The Role of Adenomatous Polyposis Coli in Slit2/Robo1 Signaling and Actin Remodeling

Home , Dynamin, Myosin light chain, Talin (protein)

Frank (Bo Wen) Pang

A thesis submitted in conformity with the requirements for the degree of Master of Medical Science

Institute of Medical Science University of Toronto

Frank Bo Wen Pang

Master of Science

Institute of Medical Science University of Toronto

2019 Abstract

A hallmark of many pathogenic diseases is localized inflammation and influx of leukocytes. The neuronal guidance cue, Slit2, acting through receptor, Robo1, constitute a signaling pathway which has inhibitory effects on leukocyte chemotaxis and adhesion by regulating Rho family

GTPase activity. To further elucidate the Slit2/Robo1 signaling pathway, a BioID assay was performed and the protein adenomatous polyposis coli (APC) was found to differentially associate with Robo1 in the presence/absence of Slit2. APC regulates Rho family GTPase activity and in cellular functions such as migration, adhesion, and spreading. We found that APC knockdown inhibited monocyte chemotaxis and adhesion on endothelial cells to the same extent as control monocytes exposed to Slit2. Additionally, APC overexpression in COS-7 cells reversed inhibitory effects of Slit2 on cell spreading. suggesting that APC functions downstream of Slit2/Robo1 signaling, and may promote a vital first step in Slit2/Robo1-induced modulation of actin remodeling.

II Acknowledgments

I am most grateful to my supervisor/mentor Dr. Lisa Robinson for her constant encouragement, guidance support through the two years. Lisa’s in-depth knowledge in science, her positive energy, and caring personality have been a constant source of inspiration and reminder to strive to be the best possible. I would also like to thank the members of my program advisory committee, Dr. Gregory Fairn, Dr. Peter Kim and Dr. Warren Lee for their encouragement, advice, and constructive criticism. I am lucky to have worked with the incredible individuals that make up the Robinson lab, and am thankful for their continued support. In particular, I am grateful to Dr. Yi Wei Huang and Dr. Vikrant Bholse for all of their help and guidance. I would also like to acknowledge the friendly environment on the 19th floor, which allowed me to meet and connect with so many wonderful researchers. I would lastly like to thank my parents for their love and support throughout my life.

III Data Attribution

The experiments presented here were performed in collaboration with a number of individuals.

The purification of Slit2 was performed by Dr. Guang Ying Liu. Preliminary BioID results were obtained in collaboration with Dr. Brian Raught with help from Dr. Yi Wei Huang and subsequent analysis was performed by Dr. Vikrant Bhosle. The APC-GFP plasmid was generously gifted by Dr. Angela Barth (Stanford University). I performed all experimental protocols and data analysis for results shown.

IV TABLE OF CONTENTS

ACKNOWLDGEMENTS ...... III DATA ATTRIBUTION ...... IV LIST OF ABREVIATIONS ...... VII LIST OF FIGURES...... X CHAPTER 1: INTRODUCTION

1.1 INFLAMMATION ...... 1 1.1.1 Leukocyte trafficking and tissue infiltration ...... 1 1.1.2 The monocyte ...... 2 1.1.3 Chemotaxis ...... 3 1.1.4 Chemoattractants...... 8 1.1.5 Chemokine receptor signaling ...... 9 1.1.6 Adhesion...... 13 1.1.7 Cell spreading ...... 17 1.2 RHO GTPASES...... 19 1.2.1 Structure and regulation ...... 19 1.2.2 Rho GTPases in actin cytoskeletal dynamics ...... 21 1.3 SLIT2/ROBO1 SIGNALING...... 24 1.3.1 Slit/Robo in development ...... 24 1.3.2 Slit/Robo in leukocyte trafficking ...... 25 1.3.3 Slit and Robo expression and structure ...... 25 1.3.4 Slit2/Robo1 signal transduction ...... 32 1.4 ADENOMATOUS POLYPOSIS COLI (APC) REGULATES CELL MIGRATION AND ADHESION ...... 35 1.4.1 Association of APC with Robo1 ...... 35 1.4.2 The initial discovery of APC ...... 38 1.4.3 Expression and structure of APC ...... 38 1.4.4 The role of APC in actin cytoskeletal remodeling ...... 44 1.5 RATIONALE, HYPOTHESIS & OBJECTIVES ...... 48 1.5.1 Rationale...... 48 1.5.2 Hypothesis ...... 49 1.5.3 Objectives ...... 49

CHAPTER 2: MATERIALS & METHODS

2.1 REAGENTS AND ANTIBODIES ...... 50 2.2 CELL CULTURE ...... 51 2.3 EXPRESSION AND PURIFICATION OF N-SLIT2 AND ΔD2-NSLIT2 ...... 51 2.4 CO-IMMUNOPRECIPITATION ...... 53 2.5 SIRNA TRANSFECTIONS ...... 53 2.6 DNA PLASMID TRANSFECTION ...... 54 2.7 IMMUNOBLOTTING ...... 54 2.8 TRANSWELL MIGRATION ASSAY ...... 55 2.9 ADHESION ...... 56 2.10 SPREADING ASSAY ...... 56 2.11 STATISTICAL ANALYSIS ...... 57

V CHAPTER 3: RESULTS

3.1 MONOCYTES AND COS-7 CELLS EXPRESS ROBO1 AND APC ...... 58 3.2 APC CONSTITUTIVELY ASSOCIATES WITH ROBO1 IN COS-7 CELLS AND APC/ROBO1 ASSOCIATION IS DECREASED AFTER N-SLIT2 TREATMENT...... 60 3.3 APC SILENCING INHIBITS CHEMOTAXIS OF U937 MONOCYTIC CELLS AND IS NOT FURTHER AFFECTED BY EXPOSURE TO N-SLIT2 ...... 64 3.4 ROBO1 SILENCING IN U937 MONOCYTIC CELLS RESULTS IN REDUCED CHEMOTAXIS THAT IS NOT FURTHER AFFECTED BY EXPOSURE TO N-SLIT2 ...... 72 3.5 APC SILENCING IN U937 CELLS INHIBITS THEIR ADHESION TO ACTIVATED ENDOTHELIAL CELLS ...... 76 3.6 N-SLIT2 TREATMENT AND APC SILENCING SIMILARLY INHIBIT SPREADING OF COS-7 CELLS ...... 79 3.7 APC OVEREXPRESSION IN COS-7 CELLS REVERSE N-SLIT2 INHIBITION OF CELL SPREADING ...... 83

CHAPTER 4: DISCUSSION, CONCLUSION & FUTURE DIRECTIONS 87

REFERENCES ...... 101

VI LIST OF ABBREVIATIONS

Abl Abelson Abs Antibodies ADAM A Disintegrin and Metalloproteinase domain-containing protein ANOVA Analysis of Variance APC Adenomatous Polyposis Coli AMER1 APC membrane recruitment protein 1 ArfGAP ADP-ribosylating factor GTPases activating protein ARHGEF4 Rho guanine nucleotide exchange factor 4 Arp2/3 Actin-related protein 2/3 Asef APC-stimulated guanine nucleotide exchange factor BirA Bifunctional Ligase BMDM Blood Monocyte Derived Macrophages CALDAG1 Calcium Diacylglycerol Guanine Nucleotide Exchange Factor I CC Cytoplasmic Conserved CCL2 C-C Motif Ligand 2 CCR2 C-C Motif Receptor 2 CNS Central Nervous System Co-IP Co-immunoprecipitation CXCL C-X-C Motif Chemokine Ligand CXCR C-X-C Motif Chemokine Receptor DAG Diacylglycerol DAPI 4’,6-diamidino-2-phenylindole DC Dendritic Cell Dvl Dishevelled DH Dbl Homology ECM Extracellular Matrix EGF Epidermal Growth Factor Ena Enabled F-actin Filamentous Actin FAP Familial Adenomatous Polyposis FBS Fetal Bovine Serum FKN Fractalkine FN Fibronectin GAP GTPase Activating Protein GAG Glycosaminoglycans GDI GDP Dissociation Inhibitors GDP Guanosine-Diphosphate

VII GDNF Glial Cell-Line Derived Neurotrophic Factor GEF Guanine Nucleotide Exchange Factor GPCR G-Protein-Coupled-Receptor GTP Guanosine-triphosphate GTPase Guanine Triphosphatase HEK293 Human Embryonic Kidney 293 Cell HRP Horse Radish Peroxidase HSC Hematopoeitc Stem Cell ICAM-1 Intercellular Adhesion Molecule-1 IF Immunofluorescence Ig Immunoglobulin IQGAP1 Ras GTPase-activating-like protein

IP3 Inositol (1,4,5)-triphosphate IRI Ischemia Reperfusion Injury KAP3 Kinesin Adaptor Protein 3 LFA-1 Leukocyte Function Associated Antigen-1 LRR Leucine Rich Repeat Mac-1 Macrophage Receptor 1, αMβ2-integrin MAPK Mitogen Activated Protein Kinase MCC Manders’ Colocalization Coefficient MCP-1 Monocyte Chemotactic Protein 1 mDia Mammalian Diaphanous Protien MLK Myosin Light-Chain Kinase MMP Matrix Metalloproteinase PAK Serine/threonine-protein kinase PBS Phosphate-Buffered Saline PBD PAK Binding Domain PCC Pearson’s Correlation Coefficient PFA Paraformaldehyde PH Pleckstrin Homology PI3K Phosphoinositide 3-kinase

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

PI(3,4,5)P3 Phosphatidylinositol (3,4,5)-triphosphate PIX PAK interacting exchange factor PKC Protein Kinase C PLC Phospholipase C PRR Pattern Recognition Receptor Robo Roundabout SD Standard Deviation

VIII SDF-1α Stromal Derived Factor 1α Slit Slit Guidance Ligand SPATA3 Spermatogenesis-associated protein 3 srGAP Slit-Robo GTPase Activating Protein TBS Tris-Buffered Saline TBS-T Tris-Buffered Saline with Tween TCF T Cell Factor Telo-HAEC Telomeric Human Aortic Endothelial Cell TM Transmembrane TNF-α Tumor Necrosis Factor α VCAM-1 Vascular Cell Adhesion Molecule-1 Vav1 Proto-oncogene vav VLA-4 Very Late Antigen-4 WASP Wiskott–Aldrich syndrome protein WAVE WASP-family verprolin homologous protein WIRS WRC Interacting Receptor Sequence

IX LIST OF FIGURES

FIGURE 1.1 CELL MIGRATION DEPENDS ON ACTIN FILAMENT STRUCTURES ...... 5 FIGURE 1.2 RHO GTPASES IN THE POLARIZED MONOCYTE ...... 7 FIGURE 1.3 GPCR INTRACELLULAR SIGNAL TRANSDUCTION ...... 11 FIGURE 1.4 LEUKOCYTE ADHESION CASCADE ...... 14 FIGURE 1.5 GPCR-DEPENDENT LFA1 ACTIVATION ...... 16 FIGURE 1.6 CELL SPREADING AND ATTACHMENT ...... 18 FIGURE 1.7 REGULATION OF RHO GTPASE ACTIVITY...... 20 FIGURE 1.8 LOCALIZATION AND FUNCTION OF ACTIN NUCLEATION FACTORS IN MAMMALIAN CELLS...... 23 FIGURE 1.9 PRIMARY STRUCTURE OF MAMMALIAN SLIT2 ...... 27 FIGURE 1.10 PRIMARY STRUCTURE OF MAMMALIAN ROBO RECEPTORS ...... 29 FIGURE 1.11 ROBO CLEAVAGE AND PROCESSING ...... 31 FIGURE 1.12 ROBO1/2 SIGNAL TRANSDUCTION ...... 34 FIGURE 1.13 ROBO1 CONSTRUCTS ...... 36 FIGURE 1.14 MODEL FOR BIOID APPLICATION ...... 37 FIGURE 1.15 ADENOMATOUS POLYPOSIS COLI DOMAINS, PROTEIN INTERACTORS AND FUNCTIONS ...... 39 FIGURE 1.16 THE Β-CATENIN DESTRUCTION COMPLEX ...... 42 FIGURE 1.17 ROLE OF THE IQGAP1-APC COMPLEX IN CELL POLARIZATION AND MIGRATION .. 46 FIGURE 2.1 STRUCTURE OF N-SLIT2 AND ΔD2-NSLIT2 ...... 52 FIGURE 3.1 MONOCYTES AND COS-7 CELLS EXPRESS ROBO1 AND APC ...... 58 FIGURE 3.2 APC CONSTITUTIVELY ASSOCIATES WITH ROBO1 IN COS-7 CELLS AND APC/ROBO1 ASSOCIATION IS DECREASED AFTER EXPOSURE TO N-SLIT2 ...... 62 FIGURE 3.3.1 APC SILENCING INHIBITS U937 MONOCYTE CHEMOTAXIS TOWARDS CXCL12 CHEMOKINE ...... 65 FIGURE 3.3.2 APC SILENCING INHIBITS U937 MONOCYTE CHEMOTAXIS TOWARDS CCL2 CHEMOKINE ...... 69 FIGURE 3.4 ROBO1 SILENCING IN U937 MONOCYTES REDUCES CHEMOTAXIS TOWARDS CXCL12 ...... 73 FIGURE 3.5 APC SILENCING IN U937 MONOCYTES REDUCES THEIR ADHESION TO TNF-Α ACTIVATED ENDOTHELIAL CELLS ...... 77 FIGURE 3.6 N-SLIT2 TREATMENT AND APC SILENCING SIMILARLY INHIBIT SPREADING OF COS-7 CELLS...... 80 FIGURE 3.7 APC OVEREXPRESSION REVERSES N-SLIT2 INHIBITION OF COS-7 CELL SPREADING ...... 84 FIGURE 4.1 PROPOSED MODEL FOR APC IN SLIT2/ROBO1 SIGNALING...... 94

X CHAPTER 1

INTRODUCTION

1.1 Inflammation

1.1.1 Leukocyte trafficking and tissue infiltration

A hallmark of many pathogenic diseases is the localized inflammation and associated influx of leukocytes. Inflammation is a localized response to injury or infection marked by capillary dilation, redness, heat and pain. Endothelial cells are activated by cytokines TNF-α and

Il-1β produced in the inflammatory response by injured or infected cells. In response, endothelial cells display enhanced surface levels of cell adhesion molecules such as intracellular adhesion molecule 1 (ICAM-1) and vascular cell adhesion molecule 1 (VCAM-1). They also secrete chemokines such as CC-chemokine ligand 2 (CCL2; monocyte chemoattractant protein 1 (MCP-

1)) and CX3CL1 (fractalkine) which promote recruitment of monocytes and other leukocytes that bear the corresponding chemokine receptors (Muller 2001; Muller, 2003).

The process of leukocyte infiltration into tissue begins with the adhesion cascade which captures circulating leukocytes from the blood stream. Leukocyte adhesion and spreading is facilitated by activated endothelial cells. Following transendothelial infiltration, leukocytes migrate towards the chemokine gradient, in a process called chemotaxis, to the site of disease.

Acute inflammation helps during infections to clear pathogens and is also an early response to tissue injury. However, continual long-term immune activation can lead to chronic inflammation and associated inflammatory diseases such as atherosclerosis and chronic kidney disease. A major player in the development of chronic inflammatory diseases is the monocyte (Ingersoll et al. 2011). Therefore, targeting monocyte recruitment, in a disease context dependent manner, can

1 potentially lead to therapeutics in inflammatory diseases where excessive cell recruitment is caused by an overactive immune response or improper resolution of an initial response which subsequently led to chronic leukocyte infiltration.

1.1.2 The Monocyte

All leukocytes are derived from multipotential hematopoietic stem cells (HSCs).

Monocytes belong in the subfamily of myeloid cells which are derived from the common myeloid progenitor. Mononuclear phagocytes are derived from dividing monoblasts in the bone marrow (Wiktor-Jedrzejczak & Gordon, 1996). They are released into systemic circulation as non-dividing monocytes and circulate for several days before entering tissues and replenishing macrophages and dendritic cells (Akagawa et al., 1996; Chapuis et al., 1997; Randolph et al.,

1998). Monocytes comprise a large reservoir of myeloid precursors since every day, half of all circulating monocytes leave the bloodstream under steady state conditions. Monocytes are most known to differentiate into macrophages when they infiltrate tissue, however, they can also form specialized cells such as DC, and tissue resident macrophages including microglia and osteoclasts (Hume et al., 2002). There is also evidence of tissue resident macrophages originating from the yolk sac during embryogenesis (Perdigeuro et al., 2014). Mononuclear phagocytes play a vital role in both the non-specific/innate and specific/adaptive immune responses. Monocyte differentiation into macrophages, which are capable of phagocytosing foreign pathogens, makes them arguably the biggest contributors to the innate immune response (Aderem & Underhill,

1999). They can also act as antigen presenting cells that interact with and activate T lymphocytes to trigger the induction of adaptive immune responses (Geissman et al., 2003).

Human monocytes have a “bean-shaped” nucleus and express CD11b, CD11c and CD14

(Muller, 2001). Circulating monocytes are morphologically heterogenous and comprise approximately 5-10% of peripheral blood leukocytes (van Furth & Cohn, 1968). Human monocytes can be categorized into three phenotypically and functionally distinct populations based on distinct expression of CD14 and CD16 (Zeigler-Heitbrock et al., 2010). Classical

(CD14++, CD16-) monocytes make up 80-90% of the human monocyte population, non-classical

(CD14+, CD16++) monocytes account for 2-10%, and intermediate (CD14++, CD16+) monocytes make up the remaining 2-5%. Though the smallest subpopulation, intermediate monocytes expand during inflammation and cytokine treatment (Zeigler-Heitbrock et al., 2010; Yang et al.,

2014).

Although monocytes routinely infiltrate tissue to replenish macrophages and DCs, their recruitment can be increased by pro-inflammatory, metabolic and immune stimuli (van Furth et al., 1985, Zhou & Tedder, 1996). Macrophages and DCs contribute to host defense against foreign pathogens and tissue homeostasis through clearance of senescent cells, tissue remodeling and repair following inflammation (Gordon et al., 1986; Gordon, 1998).

