a large family of 85 GEFs, and inactivated by an equally large family of 80 GAPs. Active GTPases interact with effector proteins to mediate a response.

12 important member of Scribble complex regulating polarity and junctions in epithelial cells.

1.3 Rho GTPases are key modulators of epithelial polarity and junctional architecture

The Rho family GTPases comprises 20 members (van Helden et al., 2012). which function as a molecular switches that cycle between an active GTP-bound form and an inactive

GDP-bound form. Rho GEFs (Guanine nucleotide Exchange Factors) are a family of proteins that catalyze activation of Rho GTPases by converting them from GDP bound form to GTP bound form (Schmidt and Hall, 2002). Once active, Rho GTPases can bind to downstream effectors and activate specific pathways depending on the effectors bound to GTPase (Fig. 1-5). Conversely, Rho GAPs (GTPase Activating Proteins) are a family of proteins that inactivate Rho GTPases by activating their intrinsic hydrolytic activity, thus converting them to GDP bound form which render them unable to bind to effectors (Moon and Zheng, 2003). Rho GTPases have been implicated in essential cellular pathways that involves actin cytoskeleton regulation including but not limited to cell migration, invasion, polarity, membrane trafficking, contractility, cell adhesion, junctional architecture, etc.

Since Rho GTPases are important for regulation and remodeling of actin cytoskeleton, the interplay between Rho GTPases and organization of epithelial cell-cell junctions is of fundamental importance.

RhoA, Rac1 and Cdc42 are extensively studied Rho GTPases (Ehrlich et al., 2002; Kovacs et al., 2002; Vasioukhin and Fuchs, 2001). Rac1 and Cdc42 are important for establishment of epithelial junctions by polymerizing actin through activation of Arp2/3 by WAVE2

(Ratheesh et al., 2012). Cdc42 allows formation of Par6-aPKC-Par3 polarity complex which regulates apicobasal cell polarity in epithelial cells. RhoA plays a fundamental role

13 in establishment of junctions through two key modulators of actin cytoskeleton: ROCK

(Rho Associated protein Kinase) and Dia1 (Diaphenous-related protein-1). ROCK and

Dia1 maintain tension at junctions, inhibit cadherin endocytosis and establish/maintain TJ barrier function (Levayer et al., 2011; Terry et al., 2011; Warner and Longmore, 2009).

The upstream and downstream regulators of Rho GTPases including GEFs, GAPs and effectors are also shown to be important for junctional architecture and polarity in epithelial cells (Levayer et al., 2011; Mack and Georgiou, 2014; Moon and Zheng, 2003; Ngok and

Anastasiadis, 2013; Ngok et al., 2014; Ratheesh et al., 2012; Terry et al., 2011; Warner and

Longmore, 2009).

1.3.1 RhoG and its function in actin cytoskeleton regulation

RhoG is a member of Rho family of GTPases that was first identified in fibroblasts cells as a growth factor stimulated gene ((Vincent et al., 1992). Evan though RhoG belongs to

Rac subfamily based on sequence and function, many GEFs, GAPS and Effectors of RhoG are different than Rac1. RhoG has been shown to be involved in multiple cellular pathways that include actin cytoskeleton modifications including cell migration, neurite outgrowth, macropinocytosis, phagocytosis, bacterial intake, transmigration of leukocyte, microtubule dynamics and invadopodia regulation (deBakker et al., 2004; Ellerbroek et al., 2004;

Goicoechea et al., 2017; Jackson et al., 2015; Katoh et al., 2006; Katoh et al., 2000; van

Buul et al., 2007). Interestingly, RhoG has never been shown to be involved in junctional architecture or polarity in epithelial cells.

1.3.2 Proximity dependent biotinylation of RhoG

The proximity dependent biotinylation (Bio-ID) is a technique used to identify interacting partners of a protein in a relatively natural cellular environment (Roux et al., 2012). Bio-

ID is based on the expression of a “bait” protein fused to a prokaryotic biotin ligase (BirA*) that when expressed in its cellular context will generate multiple biotinylated “prey” proteins based on the proximal association of the bait protein. Biotinylated proteins can then be identified using mass spectrometry approach. This technique was first employed to identify proximal proteins of nuclear human lamin A (LaA), a component of nuclear envelope (Roux et al., 2012) with a very high statistical confidence. This technique has been successfully adapted by others to identify interacting partners of various proteins

(Kim et al., 2014;Firat-Karalar et al., 2014;Elzi et al., 2014;Morriswood et al., 2013). The

Bio-ID system is extremely sensitive to proximity. It has been shown that the practical labeling radius of Bio-ID is only 10nm (Kim et al., 2014). Basically, the BioID is a screening technique to identify potential interacting partners of a protein within a small interacting radius. The interacting proteins identified through Bio-ID has to be verified by techniques like affinity pulldowns (Boukhelifa et al., 2004). We employed BioID to identify interacting partners of RhoG GTPase.

1.3.3 Structure and function of SGEF, a RhoG exchange factor

SGEF (SH3 containing Guanine nucleotide Exchange Factor) is a RhoG specific GEF

(Ellerbroek et al., 2004). It was first identified as an androgen responsive gene in prostate cancer cells (Qi et al., 2003). It is expressed differentially in multiple tissue types including Figure 1-6 A schematic of the structural organization of SGEF. SGEF (SH3 containing Guanine nucleotide Exchange Factor exchanges GTP for a GDP for Rho family GTPase RhoG. It has two nuclear localization sequences, a proline rich region, catalytic domains DH and PH, a SH3 domain and a C-terminal PDZ-Binding tail.

15 brain, lung, liver, kidney, etc (Ellerbroek et al., 2004; Qi et al., 2003). SGEF structure consists of a proline rich region (PRR) at its N-terminus, a Dbl Homology (DH) domain and a Pleckstrin Homology (PH) domain, as well as a Src Homology 3 domain near its C- terminus (Fig. 1-6). DH-PH domain of SGEF is thought to have protein-protein interaction and exchange activity (Ellerbroek et al., 2004). There are two nuclear localization sequences near N-terminus of SGEF (Qi et al., 2003). The precise role of these nuclear localization sequences is not yet completely understood. The C-terminus of SGEF also encodes a type I PDZ Binding Motif (PBM) with the sequence -ETNV which can interact with type I PDZ domains. SGEF has been implicated to function in various cellular pathways including macropinocytosis, leukocyte transmigration, dorsal ruffle formation, bacterial invasion and invadopodia dynamics (Fortin Ensign et al., 2013; Goicoechea et al.,

2017; Patel and Galan, 2006; Samson et al., 2010; van Buul et al., 2007; van Rijssel et al.,

2012; Wang et al., 2013; Wang et al., 2014). Similar to RhoG, SGEF has also never been associated with junctional architecture or polarity in epithelial cells.

1.4 PDZ domains interaction with PDZ Binding Domain (PBD)

PDZ domains were identified as modular protein-protein interaction domains in a diverse set of proteins, typically associated with cell junctions and neuronal synapses (Cho et al.,

1992; Lue et al., 1994; Willott et al., 1993; Woods and Bryant, 1991). The structure of PDZ domain consist of 80-90 amino acids (Woods and Bryant, 1991) packed with 6 β-strands and 2 α-helices (Fanning and Anderson, 1996; Harris and Lim, 2001; Lee et al., 2009). A specialized pocket at its carboxylate arm allows these PDZ domains to bind 5-7 residues at the C-terminus of target proteins which are generally referred to PBM (Cowburn, 1997;

Harris and Lim, 2001; Songyang et al., 1997). Generally, a PBM is located at the C-

16 terminus of protein, but a small number of internal PBMs (located anywhere within a protein) have also been identified to interact with PDZ domains. These internal PBMs utilizes peptide turns and folds to mimic the structure of C-terminus PBM. This allows for these internal PBM binding to PDZ domains (Brenman and Bredt, 1997; Hillier et al., 1999;

Penkert et al., 2004; Wong et al., 2003). PBMs are classified into three distinct classes based on consensus sequences of their peptides. Class I PBMs have consensus sequence

S/T – X - V/I/L (where X-any amino acid), Class II PBMs have a consensus sequence ɸ -

X - ɸ (where ɸ - a hydrophobic amino acid). All other sequences are classified into class

III PBM (Kim et al., 1995; Schneider et al., 1999; Songyang et al., 1997; Stricker et al.,

1997). Many of the PDZ domains can form homodimers and heterodimers thus increasing the local concentration of other proteins as well (Fanning et al., 2007; Im et al., 2003a; Im et al., 2003b; Utepbergenov et al., 2006; Wu et al., 2007). The scaffolding proteins of epithelial junctions and polarity are enriched in PDZ domains which in turn enable them to form multi-protein complexes required for architecture and signaling in epithelial cells.

1.5 Model systems to understand junctions in epithelial tissue

A single system is insufficient to understand the complex regulation of epithelial tissue.

For that we used three complementary and well-established model systems to understand the role of SGEF in junctions and polarity in epithelial cells.

1.5.1 Fixed MDCK cells for 2D junction analysis

MDCK epithelial cell is extensively used as a model cell line as they have clear apicobasal cell polarity, high growth rate and well characterized cell-cell junctions (Dukes et al., 2011;

Simmons, 1982). MDCK cells can polarize both in two dimensions (2D) as well as in three dimensions (3D) depending on the conditions. We are using MDCK II to study the role of

SGEF in junctions. MDCK II cells is a high passage clonal cell line derived from the parental MDCK cells (NBL-2) (Barker and Simmons, 1981; Madin and Darby, 1958).

MDCK II cells have uniform growth rate, cell shape and characteristic junctional proteins expression that is well understood and are recommended cell line of all eleven strains of

MDCK available commercially to study junctional properties of epithelial cells (Dukes et al., 2011).

1.5.2 The MDCK 3D cysts embedded in matrigel to understand junction and polarity

The structure and function of an epithelial tissue is well defined in 3D environment forming mature organs (Yamada and Cukierman, 2007). The routine lab techniques utilize much simpler 2D cell culture monolayer for its simplicity and ease of handling. In order to mimic natural growth of epithelial tissue, a 3D system was developed (McAteer et al., 1988). The

3D cultures of epithelial cells utilize an artificial or naturally derived ECM (Extracellular

Matrix) as a support rather than growing cells directly onto plastic or glass in 2D. Matrigel, a reconstituted gel of an extract containing basement membrane components, protein mixture and growth factors secreted by Engelbreth-Holm-Swarm (EHS) mouse sarcoma cells is preferred ECM support material in most of the 3D cultures (Kleinman and Martin,

2005). The components of matrigel mimics that of in vivo environment allowing cells to form 3D spheres (Pampaloni et al., 2007). Individual MDCK cells embedded in matrigel forms highly polarized 3D cysts with apical membrane facing a single fluid filled lumen, whereas its basolateral membrane faces the environment or surrounding. 3D MDCK cysts share many characteristics with the physiological development of epithelial tissue in vivo

(Debnath and Brugge, 2005; Hagios et al., 1998; O'Brien et al., 2002). This makes MDCK

18 cysts an excellent model to understand epithelial junctional properties and establishment of polarity.

1.5.3 Xenopus embryo to understand junctions and polarity in live epithelial

environment

Live Xenopus embryos are an excellent model system to understand development of junctions and polarity. Live Xenopus laevis embryos have many advantages over its other model system. First and most important, Xenopus belong to vertebrate family and thus are closer to humans than its non-vertebrate counterparts Drosophila and C. elegans. Xenopus embryos form large, highly polarized cells within 24-48 hr. after fertilization which makes it very easy for microinjection and manipulations. They grow at room temperature and thus does not require special temperature control during live imaging (Blum et al., 2015;

Stephenson and Miller, 2017; Woolner et al., 2009). The Xenopus genome is completely sequenced and functions of most of the genes are well characterized. This makes Xenopus system an amenable system to perform live imaging of epithelial junctions.

Chapter 2

Material and Methods

Cell lines

HEK 293FT cells (Thermo Scientific) were used for all the overexpression and pulldown experiments unless indicated otherwise. MDCK II cells were a gift from Dr. Ian G. Macara

(Vanderbilt University). Caco-2 cells were a gift from Dr. Scott Crawley (The University of Toledo). HEK 293FT and MDCK cells were grown in Dulbecco’s modified Eagle’s medium (DMEM) (GIBCO), containing 10% fetal bovine serum (FBS) and antibiotics

(penicillin-streptomycin). Caco-2 cells were grown in DMEM supplemented with 10 mM

L-Glutamine, containing 20% FBS and antibiotics (penicillin-streptomycin). SUM159 cells were grown in F-12 Ham’s medium containing 10% fetal bovine serum (FBS) and antibiotics (penicillin-streptomycin). All cell lines were grown at 37°C and 5% CO2. All experiments were conducted with early passage cells that were passaged no more than 15 times. Mycoplasma was tested regularly by staining with Hoechst 33342 (AnaSpec Inc).

Antibodies

SGEF polyclonal antibodies were generated in rabbits using the following antigen located at SGEF’s N-terminus (DGESEVDFSSNSITPLWRRR)(Pacific Immunologicals). The following antibodies were used in this study: RhoG (SC-26484) 1:1000 WB, Myc (9E10,

SC-131) 1:1000 WB; Afadin (SC-74433) 1:500 WB, 1:50 IF; Dlg1 (SC-9961) 1:500 WB,

1:100 IF; Scribble (SC-11049) 1:500 WB, 1:50 IF; β-catenin (SC-7963) 1:1000 WB, 1:100

IF; p120-catenin (SC-13957) 1:500 WB, 1:50 IF and pan-cadherin (SC-515872) 1:500

WB, 1:50 IF were from Santa Cruz Biotechnology; E-cadherin rr1 1:500 WB, 1:100 IF and gp135 (3F2/D8) 1:300 IF were from the Developmental Studies Hybridoma Bank at The

University of Iowa; tubulin (T9028) 1:10,000 WB was from Sigma-Aldrich; Myosin IIB

(D8H8) 1:500 WB, 1:50 IF was from Cell Signaling Technology; ZO-1 (339100) 1:1000

WB, 1:150 IF; GFP (MA5-15256) 1:10,000 WB; 6x-His Tag Monoclonal Antibody

(HIS.H8) 1:3000 WB and Cadherin-6 (2B6) 1:250 WB, 1:25 IF were from Thermo

Scientific; Myosin IIB (909901) 1:1000 WB, 1:250 IF was from Biolegend; Alexa Fluor

488 and Alexa Fluor 594-conjugated anti-mouse-IgG and anti-rabbit-IgG secondary antibodies (A11008, A11001, A11005, A32733 and R37117) and Alexa Fluor-594 (A-

11032) and Alexa Fluor-647 (A22287) conjugated to phalloidin were from Thermo

Scientific; (HRP)-conjugated anti-mouse-IgG, anti-rabbit-IgG and anti-goat-

IgG secondary antibodies were from Jackson Immunoresearch: horseradish peroxidase secondary antibodies (715-035-151, 711-035-152 and 705-035-147).

Plasmids and constructs

A list of plasmids used in the study is attached below in Table 1.

Two-hybrid screening

A Matchmaker Gold Yeast Two-Hybrid System yeast two-hybrid system (Clontech) was used to screen for SGEF-interacting proteins. FL human SGEF was cloned into the ‘bait’ vector pGBK7 and used to screen a mouse kidney pGADT7 cDNA library (>2.5 × 107 independent clones). Individual colonies containing potentially positive clones (126) were isolated, re-tested for interaction with SGEF, and false positives were discarded. The inserts from the positive clones were amplified by PCR and grouped according to their restriction pattern. Representative clones from each group were sequenced.

Lentivirus constructs and transduction pLKO.1 Blast, Addgene #26655 was used to knockdown SGEF in MDCK cells. pLKO.1

Blast lentiviral non-targeting shRNA control was from Addgene #26701. Targeting

s Table 1: Constructs 2-1. Constructs utilized in this utilized study in this study

No Construct name Tag Gene Region Expression Resistance Ref/Made by 1 pCMV_myc_SGEF_WT_FL Myc SGEF 1-871 Human Ampicillin This study 2 pCMV_myc_SGEF_DETNV Myc SGEF 1-867 Human Ampicillin This study 3 pCMV_SGEF_1-227 Myc SGEF 1-227 Human Ampicillin This study 4 pCMV_SGEF_8-250 Myc SGEF 8-250 Human Ampicillin This study 5 pCMV_SGEF_25-250 Myc SGEF 25-250 Human Ampicillin This study 6 pCMV_SGEF_50-250 Myc SGEF 50-250 Human Ampicillin This study 7 pCMV_SGEF_228-871 Myc SGEF 228-871 Human Ampicillin This study 8 pEGFP_Scribble_WT_FL GFP Scribble 1-1630 Human Kanamycin 9 pEGFP_Scribble_L-->R GFP Scribble 1-1630 Human Kanamycin Navarro et. al., 2005 10 pEGFP_Scribble_DPDZ GFP Scribble 1-724 Human Kanamycin 11 pCDNA3.1_HisA_Scribble_4PDZ His Scribble 616-1490 Human Ampicillin Petit et. al., 2005 12 pCDNA3.1_HisA_Scribble_4PDZ_M1 His Scribble 616-1490 Human Ampicillin Petit et. al., 2005 13 pCDNA3.1_HisA_Scribble_4PDZ_M2 His Scribble 616-1490 Human Ampicillin Petit et. al., 2005 14 pCDNA3.1_HisA_Scribble_4PDZ_M3 His Scribble 616-1490 Human Ampicillin Petit et. al., 2005 15 pCDNA3.1_HisA_Scribble_4PDZ_M4 His Scribble 616-1490 Human Ampicillin Petit et. al., 2005 16 pCMV_myc_SGEF_1-300 Myc SGEF 1-300 Human Ampicillin This study 17 pCMV_myc_SGEF_1-350 Myc SGEF 1-350 Human Ampicillin This study 18 pCMV_myc_SGEF_1-400 Myc SGEF 1-400 Human Ampicillin This study 19 pCMV_myc_SGEF_1-414 Myc SGEF 1-414 Human Ampicillin This study 20 pCMV_myc_SGEF_414-871 Myc SGEF 414-871 Human Ampicillin This study 21 pCMV_myc_SGEF_1-787 Myc SGEF 1-787 Human Ampicillin This study 22 pEGFP_DLG1_WT GFP DLG1 1-904 Mouse Kanamycin This study 23 pEGFP_DLG1_N-ter GFP DLG1 1-535 Mouse Kanamycin This study 24 pEGFP_DLG1_C-ter GFP DLG1 536-892 Mouse Kanamycin This study 25 pEGFP_DLG1_DGUK GFP DLG1 1-636 Mouse Kanamycin This study 26 pEGFP_DLG1_SH3 GFP DLG1 536-636 Mouse Kanamycin This study 27 pEGFP_DLG1_GUK GFP DLG1 699-893 Mouse Kanamycin This study 28 pCDNA_HA_Scribble_WT_FL HA Scribble 1-1630 Human Ampicillin Navarro et. al., 2005 29 pCMV_myc_SGEF_431-792(DH-PH) myc SGEF 431-792 Human Ampicillin This study 30 pCMV_myc_SGEF_208-425 myc SGEF 208-425 Human Ampicillin This study 31 pCMVJ3_mycSGEF_RSKP-->AAAA myc SGEF 1-871 Human Ampicillin This study 32 pCMVJ3_mycSGEF_SYQS-->AAAA myc SGEF 1-871 Human Ampicillin This study 33 pCMVJ3_mycSGEF_LITD-->AAAA myc SGEF 1-871 Human Ampicillin This study 34 pCMVJ3_mycSGEF_RSKP-->AAAA myc SGEF 1-871 Human Ampicillin This study 35 pCMVJ3_mycSGEF_FPVE-->AAAA myc SGEF 1-871 Human Ampicillin This study 36 pCMVJ3_mycSGEF_VED-->AAA myc SGEF 1-871 Human Ampicillin This study 37 pCMVJ3_mycSGEF_T-->A myc SGEF 1-871 Human Ampicillin This study 38 pCMVJ3_mycSGEF_G-->P myc SGEF 1-871 Human Ampicillin This study 39 pCS2+_mNeon_RhoG_WT mNeon RhoG 1-191 Xenopus Ampicillin This study 40 pCS2+_mNeon_SGEF_WT mNeon SGEF 1-871 Xenopus Ampicillin This study 41 pCS2+_mNeon_SGEF_1-227 mNeon SGEF 1-227 Xenopus Ampicillin This study 42 pCS2+_mNeon_SGEF_228-871 mNeon SGEF 228-871 Xenopus Ampicillin This study 43 pCS2+_mNeon_RhoG_Q61L mNeon RhoG 1-191 Xenopus Ampicillin This study 44 pCS2+_mNeon_SGEF_446/621(CD) mNeon SGEF 1-871 Xenopus Ampicillin This study 45 PLKO.1_Blast_SGEF-1122 None SGEF ShRNA -- Human Ampicillin This study 46 PLKO.1_Blast_SGEF-39.4 None SGEF ShRNA -- Human Ampicillin This study 49 pLenti_mNeon_SGEF_WT_FL mNeon SGEF 1-871 Human Ampicillin This study 50 pLenti_mNeon_SGEF_1-227 mNeon SGEF 1-227 Human Ampicillin This study 51 pLenti_mNeon_SGEF_1-400 mNeon SGEF 1-400 Human Ampicillin This study 52 pLenti_mNeon_SGEF_CD mNeon SGEF 1-871 Human Ampicillin This study 53 pLenti_mNeon_RhoG_WT mNeon RhoG 1-191 Human Ampicillin This study Arnold et. al., 54 pCS2_mcherry_a-catenin mCherry a-catenin 1-903 Xenopus Ampicillin (Unpublished data) 55 pCS2_mRFP_ZO-1 mRFP ZO-1 1-1747 Human Ampicillin Higashi et al., 2018 56 pCS2_mcherry_PLEKHA7 mCherry PLEKHA7 1-1287 Xenopus Ampicillin Higashi et al., 2018 57 pCS2+/membrane-TagBFP BFP Membrane-Tag 1-820 Xenopus Ampicillin Higashi et al., 2016 58 pCS2+/Lifeact-mRFP mRFP Lifeact 1-780 Xenopus Ampicillin Bement et al., 2015 Stephenson et. al., 59 pCS2+/SF9-mNeon mNeon SF9 1-730 Xenopus Ampicillin (Unpublished data) 60 pCS2+/mCherry-H2B mCherry H2B 1-1090 Xenopus Ampicillin Reyes et al., 2014 Arnold et. al., 61 pCS2+/vinculin-mNeon mNeon Vinculin 1-3300 Xenopus Ampicillin (Unpublished data) 23 sequences of shRNAs for canine SGEF are: SGEF SEQ#1 –

GGTGAAAAGAGGTGAGTTA (Qin et al., 2010), SGEF SEQ#2 -

CCGAAGTATGAGGTCTGCAAG. Lentiviruses were prepared by transfecting

HEK293FT cells with pLKO.1, pMD2.G and pSPAX2 constructs (pMD2.G and pSPAX2 were a gift from Didier Trono, Addgene plasmids #12259 and #12260). Cell culture medium was changed 24 h post transfection and Lentivirus were harvested 48 h post transfection. MDCK cells were infected with lentivirus particles overnight. The following day, the infection medium was removed and replaced with complete medium containing

10 μg/ml blasticidin to select for shRNA-expressing cells. Total cell lysates were subjected to Western blot analysis for protein expression as described above. For some shRNAs, single cell colonies were isolated by serial dilution. pLenti-CMV-Hygro-Dest vector from

Addgene, #17454 was used to prepare stable MDCK cells expressing mNeon-SGEF constructs as described above. For overexpression and rescue experiments MDCK cells were infected with lentivirus particles overnight and then selected with 600 ng/ml hygromycin.

Transfections and Immunoprecipitation

HEK293FT cells were transfected using standard Calcium phosphate transfection method.

Media was changed 24 hrs after transfection and cells harvested 48 hrs post transfection.

The cells cultured on 100 mm tissue culture dishes were rinsed with PBS and then scraped into a lysis buffer containing 50 mM Tris-HCl pH 7.4, 10 mM MgCl2, 150 mM NaCl, 1%

Triton X-100 and EZBlock protease inhibitor cocktail (BioVision). The supernatant was collected after centrifugation at 16,800 g for 10 min. For immunoblotting, lysates were boiled in 2X Laemmli buffer, and 20 μg of protein were resolved by SDS-PAGE. The

24 proteins were transferred onto PVDF and immunoblotted with the indicated antibodies.

Immuno complexes were visualized using the SuperSignal West Pico PLUS

Chemiluminescent HRP substrate (Thermo Scientific). For immunoprecipitation, protein concentration was measured using DC protein assay reagent (BioRad) and equal amount of protein added to respective beads (ChromoTek GFP-Trap, myc-Trap or mNeon-Trap beads). Beads were incubated with lysates for 1 h at 4ᐤC on a rotator, washed 4 times with lysis buffer, resuspended in 2X Laemmli buffer and loaded on SDS polyacrylamide gels for Western blot analysis.

3D cyst culture

The day before cyst inoculation, MDCK cells were split into a 10 cm dish at a confluency of 1:10. On the day of plating, plates were washed 2 times followed by trypsinization using

0.25% trypsin, centrifuged at 1,200 rpm followed by 2 washes and resuspended in calcium and magnesium free PBS. 2x103 cells were then plated on 100% matrigel on an 8-well glass bottom μ-slide (Ibidi) (6 μl matrigel per well). Cells were then overlaid with 300 μl of 2% matrigel in DMEM. The cysts were grown for 4 days with a change of media on day

2 followed. On Day 4 cysts were fixed with 3.7% formaldehyde and processed for immunofluorescence.

Immunofluorescence

The immunofluorescence assays were performed as described earlier (Garcia-Mata et al.,

2003). Briefly, MDCK cells grown on coverslips or transwell filters were fixed for 10 min with 3.7% paraformaldehyde, and quenched with 10 mM ammonium chloride. Cells were then permeabilized with 0.1% Triton X-100 in PBS for 10 min. The coverslips were then washed with PBS and blocked in PBS, 2.5% goat serum (Sigma), 0.2% Tween 20 for 5

25 min followed by 5 min blocking in PBS, 0.4% fish skin gelatin (Sigma), and 0.2% Tween

20. Cells were incubated with primary antibodies for 1 hr at room temperature. The coverslips were then washed with PBS, 0.2% Tween 20 and incubated with Alexa

Fluor 594 or 647 secondary antibodies for 45 min, washed as described above and mounted on glass slides in ProLong Diamond Antifade Mountant (Thermo Fisher

Scientific-P36965).

For 3D cysts, immunofluorescence was performed as previously described (O'Brien et al.,

2001). Briefly, samples were washed with PBS and fixed in 3.7% paraformaldehyde, washed, and then permeabilized with 0.2% Triton X-100 for 10 min. After blocking with

0.4% fish skin gelatin and 2.5% goat serum (30 min. each), the cysts were incubated overnight at 4°C with primary antibodies in 2.5% goat serum. Primary antibodies were used at a concentration 2X higher than that used for IF on coverslips. Cysts were then washed 3 times quickly, followed by 3 X 10 min washes with 0.2% Tween 20 in PBS.

Secondary antibodies coupled with Alexa Fluor 488, 594, or 647 were incubated with the fixed specimens for 2–3 h at room temperature. Cysts were washed 5 times with 0.2%

Tween in PBS and mounted in ProLong Diamond antifade reagent.

Transepithelial electrical resistance (TEER) measurements.

The cells were plated onto 0.4-μm polyester membrane, 12-mm Transwell filters, at 5 X

105 cells per filter (Corning). Resistance measurements were performed 3 days later in triplicate using a EVOM Epithelial Voltohmmeter (World Precision Instruments). The values represent the average of three trials minus the background resistance. Values were normalized to those of CTRL cells (100%).

Image processing and Quantifications in MDCK cells

Deconvolution microscopy: To examine the localization of actin and myosin in MDCK cells, Z-section images were captured using the HyD detectors in the Leica SP8 confocal with the PlApo CS2 N 63x/1.4 objective, and the raw data were deconvolved using

Huygens software (Scientific Volume Imaging) that accompanies the HyVolution package using Standard settings.

Zig-Zag index measurements: The zig-zag index was calculated as previously described

(Tokuda et al., 2014). Briefly, BCJ were manually traced both with freehand following exactly the lines of the junction, and with a straight line between the two TCJ encompassing the junction. The ratio of the of freehand lines (LTJ) to that of straight lines (LSt) was calculated as LTJ/LSt, and defined as the zigzag index. Around 100 cells were analyzed in total of 3 independent experiments. Two-tailed Welch’s t-tests were performed to compare control vs SGEF KD and Rescue WT cells.

Quantification of junctional E-cadherin, β-catenin, p120-catenin, Scribble and Dlg1: Line scans (6 μm line drawn perpendicular to the center of junctions) were performed using

ImageJ as follows. Brightest point projections of z-stacked images were created using

ImageJ. At least 2 fields from 2 independent experiments were used for quantification (≥

200 junctions). The intensity profiles were manually centered around the highest peak for each condition.

Quantification of Height of MDCK cells: Confocal images were captured in XZ mode using a Leica SP8 confocal microscope. Leica LAS X software was used for measurement of height at the tallest point of a cell. At least 50 cells from 10 individual images in at least

3 different experiments were used to calculate height. Two-tailed Welch’s t-tests were performed to compare control vs SGEF KD and Rescue WT cells.

Quantification ZO-1 intensities at junctions of MDCK cells: Brightest point projections of z-stacked images were created using ImageJ. A rectangle of size 4 x 1.5 μm length was aligned with its longer side parallel to the length of a junction. Average intensities were plotted using Graphpad Prism 7. Two-tailed Welch’s t-tests were performed to compare control vs SGEF KD and Rescue WT cells.

Quantification of afadin TCJ/BCJ intensity ratio : Brightest point projections of z-stacked images were created using ImageJ. A circle of 2.5 μm diameter was placed at the center of a TCJ and its corresponding BCJ. Average Afadin intensities (TCJ/BCJ) were plotted using

Graphpad Prism 7. Two-tailed Welch’s t-tests were performed to compare control vs SGEF

KD and Rescue WT cells.

Xenopus embryos and microinjections

All studies conducted using Xenopus laevis embryos strictly adhered to the compliance standards of the US Department of Health and Human Services Guide for the Care and Use of Laboratory Animals and were approved by the University of Michigan’s Institutional

Animal Care and Use Committee. Xenopus embryos were collected, in vitro fertilized, de- jellied, and microinjected with mRNAs for fluorescent probes using methods described previously (Reyes et al., 2014) Embryos were injected in the animal hemisphere at the two- cell stage (each cell injected twice) for fixed phalloidin staining experiments, and either at the two-cell stage (each cell injected twice) or at the four-cell stage (each cell injected once) for live imaging experiments and allowed to develop to gastrula stage (Nieuwkoop and

Faber stages 10 to 11). Concentrations of probes per single injection are as follows: pCS2+/Human SGEF, 87 pg; pCS2+/Lifeact-mRFP, 17 pg; pCS2+/SF9-mNeon, 74 pg; pCS2+/membrane-TagBFP, 15 pg)

The constructs and their sources are summarized in supplementary Table 2

TableTable 22:-2 Xenopus Xenopus microinjectionmicroinjection p arametersparameters

Amount mRNA or morpholino Stage injected injected pCS2+/Human SGEF 1 87 pg 4-cell stage, once per cell (Overexpression) 2 pCS2+/mNeon-SGEF 25.6 pg 4-cell stage, once per cell

3 pCS2+/mNeon-SGEF 1-227 10.8 pg 4-cell stage, once per cell pCS2+/mNeon-SGEF 228- 4 25 – 75 pg 4-cell stage, once per cell 871 5 pCS2+/mNeon-RhoG 2-cell stage, twice per cell 37 pg 6 pCS2+/Lifeact-mRFP 17 pg 2-cell stage, twice per cell

7 pCS2+/SF9-mNeon 74 pg 2-cell stage, twice per cell

4-cell stage, twice per cell (when coinjected with pCS2+/membrane-TagBFP SGEF in actin 8 (2x membrane localization 15 pg experiments) 2-cell stage, sequence of Lyn kinase) twice per cell (when coinjected with α-catenin and vinculin)

9 pCS2+/TagBFP-ZO-1 62.5 pg 4-cell stage, once per cell

10 pCS2+/PLEKHA7-mCherry 27.8 pg 4-cell stage, once per cell

11 pCS2+/vinculin-mNeon 39.2 pg 2-cell stage, twice per cell

12 pCS2+/mCherry-α-catenin 28.6 pg 2-cell stage, twice per cell

13 pCS2+/mCherry-H2B 25 pg 4-cell stage, once per cell

Xenopus F-actin staining

Gastrula-stage embryos were fixed and stained by methods described previously (Reyes et al., 2014) with the following changes: embryos were fixed with 1.5% formaldehyde, 0.25%

29 glutaraldehyde, 0.2% Triton-X100, and 0.88X MT fix buffer (1X MT buffer: 80 mM K-

PIPES, 5 mM EGTA, 1 mM MgCl2 [pH 6.8]) and blocked in Tris-buffered saline (50 mM

Tris and 150 mM NaCl [pH 7.4]) containing 10% fetal bovine serum, 5% DMSO, and 0.1%

NP40 overnight at room temperature.

Confocal Microscopy

Fluorescent confocal images were collected on either an inverted Olympus Fluoview 1000 microscope equipped with a 60X supercorrected PLAPON 60XOSC objective (NA = 1.4; working distance = 0.12 mm) and FV10-ASW software (Xenopus embryo studies), or using a Leica SP8 confocal microscope equipped with HyD detectors, a PlApo CS2 N

63x/1.4 objective, Leica LAS X software (Leica) and a Hybrid superresolution package

(HyVolution).