1.1.3 Chemotaxis

Chemotaxis is directed cell migration towards an external chemical gradient. Chemotaxis has been observed in many cell types including leukocytes during inflammation, endothelial cells during angiogenesis, spermatocytes during fertilization, and neurons during neurogenesis. All these examples help to highlight the biological significance of chemotaxis (Singer & Kupfer,

1986). Monocytes move by extending pseudopods. Chemotaxis begins first with polarization of

3 the cell in the direction along a chemoattractant gradient. Cell polarization results from preferential pseudopod extension towards areas of higher chemoattractant concentration

(Zigmond, 1974). Monocyte chemotaxis requires the coordination of motile activities between pseudopod formation at the leading edge of the cell and uropod retraction at the trailing edge.

During chemotaxis, monocytes extend short membrane protrusions called filopodia which are approximately 0.1-0.2 µm in diameter and up to 20 µm in length. Filopodial structures act as cellular tentacles which are further supported by a core bundle of actin filaments called microfilaments (Mattila & Lappalainen, 2008). In monocytes, filopodia support thin sheets of membrane-enclosed cytoplasm called lamellipodia which contain actin filaments and a meshwork of myosin II-associated microfilaments. In monocytes, as well as several other cell types, the actin network within lamellipodia, filopodia, and several other structural and regulatory proteins comprises the molecular motor driving cell migration (Jones et al., 1998)

(Figure 1.1). This process also works against cell-to-substratum adhesions called focal adhesions which utilize members of the integrin family proteins to link myosin-II containing bundles of cytoplasmic microfilaments, called stress fibers, to proteins in the extracellular matrix (ECM)

(Critchley et al., 1999). In monocytes, integrin-mediated contacts to the ECM are comprised of focal complexes and podosomes. Focal complexes are structurally similar to focal adhesions, but lack stress fibers (Allen et al., 1997). Podosomes are actin rich adhesive structures found in cells of monocytic myeloid lineage and stimulated endothelial cells (DeFife et al., 1999; Correia et al.,

1999; Moreau et al., 2003).

Figure 1.1 Cell migration depends on actin filament structures Motility is initiated by an actin-dependent protrusion of the leading edge, which is composed of lamellipodia and filopodia. These protrusive structures contain actin filaments, with elongating barbed ends orientated towards the plasma membrane. During cellular extension, new adhesions with the substratum are formed under the leading edge. Next, the nucleus and the cell body are translocated forward through actomyosin-based contraction forces that might be mediated by focal adhesion-linked stress fibres, which also mediate the attachment to the substratum. Then, retraction fibres pull the rear of the cell forward, adhesions at the rear of the cell disassemble and the trailing edge retracts. Reprinted with permission from Spring Nature, Mattila P. K., & Lappalainen, P. (2008). Filopodia: molecular architecture and cellular function. Nature Reviews Molecular Cell Biology, 9(6), 446-454. Copyright (2008)

Monocyte chemotaxis can be divided into several steps: actin-driven protrusion of lamellipodia and filopodia at the leading edge, adhesion of the leading edge to the ECM via integrin-mediated focal interactions, actomyosin-mediated cell contraction, release of contacts at the trailing edge of the cell and lastly, recycling of membrane receptors from the rear to the front end of the cell (Allen et al., 1998; Sheetz et al., 1999; Friedl, 2004; Friedl and Gilmour, 2008).

At the leading edge of migrating cells, Rac and Cdc42 activity drive formation of lamellipodia and filopodia respectively (Figure 1.2). At the cell rear, RhoA activity increases myosin light chain kinase (MLK) dependent activation of myosin, allowing for tail retraction (Figure 1.2).

This coordination of numerous molecular events is required for cell polarization and chemotaxis and is largely mediated by the actin cytoskeleton.

When circulating monocytes are exposed to chemoattractants, it leads to their activation and subsequent migration across the endothelial barrier toward the inflammatory focus. Binding of chemoattractant to its cell-surface receptor activates intracellular signaling cascades which mobilize actin cytoskeletal machinery. The generation and maintenance of cell polarity and actin cytoskeleton remodelling are necessary processes for efficient monocyte chemotaxis.

Figure 1.2 Rho GTPases in the Polarized Monocyte Rac and Cdc42 stimulate the polymerization of actin, a process necessary for the formation of lamellipodia and filopodia at the front of the cell. At the back of the cell, Rho-kinase phosphorylation results in inactivation of myosin light chain phosphatase, leading to increased myosin light-chain kinase (MLK) dependent activation of myosin. Reprinted with permission from University of Toronto, Mukovozov, I. (2010). Slit/Robo signaling in monocyte chemotaxis and function: a role in vascular inflammation. Copyright 2010.

1.1.4 Chemoattractants

Chemoattractants are a group of molecules whose gradient can be detected by leukocytes and promote directed migration of leukocytes. Several types of these mediators can recruit leukocytes towards inflammatory foci, including complement factors, bacterial components, leukotrienes and chemokines. The first chemoattractant identified was C5a, a cleaved product derived from the complement component C5 (Shin et al., 1968). Bacterial components include

N-formylpeptides such as N-formylethionyl-leucyl-phenylalanine (fMLP) which act to recruit leukocytes which express formyl peptide receptor 1 (FPR1), which include neutrophils, monocytes, eosinophils, and basophils to areas of inflammation (Williams et al., 2011).

Leukotrienes, another type of chemoattractant, are a family of eicosanoid inflammatory mediators produced in leukocytes through the oxidation of arachidonic acid (Salmon et al.,

1987).

Chemokines are a large family of small, structurally similar, peptides (Rossi et al., 2000).

They are a subcategory of cytokines whose main function is to direct chemotaxis. The majority of chemokines are secreted save for two, CX3CL1 (FKN) and CXC Chemokine Ligand 16

(CXCL16), which can exist in membrane bound and cleaved/secreted forms. In its secreted form,

CX3CL1 can potently chemoattractant leukocytes expressing its receptor CX3CR1 (Imai et al.,

1997).

Chemokine-induced signal transduction through different chemokine receptors are very similar and often redundant. The specific expression, regulation and receptor binding patterns are what dictate their functional diversity (Luster, 1998). Chemokines can be grouped into four

8 families based on the relative position of their N-terminal cysteine residues (Luster, 1998). The majority of chemokines contain four cysteine residues and fit within the α (CXC) or β (CC) chemokine families. The other two families each contain lone members. CX3CL1 is a lone member in its family because its N-terminal cysteine residues are separated by three amino acids.

Lymphotactin (XCL1), which makes up the fourth family, is a lymphocyte specific chemokine

(Kelner et al., 1994). Most chemokines bind to glycosaminoglycans (GAG) on the luminal surface of endothelial cells. This interaction keeps chemokines localized at the site of production and also enables formation of chemokine gradients for leukocyte recruitment. This is important as chemokine mutations that prevent their binding to GAG domains prevent leukocyte recruitment in vivo (Handel et al., 2005).

1.1.5 Chemokine Receptor Signaling

Chemokines interact with the G protein coupled receptors (GPCR) on the plasma membrane of monocytes/macrophages, leading to signal transduction by coupling to heterotrimeric G proteins. Heterotrimeric G proteins are composed of an α, β, and γ subunit. The

α subunit can bind to guanosine-diphosphate (GDP)/guanosine triphosphate (GTP). When bound to GDP, the α subunit interacts with β and γ subunits to form an inactive heterotrimer which binds to GPCR (Mellado et al., 2001). When the chemokine binds to GPCR, it induces a conformational change that causes the exchange of GDP to GTP on the α subunit. This causes dissociation of the α subunit from the receptor and release of the βγ heterodimer complex. This allows α and βγ subunits to freely interact with and modulate activity of target enzymes (Mellado et al., 2001). A few targets which are activated include phosphatidylinositol 3-kinase (PI3K), phospholipase C (PLC), mitogen activated protein kinase (MAPK), and adenyl cyclase (Figure

1.3). These enzymes generate secondary messengers that initiate a cascade if signaling events culminating in cytoskeletal rearrangement and leukocyte migration.

Phosphoinositide 3-kinase (PI3K) belongs to the lipid targeting subgroup of kinases and its role in chemokine receptor signaling has been thoroughly studied (Li et al., 2000; Sasaki et al., 2000; Hirsch et al., 2000, Servant et al., 2000; Jin et al., 2000; Kolsch et al., 2008). There are several isoforms of Class I PI3Ks in mammalian cells, but only a single Class IB variant has been show to interact with GPCR receptors in leukocytes (Andrews et al., 2007; Thorpe et al.,

2015). The outcome of class I PI3K activation is the phosphorylation of membrane PI(4,5)P2 which generates phosphatidylinositol (3,4,5)-triphosphate (PI(3,4,5)P3). The βγ complex also activates PI3Kγ, to generate PI(3,4,5)P3 from membrane PI(4,5)P2 (Krugmann et al., 1999). This results in recruitment of Ras guanosine triphosphates (GTPases) and activation of MAPK pathways (Kintscher et al., 2000).

Figure 1.3 GPCR Intracellular Signal Transduction Chemokine binding to G-protein coupled receptors (GPCR) induces conformational changes resulting in dissociation of α and βγ subunits. This leads to signaling across phospholipase C (PLC), phosphatidylinositol 3-kinase (PI3K), mitogen activated protein kinases (MAPKs) and Rho-family guanosine triphosphatases (Rho GTPases). Each contributes to generation of cell polarity and modulation of integrin avidity which enhances monocyte chemotaxis, spreading and adhesion. Reprinted with permission from University of Toronto, Mukovozov, I. (2010). Slit/Robo signaling in monocyte chemotaxis and function: a role in vascular inflammation. Copyright 2010.

After α subunit dissociation, the βγ complex activates PLC which cleaves phosphatidylinositol (4,5)-bisphosphate (PI(4,5,)P2) to generate inositol (1,4,5)-triphosphate (IP3) and diacylglycerol (DAG) (Figure 1.3). IP3 generation leads to mobilization of intracellular calcium stores from the endoplasmic reticulum, and coupled with DAG, activates protein kinase

C (PKC) (Li et al., 2000). PKC activation and recruitment to the plasma membrane promotes changes in the actin cytoskeleton that drive cell spreading and migration.

PI3K dependent activation of PIP3 at the cell membrane allows for recruitment of Rho

GTPases Rac and Cdc42 to the cell membrane (Ridley, 2001). The cell membrane localization of

PIP3, Rac, and Cdc42 stimulates the formation of lamellipodia and filipodia at the leading edge of migrating cells. Rho GTPases are critical regulators not only of cell migration, but also cell adhesion and spreading (Allen et al., 1997, Wojciak et al., 1999). At the back end of the cell,

Rho-kinase phosphorylation inactivates myosin light chain phosphatase, leading to increased myosin light-chain kinase (MLK) dependent activation of myosin (Nguyen et al., 1999). This results in formation of actomyosin bundles, contraction, and loss of adhesion from the substratum leading to tail retraction (Ridley, 2001; Bokoch, 2005). Cell polarity is maintained by the inhibition of leading-edge signals at the trailing edge (Fenteany & Glogauer, 2004).

Phosphatase and tensin homolog (PTEN) dephosphorylates PI(3,4,5)P3 to PI(4,5)P2 which regulates PIP3 levels (Vazquez & Devreotes, 2006). The absence of PIP3 at the trailing edge of the cell prevents activation and recruitment of Rho GTPases and subsequent actin polymerization. This allows for the formation of actomyosin bundles and tail retraction

(Worthylake & Burridge, 2001). The coordinated actin polymerization at the leading edge of cells partnered with tail retraction at the trailing edge allows for directed chemotactic migration.

1.1.6 Adhesion

Firm adhesion of monocytes to activated endothelium is required for tissue infiltration and is an integrin-dependent process (Ley et al., 2007). Integrins are large transmembrane glycoproteins that anchor a cell’s cytoskeleton to other cells or the ECM. Integrins form heterodimeric complexes composed of α and β subunits. The β2 family of integrins are only expressed in leukocytes and include Leukocyte Function Associated Antigen-1 (LFA-1 or

CD11a/CD18) and macrophage receptor 1 (MAC-1 or CD11b/CD18) which can bind both vascular cell adhesion molecule 2 (VCAM-2) and intracellular adhesion molecule 2 (ICAM-2)

(Figure 1.4). These are responsible for firm adhesion and arrest of monocytes on endothelial cells and later attachment with ECM components (Ley et al., 2007).

Chemokines are activators of integrin-mediated firm adhesion (Chan et al., 2003). GPCR stimulation leads to rapid signaling cascades that result in a net increase in average integrin affinity and valency (Chan et al., 2003; Carman & Springer., 2003). One effect of GPCR signaling cascade is the recruitment and activation of PLC. This leads to the production of IP3 and DAG followed by an increase in intracellular calcium. The combined calcium influx and

DAG activates guanine nucleotide exchange factors (GEFs), such as proto-oncogene Vav (Vav1) and calcium diacylglycerol guanine nucleotide exchange factor 1 (CALDAG1), which results in

Rho GTPase recruitment and activation (Constantin et al., 2000; Vielkind et al., 2005; Crittenden et al., 2004). Thus, Rho GTPase activity is involved in signaling cascades that connect GPCR activation to changes in integrin affinity and valency. A specific example is chemokine-mediated conformational changes

Figure 1.4 Leukocyte adhesion cascade The original three steps are shown in bold: rolling, which is mediated by selectins, activation, which is mediated by chemokines, and arrest, which is mediated by integrins. Progress has been made in defining additional steps: capture (or tethering), slow rolling, adhesion strengthening and spreading, intravascular crawling, and paracellular and transcellular transmigration. Key molecules involved in each step are indicated in boxes. ESAM, endothelial cell-selective adhesion molecule; ICAM1, intercellular adhesion molecule 1; JAM, junctional adhesion molecule; LFA1, lymphocyte function-associated antigen 1 (also known as αL β2 -integrin); MAC1, macrophage antigen 1; MADCAM1, mucosal vascular addressin cell-adhesion molecule 1; PSGL1, P-selectin glycoprotein ligand 1; PECAM1, platelet/endothelial-cell adhesion molecule 1; PI3K, phosphoinositide 3-kinase; VCAM1, vascular cell-adhesion molecule 1; VLA4, very late antigen 4 (also known as α4 β1 -integrin). Reprinted with permission from Spring Nature, Ley K., et al. (2007). Getting to the site of inflammation: the leukocyte adhesion cascade updated. Nature Reviews Immunology, 7(9), 678-689. Copyright (2007)

14 in LFA-1 which is induced by Rho GTPases RhoA and Rap-1 (Giagulli et al., 2004; Shimonaka et al., 2003). Activated Rho GTPases associate with actin binding proteins such as talin-1 and alpha-actinin to modulate integrin affinity (Sampath et al., 1998; Jones et al., 1998; Brakebusch

& Fassler, 2003) (Figure 1.5). This ultimately shows that chemokine-mediated changes in integrin affinity are dependent on interactions with the actin cytoskeleton.

Outside of GPCR signaling, the binding of ligands to integrins also induces signaling cascades (Ley et al., 2007). This outside-in signaling cascade is in contrast to GPCR signaling which is inside-out. The scaffolding protein paxillin binds integrins. Ligand-induced integrin clustering activates Src-family kinases which phosphorylate paxillin leading to the recruitment of downstream effectors such as ADP-ribosylating factor GTPases activating protein (ArfGAP) and

PAK interacting exchange factor (PIX). ArfGAP can inactivate Rac GTPases while PIX activates GTPase Cdc42 (DeMali et al., 2003). Src-family kinases also activate Vav1, a Cdc42 and Rac GEF (DeMali et al., 2003). This shows that Rho GTPases are required for the mobilization of the actin cytoskeleton to form and maintain adhesive contacts. Both GPCR- mediated and ligand-induced signaling cascades contribute to this.

Figure 1.5 GPCR-dependent LFA1 activation A putative intracellular signalling cascade from G-protein-coupled receptors (GPCRs) to affinity activation of lymphocyte function-associated antigen 1 (LFA1; also known as αL β2 -integrin) or very late antigen 4 (VLA4; also known as α4 β1 -integrin) in leukocytes, based on studies in leukocytes, platelets and transfected cell lines. Candidate molecules that remain to be confirmed are shown with question marks. The G-protein βγ subunit of the GPCR activates phospholipase C (PLC), which cleaves phosphatidylinositol-4,5- bisphosphate (PtdIns(4,5)P2 ) to produce inositol-1,4,5- trisphosphate (InsP3 ) and diacylglycerol (DAG). InsP3 triggers Ca2+ release from intracellular stores in the endoplasmic reticulum (ER), which in turn triggers Ca2+ influx from the extracellular space through the CRAC (calcium-regulated activated calcium) channel. Ca2+ activates calmodulin and may trigger one or more guanine-nucleotide-exchange factors (GEFs), such as calcium- and DAG-regulated GEFI (CALDAG-GEFI), dedicator of cytokinesis 2 (DOCK2) and VAV1. These may in turn trigger small guanosine triphosphate hydrolase enzymes (GTPases), such as RAS-related protein 1 (RAP1), RAS homologue gene-family member A (RHOA) or RAS-related C3 botulinum substrate (RAC). Finally, integrin-binding proteins activate integrins. Candidates are talin-1, alpha- actinin and L-plastin. Alternative intermediate effectors include regulator of cell adhesion and polarization enriched in lymphoid tissues (RAPL), RAP1-GTP-interacting adaptor molecule (RIAM), and cytohesin-1. It is likely that there are differences between leukocyte types, species (humans versus mice) and integrins. Reprinted with permission from Spring Nature, Ley K., et al. (2007). Getting to the site of inflammation: the leukocyte adhesion cascade updated. Nature Reviews Immunology, 7(9), 678-689. Copyright (2007).