Image Processing and Quantification in Xenopus

All images were processed and analyzed with ImageJ. En-face views shown in figures are brightest point z-projections. Side views shown are averaged projections. Videos were converted to avi files using ImageJ (JPEG compressed, 15 frames/second). Graphs and statistical analysis were done using GraphPad Prism 5.

Quantification of SGEF and RhoG localization relative to Adherens and Tight Junctions in live embryos: The apico-basal intensity profiles of mNeon-tagged SGEF and RhoG were quantified as described in (Higashi et al., 2018). Briefly, Z-Stack images of 30-40 optical slices with 0.5 µm thickness were taken from multiple embryos (see numbers in Figure legend). Each junction was quantified as follows: a 50 px X 50 px square region of interest of a straight section of the junction was cropped from the original image and reoriented, yielding a stack of 50 X-Z side views of the junction. This side view stack was flattened

30 using an average intensity projection. Because the junction is often not straight from apical to basal, the intensities at each z position were flattened into a single px using a brightest point projection. Fluorescence intensity profiles were determined for TagBFP-ZO-1 (TJ marker), PLEKHA7-mCherry (AJ marker), and either mNeon-SGEF or mNeon-RhoG for each junction. The intensity profiles from multiple junctions were then normalized and averaged to produce the graphs shown in the Figure.

Quantification of junctional FL SGEF, SGEF 1-227, and SGEF 228-871 in live embryos:

Brightest point projections of z-stack images were generated using ImageJ. From each image 14 to 20 BCJ junctions were traced with a 1 pixel wide segmented line, and mean intensities were measured. Cytosolic intensities were measured by shifting each segmented line into the cytosolic region 5-10 pixels to the side of the junction. Ratios of junctional to cytosolic SGEF were calculated. Two-tailed Welch’s t-tests were performed to compare junctional to cytosolic ratios for FL SGEF, SGEF 1-227, and SGEF 228-871.

Quantification of junctional F-actin in fixed phalloidin-stained embryos: Line scans were performed using ImageJ as follows. Brightest point projections of z-stacked images were created using ImageJ. Junctions were traced with a 5 pixel thick segmented line. Average intensities and junction lengths were measured. All junctions fully visible in each image were included. The data was graphed using Prism. Two-tailed Welch’s t-tests (single- tailed, unequal variances) were performed to compare control embryos to SGEF OE embryos using Prism.

Quantification of medial-apical F-actin in fixed phalloidin-stained embryos: Brightest point projections of z-stacked images were created using ImageJ. The entire medial-apical surface of each cell was outlined, and average intensities were measured. All cells fully

31 visible in each image were measured. The intensity data was graphed using Prism. Two- tailed Welch’s t-tests were performed to compare control embryos to SGEF OE embryos.

Quantification of junctional vinculin, α-catenin in live embryos: Line scans were performed using ImageJ as follows. Brightest point projections of z-stacked images were created using ImageJ. Junctions were traced with a 1 pixel thick segmented line, and average intensities were measured. Five to seven junctions were measured from each image. Since mRNA expression levels vary from embryo to embryo and even within each embryo, membrane probe intensities were used to normalize vinculin and α-catenin intensity data. Ratios of junctional vinculin to membrane. This data was graphed using

Prism. Two-tailed Welch’s t-tests were performed to compare normalized vinculin intensities.

Protein Expression and Purification

Polymerase chain reaction was used to amplify the human Scribble PDZ1 domain (residues

725–815) from the full-length DNA sequence. The amplified DNA was ligated into a modified pET21 vector (Novagen) that contained an N-terminal 6XHis affinity tag and a tobacco etch virus (rTEV) protease cleavage site. The nucleotide coding sequence of the pET21 -PDZ1 vector was verified by automated DNA sequencing (University of Iowa,

DNA Facility). Protein expression was conducted in BL21 (DE3) (Invitrogen) Escherichia coli cells. Typically, E. coli cells were grown at 37 °C in Luria-Bertani (LB) medium supplemented with ampicillin (100 μg/mL) under vigorous agitation until an A600 of 0.6 -

0.8 was reached. Cultures were subsequently cooled to 18°C and protein expression was induced by the addition of isopropyl 1-thio-β-d-galactopyranoside to 1 mM final

32 concentration. Induced cells were incubated for an additional 16–18 hrs at 18 °C and harvested by centrifugation.

The Scribble PDZ1 domain was purified by nickel-chelate (GE-Healthcare) and size- exclusion chromatography (GE-Healthcare). The N-terminal 6XHis affinity tag was removed by proteolysis with recombinant rTEV protease for 36 hrs at 4 °C. Undigested protein, cleaved 6XHis tag and His-tagged rTEV were separated from Scribble PDZ domains by nickel-chelate chromatography. The digested proteins were further purified with S75 size-exclusion chromatography with desired final buffer. The final yield was about 50 mg of PDZ protein (> 98% pure as judged by SDS-PAGE) from 1 L of culture.

Samples were used immediately or lyophilized and stored at –80 °C.

Synthetic SGEF peptide

The SGEF iPBM (residues 42-55: ac-KPNGLLITDFPVEDCONH2) peptide was chemically synthesized by GenScript Inc. (Piscataway, NJ) and judged to be >95% pure by analytical

HPLC and mass spectrometry. The SGEF iPBM peptide was acetylated (ac) at the N- terminus and amidated at the C-terminus. The peptide concentration was determined by absorbance measurements (A280) using the predicted extinction coefficient from the amino acid sequence.

Crystallization and Data Collection

Crystallization conditions for the free and peptide-bound forms of the Scribble PDZ1 domain were determined by the hanging-drop, vapor-diffusion method using high- throughput screens automated by a Mosquito drop setter (TTP LabTech). Equal volumes

(200 nL) of precipitant and protein (10-23 mg/mL; 20 mM Tris base, 50 mM NaCl, pH

7.5) alone or with 5-10 molar equivalents of peptide were used for crystal screens. Crystals

Table 2-3 Crystallographic Data and Refinement Statistics

Scribble Scribble PDZ1 PDZ1/SGEF-PDZ PDB ID: 6MYF peptide PDB ID: 6MYE Data Collection Statistics Temperature (K) 100 100 Wavelength (Å) 0.976 1.000 Space group C 2 2 21 P 43 21 2 Unit Cell Parameters a, b, c (Å) 39.108, 42.824, 91.234 67.89, 67.89, 44.15 , ,  (°) 90, 90, 90 90, 90, 90 Molecules per asymmetric unit 1 1 Resolution range (Å) 28.88 - 1.6 (1.657 - 1.6)a 48.01 - 1.1 (1.139 - 1.1)a I/σ(I) 18.51 (1.66)a 26.72 (1.64)a Completeness (%) 97.66 (86.00)a 99.92 (100.00)a R-merge (%) 0.07094 (0.6269)a 0.05379 (1.526)a Redundancy 6.2 (3.5)a 15.6 (12.8)a Refinement Details Resolution (Å) 1.6 1.1 Rwork/Rfree (%) 17.10/21.03 15.04/16.70 Number of atoms Protein / Peptide 697/N.A. 998/103 Solvent 99 130 B-factor average (Å2) Protein / Peptide 19.80 / N.A. 14.78 / 19.72 Solvent 30.23 27.53 Rmsd from Ideal Geometry Bond lengths (Å) 0.008 0.019 Bond angles (°) 0.99 1.85 Ramachandran Plot (% Residues) Most favored 100.00 99.01 Additionally allowed 0.00 0.99 Disallowed 0.00 0.00 Rotamer outliers (% Residues) 0.00 0.00 Clashscore 0.00 5.39 a Values in parentheses are for the highest resolution shell. of the free Scribble PDZ1 domain were obtained in condition 40 of the Index screen (0.1M

Citric acid pH3.5, 25% (w/v) PEG 3350; Hampton Research), while the Scribble PDZ1 /

SGEF-PDZpeptide complex formed crystals in condition 29 of the Index screen (60% (v/v)

Tacsimate™ pH 7.0; Hampton Research).

Initial crystal diffraction screening was achieved with a CuK rotating anode beam; full X- ray diffraction datasets for structure determination were collected using a RDI CMOS_8M

34 detector at 0.5 steps over 180° for the free crystal and over 120° for the peptide-bound form at beamline 4.2.2 at the Advanced Light Source (Berkeley, CA). The free Scribble PDZ1 domain crystallized in space group C2221 with one molecule per asymmetric unit. The

SGEF-PDZpeptide-bound complex crystallized in space group P43212 with one molecule in the asymmetric unit. Proper space group handedness was verified by analysis of the electron density.

Structure Determination and Structure Refinement

The XDS program (Kabsch, 2010a; Kabsch, 2010b) was used for indexing, integration, and scaling of the diffraction data. The program PHASER was used for the initial phasing of both the free Scribble PDZ1 structure and the Scribble PDZ1/SGEF-PDZpeptide complex structure using the previously determined Scribble PDZ1 structure (PDB ID: 2W4F) as template. Automated model building was performed using ARP/wARP. The early stages of refinement used Refmac while the later stages used the PHENIX software suite (Adams et al., 2010). Final polishing and refinement were carried in PDB_REDO (Joosten et al.,

2014) Electron density visualization and manual model building were done in Coot. Rfree values were calculated using 10% of the reflections selected randomly and not used in the refinement. The structures were refined to 1.6 Å in the free form and to 1.1 Å in the complex form. The refinement statistics are shown in Table 3. Structural alignment and figures were prepared with PyMOL (Schrodinger, LLC; Version 1.8.)

Chapter 3

Results and Discussion

3.1 RhoG knockdown and rescue with biotin ligase tagged fusion using adenoviral

expression system

To identify interacting proteins of RhoG by proximity dependent biotinylation we created a fusion protein of RhoG and BirA*. We performed a PCR of RhoG along with BirA* and cloned it into pENTR entry vector. pENTR-myc-RhoG-BirA* was then recombined with pAdeno destination vector. pAdeno-myc-RhoG-BirA* was sequenced and transfected into

HEK-A cells to obtain adenovirus expressing myc-RhoG-BirA*. High titer adenovirus

A B C

Figure 3-1. Exogenously expressed RhoG-BirA is active and biotinylated multiple proteins. A) Endogenous RhoG expressed in WT cells is shown in lane 1 and rescue with biotin ligase tagged RhoG is shown in lane 2. Anti-myc antibody detects biotin ligase alone in control cells whereas RhoG-BirA* in RhoG KD cells. (B) RhoG activity assay shows the activity of endogenous WT RhoG in control cells and exogenously expressed RhoG- BirA in cells knocked down for RhoG expressing RhoG-BirA construct. Total levels of RhoG are shown in lane 1 and 2 respectively for both cell types. (C) Lane 1 and 3 shows biotinylated proteins by control in absence and presence of biotin whereas lane 2 and 4 shows biotinylation of proteins by RhoG-BirA* in absence and in presence of biotin 24 hrs. post infection. Streptavidin conjugated HRP used for western blot

36 cloned for RhoG-BirA was used in all the experiments to express RhoG-BirA* in SUM159 cell lines. We also established BirA* adenovirus as a control.

Signaling through RhoG in cells should go through exogenously expressed recombinant fusion protein RhoG-BirA* instead of endogenous RhoG. For that we performed a RhoG knockdown in SUM159 cell lines using lentivirus expressing RhoG shRNA. As a control, we created SUM159 cell lines expressing scramble shRNA. RhoG-BirA* was expressed in SUM159 cell lines knocked down for RhoG. Fig. 3-1 A shows the knockdown efficiency by shRNA in SUM159 cells and rescue with RhoG-BirA. It can be seen from the blot that control cells expresses endogenous RhoG when probed with RhoG specific antibody whereas RhoG KD cells only expresses RhoG-Biotin ligase fusion protein.

For Rho GTPases it is important that the recombinant protein expressed exogenously is functional. Functionality of a GTPase can be shown by its binding to its downstream effectors. We used a GTPase pulldown assay to show that RhoG-BirA* is functional. This assay is based on binding to the RhoG effector ELMO, which only binds to active RhoG.

Thus only active RhoG (RhoG-GTP) will be pulled down in a GST-ELMO pulldown and not RhoG-GDP which is inactive. Fig. 3-1 B shows the activity of endogenous RhoG in

SUM159 cells expressing scramble shRNA compared to RhoG KD cell lines exogenously expressing recombinant RhoG-BirA* fusion protein. It can be seen from the blot that both endogenous and fusion protein are active to a similar extent. Thus the system is feasible and can be used for further experiment.

Once the system expressing RhoG-BirA* was ready, the next step was to check biotinylation of proteins by RhoG. Control SUM159 cells were infected with adenovirus expressing biotin ligase alone and RhoG KD cell lines were infected with RhoG-BirA*

37 adenovirus. 24hrs post infection, viruses were removed and 50M biotin was added externally to the plates. Plates were incubated with Biotin media (F12 Hams media +

Biotin) at 370C for another 24 hours Fig. 3-1 C shows that in absence of biotin very few proteins are biotinylated whereas when biotin is added externally in the media, biotinylation of protein is dramatically enhanced with several proteins of different sizes biotinylated by RhoG-BirA* and not BirA* alone.

3.2 Novel RhoG interacting membrane receptors and receptor binding proteins

identified in Bio-ID screen

Biotinylated proteins were identified using a mass spectrometry approach. Beads bound to biotinylated proteins from RhoG-BirA* expressing cells and BirA* expressing cells

(control) were sent to Dr. Ben Major’s Lab at the University of North Carolina, Chapel Hill for mass spectrometric analysis by LC/MS-MS. Biotinylation assay was repeated four times for statistical significance. The results obtained identified 347 RhoG specific potential interacting partners that were identified in RhoG-BirA* sample and not in BirA* alone. SAINT probability was used to distinguish significant hits compared to non- significant hits. SAINT (Significance Analysis of Interactome) is an algorithm that gives high probability for significant proteins. It takes into account control assay, as well as total number of hits obtained in RhoG BioID assay (Choi et al., 2011). Top hits obtained in

BioID were filtered based on its biological function. RhoG interactome was created using

Cytoscape Version 3.1.1 (Fig. 3-2). Proteins were arranged in space based on their role as membrane receptors (green) or receptor binding proteins/adaptor proteins (blue). Uniprot and Gene Ontology databases were used to create the interaction map.

Figure 3-2 An interactome of novel RhoG interacting membrane receptors and receptor binding proteins identified in Bio-ID screen. RhoG interacting proteins obtained by Bio-ID are arranged in a network. Node size corresponds to SAINT probability. Edges show interaction of these proteins amongst themselves and with RhoG based on mass spectrometry and databases Gene ontology and Uniprot. Membrane receptors are shown in green and receptor binding proteins are blue. Dark green nodes with thick margin are membrane proteins. Dark blue nodes are effectors/adaptor proteins. RhoG is shown in center as a red square.

3.2.1 Membrane receptors identified in Bio-ID screen of RhoG

RhoG is known to be activated by membrane receptors EGFR (Samson et al., 2010), G- protein coupled receptors (Condliffe et al., 2006), ICAM1 receptor in leukocyte (van Buul et al., 2007), FGF2 co-receptor syndecan-4 (Elfenbein et al., 2009) and EphA2 receptor

(Hiramoto-Yamaki et al., 2010). In our Bio-ID screen, we identified multiple membrane

39 receptors as potential RhoG interacting partners. We identified a total 40 hits for membrane receptors in the Bio-ID screen that can be potential RhoG interacting partners. RhoG is known to be activated at plasma membrane by other membrane receptors. We hypothesize that RhoG interacts and is activated by these membrane receptors. We filtered most relevant membrane receptors based on proteins identified in all 4 repeats, having average

RhoG spectral count more than ten and average control spectral count less than three.

EGFR and CD44 are the membrane receptors shown to fulfill all these criteria. Table 1 shows EGFR and CD44 with their spectral count and SAINT probabilities.

Table 3-1 Membrane receptors identified in Bio-ID screen and filtered based on criteria selected. SC- average Spectral Count of each protein in Mass spectrometry.

3.2.2 Effectors identified in the Bio-ID screen of RhoG

Few effectors have been known for RhoG including ELMO1/2, Kinectin, MLK3, PLD1

(phospholipase D1) and IQGAP2 (Katoh and Negishi, 2003; Vignal et al., 2001;

Wennerberg et al., 2002). The human genome is known to code for more than 100 effectors for other GTPases. In our Bio-ID screen we identified many potential RhoG effector hits.

We identified 112 proteins that are potential RhoG effectors. We filtered these proteins based on proteins identified in all 4 repeats, with average RhoG spectral count more than

10 and control spectral count less than 3, involved in actin cytoskeleton remodeling and protein known to bind to a receptor or a GTPase. Four proteins seem to fulfill all these

40 criteria. These includes CTNND1 (p120 catenin), FERMT2 (Kindlin-2), FMNL1 (Formin

Like protein-1) and DIAPH3 (mDia2). Table 2 shows these proteins with average spectral count in RhoG as well as control samples along with their SAINT probabilities. All these proteins are actin binding proteins or scaffolding proteins involved in actin cytoskeleton modification downstream of membrane receptors.

Table 3-2 RhoG effectors/adaptors identified in Bio-ID screen and filtered based on criteria selected. SC- average Spectral Count of each protein in Mass spectrometry.

Actin SAINT cytoskeleton Receptor OR No Gene ID Protein ID Probability Ctrl SC RhoG SC binding GTPase binding 1 FERMT2 Kindlin-2 1.00 2.67 43.33 Yes Receptor binding 2 CTNND1 p120 Catenin 1.00 1.00 35.33 Yes Receptor Binding 3 FMNL1 FMNL1 1.00 1.00 10.33 Yes GTPase binding 4 DIAPH3 mDia2 0.94 1.33 13.67 Yes GTPase binding

3.3 SGEF interacts with Scribble through a novel PDZ binding motif

We performed a yeast two-hybrid screen to identify proteins that interact with SGEF, and identified Scribble as a potential binding partner for SGEF (Fig. 3-4 A). We then confirmed the interaction by co-immunoprecipitation and western blot analysis in HEK293 cells expressing myc-SGEF WT and GFP-Scribble WT (Fig. 3-3 A-B). Since SGEF encodes a

C-terminal PDZ-binding motif (PBM) (van Buul et al., 2007), we hypothesized that the

PBM in SGEF was interacting with one of the 4 PDZ domains encoded in Scribble. Our results confirmed that the interaction was mediated by the PDZ domains in Scribble, as deletion of the 4 PDZ domains (ΔPDZ) abolished the interaction (Fig. 3-3 C). In contrast, a Scribble mutant in which the N-terminal leucine repeats (LR) region is not functional

(P305L) (Legouis et al., 2003) interacted efficiently with SGEF (Fig. 3-3 C). To map which of the PDZ domains of Scribble mediated the interaction with SGEF, we tested the interaction between myc-SGEF and a series of Scribble constructs comprising either the 41 four WT PDZ domains (4PDZ), or mutants in which each of the individual PDZ domain was inactivated by a mutations in its carboxylate binding loop (M1-M4) (Petit et al., 2005).

Our results showed that, inactivation of PDZ1 in Scribble, but not the other PDZ domains, completely abolished the interaction with SGEF, suggesting the interaction is mainly mediated by PDZ1 (Fig. 3-3 D).

Surprisingly, the deletion of the PBM in SGEF (∆ETNV) had no effect on the ability of

SGEF to bind to Scribble (Fig. 3-3 E). These results showed that SGEF’s PBM was dispensable for its interaction with Scribble’s PDZ domains, and suggested that the interaction was mediated by an internal PDZ-binding motif in SGEF. To map the Scribble binding site in SGEF we generated a series of deletion mutants in SGEF and analyzed their ability to interact with Scribble using co-immunoprecipitation. Our results confirmed our previous observations and showed that a construct comprising amino acids 228-871 of

SGEF, which includes the PBM, was unable to bind, whereas a construct comprising the first 227 amino acids (SGEF 1-227) was sufficient to interact with Scribble (Fig. 3-3 F).

Further deletion analysis narrowed down the Scribble-binding domain in SGEF to a region comprising amino acids 25-50 (Fig. 3-1 G). Alanine scanning mutagenesis of selected amino acids within this region led to defining the minimal region required for binding to residues S39-E54, a region that is highly conserved in vertebrates (Fig. 3-1 H and I). As shown in Fig 3-1 I, when amino acids LITD or FPVE were substituted by alanines, the interaction between SGEF and Scribble was completely abolished, suggesting that this region is part of the binding motif. Mutation of SYQS, located immediately upstream of

LITD, had minimal effect whereas mutating RSKP residues further upstream had no effect on binding. Interestingly, substitution of VED, which overlaps partially with FPVE

42 restored binding, although not completely (Fig. 3-1 I). In addition, a single amino acid substitution in Thr 49 (T49A) within the LITD motif, was sufficient to completely abolish the interaction (Fig. 3-3 B). Taken together, our results show that the sequence comprising residues LITDFP within the N-terminus of SGEF is essential for binding Scribble, whereas some of the residues located immediately upstream and downstream may also contribute to the interaction.

3.4 The structure of Scribble PDZ1/SGEF-PDZpeptide complex.

To obtain insight into the atomic details of the Scribble PDZ1/SGEF interaction, we sought to determine the X-ray crystal structure of the complex between Scribble PDZ1 and a peptide encoding the following SGEF amino acids: PNGLLITDFP. We obtained high- quality protein samples that yielded crystals. Crystals of the apo PDZ domain diffracted to a resolution of 1.6 Å and we determined the structure using routine methods. The Scribble

PDZ1 in complex with SGEF-PDZpeptide and ARHGEF7/β-PIX peptides also yielded crystals. Crystals of the Scribble PDZ1 domain in complex with the SGEF-PDZpeptide or

ARHGEF7/b-PIX peptide diffracted to a resolution of ~1.59 Å and 1.44 Å, respectively.

Using molecular replacement and the apo PDZ1 structure as search model we solved the structure of both complexes. A comparison of the structures for the apo Scribble PDZ1 domain and the PDZ1/ SGEF-PDZpeptide complex is shown in Fig. 3-1 J. The electron density of the SGEF peptide shows the high-quality of the structure and the ability to unambiguously trace the conformation of the peptide. This structure revealed several unique features of the Scribble PDZ1/SGEF interaction compared to typical PDZ/C- terminal ligand structures. First, 11 residues of SGEF-PDZpeptide make interactions with

43 the PDZ domain covering ~500 Å2 of accessible surface area (ASA). For context, most

PDZ/C-terminal ligand interactions are limited to 5-6 residues encompassing ~350 Å2 of

ASA. Second, the structure revealed the register of the peptide with respect to the PDZ domain, such that the P0 - Phe and P-2 - Thr in SGEF occupy the canonical S0 and S-1 pockets in Scribble PDZ1. Importantly, the structure revealed several novel interactions through pockets near the α1 helix in Scribble PDZ1 (S+1 pocket), which accommodate the

P+1 proline amino acid (Fig. 3-1 J). These features are distinct from the two published

PDZ/internal ligand structures (Hillier et al., 1999; Penkert et al., 2004)

Figure 3-3

Figure 3-3 SGEF interacts with Scribble PDZ domain through a novel PDZ binding motif. (A) Schematic representation of SGEF and Scribble constructs utilized in this figure. SH3: Src Homology 3 Domain; PBM: PDZ binding motif, LR: Leucine Rich repeats; PDZ: PSD95, Dlg1, and ZO-1 family domain. (B-G) Lysates from HEK293FT cells expressing the indicated constructs were immunoprecipitated using GFP antibodies (GFP-trap nanobodies). In all experiments, the precipitates were immunoblotted with anti-GFP antibodies to detect the immunoprecipitated protein and with anti-myc or anti-His to detect the potential interacting partner. (H) Sequence alignment comprising the Scribble binding domain of SGEF in vertebrates (aa 32 to 55 in human). (I) The indicated boxed regions withein the Scribble -binding domain in SGEF were mutagenized to Ala in FL SGEF. Lysates from HEK293FT cells expressing the myc-tagged WT-SGEF or the indicated Ala mutants were and GFP-Scribble were immunoprecipitated using GFP antibodies (GFP-trap nanobodies). The precipitates were immunoblotted with anti-GFP antibodies to detect the immunoprecipitated protein and with anti-myc to detect my-tagged SGEF mutants. Black boxes shows sequence mutated but still maintaining interaction with Scribble whereas red boxes denotes sequences mutated leading to loss of interaction with Scribble. (J) Crystal structure of the Scribble PDZ1 domain in complex with an SGEF internal PDZ binding motif peptide. The crystal structure of the apo Scribble PDZ1 domain is shown in grey, while the complex with the SGEFpeptide (SPNGLLITDFP) is shown in red (left panel). The right panel is a surface representation of the PDZ1/SGEF-PDZpeptide complex.

Figure 3-4

Figure 3-4. Mapping the binding domains of SGEF, Scribble and Dlg1 interaction.(A) Full-length SGEF was used to screen a mouse kidney cDNA library in a yeast two-hybrid system, yielding 126 positive clones. After retesting, 23 false positives were discarded. PCR and restriction digest analysis was performed on the 103 remaining clones in 19 groups. Three groups were validated as positives and were identified as Scribble, ARIP2 and LIM-domain containing protein. A representative plate showing growth and blue colored colonies, as well as a negative prey control (Lamin A) or empty vector (pGBK7) is shown in the bottom panel. (B-C) Lysates from HEK293FT cells expressing the indicated constructs were immunoprecipitated using GFP antibodies (GFP-trap nanobodies). In all experiments, the precipitates were immunoblotted with anti-GFP antibodies to detect the immunoprecipitated protein and with anti-myc to detect the interacting partner.

3.5 SGEF N-terminal region interacts with the GUK domain of Dlg1

Our previous work has shown that SGEF also interacts with Dlg1, which is a member of the Scribble polarity complex (Krishna Subbaiah et al., 2012). The interaction between

SGEF and Dlg1 was reported to involve SGEF’s PBM binding to PDZ domains 1 and 2 of

Dlg1, and an additional interaction between the SH3 domains of both SGEF and Dlg1

(Krishna Subbaiah et al., 2012). The fact that SGEF’s binding site for Scribble did not overlap with the binding site for Dlg1 raised the possibility of SGEF interacting simultaneously with both Scribble and Dlg1. This was interesting because even though

Scribble and Dlg1 are functionally associated to the Scribble complex, they do not interact directly (Ivanov et al., 2010). To confirm the interaction between SGEF and Dlg1, we took advantage of the tools and approach used in Figure 3-1 to map the Dlg1 binding site in

SGEF. As expected, both SGEF WT and SGEF 228-871, which contains the DH-PH domains, the SH3 domain and the PBM, were able to bind efficiently to Dlg1 (Fig. 3-5 A).

We also found that SGEF 1-227, which binds to Scribble, did not interact with Dlg1 (Fig.

2 A). To explore this interaction in more detail, we deleted either the PBM (SGEF

ΔETNV), or both the PBM and the SH3 domain (SGEF ΔSH3) in SGEF and tested their ability to bind Dlg1. We expected that upon deletion of these two domains the interaction between SGEF and Dlg1 would be abolished. Surprisingly, both mutants interacted efficiently with Dlg1 (Fig. 3-5 C). Since this was contrary to what had been previously published (Krishna Subbaiah et al., 2012), we conducted additional experiments to confirm or rule-out these findings. We first designed two truncation mutants of SGEF and tested them for Dlg1 binding, one encoding the N-terminal half that ends just before the start of the DH-PH domain (1-414), and the other one the C-terminal half, which includes

Figure 3-5

Figure 3-5. SGEF’s N-terminus interacts with Dlg1 GUK domain. Schematic representation of SGEF and Dlg1 constructs utilized in this study. GUK: Guanylate Kinase domain. (B-F) Lysates from HEK293FT cells expressing the indicated constructs were immunoprecipitated using GFP antibodies (GFP-trap nanobodies). In all experiments, the precipitates were immunoblotted with anti-GFP antibodies to detect the immunoprecipitated protein and with anti-myc to detect the interacting partner. (G) Sequence alignment comprising the Dlg1 binding domain of SGEF in vertebrates (aa 301-350 for human)

49 the DH-PH domain, the SH3 domain and the PBM (414-871). Our results showed that the

N-terminal half of SGEF (1-414) interacted efficiently with Dlg1, whereas the C-terminal half (414-871) showed no binding (Fig. 3-5 D). These results suggested that the binding site for Dlg1 lied between aa 227 and 414 in SGEF (Fig. 3-5 B and D). To further narrow down the binding site, we tested a series of deletion mutants covering the region between amino acids 300-400. Our results showed that SGEF 1-350 and 1-400 were able to bind to

Dlg1, whereas SGEF 1-300 was not, suggesting that the region between aa 300-350 of

SGEF was required for binding Dlg1 (Fig. 3-5 D Right panel). This region has no conserved domains or structural information, but is highly conserved in vertebrates (Fig.

3-5 G). In addition, it does not encode any obvious PBM, suggesting that the SGEF-binding site in Dlg1 is located in a region outside the PDZ domains of Dlg1.

To map the SGEF-binding domain in Dlg1, we first tested the interaction between SGEF

WT and two Dlg1 constructs, one encoding the N-terminal half including the three PDZ domains (N-term), and the other one the C-terminal half comprising the SH3 and GUK domains (C-term). Interestingly, the N-terminus showed no detectable binding, confirming that the PDZ domains of Dlg1 were not involved in the interaction (Fig. 3-5 E). In contrast, the C-terminal construct was able to bind efficiently to SGEF (Fig. 3-5 E). We confirmed these results using a Dlg1 mutant construct in which the three PDZ domains were inactivated by mutations. Our results showed that inactivating the PDZ domains abolished a previously described interaction with the RhoA GEF Net1 (Garcia-Mata et al., 2007), but had no effect in the ability of Dlg1 to bind to SGEF (Fig. 3-5 C). We then used a series of truncation mutants to further define the SGEF-binding domain in Dlg1. Our results showed that deleting the GUK domain in Dlg1 abolished the interaction with SGEF, and

50 that the GUK domain alone was able to interact with SGEF (Fig. 3-5 F). In contrast, the

SH3 by itself showed no detectable binding (Fig. 3-5 F). Overall, our results demonstrate that the PDZ and SH3 domains in Dlg1 are not required to bind to SGEF as previously reported, and define the binding domains to the GUK domain of Dlg1 interaction with a 50 amino acids region at the N-terminus of SGEF.

3.6 SGEF forms a ternary complex with Scribble and Dlg1

Our data demonstrates that Scribble and Dlg1 interact with different regions in SGEF (Fig.

1 and 2), suggesting that SGEF could bind simultaneously to Scribble and Dlg1 forming a ternary complex. To test this possibility, we co-expressed HA-Scribble and GFP-Dlg1 in

HEK293 cells in the presence or absence of myc-SGEF. We then immunoprecipitated Dlg1 using anti-GFP antibodies and immunoblotted for the three proteins. The prediction was that, if SGEF forms a ternary complex with Scribble and Dlg1, Scribble would co- precipitate with Dlg1 only when SGEF is present. As shown in Fig. 3-6 A, We did not detect any Scribble co-precipitating with Dlg1 in the absence of SGEF. However, when the three proteins were co-expressed, Scribble co-precipitated efficiently with Dlg1, demonstrating that the three proteins form a ternary complex. Using a similar approach in human epithelial cells (Caco-2) we confirmed the existence of the endogenous

Scribble/SGEF/Dlg1 ternary complex (Fig. 3 B). As further confirmation of these results, we determined that the minimum SGEF mutant that can mediate the formation of the

Scribble/SGEF/Dlg1 complex (SGEF 1-414), must contain both the Dlg1 and Scribble binding sites (Fig. 3-6 C-D). Deletion of either the Scribble or Dlg1 binding domain in

SGEF abolished the formation of the complex (Fig. 3 C-D). Overall, our results demonstrate the interaction

Figure 3-6

Figure 3-6. SGEF forms a ternary complex with Scribble and Dlg1. (A) Lysates from HEK293FT cells expressing GFP-Dlg1 and HA-Scribble in the presence or absence of myc-SGEF were immunoprecipitated using GFP antibodies (GFP-trap nanobodies). The precipitates were immunoblotted with anti-myc, anti-GFP and anti-HA antibodies as indicated. (B) Endogenous Dlg1 was immunoprecipitated from Caco2 cell lysates and immunoblotted for Dlg1, SGEF and Scribble. (C) Lysates from HEK293FT cells expressing the indicated constructs were immunoprecipitated using GFP antibodies (GFP- trap nanobodies). The precipitates were immunoblotted with anti-GFP antibodies to detect the immunoprecipitated protein and with anti-myc and anti-HA to detect the interacting partners. (D) Summary of results from panel C. (E) Cartoon representation of the ternary complex between Scribble, SGEF, and Dlg1. iPBM: internal PDZ-binding motif; DBM: Dlg Binding Motif.

between SGEF/Scribble and SGEF/Dlg1 can occur simultaneously allowing for the formation of a ternary complex between Scribble, SGEF, and Dlg1 (Fig. 3-6 E).

3.7 SGEF localizes at junctions in epithelial cells

In order to investigate the functional role of the Scribble/SGEF/Dlg1 ternary complex, we used two complementary model systems: 1) MDCK cells (MDCK II), which are the preferred mammalian model to study junction formation (Cereijido et al., 1978) and, 2)

Xenopus laevis embryos, which provide an ideal system to study cell-cell junctions and cell polarity in an intact epithelial environment during development (Blum et al., 2015;

Stephenson and Miller, 2017; Woolner et al., 2009). SGEF sequence is highly conserved in vertebrates, which allows us to use the information gained from biochemical and mammalian cell culture system to guide experiments in Xenopus embryos.

To investigate the localization of SGEF, we expressed low levels of human mNeon-SGEF

WT in MDCK cells and compared its localization to that of endogenous Scribble and Dlg1.