1.1.7 Cell Spreading

Cell spreading is the process of a cell forming passive adhesive contacts followed by cellular protrusions and extension on to the extra-cellular matrix (ECM). Much like cell adhesion and migration, cell spreading is also regulated by Rho GTPase activity (Wojciak-Stothard et al.,

1999). Cell spreading begins with cell contact to the ECM (Figure 1.6) and formation of integrin mediated focal adhesions (Cavalcanti-Adam et al., 2007; Frame & Norman, 2008). As more adhesions form, cells begin to flatten out onto the ECM and form membrane protrusions composed to lamellipodia and filopodia (Jones et al., 1998; Cavalcanti-Adam et al., 2007). Once cells are sufficiently adhered onto the ECM, they can go from passive cell spreading to active directed cell migration.

Monocyte cell spreading helps to stabilize adhesion to endothelial cells and is promoted by chemotactic stimuli (Cianciola et al., 1981). The receptor C-C motif receptor 5 (CCR5) on monocytes facilitates cell spreading ultimately leading to monocyte tissue infiltration and monocyte polarization ((Gerhardt & Ley, 2015; Montresor et al., 2012). Monocyte spreading capacity is also reflective of the cellular activation status. For example, Leishmania infection prevents monocyte activation and results in decreased monocyte spreading capacity when compared to uninfected monocytes (Figueira et al., 2015).

Cell spreading also has functional significance in macrophages (Patel et al., 2012). The capacity of macrophages to undergo phagocytosis is largely dependent on the extent of cell surface area resulting from spreading (Dupuy & Caron, 2008; Sarkar et al., 2005). Macrophages

17 that do not spread have a lower phagocytic capacity and are less capable of clearing pathogens

(Dupuy & Caron, 2008; Dias et al., 2016).

Figure 1.6 Cell spreading and attachment The early stages of cell spreading involve cell contact with the matrix and establishment of focal adhesions. Later stages involve membrane protrusion formation through actin polymerization and myosin contractions, and further focal adhesion establishment. Lastly, cells begin directed movement (crawling). The physics of early spreading are captured by a simple model balancing substrate adhesive power with the energy dissipated as the actin cortex deforms. Reprinted with permission from Elsevier, McGrath J. (2007). Cell spreading: the power to simplify. Current Biology, 17(10), 357-358. Copyright (2007).

1.2 Rho GTPases Rac and Cdc42

1.2.1 Structure and Regulation

Rho GTPases are a subfamily of the Ras superfamily of GTPases. The Rho GTPases control actin cytoskeletal dynamics. To date, over 20 Rho family GTPases have been characterized and they can be grouped into 6 different classes: Rac (Rac1, Rac2, Rac3, RhoG),

Rho (RhoA, RhoB, RhoC), Cdc42 (Cdc42Hs, G25K, TC10), Rnd (Rnd1, Rnd2, Rnd3), RhoD, and TFF (Aspenstrom, 1999; Kjoller et al., 1999). All Rho GTPases contain two main structural domains: a C-terminal ‘CAAX’ motif and a catalytic domain. The C-terminal domain can attach to membrane lipids to facilitate membrane association and subcellular localization of Rho

GTPases (Gutierrez et al., 1989; Casey et al., 1989; Fujiyama et al., 1990). The catalytic domain contains two regions (switch I and switch II) which correspond to different structural conformations in the GTP-bound and GDP-bound forms.

Rho GTPases function as molecular switches by cycling between GTP-bound and GDP- bound forms. The activity of Rho GTPases is regulated by three classes of molecules: guanine exchange factors (GEFs), GTPase activating proteins (GAPs), and GDP dissociation inhibitors

(GDIs) (Figure 1.7). When bound to GDP, GTPases are in an inactive state. GEFs facilitate the exchange of bound GDP to GTP, switching GTPases into their active states (Figure 1.7). Many

GEFs have been identified and they are characterized by the presence of a Dbl homology domain

(DH), which is capable of interacting with both switch I and switch II regions to catalyze exchange of GDP to GTP (Rossman et al., 2005). Many GEFs, such as Vav, contain a pleckstrin homology domain (PH) which allows them to bind phosphoinositides such as PIP3. This allows

Figure 1.7 Regulation of Rho GTPase Activity

This figure shows a Rho GTPase anchored to the plasma membrane. The Rho GTPase can bind either GDP or GTP. Guanine nucleotide exchange factors (GEFs) facilitate the exchange of GDP to GTP, activating the Rho GTPase. GTP-bound Rho GTPases transduce signals by binding to effector proteins. GTPase activating proteins (GAPs) increase intrinsic GTPase activity causing

GTP hydrolysis to GDP and phosphate (Pi) to occur at a faster rate. GDP-bound Rho GTPase can be sequestered by GDP dissociation inhibitors (GDIs), which bind to the GTPase, preventing plasma membrane anchoring. Reprinted with permission from Macmillan Publishers Ltd: Nature

Reviews Immunology, Tybulewicz, V. L., & Henderson, R. B. (2009). Rho family GTPases and their regulators in lymphocytes. Nature Reviews Immunology, 9(9), 630-644. Copyright (2009)

GEFs to localize to the plasma membrane where they can act locally. Thus, GEFs promote Rho

GTPase activation and facilitates their interaction with downstream effectors. The rate of GTP hydrolysis by Rho GTPases alone is relatively slow. GAPs increase the rate of hydrolysis of bound-GTP to GDP and phosphate by several orders of magnitude (Vetter et al., 2001). This results in inactivation of Rho GTPases. There have been over 30 GAPs found in humans and there exists a large diversity in primary sequences between them (Tcherkezian et al., 2007).

However, they all contain a Rho GAP domain with a conserved tertiary structure composed of α- helices and a catalytically critical ‘arginine finger’ which stabilizes formation of the transition state during GTP hydrolysis (Nassar et al., 1998). Much like GEFs, GAPs can interact with both the switch I and switch II regions on Rho GTPases which increases rate of hydrolysis of GTP resulting in Rho GTPase inactivation (Gamblin & Smerdon, 1998). Lastly, GDIs associate with

GDP-bound Rho GTPases to inhibit their activation be GEFs. GDIs have also been shown to be capable of binding to GTP-bound Rho GTPases to suppress their activity (Olofsson, 1999). In summary, the activation of Rho GTPases is regulated by outside-in signals from a variety of receptors including GPCRs, tyrosine kinase receptors, cytokine receptors and adhesion receptors.

1.2.2 Rho GTPases in Actin Cytoskeletal Dynamics

Motile cells induce actin polymerization in response to chemoattractants and Rho

GTPase activation (Carson et al., 1986; Hall et al., 1989; Howard et al., 1984). Cell migration relies on coordinated membrane protrusion at the leading edge of cells via lamellipodia and filopodia, and the retraction of the uropod at the trailing edge of the cell. Lamellipodia and filopodia extension at the leading edge requires rapid turnover of actin filaments (Symons et al.,

1991; Wang, 1985). Actin nucleation of F-actin is promoted by formins which are activated by

21 select Rho GTPases (Sit & Manser, 2011; Nicholson-Dykstra et al., 2005). The formin mammalian diaphanous protein 1 (mDia1) can only be activated by RhoA while mDia2 and mDia3 and can also be activated by Rac1 and Cdc42 (Lammers et al., 2008). Rac1 and Cdc42 also interact with Wiskott–Aldrich syndrome protein (WASP) and WASP-family verprolin homologous protein (WAVE) complexes which both drive actin related protein2/3 (Arp2/3) mediated F-actin branching (Figure 1.8; Kurisu & Takenawa, 2009). More stable actin-myosin cables can be found in the middle and rear of the cell (DeBiasio et al., 1998). Furthermore, other factors such as recycling of the plasma membrane and integrin-mediated adhesion are important for cell motility (Martenson et al., 1993; Yamada et al., 1995; Bretscher, 1996; Mitra et al.,

2005). In adhesion, Rho GTPases RhoA and Rap-1 can induce chemokine-mediated conformational changes in LFA-1 (Giagulli et al., 2004; Shimonaka et al., 2003). Activated Rho

GTPases associate with talin-1 and alpha-actinin to modulate integrin affinity to promote cellular adhesion (Sampath et al., 1998; Jones et al., 1998; Brakebusch & Fassler, 2003). Similarly in cell spreading, formation of focal adhesions is dependent on integrin affinity and valency

(Cavalcanti-Adam et al., 2007) All of these processes are dependent on coordinated mobilization of actin cytoskeleton, and are regulated by deployment of actin-binding proteins by activated

Rho GTPases.

Figure 1.8 Localization and function of actin nucleation factors in mammalian cells. Filamentous actin (F-actin; red) is nucleated and organized into branched networks by the actin- related protein 2/3 (ARP2/3) complex and its nucleation promoting factors, or is generated in unbranched forms by formins and tandem WASP homology 2 (WH2) domain-containing nucleators. Functional roles for different nucleation factors in a generic mammalian cell are depicted during phagocytosis, cell junction assembly, endocytosis, membrane ruffling and lamellipodia dynamics, filopodia formation, Golgi and tubulovesicular membrane dynamics, and stress fibre formation. Question marks indicate that the precise role for the nucleation factor is unclear. COBL, cordon-bleu; DAAM, Dishevelled-associated activator of morphogenesis; FHOD, formin homology domain; FMN, formin; FRL, formin-related in leukocytes; INF, inverted formin; JMY, junction-mediating regulatory protein; mDIA, murine Diaphanous; N- WASP, neuronal-WASP; WASP, Wiskott–Aldrich syndrome protein; WASH, WASP and SCAR homologue; WAVE, WASP-family verprolin homologue; WHAMM, WASP homologue associated with actin, membranes and microtubules. Reprinted with permission from Macmillan Publisher Ltd. Campellone, K. G., & Welch, M. D. (2010). A nucleator arms race: cellular control of actin assembly. Nature Reviews Molecular Biology, 11(4), 237-251, copyright 2010.

1.3 Slit2/Robo1 Signaling

1.3.1 Slit/Robo in development

Slits are a family of secreted proteins, which, together with their cell-surface Roundabout

(Robo) receptors, act to repel neurons during CNS development. After commissural axons cross the midline, midline glial cells express Slit to prevent axons from re-crossing the midline.

Drosophila that express mutant Slit proteins exhibit midline defects such as collapse of the regular scaffold of commissural and longitudinal axon tracts in the embryonic CNS (Rothberg et al., 1988; Rothberg et al., 1990). A similar defect was observed in Robo mutants, where projecting axon tracts would repeatedly cross the midline (Kidd et al., 1998).

Later studies have also shown a role for Slit/Robo signaling as guidance cues outside of the CNS in development. An example in Drosophila is mesoderm migration where myocyte precursors migrate away from the midline towards peripheral target sites where they fuse to form muscle fibers. In both Slit and Robo mutants, these cells do not migrate away from the midline, but instead fuse across it (Rothberg et al., 1990). Another example is pancreatic islet formation in mice where evidence has shown that Slit/Robo in signaling β-cells is required for proper formation and continual maintenance pancreatic islet architecture (Adams et al., 2018). Slit/Robo signaling in β-cells is also vital for their survival and function (Yang et al., 2013). Additionally,

Slit/Robo signaling plays a role in nephrogenesis. The proper development of the kidney depends on the formation of a structure called the ureteric bud. Formation requires the secretion of glial line-derived neurotrophic factor (GDNF) by mesenchymal cells. Both Slit and Robo knockout mice display abnormal patterns of GDNF secretion likely due to its dysregulated transcription.

Thse mice develop multiple ureteric buds and urinary collecting systems, showing the

24 importance of Slit/Robo signaling in nephrogenesis (Grieshammer et al., 2004; Liu et al., 2003).

Furthermore, mutations in Slit/Robo signaling pathway genes Slit2 and Slit Robo GTPase

Activating Protein 1 (srGAP1) were shown to confer risk for congenital abnormalities of the kidney and urinary tract (Hwang et al., 2015). In conclusion, Slit and Robo both play an important role in normal development outside of the CNS.

1.3.2 Slit2/Robo1 in Leukocyte Trafficking

The first study to demonstrate Slit-mediated inhibition of leukocyte migration utilized transwell migration assays with rat lymph node cells migrating towards CXCL12 and neutrophil- like HL-60 cells towards fMLP (Wu et al., 2001). Later studies have shown Slit2-mediated inhibition of chemotactic migration with neutrophils, macrophages, DCs, and T lymphocytes

(Tole et al., 2009; Kanellis et al., 2004; Guan et al., 2003; Prasad et al., 2007). More recently,

Slit2 was also shown in vitro by our group to have a dose dependent inhibition of monocyte chemotaxis towards chemoattractants monocyte chemotactic protein 1 (MCP-1) and stromal derived factor-1α (SDF-1α) (Mukovozov et al., 2015). Slit2 also inhibits monocyte post-adhesion stabilization of monocytes on TNF-α activated endothelial cells by inhibiting activation of Rac1

(Mukovozov et al., 2015). Therefore, Slit2 may have a therapeutic role as an inhibitor for leukocyte migration.

1.3.3 Slit and Robo expression and structure

The expression of the Slit genes has been shown in many organisms including

Drosophila, Caenorhabditis elegans, Xenopus, mice, rats and humans (Battye et al., 1999; Hao et al., 2001; Chen et al., 2000; Holmes et al., 1998; Marillat et al., 2002; Itoh et al., 1998). In

25 mammals, there are three homologs of Slit. Slit1 is expressed predominantly in the developing

CNS while Slit2 and Slit3 are expressed more systemically in organs including the lung, kidney, and heart (Yuan et al., 1999; Wu et al., 2001). Slit expression persists into adulthood suggesting a role for Slit family proteins outside of embryogenesis. In healthy human tissue, a variety of cell types have been shown to secrete Slit2 including: fibroblasts, endothelial cells, and epithelial cells (Piling et al., 2014; Brantley-Sieders et al., 2011; Zhou et al., 2011). Interestingly, expression of Slit2 by some of these cell types can become altered in pathological states. In endothelial cells, Slit2 mRNA expression decreased in proatherogenic conditions (van Gils et al.,

2013).

The Slit family of proteins, in vertebrates, contains an N-terminal signal peptide, four leucine-rich repeats (LRRs), nine epidermal growth factor (EGF) repeats and a C-terminal cysteine knot (Figure 1.9; Rothberg et al., 1988; Rothberg et al., 1990; Rothberg et al., 1992).

The EGF repeats and LRR allow Slit proteins to interact with ECM components such as glypican-1, localizing their effect and preventing extensive diffusion (Ronca et al., 2001). Slit2 can be cleaved extracellularly by metalloproteases after the fifth EGF repeat to form N-terminal

(N-Slit2) and C-terminal (C-Slit2) fragments (Brose et al., 1999; Wang et al., 1999; Brose and

Tessier-Lavigne, 2000). The N-terminal fragment includes the first 1118 amino acids which encompasses the four LRRs and the first five EGF repeats while the C-terminal fragment contains the remaining residues (Brose et al., 1999). Importantly, it is the second LRR that is necessary and sufficient for interaction with the Robo receptor and downstream signaling

(Howitt et al., 2004). Therefore, both full length Slit2 and N-terminal Slit2 fragment are capable of binding Robo to facilitate downstream signaling (Nguyen Ba-Charvet et al., 2001). The

26 cleavage of Slit2 does not eliminate its activity, but it may play a role in its diffusion, since N-

Slit2 appears to be more tightly associated with the cell membrane (Ba-Charvet et al., 2001). The fourth LRR domain allows Slit2 to homodimerize which potentiates Slit2/Robo1 signaling

(Seiradake et al. 2009). In rat neural tissue, both N-Slit2 and C-Slit2 were shown to bind heparan sulfate proteoglycan glypican-1 (Liang et al., 1999). C-Slit2 has been shown to bind to the receptor Plexin-A1 in neurons, and transduces signals causing a repulsive commissural response independent of Robo receptors (Delloye-Bourgeois et al., 2015). C-Slit2 has also recently been discovered to play a role in regulating thermogenesis in adipocytes though the receptor for it is currently unknown (Svensson et al., 2016).

Figure 1.9 Primary Structure of Mammalian Slit

Mammalian Slit protein is composed of four leucine rich repeats (LRRs), nine epidermal growth factor (EGF) repeats, a laminin G (G) domain, and a cysteine knot. Reprinted with permission from University of Toronto, Mukovozov, I. (2010). Slit/Robo signaling in monocyte chemotaxis and function: a role in vascular inflammation. Copyright 2010.

Similar to Slit proteins, the expression of Robo has been demonstrated in Drosophila, mice and humans (Kidd et al., 1998, Yuan et al., 1999). There are four isoforms of Robo in mammals. Robo1 is expressed in tissues outside the CNS, including human leukocytes (Wu et al., 2001). Robo1 expression has been demonstrated in a variety of immune cells including neutrophils, monocytes, macrophages and T-cells (Tole et al., 2009; Geutskens et al., 2010;

Mukovozov et al., 2015; Piling et al., 2014; Prasad et al., 2007). Robo2 is structurally similar to

Robo1 but its expression is different compared to Robo1. Robo3 interestingly is incapable of binding to Slit proteins and instead signals through binding with Netrin-1 and its co-receptor

Deleted in Colorectal Cancer (DCC) (Sabatier et al., 2004; Zelina et al., 2014). Robo4 is highly expressed in endothelial cells (Cai et al., 2015; Gorbunova et al., 2013). The tissue expression of

Slit and Robo is relatively complementary, suggesting a functional relationship between them in adult organisms (Yuan et al. 1999).

Robo is a single-pass type-1 receptor and a member of the Ig superfamily. The extracellular region of Robo1 contains five Ig repeats and three fibronectin type III domains. The cytoplasmic region of Robo1 contains four conserved cytoplasmic signaling motifs: CC0, CC1,

CC2, CC3 (Figure 1.10; Kidd et al., 1998; Zallen et al., 1998). Robo 2 is structurally similar to

Robo 1. Robo 3 lacks the CC1 motif and Robo 4 lacks three Ig repeats, one FNIII domain and two CC domains. Only the Ig domains of Robo are required for it to bind to Slit proteins

(Bashaw et al., 2000). Lastly, the CC3 motif of Robo is required to bind to Abelson (Abl) and to the SH3 domain of GAPs, while the CC1 motif can bind to Enabled (Ena) (Wong et al., 2001).