Both Scribble and Dlg1 are distributed throughout the lateral membrane in epithelial cells, where they co-localize with AJ and tight junctions (TJ) markers (Dow et al., 2003; Ivanov et al., 2010; Laprise et al., 2004; Navarro et al., 2005). As expected from our protein interaction studies (Fig. 3-1 to 3-5), mNeon-SGEF WT colocalized with both Scribble and

Dlg1 (Fig. 3-7 A). Our results also showed that mNeon-SGEF WT localized to the apical junctional complex (AJC)(Farquhar and Palade, 1963) and colocalizes with markers for both TJ (ZO-1) and AJ (β-catenin) (Fig. 3-7 B). In addition, mNeon-SGEF WT targeted very efficiently to the AJC in Xenopus embryos, where it colocalized with both TJ (BFP-

ZO-1) and AJ (PLEKHA7-mCherry) markers (Fig. 3-7 C). The reconstructed xz plane of these images demonstrated that SGEF signal is distributed along the lateral membrane, with the bulk of the signal concentrated at the apical region, overlapping slightly better with BFP-ZO-1 (TJ) than with mCherry-PLEKHA7 (AJ) (Fig. 3-7 C, C’). SGEF localization was better defined in Xenopus compared to MDCK cells, where mNeon-SGEF

WT signal was distributed more evenly along the lateral membrane (Fig 4 A-B).

Since SGEF is a GEF specific for the small GTPase RhoG, we expressed mNeon-RhoG to determine if it was also targeted to cell-cell junctions. We found that indeed, RhoG co-

53 localized with TJ and AJ markers in both Xenopus embryos (Fig. 3-7 D) and MDCK cells

(Fig. 3-8 A-D). However, compared to SGEF, which is concentrated at apical junctions in

Xenopus embryos (Fig 4 C’), RhoG was found to localize slightly more towards basolateral membrane (Fig. 3-7 D’).

To determine whether binding to Scribble and Dlg1 is important for targeting SGEF to

AJC, we compared the localization of mNeon-SGEF WT (SGEF WT) in Xenopus embryos and two mNeon-tagged deletion mutants of SGEF, one that binds to Scribble but not to

Dlg1 (SGEF 1-227) and another one that binds to Dlg1 but not to Scribble (SGEF 228-

871). We found that both SGEF WT and SGEF 1-227 localized efficiently to AJC where they colocalized with ZO-1 (Fig. 3-7 E-F). In contrast, SGEF 228-871 was predominantly cytosolic, with only a small fraction still targeted to junctions (Fig. 3-7 E-F). Overall, our results suggested that binding to Scribble is the main determinant for SGEF to localize at

AJC.

Figure 3-8

Figure 3-7. SGEF localizes at junctions in epithelial cells (A-B) Immunofluorescence of MDCK cells showing colocalization of mNeon-SGEF WT with Scribble and Dlg1, ZO-1 (TJ marker) and β- catenin (AJ marker). The right panels are single Z-planes along the length of the dotted yellow line. a: apical, b: basal. Scale bar: 10 μm. (C) Gastrula- stage Xenopus embryos expressing mNeon-SGEF (green), TagBFP-ZO-1 (TJ marker), and PLEKHA7-mCherry (AJ marker) were live imaged using confocal microscopy. En face views (left) are maximum intensity projections across multiple Z-planes. Side views (right) are average intensity projections along the length of the highlighted junction (see methods). a: apical, b: basal. Scale bar: XY 10 μm and XZ 1 μm. (C’) Intensity profiles of SGEF (green solid line) relative to AJs (red dotted line) and TJs (blue dotted line) along the Z-axis in Xenopus gastrula-stage epithelial cells. Note that SGEF’s peak intensity is close to ZO-1’s, but it tapers away slower basal to the apical junctions. The graph shows normalized averaged intensities fitted with a smoothed curve; error bars indicate s.d; n = 5 experiments, 18 embryos, 47 junctions. (D) Gastrula- stage Xenopus embryos expressing mNeon-RhoG (green), BFP-ZO-1 (TJ marker, blue), and PLEKHA7-mCherry (AJ marker, red) were live imaged using confocal microscopy. Maximum intensity projections of en face views and averaged side views of the highlighted junction (as in A) are shown. a: apical, b: basal. Scale bar: XY, 10 μm and XZ, 1 μm. (D’) Intensity profiles of RhoG (green solid line) relative to AJs (red dotted line) and TJs (blue dotted line). Note that RhoG expression is more basolateral compared to SGEF which is more apical. The graph shows normalized averaged intensities fitted with a smoothed curve; error bars indicate s.d. n = 3 experiments; 10 embryos; 19 junctions. (E) Gastrula-stage Xenopus embryos expressing mRFP-ZO-1 (TJ marker, magenta) and mNeon-tagged WT SGEF (top, green), SGEF 1-227 (middle, green), or SGEF 228-871 (bottom, green) were live imaged using confocal microscopy. En face views are maximum intensity projections across multiple Z-planes. Side views (right) are single Z-planes at the locations marked by yellow dotted lines. Note that WT SGEF and SGEF 1-227 appear junctional, while SGEF 228-871 appears diffusely localized. a: apical, b: basal. Scale bar: 20 μm (F) Quantification of the ratio of junctional to cytosolic (J/C) intensities of mNeon-tagged WT SGEF, SGEF 1-227, and SGEF 228-871 in Xenopus embryos. SGEF 1-227 is junctional (average J/C = 1.506) but more diffuse than mNeon-SGEF WT (average J/C = 2.133). SGEF 228-871 is not junctional (average J/C = 1.124). Error bars represent min to max with all points; n = 6 experiments, 12 embryos, 234 junctions for WT SGEF; 5, 11, 214 for SGEF 1-227; 4, 9, 180 for SGEF 228-871. Error bars represent standard error of the mean. ****p<0.00005.

Figure 3-8

Figure 3-8 RhoG localization at junctions in MDCK cells. Immunofluorescence of MDCK CTRL cells expressing WT mNeon-RhoG or GFP-RhoG stained with ZO-1(TJ marker) (A), E-cadherin and β-catenin (B-C) (AJ markers) and Scribble (D). Scale bar: 10 μm.

3.8 SGEF KD downregulates E-cadherin expression and affects adherens junction

architecture

To assess the functional role of SGEF during junction formation, barrier formation and the establishment of cell polarity, we generated stable MDCK cells expressing a previously published short hairpin RNA (shRNA) sequence targeting SGEF (Qin et al., 2010). As shown in Fig. 3-9 A, SGEF protein levels were significantly reduced in SGEF KD cells.

We first analyzed the morphology of AJ in CTRL (expressing a non-targeting shRNA) and

SGEF KD cells by immunofluorescence microscopy. Surprisingly, we found that silencing

SGEF expression led to an almost complete loss of E-cadherin from the AJ (Fig. 3-9 C).

This loss of E-cadherin at junctions was due to a decrease in E-cadherin protein levels, and not to its redistribution to different cellular locations (Fig. 3-9 B-C). Re-expression of human mNeon-SGEF WT in SGEF KD MDCK cells (Rescue WT) restored both the expression levels and proper localization of E-cadherin (Fig 5 B, C and G). Since the cytoplasmic tail of E-cadherin binds to β-catenin and p120-catenin, we also analyzed their localization and expression levels. Interestingly, the expression levels of β-catenin and p120-catenin were not affected in SGEF KD cells (Fig. 3-9 B), but their localization at the lateral membrane was disorganized compared to the CTRL cells (Fig. 3-9 C’, D and G).

The localization of both β-catenin and p120-catenin to lateral junctions was restored to normal in Rescue WT cells. The fact that some markers such as p120-catenin and β-catenin, although disorganized, were still present at cell junctions even in the absence of E-cadherin, suggested that there might be other cadherins compensating for the loss of E-cadherin.

Using a pan-cadherin antibody, which recognizes most type I and II cadherins, we found that the combined expression levels of cadherins were not changed in SGEF KD cells (Fig.

Figure 3-9

Figure 3-9. SGEF regulates adherens junction properties of epithelial cells. (A) Cell lysates from confluent CTRL and SGEF KD MDCK cells were analyzed by Western blot using anti-SGEF antibodies. Tubulin was used as a loading control. (B) Cell lysates from confluent CTRL, SGEF KD and Rescue WT MDCK cells were probed with E-cadherin, Pan- cadherin, Cadherin-6, β-catenin, p120-catenin, Scribble and Dlg1 antibodies. Tubulin was used as a loading control. (C, C’, D, E and F) Immunofluorescence showing the distribution of endogenous E- cadherin, p120-catenin, Scribble, Dlg1, β-catenin and mNeon-SGEF (green) in CTRL, SGEF KD and Rescue WT MDCK cells. The bottom panel in each set of images shows a zoomed image of the selected ROI (dotted yellow line). Note that panels C and C’ show images from same field. Confocal images are maximum projections of apical Z-planes. Scale bar: 30 μm (G) Linescan (6 μm line drawn perpendicular to center of junctions) of immunofluorescence images in panel C to F. At least 2 fields from 2 independent experiments were used for quantification (≥ 200 junctions). The intensity profiles were manually centered around the highest peak for each condition. (H) XZ view of MDCK cells from CTRL, SGEF KD and Rescue WT cells stained for E-cadherin (red), β- catenin (magenta), Nucleus (blue) and mNeon-SGEF WT (green in merge panel). Scale bar: 10 μM. (I) Quantification of height in CTRL, SGEF KD and Rescue MDCK cells. n=50 cells for each condition. Error bars represent min to max with all points. ****p<0.00005, ns: non- significant.

5 B). This indicated that one or more of the classical cadherins was upregulated in response to the loss of E-cadherin. A potential candidate is cadherin-6 (K-cadherin) which is expressed in MDCK cells and is upregulated in confluent cells (Stewart et al., 2000).

Western blot analysis showed that in SGEF KD cells the loss of E-Cadherin was accompanied by a subsequent increase in Cadherin-6 levels, which was restored to normal levels when SGEF expression was rescued (Fig. 3-9 B).

To determine whether binding to SGEF plays a role in targeting the Scribble complex to junctions and/or regulating its stability, we also analyzed the effects of silencing SGEF on the expression levels and localization of Scribble and Dlg1. Western blot analysis showed that the expression levels of Scribble and Dlg1 were not affected in SGEF KD cells (Fig.

3-9 B). In contrast, immunostaining for endogenous Scribble and Dlg1 showed that in

SGEF KD cells, the localization of Scribble was slightly more diffuse than in CTRL cells, whereas Dlg1 showed a more severe phenotype with a highly disorganized pattern at junctions (Fig. 3-9 E-G). Rescue with mNeon-SGEF WT restored the normal localization of both Scribble and Dlg1 (Fig. 3-9 E-G). As shown in Fig. 3-7 E-F, Scribble is important for recruiting SGEF to junctions, so silencing SGEF is not expected to affect Scribble localization. The small effect observed on Scribble localization might be an indirect consequence of the effects of SGEF KD on AJ structure. Taken together, these results suggest that SGEF is playing a role in targeting Dlg1 but not Scribble to the lateral membrane. Figure 3-10

Figure 3-10 SGEF regulates the establishment of junction in MDCK cells Confluent MDCK CTRL and SGEF KD cells were subjected to a calcium switch experiment, and the reassembly of cell junctions was determined by immunofluorescence with ZO-1 (TJ) and β-catenin (AJ) antibodies at 0, 1, 2, 4, 6 and 24 h after the re-addition of calcium. For a detailed protocol of the calcium switch conditions please refer to Materials and Methods. Scale bar: 50 μm.

The absence of E-cadherin in SGEF KD cells may compromise the ability of cells to assemble the lateral membrane, which plays a key role in providing mechanical stability to epithelial cells (Tang, 2017). To determine whether the loss of E-cadherin observed upon silencing SGEF was affecting the structure of the lateral membrane, we analyzed confocal xz images from CTRL, SGEF KD and Rescue WT cells stained for E-cadherin and β- catenin (Fig. 3-9 H). Interestingly, silencing SGEF had a striking effect on the height of the monolayer, which was significantly decreased. Quantification showed that CTRL cells averaged 5.94 ± 0.18 μm in height, whereas SGEF KD cells had an average height of 2.50

± 0.02 μm. Rescue with mNeon-SGEF WT restored the cell height to normal levels with an average height of 5.61 ± 0.16 μm (Fig. 3-9 I). These results suggest that the loss of E- cadherin in SGEF KD cells, may prevent the cells in the monolayer from establishing their normal columnar shape.

To determine whether SGEF KD also affects the establishment of junctions, we performed a Calcium switch assay. In the Calcium switch assay, cell-cell junctions are disrupted when cells are grown in the absence of Ca2+, due to the loss of Ca2+-dependent cadherin-mediated adhesion. Subsequent restoration of physiological levels of Ca2+ results in the synchronous de novo assembly of cell-cell junctions (Cereijido et al., 1978; Gumbiner and Simons,

1986). Our results revealed that two hours after replenishing calcium to the medium CTRL cells had already established visible AJ and TJ. In contrast, the formation of AJ and TJ in

SGEF KD cells was significantly delayed (Fig. 3-10), taking at least 6 h to establish comparable AJ and TJ.

Taken together, our results suggest that SGEF regulates the formation and maintenance of cadherin based adherens junction formation through the regulation of E-cadherin

62 expression levels. In the absence of SGEF, E-cadherin is loss, AJ components are disorganized and the lateral membrane collapses. Cadherin-6 upregulation appears to compensate the loss of E-cadherin in SGEF KD cells, and helps to maintain cell-cell adhesion and the integrity of the monolayer, although it might not be sufficient to functionally replace the loss of E-cadherin as AJ remain disorganized.

3.9 SGEF KD changes tight junction morphology and alters barrier function

It has been previously shown that the assembly of AJ leads to the formation of TJ, and that interfering with AJ formation affects the structure and function of TJ (Gumbiner et al.,

1988; Tunggal et al., 2005; Watabe et al., 1994). Since our results indicated that SGEF regulates the formation and maintenance of AJ, we then investigated whether SGEF was also required for TJ organization. We first examined the effect of silencing SGEF on the distribution of the TJ marker ZO-1. Our results revealed that SGEF depletion had a significant effect in the shape of TJ. In MDCK cells, TJ typically adopt a characteristic curvilinear/zigzag pattern (Stevenson et al., 1988), as shown for CTRL cells (Fig. 3-11 A).

In SGEF KD cells however, TJs adopted a much more linear configuration, with most TJ appearing as a virtual straight line (Fig. 3-11 A). The degree of linearity displayed by TJ can be calculated using the zigzag index, which is defined as the ratio of the length of the freehand line traced over a junction to that of a straight line traced over the same junction

(Tokuda et al., 2014). A zigzag index of 1 would indicate a straight line. The average zigzag index decreased from 1.24 in CTRL cells to 1.03 in SGEF KD cells. Re-expression of mNeon-SGEF WT in SGEF KD cells restored the normal curvilinear pattern of TJ, and the zigzag index which increased significantly to 1.16 (Fig. 3-11 A-B). In addition to the straight junctions, SGEF KD cells displayed a more uniform apical area and isometric

Figure 3-11

Figure 3-11 SGEF KD regulates tight junction architecture and permeability (A) Confocal images showing maximum projection of apical Z-planes in CTRL, SGEF KD and Rescue WT MDCK cells stained for endogenous ZO-1 and mNeon-SGEF (green). The bottom panels shows a zoomed image of the selected ROIs (dotted yellow line). Scale bar: 20 μm. (B-D) Quantification of Zigzag index, apical cell area and axial ratio in CTRL, SGEF KD and Rescue WT cells. 2 fields from 2 independent experiments were used for quantification. (N = at least 75 cells for zigzag index, N = 100 for area and N=150 for axial ratio). Error bars represent min to max values. (E) Transepithelial Electrical Resistance (TEER) of CTRL, SGEF KD and Rescue WT cells is plotted. Data represents the average of 3 experiments performed in duplicates. Control was normalized to 1 and data was plotted relative to control. Error bars represent S.E.M. *p<0.05, **p<0.005, ***p<0.0005, ****p<0.00005, ns: non-significant. shape when compared to CTRL cells (Fig. 3-11 A). This was confirmed by measuring the apical area, which showed a small but significant increase in SGEF KD cells (Fig. 3-11 C).

The apical area measurements for individual SGEF KD cells were distributed within a narrower range when compared to CTRL and Rescue WT cells, which indicates a more uniform size (Fig. 3-11 C). In addition, the axial ratio (major/minor axis aspect ratio) also decreased significantly when SGEF was silenced, which is an indication of a more isometric shape (Fig. 3-11 C and D). Both parameters were rescued by re-expressing mNeon-SGEF WT (Fig. 3-11 C and D). We next determined whether the alterations observed in TJ architecture affected paracellular permeability, by analyzing the

Transepithelial Electrical Resistance (TEER), which measures the charge-selective permeability of small solutes in confluent monolayers grown in semi permeable filters

(Shen et al., 2011), and provides an indication of TJ strength (Anderson and Van Itallie,

2009). We measured TEER in confluent CTRL, SGEF KD and Rescue WT MDCK cells, and found that it was significantly reduced in SGEF KD cells compared to CTRL and

Rescue WT cells (Fig. 3-11 E). Taken together, these results suggest that SGEF regulates the architecture of tight junctions, which affects both the size and shape of epithelial cells, as well as their barrier function.