Figure 1.10 Primary Structure of Mammalian Robo Receptors

The Robo1/2 receptors are composed of five immunoglobulin (Ig) repeats, three fibronectin (FN) type III, a transmembrane domain (TM) and four conserved cytoplasmic (CC) signaling motifs.

Robo 3 lacks the CC1 motif. Robo 4 lacks three Ig repeats, one FNIII domain and two CC domains.

Robo1 is capable of forming homodimers and even compact tetrameric dimer-of-dimers

(Aleksandrova et al., 2018). This dimerization is mainly mediated by central Ig domains and does not seem to impact binding between monomeric or dimeric Slit2 (Aleksandrova et al.,

2018). Robo1 dimerization potentiates downstream signaling. Robo1 undergoes proteolytic cleavage from the cell surface by a disintegrin and metalloproteinase domain-containing protein-

10 (ADAM10) (Figure 1.11; Seki et al. 2010). The evidence of this is the detection of Robo1 N- terminal fragment in conditioned media of cancer cell lines and patients with hepatocellular carcinomas (Ito et al., 2006). The proteolytic site is between Glu852 and Glu853, 10 residues away from the plasma membrane (Seki et al., 2010). After cleavage, the remaining C-terminal fragment of Robo1 is subsequently cleaved by γ-secretase to form soluble fragment (Figure

1.10). Although the function is unknown, the soluble Robo1 fragment translocates to the nucleus

(Seki et al., 2010). Therefore, the responsiveness of a cell towards Slit2 depends on surface level expression of Robo1 receptor.

Figure 1.11 Robo cleavage and processing Slit-Robo binding creates tension in Robo juxtamembrane domain. This reveals a cleavage site for ADAM10 to cleave Robo ectodomain. The remaining fragment is further processed by γ- secretase, releasing the C-terminal fragment. Reprinted with permission from Rockfeller University Press, Blockus H. & Chedotal A., (2016). Slit-Robo signaling. Development, 143, 2037-3044. Copyright (2016).

1.3.4 Slit2/Robo1 Signal Transduction

Slit2/Robo1 signaling through two pathways leads to mobilization and remodeling of cytoskeleton: Ena protein and Rho GTPases. Both require the CC motifs in the cytoplasmic domain of Robo1 to signal.

Ena and its mammalian homolog Mena are members of a protein family which links signal transduction to localized remodeling of actin cytoskeleton by binding to profilin, an actin binding protein which regulates actin polymerization (Lanier et al., 1999; Wills et al., 1999). Ena is a substrate for Ableson (Abl) kinase and both are able to bind to Robo (Figure 1.12; Gertler et al., 1989). Ena binds to the CC1 motif on Robo1 while Abl binds to CC3 (Bashaw et al., 2000).

Abl and Ena play opposite roles in Robo signal transduction with Ena playing a vital role in

Robo’s repulsive output and Abl antagonizing Robo signaling (Bashaw et al., 2000).

The second pathway through which Slit2/Robo1 mediates cell repulsion is through modulation of Rho GTPase activity. In many cell types, it has been shown that Slit2/Robo1 signaling results in recruitment of srGAP and inactivation of RhoGTPases Rac and Cdc42.

Additionally, Vilse, a conserved GAP for Rac and Cdc42, has been shown to mediate Robo repulsion in tracheal cells and axons (Lundstrom et al., 2004). Slit-Robo (sr)GAPs are composed of several functional domains: an F-BAR domain, RhoGAP domain, and SH3 domain and a functionally unknown carboxyl-terminal region (Itoh et al., 2005; Tsujita et al., 2006;

Tcherkezian et al., 2007). The SH3 domain of srGAPs allow them to interact with the proline rich CC3 motif of Robo1 (Li et al., 2006; Wong et al 2001; Rao et al., 2002). In neurons, interaction between Slit2 and Robo1 increases recruitment of srGAP to Robo1 CC3 motif and

32 increases intrinsic activity of GAP activity on Cdc42 leading to its inactivation (Wong et al.,

2001; Rao et al., 2002). In neutrophils, Slit2/Robo1 signaling inhibits chemoattractant-mediated activation of Rac and Cdc42 (Tole et al., 2009). Finally, srGAP1 has been shown to inhibit lamellipodial dynamics and cell migration by modulating Rac1 activity (Yamazaki et al., 2013).

Thus, Slit2/Robo1 signaling results in recruitment of srGAPs and inactivation of Rho GTPases

Rac and Cdc42.

More recently, a functional domain was identified on a diverse set of receptors including

Robo1, netrin receptors, and GPCRs (Chen et al., 2014). This conserved peptide motif called the

WRC interacting receptor sequence (WIRS) directly binds to a conserved surface on wave regulatory complex (WRC) (Figure 1.12). This suggests that Robo1 may interact directly with the WRC and actin cytoskeleton via the WIRS motif.

Figure 1.12 Robo1 Signal Transduction Enabled protein binds to Robo-1 and may contribute to Slit-mediated repulsion. Abelson kinase phosphorylates intracellular domains of Robo and antagonizes Robo function. Ligation of Robo- 1 by Slit2 results in the recruitment of srGAPs to the plasma membrane. srGAPs convert active GTP-bound forms of Cdc42 and Rac to their inactive, GDP-bound counterparts, thereby inhibiting the dynamic actin polymerization required for chemotaxis and preventing cell migration. A conserved peptide motif, the WRC interacting receptor sequence (WIRS), directly binds to a conserved surface on the WRC, linking the activity of cell surface Robo receptors to actin-nucleating machinery. Reprinted with permission from University of Toronto, Mukovozov, I. (2010). Slit/Robo signaling in monocyte chemotaxis and function: a role in vascular inflammation. Copyright 2010.

1.4 Adenomatous Polyposis Coli (APC) regulates cell migration and adhesion

1.4.1 Association of APC with Robo1

The full spectrum of Slit2/Robo1 effects cannot be fully explained by our current knowledge of downstream signaling interactors. We took an unbiased approach to discover novel interactors of

Robo1 in the presence and absence of Slit2 using Biotin Ligation Assay (BioID). For the BioID, our lab generated HEK293T cells stably expressing a full length Robo1 or a Robo1 mutant which lacks the cytosolic tail (NTM) (Figure 1.13). BioID assays were performed in collaboration with Dr. Brian Raught (Figure 1.14). Biotinylated proteins were isolated and identified with mass spectrometry. Adenomatous polyposis coli (APC) was found to constitutively associate with Robo1. APC/Robo1 association was abolished following exposure to Slit2. Additionally, no APC/Robo1 association was observed with the Robo1 NTM mutant protein irrespective of Slit2 exposure. This was an interesting observation since much like

Slit2/Robo1 signalling, APC also plays an important role in actin dependent processes including cell migration, adhesion and spreading (Noritake et al., 2005; Etienne-Manneville, 2009; Juanes et al., 2017; Mimori-Kiyosue et al., 2000).

Human Robo1 FL

Ig FNI T repeats II M

Human Robo1 NTM

Ig FNI T repeats II M Figure 1.13 Robo1 Constructs

The two Robo1 constructs used to make stably transfected cell lines. Robo1 full length (FL) is the wild type with all regions intact. Robo1 N-terminal mutant (NTM) lacks the cytosolic tail of

Robo1, thus, preventing internal Robo1 signaling.

Figure 1.14 Model for BioID application

Expression of promiscuous biotin-ligase fusion protein leads to selective protein biotinlyation in a proximity dependent fashion. These proteins can be specifically captured from lysed cells and identified with mass spectrometry. Reprinted with permission from Rockfeller University Press,

Roux K et al., (2012). A promiscuous biotin ligase fusion protein identifies proximal and interacting proteins in mammalian cells Journal of Cell Biology, 196(6), 801-810. Copyright (2012).

1.4.2 The initial discovery of APC

Adenomatous polyposis coli (APC) was first discovered in the context of familial adenomatous polyposis (FAP) and colorectal cancers (Bodmer et al., 1987; Kinzler et al., 1991).

FAP is an autosomal dominant inherited disease characterized by the presence of adenomatous polyps in the colon as a precursor, and eventually leading to the development colorectal cancers.

Patients who had FAP were found to possess a germline mutation in the APC gene. This mutation results in a C-terminal truncation of the APC protein (Prosser et al., 1994). Although many truncation mutations have been recorded in patients, the two most prevalent nonsense mutations occur at codon numbers 1061 and 1309. These mutations truncate the APC protein by over 50%. Sporadic somatic mutations in APC gene have also been observed with the mutations primarily occurring between codons 1250 and 1550 (Beroud and Soussi, 1996). As such, full length APC was determined to possess tumor suppression properties (Solomon et al., 1987).

1.4.3 Expression and structure of APC

APC is a large (~311kDa), multi-domain, cytosolic protein which plays a role in many cellular functions in the nucleus and cytoplasm. APC is highly expressed in most tissues and cell types, with the lowest expression levels seen in muscle and adipose tissue. The function APC plays is largely dependent on its localization within the cell and the proteins it interacts with. The

APC protein, from N to C-terminus consists of an oligomerization domain, an armadillo repeat region, a β-catenin-binding region (consisting of 15-amino acid repeats, 20-amino acid repeats, and SAMP repeats), a basic region, an EB1-binding domain, and lastly, a Disks large homolog

(DLG) binding domain (Figure 1.15).

Figure 1.15 Adenomatous Polyposis Coli domains, protein interactors and functions This figure outlines the domains and their functions of full-length APC. The armadillo (Arm) repeats bind Asef1/2 and IQGAP1 which modulate activity of Rho GPTases to stimulate cell migration and adhesion. The β-catenin-binding region binds to β-catenin and Axin to facilitate β- catenin degradation which controls both cell proliferation, chromosomal segregation and adhesion. The Basic region binds to microtubules and helps with kinetochore function and chromosomal segregation. The end binding protein (EB) 1 binding domain binds EB1 to stabilize microtubules which function in chromosomal segregation and cell migration. Reprinted with permission from Company of Biologists Ltd: Journal of Cell Science, Aoki K., & Taketo M., (2007). Adenomatous Polyposis Coli (APC): a multi-functional tumor suppressor gene Journal of Cell Science, 120, 3327-3335. Copyright (2007).

At the very N-terminus of APC lies the oligomerization domain which allows APC to form homo-dimers (Su et al., 1993) (Figure 1.15). Within this domain, amino acids 6-57 are essential for APC oligomerization (Joslyn et al., 1993). Dimers can form between full length and

C-terminal truncated APC, which may elicit a dominant negative effect in APC function (Cottrell et al., 1992; Bodmer, 1999; Su et al., 1993).

The armadillo (Arm) repeat domain of APC is located near the N-terminus of the APC protein (Figure 1.15). This 7-repeat domain is highly conserved across species and retained in mutant, C-terminally truncated, APC proteins. It is capable of binding to APC-stimulated guanine nucleotide exchange factors (Asef) 1 and 2. Asef1/2 are GEFs which specifically activate Rho GTPases Rac1 and Cdc42 (Kawasaki et al., 2000). A different, currently unknown region in the Arm repeat domain also allows APC to interact with Ras GTPase-activating-like protein 1 (IQGAP1). Contrary to its name, IQGAP1 has no GAP activity and actually binds to the active states of Rho GTPases Rac1 and Cdc42 to stabilize them (Watanabe et al., 2004;

Noritake et al., 2005).

The adjacent following domain of APC is referred to as the β-catenin binding region

(Figure 1.15). APC’s β-catenin binding region can be further sub-divided into 15-amino acid repeats, 20-amino acid repeats and SAMP repeats. The 15-amino acid repeats occur between residues 1020 and 1169 and provide binding sites for β-catenin (Rubinfeld et al., 1993). They are unique to APC and do not resemble β-catenin binding sites in the cadherin protein family

(Ozawa et al., 1989). The binding of β-catenin to the 15-amino acid repeats, does not mark them for subsequent degradation. This is shown as cells expressing the C-terminal APC truncation

40 mutatant, which still possess the 15-amino acid repeats, can bind β-catenin, but can’t mark it for degradation (Figure 1.15; Rubinefeld et al., 1993; Su et al., 1993). The 20-amino acid repeats carry a signature TPXXFSXXXSL sequence (Figure 1.14; Groden et al., 1991). The binding of

β-catenin to 20-amino acid repeats occur after it has been phosphorylated by Casein Kinase 1

(CK1) and glycogen synthase kinase (GSK)-3β (Munemitsu et al., 1995; Liu et al., 2002; Stamos

& Weis, 2013). Lastly, the SAMP repeats in the β-catenin binding region allow APC to bind to the regulator of G protein signaling (RGS) domain of Axin (Figure 1.14; Kishida et al., 1998).

Axin acts as a scaffolding protein in the formation of the multiprotein complex with APC and β- catenin, which facilitates phosphorylation of both APC and β-catenin by GSK-3β. APC phosphorylation enhances its capacity to bind β-catenin and ultimately results in greater β- catenin degradation (Ikeda et al., 2000; Kawahara et al., 2000).

APC is a vital component of the β-catenin destruction complex. The β-catenin destruction complex is composed of a complex of proteins consisting of APC, Axin, GSK-3β, and CK1 and is responsible for regulating intracellular levels of β-catenin (Figure 1.16). Wnt signaling transduces signals through the cytoplasmic protein Dishevelled (Dvl) which forms a complex with Axin, preventing it from complexing with APC and β-catenin (Noordermeer et al., 1994).

This inhibition of the destruction complex allows accumulation of intracellular β-catenin.

Intracellular β-catenin concentrations plays two main roles. One role involves its nuclear translocation where β-catenin can act as a transcriptional activator (Smalley et al., 1999). β- catenin can interact with T-cell factor (TCF) family of transcription factors to increase transcription of wnt signaling genes (Behrens et al., 1996). The second role is β-catenin’s association with cadherins to mediate intracellular adhesion. In epithelial cells, E-cadherin is

41 responsible for cell-cell adhesion. Binding of β-catenin is essential for this function as it links E- cadherin to α-catenin and the actin network (Ozawa and Kemler, 1990; Jou et al., 1995).

Figure 1.16 The β-catenin destruction complex In the absence of signaling, the destruction complex targets cytosolic β-catenin for destruction. Reprinted with permission from Cell Press: iScience, Mukherjee A., et al. (2018). Understanding How Wnt Influences Destruction Complex Activity and β-Catenin Dynamics. iScience, 6, 13-21. Copyright (2018).

After the β-catenin binding region is the basic domain, situated between amino acids

2200 and 2400, and aptly named for the large proportion of arginine and lysine residues (Groden et al., 1991) (Figure 1.15). This domain also contains a high percentage of proline residues, which combined with the high proportion of basic residues allow APC to bind microtubules.

APC fragments containing the basic domain have been shown to bind tubulins and stimulate their polymerization in vitro (Munemitsu et al., 1994). APC binding to microtubules through the basic domain to regulates kinetochore function and ultimately chromosomal segregation (Kaplan et al., 2001; Fodde et al., 2001; Dikovskaya et al., 2007).

The end-binding protein 1 (EB1) binding domain is located after the basic domain, close to the C-terminus of APC (Figure 1.15). The EB1 domain contains a binding site for EB1 which has been found to closely associate with the centromere, mitotic spindles and plus ends of microtubules at all stages of the cell cycle (Su et al., 1995; Berrueta et al., 1998; Morrison et al.,

1998; Juwana et al., 1999). EB1 binding to plus ends of microtubules and interaction with the dynactin complex occurs independently of APC (Berrueta et al., 1998; Berrueta et al., 1999).

When the EB1 domain of APC is removed from APC, it is still capable of binding onto microtubules through its basic domain, however it does so indiscriminately. This suggests that

EB1 links APC to localization to the plus ends of microtubules and may potentially facilitate interaction with other specific sites of the plasma membrane (Askham et al., 2000). Although C- terminal truncation mutations in APC often lack an EB1 domain, somatic mutations in EB1 gene have not been found in colorectal tumors (Jais et al., 1998). Also, in vivo experiments in mice expressing APC lacking the EB1 domain did not increase risk of gastrointestinal tumors. These

43 results suggest that EB1 domain is not responsible for the tumor suppressor properties of APC

(Smits et al., 1999).

At the C-terminus of APC is the disks large homolog (DLG) binding domain (Figure

1.15). Within the last 72 amino acids of APC contains a binding site for the DLG protein

(Matsumine et al., 1996). APC binds to DLG through this domain and this association can be abolished by removing the final 72 amino acids of APC. Overexpression experiments have suggested that APC-DLG interactions suppress cell cycle progression from G0/G1 to S phase independently of β-catenin’s effects on the cell cycle (Ishidate et. Al., 2000). In summary, the

APC protein contains several domains, each with different functions in the cell. The two domains which allow APC to regulate cytoskeletal remodeling are the armadillo repeats region and the

EB1 binding domain.

1.4.4 The role of APC in actin cytoskeletal remodeling

Cytoskeletal remodeling is important for many different cellular functions including migration, adhesion and spreading to name a few. The role of APC in actin cytoskeletal remodeling can be divided into two parts: regulating actin polymerization and regulating microtubule dynamics.

The localization of APC to the leading edge of cells occurs in two steps. This localization is dependent on the microtubule network but not the actin network (Nathke et al., 1996; Juanes et al., 2017). APC first reaches the plus ends of microtubules by interacting with kinesin adapter protein (KAP) 3 through the armadillo repeats region (Barth et al., 2008). From the plus ends of

44 microtubules, APC is shuttled to the plasma membrane by the protein APC membrane recruitment protein (AMER) 1 which it also interacts with through the armadillo repeats region

(Grohmann et al., 2007). The interactor which maintains APC localization at the leading edge of migrating cells is currently unknown. However, at the leading edge, APC interacts with proteins which function in actin polymerization and microtubule stability to promote membrane protrusion and polarized cell migration.

APC associates with the scaffolding protein IQGAP1 through its armadillo repeats region

(Watanabe et al., 2004). IQGAP1 is capable of binding to the activated GTP-bound forms of

Rho-family GTPases Rac1 and Cdc42 to stabilize and maintain their active states (Figure 1.16;

Watanabe et al., 2004; Noritake et al., 2005). This facilitates the formation of lamellipodia and filopodia, essential components in cell migration. APC is required for facilitating IQGAP1 localization to the leading edge of migrating cells. When either APC or IQGAP1 are knocked down in epithelial cells, polarized migration of cells was reduced (Noritake et al., 2005).

Figure 1.17 Role of the IQGAP1-APC complex in cell polarization and migration Directional cell migration is usually initiated in response to extracellular cues. Extracellular signals, including growth factors and chemokines, activate Rac1 and Cdc42 through their receptors and certain GEFs at leading edges. Activated Rac1 and Cdc42 induce the polymerization of actin filaments through their effectors. Activated Rac1 and Cdc42 also mark spots where IQGAP1 crosslinks actin filaments. There, APC is recruited through IQGAP1 to actin filaments. IQGAP1 captures the plus-ends of microtubules through CLIP-170. APC then directly and/or indirectly stabilizes microtubules, which are necessary for stable actin meshwork at leading edges. Reprinted with permission from Company of Biologists Ltd: Journal of Cell Science, Noritake J et al. (2005). IQGAP1: a key regulator of adhesion and migration. Journal of Cell Science, 118, 2085-2092. Copyright (2005).

APC recruits GEFs to the leading edge of migrating cells through interactions with its armadillo repeats region (Kawasaki et al., 2000; Kawasaki et al., 2003; Hamann et al., 2007).

Asef1 is capable of binding to Rac GTPases, however, there is contention as to whether Asef1 also facilitates Rac GTPases transition to its active form (Hamann et al., 2007). However, both

Asef1 and its relative, Asef2, are capable of binding and activating Cdc42 GTPases (Hamann et al., 2007). As APC’s interaction with Asef1/2 occur through the armadillo repeats regions, the interaction is maintained in C-terminal truncation mutants of APC as well. Interestingly, the interaction between APC and Asef1/2 are stronger with the C-terminal mutant compared to full length APC (Kawasaki et al., 2003). This could in part explain the aberrant migration of cancer cells that possess APC truncation mutations.

APC’s C-terminus contains an EB1 binding domain (Figure 1.17). This domain allows

APC to bind EB1 which together stabilize and polymerize microtubules (Nathke et al., 1996,

Morrison et al., 1998). Experiments with siRNA targeting APC have shown that APC depletion decreases overall microtubule stability as well as migration and cell protrusion formation

(Kroboth et al., 2007). APC and EB1 have also been shown to function downstream of Rho and formin mDia in microtubule stabilization (Wen et al., 2004). In summary, APC stimulates cell migration through several pathways.

1.5 Rationale, Hypothesis & Objectives

1.5.1 Rationale

Slit2/Robo1 signaling inactivates Rho-family GTPases Rac and Cdc42 which are responsible for formation of lamellipodia and filopodia at the leading edge of migrating cells.

This results in functional changes in monocytes reflected in reduced chemotaxis and adhesion to activated endothelial cells (Mukovozov et al., 2015). We observed that not all effects of Slit2 can be explained by this. To gain more insight into downstream players of Slit2/Robo1 signaling, our group performed BioID experiments using HEK293 cells stably expressing full length Robo1 or a mutant species of Robo1 lacking the intracellular domain. An interesting observation we found was that the cytosolic protein APC constitutively associates with the cytoplasmic tail of the

Robo1 receptor. The association between Robo1 and APC is lost following exposure to Slit2. No association was observed between APC and the Robo1 mutant lacking the intracellular domain.

APC, through its Arm domain is also involved in cellular migration and adhesion by interacting with Asef1/2, Rho GEFs which activate Rac1 and Cdc42, and IQGAP1, a scaffolding protein that binds and stabilizes the active states of Rac1 and Cdc42 (Kawasaki et al., 2003; Watanabe et al., 2004). Since Slit2 differentially affects the association between Robo1 and APC, and

Slit2/Robo1 and APC both affect Rho GTPase activity and actin remodeling, we reasoned that

Slit2 may exert its effects through APC.

1.5.2 Hypothesis

We hypothesized that Slit2/Robo1 signaling exerts its effects by dissociating APC from Robo1, thereby preventing cell migration, adhesion, and spreading.

1.5.3 Objectives

The objectives of this study are:

1. To confirm, in an endogenous system, the differential association of APC and Robo1

before and after exposure to Slit2.

2. To determine how APC interacts with Slit2/Robo1 to influence monocyte

chemotaxis.

3. To determine how APC interacts with Slit2/Robo1 to influence monocyte adhesion

on activated endothelial cells.

4. To determine how APC interacts with Slit2/Robo1 to affect cell spreading.

CHAPTER 2

METHODS

2.1 Reagents and antibodies

The following primary antibodies were used for immunoblotting, co- immunoprecipitation, and immunofluorescence microscopy: anti-human Robo-1(PA5-29917

ThermoFisher Scientific) (MAB71181, R&D Systems) (600-401-692, Rockland Antibodies &

Assays), anti-human APC (PA5-30580, ThermoFisher Scientific) (ab15270 Abcam), and ß-actin

(MA5-15739, ThermoFisher Scientific). The following secondary antibodies were used for immunoblotting and immunofluorescence microscopy: horseradish peroxidase-conjugated goat anti-rabbit IgG (111-035-003, Jackson ImmunoResearch Laboratories Inc), horseradish peroxidase-conjugated goat anti-mouse IgG (115-035-003, Jackson ImmunoResearch

Laboratories Inc), Alexa-Fluor 488 conjugated mouse IgG2a (557703, BD BioScience), and

Alexa Fluor 594 donkey anti-rabbit IgG (A21207, Life Technologies). The following antibody was used as control for co-immunoprecipitation assays: Rabbit IgG (I5006, Sigma-Aldrich).

Protein knockdown experiments were performed with the following siRNAs: Robo1 siRNA (s12092, Ambion by Life Technologies, sense sequence:

GAUCAUACCUUGAAAAUUAtt), APC siRNA (s1433, Ambion by Life Technologies, sense sequence (5’->3’): GGAUCUGUAUCAAGCCGUUtt), and siControl siRNA (D-001212-01-20,

Dharmacon RNA Technologies).

Chemokines used for transwell migration assays were human recombinant monocyte chemotactic protein-1 (MCP-1, CCL2) (78087, Stemcell Technologies Inc) and human recombinant stromal derived factor-1α (SDF-1α, CXCL12) (300-28A, PeproTech).

2.2 Cell Culture

Human histiocytic lymphoma (U-937) monocytic cells were cultured in RPMI-1640

(250-000-CL, Wisent Bioproducts) supplemented with 5% FBS. Human arterial endothelial cells

(Telo-HAECs) were grown in endothelial basal medium 2 (EBM-2) (CC-3156, Lonza) supplemented with Clonetics® EGM-2 SingleQuots®. These include: 10mL FBS, 2mL of recombinant human fibroblast growth factor-B, 0.5mL of ascorbic acid, 0.5mL of recombinant human vascular endothelial growth factor, 0.5mL of recombinant human epidermal growth factor, 0.5mL heparin, 0.2mL hydrocortisone, 0.5mL recombinant insulin-like growth factor-1, and 0.5mL gentamicin sulfate amphotericin-B for 500mL of EBM-2. African green monkey kidney fibroblast (COS-7) cells were cultured in Dulebecco’s modification eagle’s medium

(DMEM) (319-005-CL, Wisent Bioproducts), supplemented with 5% FBS.

2.3 Expression and Purification of N-Slit2 and ΔD2-NSlit2

Human embryonic kidney 293-6E cell line were transfected with cDNA to transiently express bioactive N-terminal human Slit2 (N-Slit2) or bio-inactive ΔD2-NSlit2, a Slit2 mutant which lacks the D2 domain required to bind Robo-1. Both forms of N-Slit2 contained a His tag at their carboxyl terminus which was used for N-Slit2 purification (Figure 2.1; Tole et al., 2009;

Patel et al., 2012; Chaturvedi et al., 2013). Following purification, Slit2 was aliquoted, snap frozen and stored at -80 °C for future use. Aliquots were never re-frozen or used after storage at

4 °C. In the experiments described below, Slit2 was generally used at a concentration of 30nM diluted in basal media. The bioactivity of N-Slit2 was quantified with cell spreading assays, described below, using RAW 264.7 cells.

N-Slit2 ~140kDa ΔD2-NSlit2 ~120kDa

Figure 2.1 Structure of N-Slit2 and ΔD2-NSlit2

N-Slit2 is the N-terminal fragment of the Slit2 protein containing the 4 LRR domains (D1-4) and the first 5 EGF repeats. ΔD2-NSlit2 is a bio-inactive Slit2 mutant where the D2 domain is replaced with a linker, preventing interaction with Robo-1.

2.4 Co-Immunoprecipitation

COS-7 cells were grown in T-75 flasks to full confluency. Cells were lifted using trypsin for 5 minutes at 37ºC, 5% CO2. COS-7 cells were then centrifuged at 300G for 5 minutes. Cell pellets were resuspended in basal media or basal media supplemented with 30nM NSlit-2 for 15 minutes at 37ºC, 5% CO2. Cells were then centrifuged again and lysed for 15 minutes with

200µL of lysis buffer (40mM Tris pH 7.5, 20% glycerol, 1% DDM, 100mM NaCl, and 2x proteinase inhibitor mix). Cell lysates were centrifuged at 16,400G for 20 minutes at 4ºC.

Supernatant was collected and 30µL was taken to prepare western blot sample for total lysate.

The remainder of the supernatant was precleared with 40µL of Protein G Sepharose beads

(P3296-5ML, Sigma-Aldrich) for 30 minutes at 4ºC. Beads were centrifuged for 1 min at 2300G at 4ºC. Each treatment was equally separated into 1.5mL Eppendorf tubes containing either APC antibody with 40µL of beads or rabbit IgG control antibody with 40µL of beads. Tubes were rotated at 4ºC for 2 hours. Beads were centrifuged for 2.5 minutes at 2300G and supernatant was collected. Beads were then washed with 1mL of PBS. This step was repeated two more times.

Beads were then boiled with 1X SDS loading buffer for 5 minutes at 95 ºC. Beads were then spun down again for 2 minutes at 2300G, 4ºC and supernatant was collected.

2.5 siRNA transfections

U937 cells were transfected in a 6 well plate at cell density of 5x105 cells/well. Each well was transfected with 4.5ρmol of siRNA and 9µL of Lipofectamine RNAiMAX transfection reagent (13778150, ThermoFisher Scientific) in serum-free RPMI-1640 media using the protocol provided by Invitrogen. Medium was changed 4 hours after transfection and cells were transfected in two 48-hour periods. COS-7 cells were transfected in a 6 well plate at cell density

53 of 1x106 cells/well. Each well was transfected with 3pmol of siRNA and 9µL of Lipofectamine

RNAiMAX transfection reagent in serum-free DMEM media using the protocol provided by

Invitrogen. Medium was changed 4 hours after transfection and cells were transfected in twice,

48 hours apart.

2.6 DNA Plasmid Transfection

COS-7 cells were seeded in a 6 well plate and grown to approximately 85% cell density.

Cells were co-transfected with pEGFP-C1-APC (supplied by Angela Barth, Stanford University), an APC protein C-terminally tagged with GFP, and control pEGFP-C1 plasmids at 1µg of

DNA/well, using LipoD293 transfection reagent (SL-100668, SignaGen Laboratories) in basal

DMEM media. Cells were incubated for 4 hours in at 37ºC, 5% CO2. Transfection media was then replaced with full DMEM media and cells were allowed to grow for 24 hours in at 37ºC, 5%

CO2. Cells were lifted with trypsin and used for cell spreading assays.

2.7 Immunoblotting

U937 and COS-7 cells were pelleted and lysed with ice-cold lysis buffer (ab156034,

Abcam). Cell debris was pelleted by centrifugation at 16,400G for 10 minutes. Protein samples were added to SDS loading buffer and boiled at 95ºC for 5 minutes. Protein samples were loaded and separated through an 8% acrylamide gel (acrylamide/bis solution 37.5:1) in SDS running buffer (880-570-LL, Wisent Bioproducts). Protein gels were electro-transferred to poly- vinyldene fluoride (PVDF) membranes in transfer buffer (880-560-LL, Wisent Bioproducts) for

2 hours at 250 amperes on ice. Membranes were blocked for 1 hour in 5% skim milk in TBS with 1% Tween (TBS-T). Membranes were probed overnight for APC, Robo1, and β-actin.

Membranes were incubated with host specific, HRP conjugated secondary antibodies for an hour. HRP substrate (WBLUC0500, Millipore Corporation) was added to the membrane before immunoreactive bands were visualized by enhanced chemiluminescence (BioRad Chemidoc MP

Imaging System). ImageLab software was used for densitometry analysis and subsequent statistical analysis was performed using Prism.

2.8 Transwell Migration Assay

U937 cells were transfected using the siRNA transfection protocol above. U937 cells

(1x106 cells/mL, 100µL/condition) were incubated with ΔD2-NSlit2 negative control or Slit2 at

30nM for 15 minutes at 37°C and 5% CO2. The cells were then loaded into the top chamber of a

5µm Transwell insert (3421, Corning Life Sciences) designed for a 24-well plate. A glass coverslip was placed in the bottom well. The bottom chamber was filled with 600µL of serum- free RMPI-1640 alone or with 12.5ηmol of SDF-1α or 12nmol of MCP-1 chemoattractant in the presence or absence of N-Slit2. Monocytes were allowed to migrate into the bottom chamber for

3.5 hours at 37°C and 5% CO2. Following incubation, the monocytes were rapidly centrifuged onto the coverslips at 200g, fixed with 4% PFA, washed with PBS and labeled with DAPI dye for visualization of cell nuclei. A Quorum Spinning Disk Confocal microscope (Leica DMi8) was used to take representative high-power (40X) images and total number of cells was counted in at least 10 random fields. The data represent the mean values ± SEM from at least 4 independent experiments.

2.9 Adhesion

U937 cells were transfected using the siRNA transfection protocol above. TeloHAEC endothelial cells were seeded (~1x104 cells/well) in a 96-well tissue culture plate and grown to confluence. Once confluence was confirmed using light microscopy, the wells were aspirated and replenished with endothelial basal medium 2 alone or with 20ng/mL TNF-α (T 0157, Sigma-

Aldrich). The plates were then incubated at 37°C and 5% CO2 for 4 hours. U937 cells were simultaneously labeled with Calcein AM viability dye (354217, BD Biosciences) at 37°C and

5% CO2 for 30 minutes. After labeling, monocytic U937 cells were washed with PBS, pelleted

(400g, 5 min) and resuspended in serum free RPMI-1640 at 1x106 cells/mL. U937 cells were then incubated with PBS in the presence of absence of Slit2 (30nM) at 37°C and 5% CO2 for 15 minutes. The U937 cells were allowed to settle onto the endothelial cell monolayer

5 (1x10 cells/well) and incubated at 37°C and 5% CO2 for 30 minutes. The plates were then centrifuged (100g, 2min) upside down to remove non-adherent cells. A fluorescent plate reader

(Molecular Devices SpectraMax Gemini EM) was used to measure the fluorescence intensity of each well (494nm excitation, 517nm emission for Calcein AM). Fluorescence intensities were normalized to the unstimulated condition. The data represent the mean value ± SEM from at least

4 independent experiments.

2.10 Spreading Assay

Glass coverslips were placed in a 12-well clear bottom tissue culture plate and coated with fibronectin (10 838 039 001, Sigma-Aldrich) for 2 hours at 37ºC. Fibronectin was removed and coverslips were rinsed with PBS. COS-7 cells were incubated in serum-free DMEM media

5 for 1 hour at 37ºC, 5% CO2. Cells (1x10 cells/mL, 500µL/condition) were then incubated with

PBS vehicle, Slit2, or ΔD2-NSlit2 at a concentration of 30nM for 15 minutes at 37ºC, 5% CO2.

Cells were then seeded onto the coated coverslips and allowed to adhere and spread for 1 hour at

37ºC, 5% CO2. Following incubation, cells were fixed and permeabilized with 4% PFA and

0.3% TRITONTM X100 Surfactant for 10 minutes at room temperature. Cells were then washed with PBS and F-actin was labeled with phalloidin 488nm and nuclear labeling with DAPI dye for

20 minutes each. Coverslips were mounted with Dako mounting media and images were taken with a Quorum Spinning Disk Confocal microscope. At least 10 random fields were taken and cell surface area was quantified with Volocity software. The data represent the mean values ±

SEM from at least 3 independent experiments.

2.11 Statistical Analysis

One-way ANOVA followed by Tukey correction for multiple comparisons was performed using Prism software to analyze data with more than 2 treatment. Unpaired, non- parametric T-test (Wilcoxon) was used to analyze data with only two treatment groups.

Significant difference was considered for p<0.05. Graphic representations show a mean ± SEM as variance bars. All statistical analysis was performed with Prism 6.

CHAPTER 3

RESULTS

3.1 Monocytes and COS-7 cells express Robo1 and APC

Immunoblotting confirmed Robo1 and APC expression in human U937 monocytic cells.

Immunofluorescence labeling confirmed the presence of the Robo1 receptor and APC in human

U937 monocytic cells as well (Figure 3.1A, Figure 3.1B). Immunoblotting confirmed Robo1

and APC expression in COS-7 cells (Figure 3.1A). For both U927 and COS-7 cells, two bands

were seen for APC. For COS-7 cells, two bands were also seen for Robo1. In both instances, the

second band could represent a degradation product.

Figure 3.1 A U937 COS-7

APC ~300kDa-

Robo1 ~200kDa-

B APC Robo1

Figure 3.1 Monocytes and COS-7 cells express Robo1 and APC. Total cell lysates were extracted from U937 monocytes and COS-7 cells and immunoblotting performed. Samples were run on an 8% polyacrylamide gel, transferred onto a PVDF membrane and probed for using anti- human APC and Robo1 antibodies. Bands were seen at approximately 300kDa and 200kDa indicated by the protein ladder. These bands correspond to the sizes of APC and Robo1 respectively (A). Immunoblot results of COS-7 cell lysate samples show double bands present at approximately 300kDa and 200kDa indicated by the protein ladder (A). The double bands are potentially splice variants of APC and Robo1. Protein expression was further validated in U937 monocytes using immunofluorescence staining against Robo1 (green) and APC (red) using anti- human Robo1 and APC antibodies (B).