3.10 SGEF KD stimulates actomyosin contractility

The straight TJ phenotype displayed by SGEF KD cell bears a striking resemblance to that induced by silencing ZO-1 and ZO-2 (Choi et al., 2016; Fanning et al., 2012). Silencing

ZO-1 and ZO-2 promotes the assembly of a highly organized actomyosin array at the apical junction, which results in an increase in the tension at the junctions (Choi et al., 2016;

Fanning et al., 2012). We found that in SGEF KD cells myosin IIB was also arranged periodically in apical arrays along with ZO-1 (Fig. 3-12 A). In contrast, CTRL and Rescue

Figure 3-12

Figure 3-12. SGEF KD stimulates actomyosin contractility (A) Confocal images showing maximum projection of apical Z-sections in CTRL, SGEF KD and Rescue WT MDCK cells stained for endogenous ZO-1 (green), myosin IIB (red) and mNeon-SGEF (magenta, in Rescue). mNeon signal is shown in magenta in Rescue panel to maintain color consistency. Scale bar: 5 μm. (B) Confocal images showing CTRL, SGEF KD and Rescue WT MDCK cells stained for endogenous F-actin using phalloidin (green) and myosin IIB (red). Left panel: maximum projection of apical Z-planes; Right panel: maximum projection of basal Z-planes. Images were processed using the HyVolution deconvolution package (see Methods). Scale bar 0.5 μm. (C ) Quantification of intensities of ZO-1 at junctions measured using a rectangle of 2 x 3 μm placed along bicellular junctions. At least 2 fields from 2 independent experiments were used for quantification (≥ 100 junctions). (D) Total cell lysates from confluent CTRL, SGEF KD and Rescued WT MDCK cells were immunoblotted with ZO-1, myosin IIB, and Afadin antibodies. Tubulin was used as a loading control. (E) Maximum projection of confocal images showing the localization of endogenous Afadin in CTRL, SGEF KD and Rescue WT cells. Scale bar: 5 μm. (F) Quantification of the ratio of TCJ over BCJ intensity of Afadin was measured as described in Methods. At least 3 fields from 2 independent experiments were used for quantification (≥ 200 junctions). Error bars represent S.E.M. ****p<0.00005, ns: non-significant. WT cells showed a more diffused myosin pattern that was only weakly associated with 66 junctions (Fig. 3-12 A). To further characterize the actomyosin arrangement at junctions we stained the cells for F-actin and myosin IIB and imaged them using Hyvolution superresolution technology to obtain a higher level of detail. In CTRL cells very little myosin

IIB can be found colocalizing with actin at BCJ, but at TCJ the signal is more concentrated as has been previously shown (Fig. 3-12B, white arrowheads) (Fanning et al., 2012). In contrast, SGEF KD cells show an enlarged actomyosin array distributed periodically along the length of the BCJ. (Fig 7 B, yellow arrowheads). In addition, silencing SGEF promoted a dramatic increase in the number and thickness of stress fibers at basal z-sections, something that was not observed in ZO-1/2 KD cells (Choi et al., 2016; Fanning et al.,

2012). These stress fibers were also abundantly decorated with myosin IIB, when compared to those in CTRL cells (Fig. 3-12 B and Fig. 3-13). Re-expression of mNeon-

SGEF WT in SGEF KD cells restored the normal apical and basal actomyosin pattern (Fig.

3-12 B).

We did not observe a significant difference in the the expression levels of total myosin in

SGEF KD cells, suggesting that the phenotype observed was mainly driven by increased myosin rearrangement (Fig. 3-12 D). Treatment with both the myosin inhibitor blebbistatin and ROCK inhibitor Y27632 disrupted the apical actomyosin array, as well as the basal stress fibers (Fig. 3-13 A and A’), although blebbistatin had a stronger effect than Y27632.

This could be due to the fact that cells were treated with the inhibitors for 16 hrs, which in some cases can elicit compensatory effect (Choi et al., 2016). Nevertheless, this suggests that the increase in contractility observed in response to SGEF depletion is mediated by a

ROCK/myosin pathway.

The similarities between the SGEF KD and ZO-1/2 KD phenotypes suggest that SGEF and

ZO-1 may be operating within the same pathway. Interestingly, analysis of ZO-1 fluorescence intensity at TJ showed that it decreased significantly when SGEF was silenced and was rescued by SGEF re-expression (Fig. 3-12 C). A similar result was obtained when

Figure 3-13

Figure 3-13 SGEF signals through ROCK/myosin pathway to induce contractility in epithelial cells. Monolayers of CTRL and SGEF KD cells were treated with DMSO (vehicle), 100 mM of Blebbistatin or 30 mM of Y27632 for 16 hrs and subsequently stained with antibodies specific for myosin and phalloidin to detect F-actin. Apical (A) and basal (A’) confocal maximum projected z-sections are shown separately to visualize apical actomyosin arrays and basal stress fibres. Scale bar: 30 μm.

we analyzed the levels of ZO-1 by Western blotting (Fig. 3-12 D). These results place

SGEF upstream of ZO-1, regulating its expression levels. We also analyzed the expression

68 and localization of afadin, an adapter protein that interacts with cytoskeleton and junctional proteins (Takai and Nakanishi, 2003). In ZO 1/2 KD cells, afadin is recruited to AJ and is especially enriched at tricellular junctions (TCJ) (Choi et al., 2016). In contrast, in SGEF

KD cells both the expression levels of afadin and its localization at AJ are reduced (Fig. 3-

12 E). However, the reduction observed in afadin targeting to AJ is not uniformly distributed along AJ, as afadin signal intensity remained high at TCJ, but was dramatically reduced at bicellular junctions (BCJ) (Fig. 3-12 E). Quantification of the ratio of intensity between TCJ and BCJ, showed a significant increase when SGEF is KD confirming the enrichment at TCJ (Fig. 3-12 F). Both the expression and localization of afadin were rescued to normal levels upon re-expression of mNeon-SGEF WT (Fig. 3-12 D-F). These result suggest that silencing SGEF and ZO-1/2 elicit a similar but not identical response, redistributing afadin to TCJ in response to an increase in tension (Choi et al., 2016).

Taken together, our results suggest that SGEF might function in a similar pathway as ZO

1/2 and Afadin to regulate apical TJ architecture by increasing tension and the regulating actomyosin contractility upon SGEF KD.

3.11 SGEF overexpression promotes apical constriction in Xenopus embryos

As shown in Fig 4, SGEF localizes to apical junctions when expressed in Xenopus embryos. We noticed that when SGEF was expressed at low levels, there were no major differences in the general appearance of the cell-cell junctions. However, in cells expressing higher levels, the area of the apical surface was significantly smaller. To confirm this gain of function phenotype, we overexpressed 3xGFP-SGEF at a high level in

Xenopus embryos and analyzed the effects on TJ using mRFP-ZO-1 as a marker. First, we found that SGEF OE cells were frequently apically constricted (yellow arrows in Fig. 3-14

A). Live imaging analysis showed that 3xGFP-SGEF expressing cells constrict over time, whereas control cells remain quite stable over that time frame (Fig. 3-14 B). Live imaging analysis of mRFP-ZO-1 dynamics showed that in CTRL cells ZO-1 is quite stable over time, whereas in SGEF OE cells ZO-1 was very dynamic at junctions (Fig. 3-14 C).

Quantification confirmed that SGEF OE cells had smaller apical areas due to apical constriction when compared to CTRL cells (Fig. 3-14 D).

Apical actomyosin activity is known to be responsible for contractile movement during embryogenesis and other developmental stages (Munjal and Lecuit, 2014; Roper, 2013).

As SGEF OE cells were highly contractile at apical region, we analyzed the F-actin levels in Xenopus embryos. In Xenopus we found that F-actin levels were significantly increased in both junctional as well as medial apical region in SGEF OE cells (Fig. 3-14 E, E’ and

E’’). We also analyzed the effects of overexpressing mNeon-SGEF WT on the localization of F-actin and myosin II in live Xenopus embryos by co-expressing an F-actin probe

(Lifeact-mRFP) and a myosin II intrabody (SF9-mNeon). The control image shown is from a control region of a mosaic SGEF OE embryo. Compared to CTRL, SGEF OE resulted in increased and reorganized junctional and medial-apical F-actin and myosin; F-actin was broader at cell-cell junctions, whereas myosin was reorganized into a strong band interior to cell-cell junctions and a broad diffuse belt extending to both sides of the junction (Fig.

3-14 F).

α-catenin links the AJ to the actin cytoskeleton (Buckley et al., 2014). When α-catenin senses increased junctional tension generated by the actomyosin cytoskeleton, it undergoes a conformational change, recruiting vinculin and thus strengthening the AJ’s connection to

Figure 3-14

Figure 3-14. SGEF regulates apical constriction in epithelial cells (A) Gastrula-stage Xenopus embryos expressing mRFP-ZO-1 (TJ marker) with either GFP (CTRL) (top), or 3xGFP-SGEF overexpressed (OE) at high levels (bottom). Yellow arrows point to apically constricted cells. Scale bar: 20 μm. (B) Time lapse of CTRL and SGEF OE of a single cell over a period of 24 mins. Note that SGEF OE cell constricts apically whereas CTRL cell stays same in apical area. (C) Time projection of ZO-1 signal over a 203 sec interval shows that junctions in SGEF OE cells are more dynamic than in controls. Scale bar: 20 μm. (D) The apical surface area of SGEF OE cells is significantly smaller than control cells, and some SGEF OE cells exhibit severe apical constriction. Graph shows mean +/- S.E.M. Control, n = 132 cells, 3 embryos, 2 experiments; SGEF OE, n = 147 cells, 3 embryos, 2 experiments. (E) Control, and SGEF OE gastrula-stage Xenopus embryos were fixed and stained with phalloidin to reveal F-actin. Images in the top row were taken with lower laser power optimized for viewing cell-cell junctions, and images in the bottom row were taken with higher laser power optimized for viewing medial-apical actin. Scale bar: 10 μm (E’) F- actin intensity at TCJ was quantified from fixed phalloidin stained embryos. n = control: 3 experiments, 11 embryos, 288 junctions; SGEF KD: 3, 12, 178; SGEF OE: 2, 13, 304. Graph shows a dot plot with the mean +/- SEM indicated. (E’’) F-actin intensity at BCJ was quantified from fixed phalloidin stained embryos. n = control: 3 experiments, 7 embryos, 50 cells; SGEF KD: 3, 12, 41; SGEF OE: 3, 8, 50. Graph shows a dot plot with the mean +/- S.E.M indicated. (F) Control and SGEF OE embryos expressing an F-actin probe (Lifeact-mRFP, magenta in merge) and a myosin II intrabody (SF9- mNeon, green in merge) were live imaged by confocal microscopy. The control image shown is from a control region of a mosaic SGEF OE embryo. Scale bar: 10 μm. (G) Control and SGEF overexpressing (OE) embryos co-expressing mNeon-Vinculin, mCherry-alpha- catenin, and BFP-membrane. Scale bar: 10 μm. (H) Scatter plot comparing junctional intensities of vinculin (normalized to membrane probe intensities) in control and SGEF OE embryos. n = 3 experiments, 8 embryos, 63 junctions for control embryos; 3 experiments, 8 embryos, 63 junctions for SGEF OE embryos. Confocal images in (A), (B), (E), (F) and (G) are maximum projections of apical sections. Error bars represent S.E.M. ****p<0.00005.

F-actin (Yonemura et al., 2010). Therefore, the recruitment of fluorescently-tagged vinculin to junctions can be used as a readout for increased junctional tension (Hara et al.,

2016; Higashi et al., 2016). In control Xenopus embryos, mNeon-Vinculin was weakly

72 recruited to cell-cell junctions, whereas in SGEF OE embryos mNeon-Vinculin was strongly recruited to junctions and was particularly increased near cell vertices (Fig. 3-14

G and H). These results indicated that SGEF plays a role in the regulation of apical actomyosin constriction in a process that involves the recruitment of vinculin to the junctions.

3.12 The nucleotide exchange activity of SGEF is required for junctional maintenance

whereas its scaffolding activity is required for apical contractility

SGEF activates RhoG, a Rho GTPases related to Rac that plays a role in a variety of cellular processes, including cell migration, invasion, macropinocytosis and neurite outgrowth

(Ellerbroek et al., 2004; Jackson et al., 2015; Katoh et al., 2006; Katoh et al., 2000; Valdivia et al., 2017; van Buul et al., 2007; Wyse et al., 2017). Our results suggest that SGEF is playing a role in both the formation and maintenance of cell-cell junctions, as well as in the regulation of apical contractility. How is SGEF orchestrating these processes? One possibility is that SGEF is recruited to junctions in order to activate RhoG locally.

Alternatively, SGEF could function as a scaffold, to mediate the formation of the

Scribble/SGEF/Dlg1 ternary complex. In order to explore these possibilities, we first analyzed the effect of silencing SGEF on the activity levels of RhoG in MDCK cells. Using a RhoG activity pulldown assay (van Buul et al., 2007), we found that the activity of RhoG was significantly reduced when SGEF expression was silenced. Active RhoG levels returned to normal when SGEF expression was rescued with mNeon-SGEF WT (Rescue

Figure 3-15

Figure 3-15. The exchange activity of SGEF is required for junctional maintenance whereas scaffolding activity of SGEF is required for apical contractility (A) Active RhoG was precipitated from total lysates of CTRL, SGEF KD, Rescue mNeon-SGEF WT and Rescue catalytic dead (CD) mNeon-SGEF using GST–ELMO and immunoblotted with anti-RhoG antibodies. (B) For quantification, active RhoG levels were normalized to total RhoG levels. Data are mean ± S.E.M. of three independent experiments. (C) Lysates from CTRL, SGEF KD, and SGEF KD cells rescued with either mNeon- SGEF 1-227, mNeon-SGEF 1-400 or mNeon-SGEF CD were probed for E-cadherin, β-catenin, ZO-1 and myosin IIB antibodies. Tubulin was used as a loading control. (D-E) Confluent MDCK CTRL, SGEF KD, and SGEF KD cells rescued with either mNeon- SGEF 1-227, mNeon-SGEF 1-400 or mNeon-SGEF CD were stained for endogenous E-cadherin and β-catenin and mNeon- SGEF (green). Confocal images are maximum projections of apical Z-planes. Scale bar: 5 μm. (F) XZ view of MDCK cells from CTRL, SGEF KD and SGEF KD cells rescued with mNeon-SGEF 1-227, mNeon-SGEF 1-400 or mNeon-SGEF CD stained for F- actin (magenta), Nucleus (Hoechst) and mNeon-SGEF (green). Scale bar: 10 μm. (H) Linescan (6 μm line drawn perpendicular to center of junctions) of immunofluorescent images in panel D. At least 2 fields from 2 independent experiments (≥ 150 junctions) were used for quantification. The intensity profiles from were manually centered around the highest peak for each condition. (I) Quantification of height in CTRL, SGEF KD and SGEF KD cells rescued with mNeon-SGEF 1-227, mNeon-SGEF 1-400 or mNeon- SGEF CD cells. n=50 cells for each condition. Error bars represent min to max values with all points. (J) Quantification of Zigzag index of CTRL, SGEF KD and SGEF KD cells rescued with mNeon-SGEF 1-227, mNeon-SGEF 1-400 or mNeon-SGEF CD. At least 2 fields from 2 independent experiments (≥ 200 junctions) were used for quantification. ****p<0.00005, ns, non-significant.

WT), but not when rescued with a catalytic dead (CD) mutant of SGEF (R446A, N621A)

(Rescue CD) (Ellerbroek et al., 2004) (Fig. 3-15 A and B). These results indicate that modulating the expression levels of SGEF in MDCK cells has a significant effect on the endogenous levels of active RhoG.

To explore the contribution of the catalytic and scaffolding activity of SGEF, we generated stable cell lines expressing three different SGEF mutants in the background of SGEF KD cells and tested their ability to rescue the main phenotypes described in Fig. 3-9 to 3-12 for

SGEF KD cell, i.e. the loss of E-cadherin, straight TJ and the increased actomyosin contractility. First, SGEF 1-227, a construct that binds to Scribble but not to Dlg1 (see Fig.