3.2 APC constitutively associates with Robo1 in COS-7 cells and APC/Robo1 association is decreased after N-Slit2 treatment

An unbiased screen for interactors of Robo1 using BioID was performed in collaboration with Dr. Brian Raught (University Health Network). Two different Robo1 expression plasmids were used to generate cell lines stably expressing different species of Robo1, namely full-length

Robo1 (wildtype) and Robo1 N-terminal mutant (NTM). Robo1 NTM lacks a cytosolic tail

(Figure 1.13). A mutated bifunctional ligase (BirA*) protein was added to the C-terminus of both

Robo1 expression plasmids. The cDNA protein plasmids were stably transfected in HEK293 cells, which endogenously express APC and very low levels of Robo1. BioID was performed on stably transfected HEK293 cells in the presence and absence of Slit2 treatment. APC was found to constitutively associate with Robo1 and this association was lost after Slit2 treatment. The

BioID observations were further confirmed with co-immunoprecipitation using the same stably transfected HEK293 cells with and without Slit2 treatment by pulling down FLAG-tagged

Robo1 with anti-FLAG antibody and immunoblotting for Robo1 and APC.

However, stably transfected cell lines greatly overexpress the transfected protein which may artificially influence protein subcellular localization and trafficking (Prelich, 2012). To verify our findings, co-immunoprecipitation experiments were repeated in an endogenous system.

Co-immunoprecipitation experiments were performed using COS-7 cells which endogenously express both Robo1 and APC at moderate levels (Figure 3.1C). Cells were incubated with N-Slit2 (30nM) or control medium for 15 minutes prior to cell lysis. Proteins

60 from N-Slit2 and control-treated cell lysates were pulled down with wither anti-APC Ab or non- specific anti-Rabbit IgG Ab. Samples were run with total lysate controls on a polyacrylamide gel and probed with anti-APC and anti-Robo1 Ab. No bands were observed in lanes containing samples pulled down with non-specific anti-Rabbit IgG Ab (Figure 3.2). Bands at the approximate size of APC (~300kDa) were present in the following lanes: total lysate, anti-APC antibody pull down of control-treated cells, and anti-APC antibody pull down of N-Slit2 treated cells (Figure 3.2). Bands at the approximate size of Robo1 (~200kDa) were also present in all the lanes just mentioned (Figure 3.2). However, densitometry analysis showed a significant reduction in Robo1 co-immunoprecipitation in lysates from cells treated with N-Slit2 as compared with lysates from control-treated cells (P<0.001). This observation is consistent with

BioID and past co-immunoprecipitation results.

APC

NSlit2

Figure 3.2 A

Slit2 IgGSlit2 APC -Slit2 Total - - N Untreated NTotal UntreatedUntreated IgG NAPC 6. 1. 2. 3. 4. 5.

~300kDa IP: APC

~200kDa IB: Robo1

B Robo1

*** 1.25

1.00

0.75

0.50

Relative Intensity 0.25

0.00 Untreated N-Slit2

Figure 3.2 APC constitutively associates with Robo1 in COS-7 cells and APC/Robo1 association is decreased after exposure to N-Slit2. COS-7 cells were treated with either basal medium (control) or basal medium containing 30nM of N-Slit2. Cells were then lysed and centrifuged with supernatant being collected. Cell lysate samples were pre-cleared for 30 minutes with Protein G Sepharose beads alone. Lysates were then equally divided into protein pull downs with anti-APC Ab or anti-rabbit IgG Ab in conjunction with Protein G beads for 2 hours. Samples were washed three times with PBS. Protein pull downs were made into western blot samples, ran, and probed for with anti-APC and anti-Robo1 Ab. No bands were present in the anti-rabbit IgG pulldown lanes. Bands were present at the approximate size of APC and

Robo1 in the anti-APC pulldown lanes in both the control and N-Slit2 treated conditions.

However, a fainter Robo1 band was present in the N-Slit2 treatment lane (A). This was also quantified by densitometry and normalizing to respective APC bands (B). Three independent experiments were performed and statistical analysis was done using paired, non-parametric t-test.

3.3 APC silencing inhibits chemotaxis of U937 monocytic cells and is not further affected by exposure to N-Slit2

We previously demonstrated that N-Slit2 causes a dose dependent inhibition of monocyte chemotaxis towards chemokines CCL2 and CXCL12 (Mukovozov et al., 2015). Transwell migration assays were performed using U937 monocytes in which APC had been silenced. APC knockdown was confirmed by immunoblotting (Figure 3.3A, P<0.001). APC knockdown did not affect Robo1 expression (Figure 3.3A).

U937 monocytes failed to migrate in the absence of the chemokine CXCL12 (SDF-1α)

(Figure 3.3B, C). When CXCL12 chemokine was added to the bottom chamber of the transwell in the presence of bio-inactive ΔD2-Slit2, the migration of scrambled siRNA control transfected

U937 monocytes to the lower chamber was significantly increased (P<0.0001). Scrambled siRNA transfected U937 monocytes that were exposed to bioactive N-Slit2 exhibited significantly reduced chemotaxis towards the chemokine CXCL12 when compared with ΔD2-

Slit2 treated cells (Figure 3.3B, C; P<0.0001). U937 monocytes transfected with APC siRNA exhibited significant decreased chemotaxis towards CXCL12 both in the presence and absence of

N-Slit2 treatment (Figure 3.3B, C; P<0.0001). N-Slit2 treatment in conjunction with APC knockdown did not further inhibit chemotactic migration (Figure 3.3B, C). These data suggest that the cytosolic protein APC is vital for monocyte chemotaxis towards CXCL12.

APC

Figure 3.3.1 ****

A 1.00 Scr APC 0.75 siRNA siRNA 0.50

APC ~300kDa Relative Intensity 0.25 z 0.00 siScr siAPC Robo1 ~250kDa Robo1

β-Actin ~40kDa 1.25 1.00

0.75 0.50

Relative Intensity 0.25

0.00 siScr siAPC

No SDF-1⍺ SDF-1⍺

DAPI

siScr + ΔD2-NSlit2 siAPC + ΔD2-NSlit2 siScr + ΔD2-NSlit2 siAPC + ΔD2-NSlit2

siScr + NSlit2 siAPC NSlit2 siScr + NSlit2 siAPC NSlit2

50 **** **** ****

Cell Number/FOV 10

Figure 3.3.1 APC silencing inhibits U937 monocyte chemotaxis towards CXCL12 chemokine. U937 human monocytic cells were transfected with control scrambled or APC- specific siRNA and knockdown of APC was confirmed by immunoblotting (A; n=4; P<0.001).

APC knockdown did not affect Robo1 expression (A). Transfected monocytes were treated for

15 minutes with N-Slit2 or bio-inactive ΔD2-Slit2 and migration assays performed using a 5µm mesh transwell. CXCL12 was put in the bottom chamber. After 3 hours, cells which had migrated to the bottom chamber were centrifuged onto coverslips, fixed with 4% paraformaldehyde, permeabilized with 0.3% TRITONTM X100 Surfactant, and nuclei labeled with DAPI. Cells were counted from 10 random fields of view on a Leica DMi8 spinning disk confocal microscope (B). Results from three independent experiments were averaged and plotted

(C). Statistical significance was confirmed through one-way ANOVA followed by Tukey correction for multiple comparisons (n=3; ****P<0.0001).

To verify that the observations above were not specific to CXCL12, we repeated transwell migration assays, but using the chemokine CCL2 (MCP-1) instead. Similar results were obtained compared to experiments performed with CXCL12. U937 monocytes did not migrate without addition of CCL2 irrespective of siRNA transfection or Slit2 treatment (Figure

3.4A, B). When the chemokine CCL2 was added to the bottom chamber of the transwell in the presence of bio-inactive ΔD2-Slit2, the migration of scrambled siRNA control-transfected, U937 monocytes displayed significantly increased chemotaxis (Figure 3.4 A, B; P<0.001). Control- transfected U937 monocytes that were treated with N-Slit2 exhibited significantly reduced migration towards CCL2 when compared with ΔD2-Slit2 treated cells (Figure 3.4A, B;

P<0.001). When U937 monocytes were transfected with APC siRNA, a reduction in chemotaxis towards CCL2 was observed both in the presence and absence of N-Slit2 treatment (Figure 3.4A,

B; P<0.001). N-Slit2 treatment did not further inhibit chemotaxis of cells deficient in APC

(Figure 3.4A, B). These data suggest that the cytosolic protein APC is a vital for monocyte chemotactic migration towards CCL2. These results further suggest that knocking down APC and exposure to N-Slit2 may inhibit cell chemotaxis through a similar mechanism.

Figure 3.3.2 A

No MCP-1 MCP-1

DAPI

siScr + ΔD2-NSlit2 siAPC + ΔD2-NSlit2 siScr + ΔD2-NSlit2 siAPC + ΔD2-NSlit2

siScr + NSlit2 siAPC + NSlit2 siScr + NSlit2 siAPC + NSlit2

**** 50 **** ****

Cell Number/FOV 10

Figure 3.3.2 APC silencing inhibits U937 monocyte chemotaxis towards CCL2 chemokine.

U937 human monocytic cells were transfected with control scrambled or APC-specific siRNA.

Transfected monocytes were treated for 15 minutes with N-Slit2 or bio-inactive ΔD2-Slit2 and migration assays performed using a 5µm mesh transwell. CXCL12 was put in the bottom chamber. After 3 hours, cells which had migrated to the bottom chamber were centrifuged onto coverslips, fixed with 4% paraformaldehyde, permeabilized with 0.3% TRITONTM X100

Surfactant, and nuclei labeled with DAPI. Cells were counted from 10 random fields of view on a Leica DMi8 spinning disk confocal microscope (A). Results from three independent experiments were averaged and plotted (B). Statistical significance was confirmed through one- way ANOVA followed by Tukey correction for multiple comparisons (n=3; ****P<0.0001).

3.4 Robo1 silencing in U937 monocytic cells results in reduced chemotaxis that is not further affected by exposure to N-Slit2

Previous transwell migration assays have established the requirement of APC for monocyte chemotactic migration. BioID and co-immunoprecipitation results have shown that

APC constitutively associates with Robo1 and that the association between APC and Robo1 is lost after Slit2 binds Robo1. Combining these results, we hypothesized that the constitutive association between APC and Robo1 helps to localize APC to the plasma membrane, and is required for monocyte chemotaxis.

Transwell migration assays were performed using U937 cells in which Robo1 was knocked down using specific siRNA. Robo1 knockdown was confirmed with immunoblotting

(Figure 3.5A, P<0.001). Robo1 knockdown did not affect APC expression (Figure 3.5A).

In the absence of CXCL12, U937 monocytes exhibited minimal migration (Figure 3.5B,

C). In the presence of CXCL12, significant migration was observed in control scrambled siRNA- transfected U937 monocytes exposed to bio-inactive ΔD2-Slit2 (Figure 3.5B, C; P<0.0001).

When control scrambled siRNA-transfected monocytes were exposed to bioactive N-Slit2, chemotaxis was significantly reduced (Figure 3.5B, C; P<0.0001). In the presence of bio-inactive

ΔD2-Slit2, cells in which Robo1 was knowcked down showed significantly less chemotaxis than cells transfected with scrambled, non-targeting siRNA (Figure 3.5B, C; P<0.0001). Exposure to

N-Slit2 did not further reduce migration (Figure 3.5B, C). These results demonstrate that expression of APC alone is not enough for monocyte chemotaxis, but rather that is is also required.

Figure 3.4 Robo1

*** A 1.25 Scr Robo1 siRNA siRNA 1.00 0.75 Robo1 ~250kDa 0.50

Relative Intensity 0.25 0.00 APC ~310kDa Scr siRNA Robo1 siRNA

APC β-Actin ~40kDa 1.25 1.00 0.75 0.50

Relative Intensity 0.25 0.00 Scr siRNA Robo1 siRNA

B No SDF-1⍺ SDF-1⍺

DAPI

siScr + ΔD2-NSlit2 siRobo1 + ΔD2-NSlit2 siScr + ΔD2-NSlit2 siRobo1 + ΔD2-NSlit2

siScr + N-Slit2 siRobo1 N-Slit2 siScr + N-Slit2 siRobo1 N-Slit2

C 50 **** **** **** 40

Cell Number/FOV 10

Figure 3.4 Robo1 silencing in U937 monocytes reduces chemotaxis towards CXCL12.

U937 human monocytic cells were transfected with control scrambled or Robo1-specific siRNA and knockdown of Robo1 was confirmed by immunoblotting (A; n=3; ***P<0.001). APC expression was also looked at by immunoblotting (A). Transfected monocytes were treated for

(C). Statistical significance was confirmed through one-way ANOVA followed by Tukey correction for multiple comparisons (n=3; ****P<0.0001).

3.5 APC silencing in U937 cells inhibits their adhesion to activated endothelial cells

We previously showed that Slit2/Robo1 signaling reduces monocyte adhesion to TNF-α activated endothelial cells (Mukovozov et al., 2015). Since cell adhesion requires dynamic actin remodeling, we hypothesized that APC may play a role in monocyte adhesion onto TNF-α activated endothelial cells.

Telo-HAECs were incubated with TNF-α for 4 hours to simulate inflammation and increase endothelial expression of adhesion molecules such as ICAM-1 and VCAM-1 (Rothlein et al., 1986; Neish et al., 1995). U937 cells were transfected with APC specific siRNA or control non-targeting scrambled siRNA. U937 cells were then incubated with N-Slit2 or ΔD2-Slit2 and their ability to adhere to TNF-α telo-HAECs examined.

When Telo-HAECs were not activated with TNF-α, there was minimal monocyte adhesion irrespective of siRNA transfection and Slit2 treatment (Figure 3.6). Scramble siRNA- transfected U937 cells displayed increased adhesion to activated Telo-HAECs (Figure 3.6;

P<0.0001). When the scrambled siRNA-transfected monocytes were instead treated with N-Slit2, adhesion onto TNF-α activated endothelial cells was significantly decreased (Figure 3.6;

P<0.05). When APC was first silenced in monocytes, cell adhesion to TNF-α activated endothelial cells were significantly decreased, in the presence of bio-inactive ΔD2-Slit2 or bioactive N-Slit2 (Figure 3.6, P<0.05). When APC was knocked down, N-Slit2 did not further reduce monocyte adhesion to activated endothelial cells (Figure 3.6). These data suggest that

APC plays a vital role in monocyte adhesion to TNF-α activated endothelial cells.

Figure 3.5

600 *

**** * 500

400

300

200

(%) Adhesion Relative 100

Figure 3.5 APC silencing in U937 monocytes reduces their adhesion to TNF-α activated endothelial cells. U937 cells were transfected with control non-targeting scrambled siRNA or

APC-specific siRNA. Monocytes were incubated with a fluorescent cell permeant dye Calcein

AM for 30 minutes then with of N-Slit2 or ΔD2-Slit2 for 15 minutes. Monocytes were then seeded into a 96 well plate onto a monolayer of TNF-α activated telo-HAECs. After 30 minutes, non-adhered monocytes were removed by centrifugation and relative adhesion was measured in a fluorescent plate reader at 494nm excitation, 517nm emission. Mean values from three independent experiments were plotted and statistical analysis was performed with one-way

ANOVA followed by Tukey correction for multiple comparisons (*P<0.05, ****P<0.0001).

3.6 N-Slit2 treatment and APC silencing similarly inhibit spreading of COS-7 cells

COS-7 cells were chosen for protein overexpression experiments because they are more easily transfected compared to immune cells. Actin remodeling can be measured in COS-7 cells through cell spreading, a process akin to cell migration and adhesion and it involves the establishment of initial adhesion before cellular lamellipodia and filopodia protrusions expand and promote cell spreading (Cuvelier et al., 2007). Since cell spreading requires the modulation of Rho-family GTPase activity, we hypothesized that APC mediates spreading of COS-7 cells.

COS-7 cells were transfected with APC-specific siRNA or control non-targeting scrambled siRNA and APC knockdown was confirmed by immunoblotting (Figure 3.7A,

P<0.001). Transfected cells were incubated with N-Slit2 or bio-inactive ΔD2-Slit2, then seeded onto fibronectin coated coverslips for 1 hour before being fixed and cell spreading measured.

Cell spreading was quantified by measuring maximal cell area and nuclear size.

COS-7 cells transfected with control scrambled siRNA spread robustly in the presence of

ΔD2-Slit2 (Figure 3.7 B, C). In the presence of N-Slit2, a 50% reduction in cell spreading was observed (Figure 3.7B, C; P<0.0001). When APC was knocked down, cell spreading was significantly inhibited (Figure 3.7B, C; P<0.001), to levels comparable to scrambled siRNA transfected cells exposed to N-Slit2 (Figure 3.7B, C; P<0.0001). A similar reduction in cell spreading was observed with N-Slit2 as with ΔD2-Slit2 (Figure 3.7B, C; P<0.0001). Neither transfection nor Slit2 treatments affected cell growth as quantified by cell nucleus size (Figure

3.7D; Edens et al., 2013). Together, these results suggest that APC is required for spreading of

COS-7 cells.

Figure 3.6

Scr APC siRNA siRNA 1.25 *** APC ~300kDa 1.00

0.75 0.50 β-Actin ~40kDa Relative Intensity 0.25

0.00 siScr siAPC

Basal N-Slit2 ΔD2-Slit2 Phalloidin DAPI Phalloidin DAPI

siScr

siAPC

Cell Area

**** 5000 **** ****

4000

) 2

m 3000 µ

2000

Area ( 1000

0 Basal NSlit2 ΔD2 Basal NSlit2 ΔD2

siScr siAPC

Nucleus Size

300

250 )

2 200 m

µ 150

Area ( 100

0 Basal NSlit2 ΔD2 Basal NSlit2 ΔD2 siScr siAPC

Figure 3.6 N-Slit2 treatment and APC silencing similarly inhibit spreading of COS-7 cells.

COS-7 cells were transfected with control non-targeting scrambled siRNA or APC-specific siRNA and APC knocked was confirmed by immunoblotting (A, (***P<0.001). Transfected

COS-7 cells were treated for 15 minutes N-Slit2 or bio-inactive ΔD2-Slit2 and seeded onto fibronectin coated coverslips. After 1 hour, adhered cells were fixed with 4% paraformaldehyde and permeabilized with 0.05% TRITONTM X100 Surfactant. Images of 15 cells were taken per treatment using a Leica DMi8 spinning disk confocal microscope (B). Total cell area and nuclear size were measured from 3 independent experiments with Volocity (C, D). Statistical analysis was performed with one-way ANOVA followed by Tukey correction for multiple comparisons

(****P<0.0001).

3.7 APC overexpression in COS-7 cells reverse N-Slit2 inhibition of cell spreading

Slit2/Robo1 decreases association between Robo1 and APC (Figure 3.2) and increases interaction between Robo1 and Slit-Robo GTPase activating proteins (srGAPs) (Wong et al.,

2001). Previous results show that APC is required for spreading of COS-7 cells (Figure 3.7). The next step is to determine if APC overexpression would the reverse N-Slit2 inhibition of cell spreading. Our hypothesis is that overexpressing APC in COS-7 cells will prevent N-Slit2 inhibition of cell spreading by maintaining APC-Robo1 association even in the presence N-Slit2.

COS-7 cells were transfected with control pEGFP-C1 plasmid or pEGFP-C1-APC plasmid. Transfected cells were incubated with N-Slit2 or bio-inactive ΔD2-Slit2, then seeded onto fibronectin coated coverslips for 1 hour before being fixed and cell spreading measured.

Cell spreading was quantified by measuring maximal cell area and nuclear size.

COS-7 cells transfected with control pEGFP-C1 spread robustly in the presence of ΔD2-

Slit2 (Figure 3.8 A, B). In the presence of N-Slit2, a significant reduction in cell spreading was observed (Figure 3.8A, B; P<0.0001). When APC was overexpressed, cell spreading was no longer inhibited by N-Slit2 (Figure 3.8A, B). Neither transfection nor Slit2 treatments affected cell growth as quantified by cell nucleus size (Figure 3.8C). Together, these results suggest that

APC functions downstream of Slit2/Robo1 signaling and is required for spreading of COS-7 cells.

Figure 3.7 A Basal N-Slit2 ΔD2-Slit2

GFP

Phalloidin

DAPI GFP

GFP-APC

B Cell Area

**** **** **** 3000

2500

)

2 2000

m µ 1500

Area ( 1000

500

0 Basal NSlit2 ΔD2 Basal NSlit2 ΔD2

GFP GFP-APC

Nucleus Size

300

250

)

2 200

m µ 150

Area ( 100

0 Basal NSlit2 ΔD2 Basal NSlit2 ΔD2

GFP GFP-APC

Figure 3.7 APC overexpression reverses N-Slit2 inhibition of COS-7 cell spreading.

COS-7 cells were transfected with pe-GFP-C1 or APC-pe-GFP-C1. Transfected COS-7 cells were treated for 15 minutes N-Slit2 or bio-inactive ΔD2-Slit2 and seeded onto fibronectin coated coverslips. After 1 hour, adhered cells were fixed with 4% paraformaldehyde and permeabilized with 0.05% TRITONTM X100 Surfactant. Images of 15 cells were taken per treatment using a

Leica DMi8 spinning disk confocal microscope (B). Total cell area and nuclear size were measured from 3 independent experiments with Volocity (C, D). Statistical analysis was performed with one-way ANOVA followed by Tukey correction for multiple comparisons

(****P<0.0001).

CHAPTER 4

DISCUSSION, CONCLUSIONS & FUTURE DIRECTIONS

The aim of this project was to determine the functional role of APC in Slit2/Robo1 signaling and the bigger scope of actin cytoskeletal remodeling. The project was based on novel

BioID observations showing that APC constitutively associates with the cytosolic tail of Robo1 receptor and that this association is lost when cells were exposed to Slit2. For the project, we wanted to determine if the differential association of APC and Robo1 had a functional significance and if so, could help to further elucidate the Slit2/Robo1 signaling pathway. To study APC in the context of Slit2/Robo1 signaling, we modulated APC expression in cells and looked at several functional readouts that have been well established to be affected by

Slit2/Robo1 signaling. These actin-remodeling dependent cellular processes included chemotaxis and adhesion in U937 monocytic cells and cell spreading in COS-7 cells. These cell lines were chosen because of their endogenous expression of both Robo1 and APC.

Before performing any functional assays with manipulation of APC expression, we first wanted to confirm the observations seen from the BioID with an endogenous system. The reason for this is because overexpression in cells can potentially introduce artificial protein interactions which would not normally be seen otherwise. For this, we used COS-7 cells which we showed had high endogenous expression of both APC and Robo1. Our co-immunoprecipitation experiments were consistent with results using HEK293 cells stably expressing Robo1. We observed constitutive association between APC and Robo1, which was significantly reduced after exposure to N-Slit2 (P<0.001).

This significant decrease in APC-Robo1 association could potentially be caused by a conformational change in the cytosolic tail of Robo1 following binding of Slit2 to the extracellular domain of Robo1. A conformational change could make the binding site on the cytosolic tail of Robo1 less accessible for APC, thus decreasing their association. Although it is currently unknown if Slit2/Robo1 binding causes any conformational change to Robo1’s cytoplasmic tail, past literature has shown that the extracellular domain of Robo1 undergoes a conformational change after binding to Slit2. After Slit2 binding, Robo1 undergoes a conformational change that reveals a cleavage site 10 residues away from the plasma membrane

(Seki et al., 2010). This site allows Robo1 to undergo proteolytic cleavage by matrix metalloprotease ADAM10 (Ito et al., 2006).

We attempted to visualize and quantify differential co-localization between APC and

Robo1 in the presence and absence of Slit2 using immunofluorescence microscopy. The colocalization for APC and Robo1 was quantified using two metrics: the Pearson’s correlation coefficient (PCC) and the Manders’ colocalization coefficient (MCC). MCC is a measure of the degree two proteins are simultaneously present within a certain area (Dunn et al. 2011). One advantage of using MCC is that it measures co-occurrence of two fluorescent signals independent of signal proportionality. This means if one protein is more highly expressed than the other, the difference in fluorescent intensity won’t impact the readout. One disadvantage of

MCC is that fluorescence values used for analysis can almost never be taken from the original image. Images used for MCC analysis must first be formatted to remove all background fluorescence in order to distinguish between areas of fluorescence and non-fluorescence (Dunn et al. 2011). PCC is a measure of how much a change in expression of one protein effects the

88 change in expression of another protein (Dunn et al. 2011). An advantage to using PCC is that it can be used to quantify images without first formatting the image to remove background fluorescence. This would help quantify images where signals are relatively weak and would likely be removed entirely with formatting. A major disadvantage of PCC is that it is not as reliable for quantifying colocalization between two proteins that are compartmentalized because the intensity ratios of the two proteins would vary more greatly. Another disadvantage of using

PCC is that it only measures the colocalization of protein A to protein B while MCC additionally quantifies the colocalization of protein B to protein A (Dunn et al. 2011). In our results, we found inconsistencies between PCC and MCC led to inconclusive analysis. An explanation for the inconsistency could be the differential compartmentalization of Robo1 and APC which makes PCC less reliable. Another possible explanation is could be the antibodies we used to probe for Robo1 and APC. Our Robo1 antibody was specific for an extracellular domain of the

Robo1 receptor. This could have provided enough distance across the plasma membrane between the fluorescence of Robo1 and the fluorescence of cytoplasmic APC to prevent an accurate readout of colocalization. Although colocalization analyses using IF may hint at the presence of protein-protein colocalization, it is often paired with other methods of quantifying colocalization

(Dunn et al. 2011).

An alternative method to confirm association between APC and Robo1 would be with the proximity ligation assay (P-LISA) (Bellucci et al., 2014). Previous studies have shown that the

P-LISA is an effective assay to study Robo1 interactions with itself and cytoplasmic proteins

(Alexsandrova et al., 2018; Samelson et al., 2015). Primary antibodies specific for APC and

Robo1 would be used to recognize the two proteins in a cell. Secondary antibodies coupled with

89 complementary oligonucleotides would be subsequently added. If the complementary oligonucleotides are in close proximity, they can be ligated into circular DNA which can be amplified by DNA polymerase using fluorochrome coupled oligonucleotides. The resulting fluorescence would visually confirm APC-Robo1 association. This method has an advantage over co-immunoprecipitation because the cells are not lysed before close-proximity proteins are detected. This more convincingly shows that protein-protein interactions occur endogenously, while in co-immunoprecipitation assays, protein-protein interactions, in the lysed cell supernatant, may occur between proteins that would otherwise be separated compartmentally.

Additional assays that can also be used to quantify protein-protein interactions include chemically crosslinking interacting proteins and label transfer protein interaction analysis.

We showed that APC signaling promotes monocyte chemotaxis. When APC was silenced, monocytes exhibited significantly less chemotaxis. These results agree with past literature which showed that APC plays a vital role in directed cell migration (Aoki & Taketo,

2007). In cells expressing a truncated form of APC, cell migration was observed to be abhorrent and unstable (Kawasaki et al., 2003). This suggests that the both the N-terminus of APC, which allows it to interact with proteins that regulate Rho GTPase activity, and the C-terminus of APC, which contains sites which allow it to bind and stabilize microtubules, are both important for the proper function of directed cell migration (Aoki & Taketo, 2007). The role of APC in directional cell migration is divided into its role in promoting actin cytoskeletal dynamics and its role in stabilizing microtubules. APC localizes at the membrane protrusions of migrating cells where it helps to localize the adaptor protein IQGAP1 to these membrane protrusions (Aoki & Taketo,

2007). The protein IQGAP1 can bind to the active (GTP-bound) forms of Rho GTPases Rac1

90 and Cdc42 to stabilize their activity. The Rho GTPases Rac1 and Cdc42 promote the formation of lamellipodia and filopodia respectively (Watanabe et al., 2004; Noritake et al., 2005). APC also binds to the protein EB1 to stabilize the plus ends of microtubules (Askham et al., 2000).

Microtubules extend into membrane protrusions to fortify their structure and APC’s role in microtubule stability is vital for migration at the leading edge of cells.

In our results, we found the N-Slit2 exposure did not further reduce the migration APC- deficient monocytes, suggesting that Slit2 may induce its effects through a pathway that involves

APC. We also confirmed with immunoblotting that APC knockdown did not affect Robo1 expression, meaning the lack of response in APC silenced cells was caused by the absence of

APC alone. To ensure that our observations were not chemoattractant dependent, we performed transwell migration assays with APC knockdown using two different chemoattractants: CXCL12 and CCL2. Results were similar for both. Our results suggest that either APC acts downstream from Robo1, or that Robo1 and APC act through parallel pathways that both require Slit2 to exert its effect.

Our next step would be to quantify levels of active Rho GTPases Rac1 and Cdc42 in

APC silenced cells before and after exposure to a chemoattractant. This could be performed with a Pak binding domain (PBD)-pull down assay or small GTPase activation assay (G-LISA) (Tole et al., 2009; Mukovozov et al., 2015). If the inhibition of chemotaxis is the result of APC depletion, we would expect to see significantly lower levels of active Rac1 and Cdc42 in APC silenced cells. If active Rac1 and Cdc42 levels are unchanged, then this would suggest that

APC’s role in microtubule stability may play a much larger role within its function to promote

91 directed cell migration. This can be further verified by quantifying microtubule stability using immunofluorescence.

In our next step, we wanted to determine whether APC’s differential association with

Robo1 played a functional role. Using Robo1-deficient monocytes, we showed that Robo1 also plays a vital role in promoting monocyte chemotaxis. When Robo1 was silenced, monocytes exhibited significantly less chemotaxis even though APC expression in these monocytes remained unchanged. These observations fall in line with previous studies that have shown pro- migratory roles for Robo1 and Robo4 in non-hematopoietic cells (Grone et al., 2006). This also suggests that APC expression alone is insufficient for monocyte chemotaxis, APC also needs to be associated with Robo1. These observations fall in line with previous literature which showed that inhibiting kinesin-1 and kinesin-2, proteins that bind and bring APC to the plus ends of microtubules, prevented APC from localizing in membrane protrusions of actively migrating cells, thus reducing migratory capacity (Ruane et al., 2016; Etienne-Manneville, 2009). A similar effect was obtained by removing the EB-1 binding domain from APC, which prevented it from binding to the plus ends to microtubules to stabilize membrane protrusions (Barth et al., 2002).

APC localization at the plasma membrane can also promote chemotaxis through its interaction with GEFs Asef1/2 which bind and activate Rho GTPases Rac1 and Cdc42 (Kawasaki et al.,

2000). This suggests that Robo1’s role in chemotaxis is to keep APC at the membrane for it to promote directed cell migration. Another possible reason for Robo1 surface expression could be to bind to chemokine receptors, such as CXCR4, to regulate their downstream signaling (Prasad et al., 2007). This interaction can occur in the absence of Slit2, allowing Robo1 to inhibit chemokine signaling in regions of a cell less exposed to the chemokine gradient. This could better direct the cell towards the chemokine gradient.

Our next step would be to quantify levels of active Rho GTPases Rac1 and Cdc42 in

Robo1 silenced cells before and after exposure to a chemoattractant. Similar to APC silenced cells, experiments could be performed with a PAK binding domain (PBD)-pull down assay or a small GTPase activation assay (G-LISA). If we observed significantly lower levels of active

Rac1 and Cdc42 in Robo1 silenced cells, it would further support the idea that APC’s association with Robo1 is vital for its function in promoting Rho GTPase activation at the leading edge of migrating cells. Otherwise, if active Rac1 and Cdc42 levels are unchanged, then this would suggest that Robo1’s role in localizing APC at the membrane is more important for APC’s role in microtubule stability may play a much larger role within its function to promote directed cell migration. This can be further verified by quantifying microtubule stability.

Based on our results with using APC-deficient and Robo1-deficient monocytes, our proposed model for APC/Robo1 interactions in chemotaxis is as follows: in the absence of

Slit2/Robo1 signaling, APC is constitutively associated with Robo1 (Figure 4.1). This allows

APC to localize and interact with the adapter protein IQGAP1 at the plasma membrane. IQGAP1 can bind and stabilize the active (GTP-bound) states of Rho GTPases Rac1 and Cdc42. Active

Rac1 and Cdc42 promote directed cell migration through lamellipodia and filopodia formation respectively. When Slit2 binds to Robo1, the association between APC and Robo1 is lost (Figure

4.1). Additionally, srGAPs are recruited and bind to the CC3 domain of Robo1’s cytoplasmic tail. Rho GTPases Rac1 and Cdc42 can bind to srGAPs which hydrolyse their bound GTP to

GDP, thus inactivating the Rho GTPases and inhibiting directed cell migration.

Proposed Model

No N-Slit2 Exposure

Cell migration

N-Slit2 Exposure

IQGAP1

Figure 4.1 Proposed model for APC in Slit2/Robo1 signaling

In the absence of Slit2/Robo1 signaling, APC is constitutively associated with Robo1. This allows APC to interact with the adapter protein IQGAP1 at the plasma membrane. IQGAP1 can bind and stabilize the Rho GTPases Rac1 and Cdc42 in their active (GTP-bound) states. Active

Rho GTPases promote cell migration through lamellipodia and filopodia formation. When Slit2 binds to Robo1, the association between APC and Robo1 is lost. Additionally, srGAPs are recruited and bind to the CC3 domain of Robo1’s cytoplasmic tail. Rho GTPases Rac1 and

Cdc42 can bind to srGAPs which hydrolyse their bound GTP to GDP, thus inactivating the Rho

GTPases and inhibiting cell migration

We next showed that APC plays a vital role in monocyte adhesion. When APC was silenced, monocytes exhibited significantly less adhesion to activated endothelial cells. This observation falls in line with past literature which showed APC promoting actin dependent cell adhesion and maintaining adhesive contacts through promoting Rho GTPase activity (Harris &

Nelson, 2010; DeMali et al., 2003). Exposing N-Slit2 to APC-deficient monocytes did not further reduce their adhesion, suggesting that like in chemotaxis, Slit2 may induce its effects through a pathway that involves APC. Similar to chemotaxis, adhesion involves Rho GTPase activity However, cell adhesion, unlike chemotaxis, is an integrin dependent process. Although not much is currently known about APC’s interactions with integrin, interactions between APC and α3 integrin have been demonstrated to stabilize focal adhesions (de Jesus Perez et al., 2012).

This could provide an explanation for APC’s role in monocyte adhesion.

Conclusions made from results can be further strengthened by repeating the adhesion assays using APC-silenced monocytes with ICAM-1 and VCAM-1 coated wells instead of a monolayer of activated endothelial cells. Coating wells with these adhesion molecules rather than stimulating their expression in endothelial cells decreases variance in the level of adhesion molecule expression across wells. This would make the true effect in these results more consistent. Another upcoming experiment would be to look at the effect of Robo1 knockdown in adhesion. We have shown Robo1 possesses a vital role in chemotaxis, if we subsequently find that Robo1 silencing also significantly inhibits monocyte adhesion, this would help solidify the idea of APC needing to associate to Robo1 in order to exert its function. Additionally, this result would strengthen the novel finding of APC functioning downstream of Slit2/Robo1 signaling.

In results not presented, we attempted to transfect U937 monocytes with a plasmid of similar size to GFP-tagged APC and yielded very poor transfection efficiency (<1%).