3-5), so it cannot form a ternary complex (no scaffolding activity) and has no exchange activity. Second, SGEF 1-400, a construct that expresses only the minimum region required to bind Scribble and Dlg1, so it has scaffolding activity (can form a ternary complex, see

Fig 3), but has no catalytic activity. Finally, SGEF CD, a catalytic dead FL SGEF that contains the binding sites for both Scribble and Dlg1 (scaffolding), as well as all the other domains in SGEF. Interestingly, none of the three constructs tested were able to rescue the loss of E-cadherin observed in SGEF KD cells (Fig. 3-15 C, D and H). This suggested that the catalytic activity of SGEF and thus RhoG activation is required for the regulation of E- cadherin expression. Similar results were observed for β-catenin, which loses its tight localization to junctions in SGEF KD cells and could not be rescued by any of the three constructs tested (Fig. 3-15 E). As none of the SGEF mutants tested (1-227, 1-400 and CD) rescued the loss of E-cadherin or the disorganized β-catenin pattern at AJ, we hypothesized that they would not be able to rescue the reduced height phenotype observed in SGEF KD cells either. As expected, neither of the three SGEF mutants rescued the cell height defect.

The average height in cells expressing each of the three SGEF mutants was within the same range of the height measured in SGEF KD cells, approximately 2X shorter than that of

CTRL cells cells (CTRL- 5.95 ± 0.18 μm, SGEF KD- 2.5 ± 0.02 μm, Rescue 1-227- 2.99

± 0.065 μm, Rescue 1-400- 2.57 ± 0.049 μm , Rescue CD- 3.009 ± 0.064 μm) (Fig. 3-15

F and I).

Interestingly, the mutants that can form a ternary complex but have no catalytic activity

(SGEF 1-400 and SGEF CD), were able to rescue the straight TJ phenotype (Fig. 3-15 G and J). Quantification of the linearity of the TJ showed that, although not completely rescued to the levels observed in CTRL cells, the zigzag index increased significantly in both Rescue 1-400 and Rescue CD cells when compared to SGEF KD (Fig. 3-15 J). Both mutants were also able to rescue the increase in contractility observed in SGEF KD cells, reverting the formation of the apical actomyosin array to a more diffuse myosin staining around junctions like the one found in CTRL cells (Fig. 3-15 G).

Overall, our results demonstrate that the regulation of E-cadherin-mediated AJ formation and maintenance requires the exchange activity of SGEF, whereas the ability of SGEF to function as a scaffold by forming the Scribble/SGEF/Dlg1 ternary complex is important to regulate actomyosin contractility and normal junctional architecture.

3.13 SGEF KD does not affect polarity but impairs lumen formation in 3D MDCK

cysts

When MDCK cells are embedded in a 3D ECM such as matrigel or collagen, they form a polarized spherical cyst with a fluid-filled hollow lumen surrounded by a monolayer of polarized cells (O'Brien et al., 2002). The organization of these cysts resembles that of epithelia in vivo, and thus, cyst development provides a model system for the formation of epithelial morphogenesis in vitro. We used this system to test the potential role of SGEF in the establishment of apicobasal polarity and cyst morphogenesis. First, we determined whether SGEF was required for the establishment of apicobasal polarity in 2D MDCK

77 cultures, where cells form a columnar monolayer with a well defined apical and basal membrane. Our results showed that in SGEF KD cells, the apical membrane marker gp135/podocalyxin (Ojakian and Schwimmer, 1988), targeted efficiently to the apical surface (Fig. 3-16 A). These results suggested that even though silencing SGEF induced dramatic changes in TJ and AJ architecture (Fig. 3-9 and 3-11), it did not affect apicobasal polarity. We then generated cysts by embedding CTRL, SGEF KD and Rescue WT cells in matrigel and allowing them to grow for 4 days. We stained the cysts with different markers, including β-catenin and E-cadherin (AJ), gp135 (polarity), and F-actin. Our results showed that in CTRL cells most cysts had a single open lumen that was properly polarized (65%), with β-catenin and E-cadherin decorating the lateral junctions, and gp135 and F-actin concentrated at the apical surface (towards the luminal surface inside of the cyst) (Fig. 3-14 B and C). The rest of the CTRL cysts contained more than one lumen

(multiple), which were all open (35%) (Fig. 3-16 B, C and D). In contrast, 77% of the

SGEF KD cysts were severely disorganized with no obvious central lumen. Instead of a single open lumen, SGEF KD cysts displayed multiple gp135-positive patches, the majority of which were closed, or, when open, had a very small lumen (Fig. 3-14 B and

D). This suggested that despite the severity of the SGEF KD phenotype, the establishment of apicobasal polarity was affected, as gp135 was properly targeted to the apical membrane

(Fig. 3-16 B). β-catenin still localized to lateral membranes in SGEF KD cysts (Fig. 3-14

B), but E-cadherin was

Figure 3-16

Figure 3-16. SGEF does not affect polarity but regulates lumen formation in 3D MDCK cysts (A) Immunofluorescence of MDCK CTRL and SGEF KD cells using gp135 (green), actin (magenta) and nucleus (hoechst). Scale bar: 10 μm. (B-C) MDCK CTRL, SGEF KD and Rescue WT cells were plated on matrigel to form 3D cysts. Cysts were fixed and stained for β-catenin (red), gp135 (green) and nuclei (blue) in panel (B) and E-cadherin (green), phalloidin (red) and nuclei (blue) in panel (C). For detailed protocol of growing and staining cyst please see materials and methods. Scale bar: 5 μm (D) Cysts from CTRL, SGEF KD, Rescue WT and Rescue CD were classified based on the number of cysts (single or multiple) and the phenotype of the lumen (open or closed). 3 independent experiments were counted for each condition (≥200 cysts/condition). Images in (B) and (C) are single Z-section corresponding to the center of the cyst.

significantly downregulated (Fig 3-14 C), which was consistent with our observations in

2D cultures. Re- -expression of mNeon-SGEF WT (Rescue WT) restored the normal phenotype. The majority of the Rescue WT cysts had open lumens (75%), either single

(35%), or multiple (40%) (Fig. 3-16 B, C and D), and E-cadherin expression was also restored to normal levels (Fig. 3-16 C). These results indicate that, even though SGEF does not play a major role in the establishment of polarity, it regulates both the ability of cysts to form an open lumen, as well as the number of lumens formed in each cyst.

To explore the contribution of the catalytic and scaffolding activities of SGEF during cyst formation, we also generated cysts using the stable Rescue CD cells described in Figure 9, which express a full length catalytic dead mutant of SGEF in SGEF-KD cells. Our results showed that, in agreement with the results obtained in 2D monolayers, the expression of

E-cadherin was not rescued in Rescue CD cysts, confirming the requirement of the exchange activity for the regulation of E-cadherin levels (Fig. 3-16 C). In addition, expression of SGEF CD did not rescue the ability of the cysts to form a single central lumen

(Fig. 3-16 C and Fig. 3-17). Quantification showed that the number of cysts that had a single lumen decreased from 65% in CTRL cells to 13% in SGEF KD cells. Rescue WT increased the number of single lumens to 36%, whereas in Rescue CD cysts, only 14% had a single lumen (Fig. 3-16 D, red+dark green bars). To our surprise, the scaffolding activity of SGEF appeared to be required during lumen opening, as SGEF CD expression significantly restored the number of cysts that formed open lumens (Fig. 3-14 C and Fig.

3-17). Quantification demonstrated that the number of cysts with an open lumen decreased

80 from 100% in CTRL cells to 23% in SGEF KD cells. Re-expression of SGEF WT and

SGEF CD restored the number of cysts with open lumens to

Figure 3-17

Figure 3-17 Catalytic activity of SGEF is required for the lumen formation in MDCK cyst grown in matrigel. MDCK CTRL, SGEF KD and rescue mNeon-SGEF CD cells were plated on matrigel to form 3D cysts. Cysts were fixed and stained for gp135 (green), phalloidin (red) and nuclei (Hoechst). mNeon-SGEF CD is pseudo-colored in magenta. Quantification in Fig 10D. For detailed protocol of

growing and staining cyst please see Materials and Methods. Scale bar: 10 μm

77%, 59% respectively (Fig. 3-16 D, red + orange bars). Overall, these results suggest that

SGEF plays a role during lumen formation in 3D cysts. The catalytic activity of SGEF is important for formation of a cyst with a single lumens, which may depend on the regulation of E-cadherin expression (see Discussion). The scaffolding activity, on the other hand, is required for the formation of a fluid-filled open lumen.

Chapter 4

Discussion

The formation of a polarized epithelium requires the coordinated action of three highly conserved protein complexes; PAR, Crumbs and Scribble (Bilder et al., 2003). These polarity protein complexes act as scaffolds recruiting other proteins in response to both intracellular and extracellular cues, which ultimately result in the reorganization of the cytoskeleton and vesicular trafficking that leads to the establishment of cellular asymmetry

(Rodriguez-Boulan and Macara, 2014).

The Scribble complex was initially identified in Drosophila as a critical regulator of epithelial polarity (Bilder and Perrimon, 2000; Gateff and Schneiderman, 1974; Mechler et al., 1985; Woods and Bryant, 1991) and was later shown to be involved in the regulation of other cellular processes, including cell-cell adhesions, asymmetric cell division, vesicular trafficking, cell migration, and planar-cell polarity (Elsum et al., 2012). The

Scribble complex is comprised of three proteins, Scribble, Dlg and Lgl, which are thought to function in a common pathway (Elsum et al., 2012). However, the molecular mechanism that regulate their functions are poorly characterized.

In this study, we show that SGEF, a RhoG-specific GEF, forms a ternary complex with two of the members of Scribble polarity complex, Scribble and Dlg1. The formation of this ternary complex positions SGEF at cell-cell junctions where it regulates the formation and maintenance of AJ, as well as actomyosin contractility and barrier function at TJ. SGEF

83 does not appear to affect apicobasal polarity in 3D cysts but it plays a role during lumen formation.

Our results provide a detailed map of the interaction between these three proteins. SGEF interacts with Scribble PDZ1 domain through a novel internal PBM. Although most PBMs are typically located at the extreme C-terminus of a protein (Songyang et al., 1997), a small number of internal PBMs have been identified (Brenman and Bredt, 1997; Hillier et al.,

1999; London et al., 2004; Penkert et al., 2004; Wong et al., 2003). In contrast to the C- terminal PBMs, a consensus motif for the internal PBMs identified so far has not been defined. The structure of the Scribble PDZ1-SGEF interaction is also very different and makes much more extensive contacts compared to the two previously published

PDZ/internal ligand structures (Hillier et al., 1999; Penkert et al., 2004). Interestingly,

SGEF encodes a second PBM at its C-terminus (type I). Our results show that it does not interact with any of the 7 PDZ domains encoded by Scribble and Dlg1, suggesting it may function to recruit other interacting partners to the Scribble complex. It would be interesting to further understand scaffolding activity of SGEF by characterizing the interacting partners of SGEF C-terminus PBM. This would help us understand the existence of multimeric protein complex formation (if any) through SGEF. Also, Lgl is another member of the Scribble polarity complex that has been shown to bind Scribble

(Kallay et al., 2006). It will be interesting to determine if SGEF mediates the formation of this quaternary protein complex Scribble/SGEF/Dlg1/Lgl. By forming such multimeric complex, SGEF could bring together Scribble polarity complex members and other signaling/structural proteins for regulating polarity and junctions in epithelial cells.

Here, we also showed that Dlg1 GUK domain binds to SGEF peptide 300-350 residues at its N-terminus. Our preliminary unpublished data narrowed down the binding site of Dlg1’s

GUK domain to amino acids 300-330 in SGEF. The GUK domain of Dlg1 has been shown to bind phosphorylated serines of LGN protein with a conserved motif -RRXS- (where X is any amino acid) (Mori et al., 2013; Zhu et al., 2011). Interestingly, SGEF peptide within region of 300-330 residues is Serine rich encoding an inverted SXRR conserved motif. It would be interesting to understand if SGEF also utilizes this conserved motif and phosphorylation of specific Serine residues for the interaction with Dlg1 GUK domain.

SGEF belongs to a small sub-family of six related GEFs called Ephexins (Rossman et al.,

2005), but the Scribble and Dlg1 binding sites are only found in SGEF. Both binding interfaces are located at SGEF’s N-terminus and are highly conserved in vertebrates. SGEF orthologs do not seem to be present in Drosophila or other invertebrates. The closest homolog in Drosophila is Ephexin, which lacks the N-terminal Scribble/Dlg1 binding region and is more closely related to Ephexin 4 in humans. Interestingly, the formation of a ternary complex comprising Scribble and Dlg appeared at least one more time during evolution. In Drosophila, a protein called GUK-holder (GUKH) also forms a ternary complex with Dlg1 and Scribble (Mathew et al., 2002). Like SGEF, GUKH also binds to the GUK domain of Dlg1 and to PDZ1 in Scribble, although through a C-terminal PBM

(Caria et al., 2018; Mathew et al., 2002). However, in contrast to SGEF, GUKH functions exclusively as a scaffold and has no known enzymatic activity. GUKH plays a role in the development of Drosophila epithelial tissues, and silencing GUKH enhances the defects caused by Scribble or Dlg depletion in eyes and wings (Caria et al., 2018). Two GUKH orthologs have been identified in humans, Nance-Horan syndrome (NHS) and NHSL1

(Katoh and Katoh, 2004). NHS proteins localize to cell junctions and also associate with

Scribble and the Dlg family member PSD95 (Dlg4). However, the ability of NHS to mediate the formation of a ternary complex has not been demonstrated (Sharma et al.,

2006) (Walsh et al., 2011).

It is tempting to speculate that the function of Drosophila GUKH is conserved in humans in the NHS family, and SGEF regulates a different function that appeared later during vertebrate evolution. It also raises the possibility in vertebrates of different ternary complexes co-existing in the same cell, and possibly regulating different processes. This could provide important evidence for two different scaffolding proteins, SGEF and NHS forming distinct protein complexes with Scribble and Dlg1 that are regulated spatiotemporally during development.

In mammalian cells, both Scribble and Dlg1 are targeted to cell-cell junctions where they are distributed throughout the lateral membrane and co-localize with both AJ and TJ markers (Dow et al., 2003; Ivanov et al., 2010; Laprise et al., 2004; Navarro et al., 2005).

Our results show that SGEF targets to cell-cell junctions in a Scribble-dependent manner, where it also co-localizes with AJ and TJ markers. When SGEF expression is silenced, the most striking phenotype is an almost complete loss of E-cadherin expression, which is accompanied by disorganized AJ, a collapse of the lateral membrane and a three-fold decrease in cell height. Silencing Scribble or Dlg1 in mammalian cells also affects AJ architecture in a similar fashion, although the effects are typically milder and are not associated with significant E-cadherin downregulation (Hendrick et al., 2016; Laprise et al., 2004; Qin et al., 2005). Surprisingly, neither β-catenin nor p120-catenin are

86 downregulated upon SGEF KD even in the absence of E-cadherin, and cells can still form a monolayer. Moreover, β-catenin and α-catenin are still localized to AJ when E-cadherin expression is silenced to almost undetectable levels (Capaldo and Macara, 2007). This could be a result of compensation by other cadherins, which are upregulated in the absence of E-cadherin (Tinkle et al., 2004; Tunggal et al., 2005). We found that Cadherin-6, which is expressed at high levels in MDCK cells (Stewart et al., 2000), is upregulated in SGEF

KD cells. This may contribute to maintaining the monolayer integrity in the absence of E- cadherin, although it is not sufficient to restore normal AJ architecture. As in E-cadherin

KD cells (Capaldo and Macara, 2007), SGEF KD affects primarily the establishment of junctions but not the maintenance of already established junctions. Interestingly, downregulation of E-cadherin expression is a hallmark of epithelial-to-mesenchymal transition (EMT) (Kalluri and Weinberg, 2009). Despite a drastic decrease in E-cadherin levels, we see no phenotypic indication of EMT in SGEF KD cells. This is not completely unexpected, as studies in normal cells have shown that E-cadherin loss is not always sufficient to induce EMT (Chen et al., 2014). It is possible that the partial compensation by

Cadherin-6 and/or other Cadherins plays a role in preventing EMT. On the other hand, even though it is evident that SGEF KD cells are not completely mesenchymal, recent findings indicate that Epithelial to Mesenchymal transition is a complex process involving multiple steps and that complete EMT is not essential for metastasis ((Aiello et al., 2017;

Jolly et al., 2018; Ye et al., 2015; Zheng et al., 2015)). Moreover, a complete switch from

Epithelial to Mesenchymal transition is a rare event during development or cancer

(Campbell, 2018; Jolly et al., 2018). Instead, cells that undergo EMT pass through multiple stages that are intermediate between epithelial and mesenchymal phenotypes (Grigore et

87 al., 2016; Lambert et al., 2017). The loss of E-cadherin displayed by SGEF KD cells could be one of those intermediate stages. It requires further investigation to determine if SGEF

KD cells show characteristics of partial EMT.

Besides its role in regulating AJ structure, SGEF also regulates the structure and function of TJ. In SGEF KD, TJ lose their typical curvilinear aspect and become very straight, with cells adopting a very isometric polygonal shape. In addition, a highly contractile actomyosin array is recruited to TJ in SGEF KD cells, which increases tensions at apical junctions. A strikingly similar TJ phenotype has been described in ZO-1 KD (Tokuda et al., 2014; Van Itallie et al., 2009) and ZO-1/ZO-2 double KD in MDCK cells (Choi et al.,

2016; Fanning et al., 2012), as well as during Shroom overexpression (Hildebrand, 2005).

Both ZO-1/ZO-2 and Shroom function upstream of ROCK (Choi et al., 2016; Hildebrand,

2005), which suggest that SGEF may also operate directly or indirectly in a pathway that regulates ROCK recruitment and/or activation. Afadin is an adapter protein that links actin cytoskeleton to AJ and ZO-1 and modifies cell shape by transmitting contractile forces generated by actomyosin contraction during germband extension in Drosophila development (Sawyer et al., 2011). As afadin has been shown to bind both TJ proteins as well as actin cytoskeleton, it will be interesting to understand if SGEF/Scribble/Dlg1 ternary complex regulates actomyosin contractility and apical tight junction architecture through Afadin.

Notably, silencing SGEF has a much broader and more severe impact on both AJ and TJ than the above mentioned studies, with loss of E-cadherin, disorganized AJ, short cell height, and TEER reduction, which have not been observed when Scribble, Dlg1 or ZO-

1/ZO-2 were silenced (Choi et al., 2016; Fanning et al., 2012; Hendrick et al., 2016; Laprise et al., 2004; Qin et al., 2005), or upon Shroom overexpression (Hildebrand, 2005). This suggests that SGEF may function in other pathways in parallel to those in which

Scribble,Dlg and ZO1 are involved. Alternatively, it can be attributed to differences in the efficiency of KD, or to the use of different cell lines in the previous studies that can result in milder phenotypes.

SGEF has been shown to direct actin cytoskeleton remodeling at both the dorsal and ventral surface of the cells, but never at cell-cell junctions (Ellerbroek et al., 2004; Goicoechea et al., 2017; Patel and Galan, 2006; van Buul et al., 2007). Our study delineates two very well-defined roles for SGEF, which can be classified according to their dependence on

SGEF’s nucleotide-exchange activity. The regulation of E-cadherin stability and AJ architecture requires the nucleotide-exchange activity of SGEF, suggesting it is mediated by RhoG. A knockout of RhoG will be useful in understanding its role in junction formation, downstream of SGEF. It will also be interesting to understand if RhoG is regulated spatiotemporally during establishment of junctions in epithelial cells. On the other hand, SGEF-mediated regulation of TJ is independent on its exchange activity, and only requires the ability of SGEF to form a ternary complex with Scribble and Dlg1. The molecular mechanisms that control TJ architecture and properties in SGEF KD are not known. It will be insightful to understand how SGEF regulates these two processes, possibly through various upstream/downstream signaling cues. Identifying SGEF interacting partners, will be helpful in deciphering molecular mechanism through which

SGEF ternary complex regulates apical contractility. The regulation of AJ architecture through RhoG could be better understood by finding the downstream effectors of RhoG. A

89 potential candidate connecting RhoG activity and AJ formation is the RhoG effector

ELMO2. ELMO2 is expressed in MDCKs and has been shown to play a role, together with the Rac1 GEF DOCK1, in recruiting E-cadherin to junctions (Toret et al., 2014). However, silencing either ELMO2 or DOCK1 only delays junction formation and has no effect on

E-cadherin expression levels, which suggest that SGEF and RhoG may function through a yet to be characterized pathway. This necessitates a screen for other RhoG effectors acting downstream of RhoG in regulating junctional architecture. Moreover, characterization of

GAPs for RhoG that regulates its activity at junctions will be helpful in understanding its spatiotemporal regulation at junctions.

Regarding the role of the Scribble/SGEF/Dlg1 ternary complex in the regulation of TJ, virtually nothing is known. Scribble has been shown to interact with ZO-1/ZO-2 suggesting that the ternary complex may be physically linked to TJ (Ivanov et al., 2010; Metais et al.,

2005). Our future efforts are aimed to characterize the downstream signaling pathways that connect SGEF with AJ and TJ function.

Overexpression of SGEF, on the other hand, induces a different contractility phenotype, with a dramatic accumulation of actin and myosin at the apical belt, which leads to apical contraction and reduced apical cell area. At the apical surface, actin polymerization and actomyosin contractility are regulated by the coordinated actions of several GTPases, including RhoA, Rac1 and Cdc42. This requires a tight control of the spatial and temporal activation pattern of each of the GTPases involved. The molecular mechanisms controlling these processes are still not well characterized, but several RhoGEFs and RhoGAPs are identified to modulate this process (Aijaz et al., 2005; Holeiter et al., 2012; Ngok and

Anastasiadis, 2013; Nishimura et al., 2012; Ratheesh and Yap, 2012; Sousa et al., 2005;

Terry et al., 2011; Tripathi et al., 2012; Zebda et al., 2013). Silencing the expression of these GEFs and GAPs alters junction integrity or interferes with contractility suggesting that multiple GEFs and GAPs regulate different functions at cell junctions (Ngok et al.,

2014; Ratheesh et al., 2013). Interestingly, the RhoA-GAP DLC3 and the Rac1-GEF β-

PIX interact with Scribble to locally control RhoA and Rac activity along the lateral membrane of epithelial cells (Audebert et al., 2004; Hendrick et al., 2016). It is possible that SGEF’s interaction with Scribble and Dlg1 also contributes to the regulation of the balance of the different GTPases that function at cell junctions and control contractility.

This can be mediated by RhoG, which can act both upstream and in parallel of Rac1 and

Cdc42 (Brugnera et al., 2002; Gauthier-Rouviere et al., 1998; Katoh and Negishi, 2003;

Samson et al., 2010; Wennerberg et al., 2002). Further studies are needed to understand the molecular mechanisms that control these processes.

Contrary to our expectations, silencing SGEF does not affect apicobasal polarity when tested in 3D cysts. However, in the absence of SGEF, MDCK cysts display two distinct and very severe phenotypes. First, instead of a single central lumen, most SGEF KD cysts display multiple lumens, and, second, most of these lumens are either completely closed or very small. These results agree with previous RNAi screen which identified SGEF as one of 5 human RhoGEFs with defects in lumen formation (Zhang et al., 2015). The formation of cysts with multiple lumens may be caused by the loss of E-cadherin, as previous work has shown that E-cadherin KD cysts have multiple small lumens (Jia et al., 2011). ZO-1, which is also reduced in SGEF KD, may also contribute to the phenotype, as the average number of lumens increase in ZO-1-depleted cysts (Odenwald et al., 2017). Additionally,

Dlg1 KD also promotes multiple lumens, suggesting that the interaction between SGEF

91 and Dlg1 may be important to specify a single lumen (Awad et al., 2013). Second, the formation of an open lumen is severely impaired in SGEF KD cysts, which form either very small or completely closed lumens. In MDCK cells grown in matrigel, the formation of an open lumen requires the polarized delivery of secretory vesicles which are targeted to the site of lumen initiation where they fuse with the plasma membrane (Sigurbjornsdottir et al., 2014). The mechanism by which SGEF regulates the formation of an open lumen is not known, but previous work suggest it may be related to its ability to bind Scribble, as

Scribble function has been associated with lumen opening. When Scribble is silenced, cysts form with closed lumens in vitro (Hendrick et al., 2016; Yates et al., 2013), and in vivo, lungs from the Scribble mouse mutant Circletail (Crc) are abnormally shaped with fewer airways, that often lack a visible, open lumen (Yates et al., 2013). Again, as observed with the results in 2D monolayers, the scaffolding and catalytic activity of SGEF seem to independently regulate the two main cysts phenotypes observed; the scaffolding activity is sufficient to mediate lumen opening, whereas the catalytic activity is required for the regulation of lumen number.

Chapter 5

Conclusions and Future Perspective

5.1 Conclusions

In this study we show that SGEF, a RhoG specific GEF forms a ternary complex with two members of the Scribble polarity complex, Scribble and Dlg1, which do not interact directly otherwise. We identified two separate roles of SGEF in epithelial cells: 1- SGEF ternary complex with Scribble and Dlg1 regulates apical actomyosin contractility and architecture of TJs, 2- SGEF exchange activity regulating RhoG GTPase for the proper architecture of AJ.

Firstly, we mapped the binding domains of the ternary complex formation by truncation mutant analysis. Our studies showed that SGEF utilizes a novel internal PDZ Binding

Motif (PBM) to interact with PDZ1 of Scribble. At the same time SGEF can interact with

GUK domain of Dlg1 utilizing an unstructured 30 aa motif at its N-terminus. We also crystallized the SGEF peptide in complex with PDZ1 of Scribble. This crystal structure provided many new insights into PDZ domain-PDZ Binding Motif interaction field, that were previously unknown.

Next, we found that SGEF localizes to cell-cell junctions of epithelial cells in a Scribble dependent fashion. Our results imply that during junction formation Scribble localizes at junction first followed by SGEF and then Dlg1. RhoG also localizes to junction where possibly it gets activated by SGEF. SGEF targeting to junctions is important as in the absence of SGEF, architecture of both AJs as well as TJs is disorganized. In MDCK cells, a knockdown of SGEF leads a reduction in total ZO-1 levels at TJs. The architecture of

TJs also gets disorganized such that otherwise normal curvilinear TJ are replaced by straight junctions along with increased permeability of epithelial sheet when SGEF is silenced. Straightening of apical TJs is accompanied by increased Myosin IIB localization to apical actin belt. Myosin IIB along with actin increases tension and contractility in SGEF

KD cells. The straight junction phenotype as well as actomyosin contractility can be rescued by full length WT-SGEF. Interestingly, only the scaffolding activity of SGEF to form a ternary complex with Scribble and Dlg1 are required for the rescue of straight junction and apical contractility phenotype.

At the adherens junction, silencing of SGEF leads to disorganization of lateral membrane of MDCK cells. This is evident by an almost complete loss of E-cadherin, not only from junctions but a reduction in total E-cadherin levels in cells silenced for SGEF. As E- cadherin binds α and β-catenins at its C-terminus, loss in E-cadherin also leads to diffused localization of β-catenin and p120-catenin at junctions in SGEF KD cells rather than precise localization in CTRL cells. Further, silencing of SGEF leads to a dramatic collapse of lateral membrane with cells acquiring a flat phenotype displayed by reduction in height.

As an indirect consequence of lateral AJ disorganization, localizations of Scribble and Dlg1 are also diffused at lateral membrane. Interestingly, only full length SGEF with catalytic activity could rescue this loss of function phenotype displayed by loss of E-cadherin and disorganized adherens junction. It is not evident if the stabilization of AJ requires both the scaffolding activity and the exchange activity of SGEF but it is clear that the exchange activity of SGEF to activate RhoG at junctions is required for stabilization of E-cadherin based AJ in epithelial cells. It will be interesting to understand if the C-terminal PDZ tail of SGEF has any role in stabilization of junctions in epithelial cells.

On the other hand, overexpression of SGEF in live Xenopus embryos displays increased apical contractility and reduced apical cell area. This increase in contractility is due to increased actin and myosin localization at apical and medial-apical planes of SGEF OE embryos. Overexpression of SGEF also demonstrates an increased localization of vinculin at junctions. Vinculin is a protein that gets recruited at junction by a-catenin during increased tension. This provides useful information that SGEF overexpressing cells have increased tension thus recruiting Vinculin to apical junctions.

We also analyzed the role of SGEF in 3D MDCK cysts formation. Apparently, SGEF does not play a role in maintaining polarity of MDCK cells as the polarity markers gp135 still localizes to apical membrane domain whereas β-catenin still localizes to basolateral membrane domain, although disorganized. However, our results demonstrate that SGEF regulates lumen formation such that in absence of SGEF MDCK cyst have multiple or no lumen. Interestingly, the catalytic activity of SGEF regulates a single lumen formation whereas its scaffolding activity regulates openness of lumen or fluid filled lumen. This provides a clue for the role of SGEF in transport of fluid across the membrane to form fluid filled hollow lumen.

Overall, this study demonstrate that SGEF is an important part of Scribble polarity complex regulating both, the establishment of epithelial TJ and AJ as well as actomyosin based contractility. The scaffolding activity of SGEF as well as its exchange activity plays an important role for its function in epithelial cells. It needs further investigation to identify upstream and downstream regulators of SGEF and RhoG.

5.2 Future directions

To our knowledge this is the first study showing the involvement of SGEF in junction formation and contractility in epithelial cells. There are many unanswered questions that require further investigation.

To begin with, we showed that SGEF has a classical C-terminus PDZ binding tail that does not interact with any of the 7 PDZ domains encoded by Scribble and Dlg1, suggesting it may function to recruit other interacting partners to the Scribble complex. It would be interesting to characterize the binding partners of SGEF C-terminus PBM and also if any of those binding partners of SGEF C-terminus PBM are involved in maintenance of polarity and junctions in epithelial cells.

Also, we showed that SGEF forms a ternary complex with Scribble and Dlg1. As Lgl is also a part of Scribble polarity complex it is tempting to speculate that SGEF could further form a Scribble/Dlg1/Lgl/SGEF quaternary complex.

We narrowed down the GUK domain of Dlg1 binding site in SGEF to 30 amino acids (300-

330 aa) at the N-terminus of SGEF. The GUK domain of Dlg1 has been shown to bind peptides rich in Serine residues with a consensus sequence -RRXS- (where X is any amino acid). Interestingly, the GUK binding domain of SGEF is also rich in Serines with a putative inverted consensus sequence -SXRR- at its N-terminus. It will be interesting to understand if any of these conserved residues are important for the interaction. Also, it has been shown that Serine residues in LGN peptide gets phosphorylated and this phosphorylation plays an important role in its interaction with Dlg1 GUK domain. It will be interesting to unravel the phosphorylation status of Serine residues in SGEF peptide. So it will be of interest to identify kinase and phosphatase that could phosphorylate or dephosphorylate SGEF respectively. This could add one more step of regulation of SGEF

96 activity in vivo. Further, we can find out if this phosphorylation plays any role in ternary complex formation with Scribble and Dlg1, junctional architecture and polarity of epithelial cells.

The mammalian protein NHS has also been shown to bind both Scribble and Dlg1, not at the same time though. There is an intriguing possibility that NHS also forms a ternary complex with Scribble and Dlg1. This raises the possibility of two ternary complexes of

Scribble and Dlg1 with either SGEF or NHS, co-existing in epithelial cells. Further, we can investigate if these two ternary complexes co-exist or they form complexes at different times during junction formation, probably at different location in epithelial cells.

The SGEF KD cells showed a loss of E-cadherin. This could be one of the intermediate stages of EMT. It will be interesting to find out if SGEF KD cells show characteristics of partial EMT by looking at other markers for EMT (reviewed in (Lamouille et al., 2014;

Wu et al., 2016)). Further, it will be interesting to understand if silencing of Scribble/Dlg1 could push SGEF KD towards more mesenchymal phenotype.

As RhoG activation by SGEF is required for the junctional architecture, it is tempting to speculate that the knockdown/knockout of RhoG should disrupt AJ architecture in a similar way as SGEF KD but not the apical contractility. Also, Constitutively active and dominant negative versions of RhoG could be helpful in gaining further insights into this complex mechanistic pathway of junctions’ establishment in epithelial cells. A biosensor of RhoG could serve as a great tool for investigating spatiotemporal activation of RhoG during junctions’ establishment.

To understand the role of RhoG in junctions, a screen to characterize other RhoG effectors acting downstream of RhoG in regulating junctional architecture will be helpful. Moreover,

97 characterization of GAPS for RhoG that regulates its activity at junctions will be helpful in understanding its spatiotemporal regulation at junctions.

It is still not clear how a knockdown of SGEF recruits Myosin IIB to apical tight junctions and an overexpression of SGEF also recruits myosin IIB to apical junctional complex and to regulate contractility. It will be interesting to understand mechanism of this regulation of myosin IIB at apical domain of epithelial cells by SGEF.

Lastly, a knockdown of Scribble, Dlg1 or Lgl in mammalian cells does not show a severe phenotype (Choi et al., 2016; Fanning et al., 2012; Hendrick et al., 2016; Laprise et al.,

2004; Qin et al., 2005) displayed by knock down of these proteins in live Drosophila or C. elegans system. To understand the role of these proteins precisely CRISPR/Cas9 can be used to knockout Scribble, Dlg1 or Lgl in mammalian epithelial cells. further, a knockout of these proteins in SGEF KD background will provide valuable information for their role as scaffolds in epithelial junction and polarity.

As SGEF is important for lumenogenesis in MDCK 3D cysts grown in matrigel, it will be interesting to understand how SGEF regulates a hollow single lumen formation. The role of SGEF in vesicular trafficking, vesicle fusion and transport of vesicles across the plasma membrane will be useful in understanding lumenogenesis.

Author Contribution

Sahezeel Awadia designed and performed experiments for pulldown analysis and

Immunofluorescence of MDCK cells, western blots, TEER and quantifications. Torey

Arnold and Farah Huq performed Xenopus experiments, Titus Hou and Young Joo Sun performed Crystallography and NMR studies, Silvia Goicoechea performed RhoG assays

98 and western blots for total protein levels, Gabriel Kreider-Letterman helped in immunofluorescence and western blots in MDCK cells.

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