Transfection of U937 monocytes were performed using Neon® transfection system, Amaxa® electroporation, and LipoD293TM transfection reagent. All yielded similarly low transfection efficiency, which would have provided insufficient cell numbers to perform chemotaxis or adhesion assays. We switched from the monocyte cell line U937 to COS-7 cells because they are more easily transfected by large sized plasmids. Transfecting COS-7 cells with our GFP-tagged

APC plasmid yielded a transfection efficiency of approximately 10% which provided us with a sufficient number of cells for cell spreading assays.

Our experiments with COS-7 cells showed that exposure to Slit2 significantly inhibited cell spreading (P<0.001). Additionally, we showed that APC knockdown also significantly inhibited cell spreading (P<0.001) to approximately the same extent as control cells exposed to

N-Slit2. Our results suggest that APC may act downstream of Slit2/Robo1 signaling to regulate cell spreading. These observations fall in line with previous studies which have documented the role of APC in cell spreading. In spreading epithelial cells, APC has been shown migrate along microtubules and localize at the tip regions of cellular extensions (Mimori-Kiyosue et al., 2000).

N-Slit2-induced-inhibition of cell spreading falls in line with results obtained by our group. We performed cell spreading assays using RAW264.7 murine macrophages and found the reduction in cell spreading after N-Slit2 exposure to be comparable to that of COS-7 cells after

N-Slit2 exposure (Bhosle et al., submitted 2019). Additionally, other groups have shown APC localizes in membrane protrusions, where it has been implicated in promoting protrusion

97 formation and cell motility (Mimori-Kiyosue et al., 2000; Oldenwald et al., 2013). This suggests that APC needs to be in membrane protrusions for spreading which falls in line with our observations of APC localizing at the plasma membrane via association with Robo1. We found that exposure to N-Slit2 disrupted APC/Robo1 association which could prevent APC from localizing in membrane protrusions, thus, inhibiting cell spreading.

To verify these findings, we could repeat cell spreading assays with APC knockdown using RAW 264.7 cells in the presence or absence of N-Slit2. If similar inhibition of cell spreading is observed in APC silenced cells, this would increase the robustness of our result. A limitation is the difficulty efficiently transfecting RAW264.7 cells. A possible solution is using lentivirus as a transfection reagent, which previous studies have shown, to dramatically increase much transfection efficiency in immune cells (Swainson et al., 2008).

Our rationale for performing APC overexpression experiments is based on two observations. Firstly, we showed that exposure to Slit2, significantly reduces association between

APC and Robo1. A possible explanation could be that Slit2/Robo1 interaction causes a conformational change in Robo1’s cytoplasmic tail which makes APC/Robo1 association unfavorable. Secondly, other groups have shown that Slit2/Robo1 interactions have the downstream effect of increasing the association between Robo1 and srGAPs (Wong et al., 2001).

This suggests that the association between APC and Robo1 as well as the association between

Robo1 and srGAPs may be competitive in nature. We hypothesized that APC overexpression, would therefore overcome the unfavorable conditions brought upon by Slit2/Robo1 binding and allow APC-Robo1 association to occur even in the presence of these inhibitory signals.

We showed that APC overexpression reverses N-Slit2 induced inhibition of cell spreading. This further supports the idea of competitive binding between APC and srGAP for the cytoplasmic tail of Robo1. N-Slit2 exposure in APC-overexpressed cells likely did not dissociate

APC from Robo1, preventing membrane localization of srGAP to inactivate Rho GTPases Rac1 and Cdc42. Cells that overexpressed APC did not exhibit increased spreading compared to control cells. This indicates that APC overexpression does not hyperactivate its downstream signaling effectors, but rather maintains it at a controlled active state even in the presence of inhibitory signals like Slit2/Robo1 binding. These observations fall in line with previous studies which have shown that by overexpressing wild type APC in cells APC-mutant cells, cellular functions such as migration and adhesion were restored, and also no hyperactivated (Kawasaki et al., 2003).

Since we showed that APC overexpression reverses Slit2 inhibitory effects on cell spreading, our next experiments would be to determine if APC overexpression reversed Slit2 effects in other assays. These other actin remodelling assays could include adhesion or chemotaxis. However, these assays would require a near homogenous population of cells overexpressing APC. Therefore, GFP-tagged APC plasmid transfection protocols would need to be optimized to yield a higher transfection efficiency in monocytes. Additionally, cell sorting for

GFP-positive cells would need to be performed to obtain a homogenous cell population for these assays.

In conclusion, the showed that the novel association of APC and Robo1 has functional relevance in several processes dependent on actin remodeling. When Slit2 binds Robo1, APC’s constitutive association with Robo1 is lost, removing APC from membrane protrusions where it can normally promote actin remodeling processes including chemotaxis, adhesion and spreading.

APC likely exerts these effects by interacting with proteins such as Asef1/2 and IQGAP1 to promote and stabilize Rho GTPase activity. Additionally, following Slit2/Robo1 binding, srGAPs are recruited and bind to Robo1 leading to Rho GTPase inactivation. The effects of Slit2 inhibition on cell spreading can be reversed by overexpressing APC which suggests competitive binding nature of APC and srGAP onto Robo1.

Currently, many anti-inflammatory agents broadly suppress immune activation and function, which may leave the host susceptible to infection. Slit2/Robo1 signaling also has immunosuppressive effects and targeted local delivery of Slit2, or the equivalent of Slit2 signaling, could be utilized to prevent localized leukocyte cell recruitment and the associated tissue damage, while maintaining overall function of the immune system in the host. Our project has helped to further elucidate the Slit2/Robo1 signaling pathway. In order to further develop

Slit2 or Slit2-related peptides as therapies to inhibit pathologic inflammation, a fulsome understanding of how Slit2 signals through Robo-1 is needed.

100

References

Adams, M.T., Gilbert, J.M., Hinojosa Paiz, J., Bowman, F.M. & Blum, B. Endocrine cell type sorting and mature architecture in the islets of Langerhans require expression of Roundabout receptors in beta cells. Sci Rep 8, 10876 (2018).

Aderem, A. & Underhill, D.M. Mechanisms of phagocytosis in macrophages. Annu Rev Immunol 17, 593-623 (1999).

Akagawa, K.S. et al. Generation of CD1+RelB+ dendritic cells and tartrate-resistant acid phosphatase- positive osteoclast-like multinucleated giant cells from human monocytes. Blood 88, 4029-4039 (1996).

Aleksandrova, N. et al. Robo1 Forms a Compact Dimer-of-Dimers Assembly. Structure 26, 320-328 e324 (2018).

Allen, W.E., Jones, G.E., Pollard, J.W. & Ridley, A.J. Rho, Rac and Cdc42 regulate actin organization and cell adhesion in macrophages. J Cell Sci 110 ( Pt 6), 707-720 (1997).

Allen, W.E., Zicha, D., Ridley, A.J. & Jones, G.E. A role for Cdc42 in macrophage chemotaxis. J Cell Biol 141, 1147-1157 (1998).

Andrews, S., Stephens, L.R. & Hawkins, P.T. PI3K class IB pathway. Sci STKE 2007, cm2 (2007).

Askham, J.M., Moncur, P., Markham, A.F. & Morrison, E.E. Regulation and function of the interaction between the APC tumour suppressor protein and EB1. Oncogene 19, 1950-1958 (2000).

Aspenstrom, P. The Rho GTPases have multiple effects on the actin cytoskeleton. Exp Cell Res 246, 20- 25 (1999).

Barleon, B. et al. Migration of human monocytes in response to vascular endothelial growth factor (VEGF) is mediated via the VEGF receptor flt-1. Blood 87, 3336-3343 (1996).

Barth, A.I., Caro-Gonzalez, H.Y. & Nelson, W.J. Role of adenomatous polyposis coli (APC) and microtubules in directional cell migration and neuronal polarization. Semin Cell Dev Biol 19, 245-251 (2008).

Bashaw, G.J., Kidd, T., Murray, D., Pawson, T. & Goodman, C.S. Repulsive axon guidance: Abelson and Enabled play opposing roles downstream of the roundabout receptor. Cell 101, 703-715 (2000).

Battye, R., Stevens, A. & Jacobs, J.R. Axon repulsion from the midline of the Drosophila CNS requires slit function. Development 126, 2475-2481 (1999).

Bazan, J.F. et al. A new class of membrane-bound chemokine with a CX3C motif. Nature 385, 640-644 (1997).

101

Behrens, J. et al. Functional interaction of beta-catenin with the transcription factor LEF-1. Nature 382, 638-642 (1996).

Bellucci, A., Fiorentini, C., Zaltieri, M., Missale, C. & Spano, P. The "in situ" proximity ligation assay to probe protein-protein interactions in intact tissues. Methods Mol Biol 1174, 397-405 (2014).

Beroud, C. & Soussi, T. APC gene: database of germline and somatic mutations in human tumors and cell lines. Nucleic Acids Res 24, 121-124 (1996).

Berrueta, L. et al. The adenomatous polyposis coli-binding protein EB1 is associated with cytoplasmic and spindle microtubules. Proc Natl Acad Sci U S A 95, 10596-10601 (1998).

Berrueta, L., Tirnauer, J.S., Schuyler, S.C., Pellman, D. & Bierer, B.E. The APC-associated protein EB1 associates with components of the dynactin complex and cytoplasmic dynein intermediate chain. Curr Biol 9, 425-428 (1999).

Bienz, M. & Hamada, F. Adenomatous polyposis coli proteins and cell adhesion. Curr Opin Cell Biol 16, 528-535 (2004).

Bodmer, W.F. et al. Localization of the gene for familial adenomatous polyposis on chromosome 5. Nature 328, 614-616 (1987).

Bokoch, G.M. Regulation of innate immunity by Rho GTPases. Trends Cell Biol 15, 163-171 (2005).

Brakebusch, C. & Fassler, R. The integrin-actin connection, an eternal love affair. EMBO J 22, 2324- 2333 (2003).

Brantley-Sieders, D.M. et al. Angiocrine factors modulate tumor proliferation and motility through EphA2 repression of Slit2 tumor suppressor function in endothelium. Cancer Res 71, 976-987 (2011).

Brose, K. et al. Slit proteins bind Robo receptors and have an evolutionarily conserved role in repulsive axon guidance. Cell 96, 795-806 (1999).

Brose, K. & Tessier-Lavigne, M. Slit proteins: key regulators of axon guidance, axonal branching, and cell migration. Curr Opin Neurobiol 10, 95-102 (2000).

Cai, H. et al. Roundabout4 suppresses glioma-induced endothelial cell proliferation, migration and tube formation in vitro by inhibiting VEGR2-mediated PI3K/AKT and FAK signaling pathways. Cell Physiol Biochem 35, 1689-1705 (2015).

Carman, C.V. & Springer, T.A. Integrin avidity regulation: are changes in affinity and conformation underemphasized? Curr Opin Cell Biol 15, 547-556 (2003).

Casey, P.J., Solski, P.A., Der, C.J. & Buss, J.E. p21ras is modified by a farnesyl isoprenoid. Proc Natl Acad Sci U S A 86, 8323-8327 (1989).

102

Cavalcanti-Adam, E.A. et al. Cell spreading and focal adhesion dynamics are regulated by spacing of integrin ligands. Biophys J 92, 2964-2974 (2007).

Chan, J.R., Hyduk, S.J. & Cybulsky, M.I. Chemoattractants induce a rapid and transient upregulation of monocyte alpha4 integrin affinity for vascular cell adhesion molecule 1 which mediates arrest: an early step in the process of emigration. J Exp Med 193, 1149-1158 (2001).

Chan, J.R., Hyduk, S.J. & Cybulsky, M.I. Detecting rapid and transient upregulation of leukocyte integrin affinity induced by chemokines and chemoattractants. J Immunol Methods 273, 43-52 (2003).

Chapuis, F. et al. Differentiation of human dendritic cells from monocytes in vitro. Eur J Immunol 27, 431-441 (1997).

Chen, B. et al. The WAVE regulatory complex links diverse receptors to the actin cytoskeleton. Cell 156, 195-207 (2014).

Chen, J.H. et al. Embryonic expression and extracellular secretion of Xenopus slit. Neuroscience 96, 231-236 (2000).

Coleman, H.A., Labrador, J.P., Chance, R.K. & Bashaw, G.J. The Adam family metalloprotease Kuzbanian regulates the cleavage of the roundabout receptor to control axon repulsion at the midline. Development 137, 2417-2426 (2010).

Constantin, G. et al. Chemokines trigger immediate beta2 integrin affinity and mobility changes: differential regulation and roles in lymphocyte arrest under flow. Immunity 13, 759-769 (2000).

Correia, I., Chu, D., Chou, Y.H., Goldman, R.D. & Matsudaira, P. Integrating the actin and vimentin cytoskeletons. adhesion-dependent formation of fimbrin-vimentin complexes in macrophages. J Cell Biol 146, 831-842 (1999).

Cottrell, S., Bicknell, D., Kaklamanis, L. & Bodmer, W.F. Molecular analysis of APC mutations in familial adenomatous polyposis and sporadic colon carcinomas. Lancet 340, 626-630 (1992).

Critchley, D.R. et al. Integrin-mediated cell adhesion: the cytoskeletal connection. Biochem Soc Symp 65, 79-99 (1999).

Crittenden, J.R. et al. CalDAG-GEFI integrates signaling for platelet aggregation and thrombus formation. Nat Med 10, 982-986 (2004).

Cuvelier, D. et al. The universal dynamics of cell spreading. Curr Biol 17, 694-699 (2007).

Cybulsky, M.I. & Gimbrone, M.A., Jr. Endothelial expression of a mononuclear leukocyte adhesion molecule during atherogenesis. Science 251, 788-791 (1991). de Jesus Perez, V.A. et al. Loss of adenomatous poliposis coli-alpha3 integrin interaction promotes endothelial apoptosis in mice and humans. Circ Res 111, 1551-1564 (2012).

103

DeFife, K.M., Jenney, C.R., Colton, E. & Anderson, J.M. Cytoskeletal and adhesive structural polarizations accompany IL-13-induced human macrophage fusion. J Histochem Cytochem 47, 65-74 (1999).

Delloye-Bourgeois, C. et al. PlexinA1 is a new Slit receptor and mediates axon guidance function of Slit C-terminal fragments. Nat Neurosci 18, 36-45 (2015).

DeMali, K.A., Wennerberg, K. & Burridge, K. Integrin signaling to the actin cytoskeleton. Curr Opin Cell Biol 15, 572-582 (2003).

Dias, M. et al. Role of SpdA in Cell Spreading and Phagocytosis in Dictyostelium. PLoS One 11, e0160376 (2016).

Dickinson, R.E. & Duncan, W.C. The SLIT-ROBO pathway: a regulator of cell function with implications for the reproductive system. Reproduction 139, 697-704 (2010).

Dikovskaya, D. et al. The adenomatous polyposis coli protein contributes to normal compaction of mitotic chromatin. PLoS One 7, e38102 (2012).

Dunn, K.W., Kamocka, M.M. & McDonald, J.H. A practical guide to evaluating colocalization in biological microscopy. Am J Physiol Cell Physiol 300, C723-742 (2011).

Dupuy, A.G. & Caron, E. Integrin-dependent phagocytosis: spreading from microadhesion to new concepts. J Cell Sci 121, 1773-1783 (2008).

Etienne-Manneville, S. APC in cell migration. Adv Exp Med Biol 656, 30-40 (2009).

Fearnhead, N.S., Britton, M.P. & Bodmer, W.F. The ABC of APC. Hum Mol Genet 10, 721-733 (2001).

Fenteany, G. & Glogauer, M. Cytoskeletal remodeling in leukocyte function. Curr Opin Hematol 11, 15-24 (2004).

Fodde, R., Smits, R. & Clevers, H. APC, signal transduction and genetic instability in colorectal cancer. Nat Rev Cancer 1, 55-67 (2001).

Frame, M. & Norman, J. A tal(in) of cell spreading. Nat Cell Biol 10, 1017-1019 (2008).

Friedl, P. Prespecification and plasticity: shifting mechanisms of cell migration. Curr Opin Cell Biol 16, 14-23 (2004).

104

Friedl, P. & Gilmour, D. Collective cell migration in morphogenesis, regeneration and cancer. Nat Rev Mol Cell Biol 10, 445-457 (2009).

Fujiyama, A. & Tamanoi, F. RAS2 protein of Saccharomyces cerevisiae undergoes removal of methionine at N terminus and removal of three amino acids at C terminus. J Biol Chem 265, 3362-3368 (1990).

Gamblin, S.J. & Smerdon, S.J. GTPase-activating proteins and their complexes. Curr Opin Struct Biol 8, 195-201 (1998).

Geissmann, F., Jung, S. & Littman, D.R. Blood monocytes consist of two principal subsets with distinct migratory properties. Immunity 19, 71-82 (2003).

Gertler, F.B., Bennett, R.L., Clark, M.J. & Hoffmann, F.M. Drosophila abl tyrosine kinase in embryonic CNS axons: a role in axonogenesis is revealed through dosage-sensitive interactions with disabled. Cell 58, 103-113 (1989).

Geutskens, S.B., Hordijk, P.L. & van Hennik, P.B. The chemorepellent Slit3 promotes monocyte migration. J Immunol 185, 7691-7698 (2010).

Giagulli, C. et al. RhoA and zeta PKC control distinct modalities of LFA-1 activation by chemokines: critical role of LFA-1 affinity triggering in lymphocyte in vivo homing. Immunity 20, 25-35 (2004).

Gorbunova, E.E., Gavrilovskaya, I.N. & Mackow, E.R. Slit2-Robo4 receptor responses inhibit ANDV directed permeability of human lung microvascular endothelial cells. Antiviral Res 99, 108-112 (2013).

Gordon, S. The role of the macrophage in immune regulation. Res Immunol 149, 685-688 (1998).

Gordon, S. et al. Localization and function of tissue macrophages. Ciba Found Symp 118, 54-67 (1986).

Grage-Griebenow, E., Flad, H.D. & Ernst, M. Heterogeneity of human peripheral blood monocyte subsets. J Leukoc Biol 69, 11-20 (2001).

Grieshammer, U. et al. SLIT2-mediated ROBO2 signaling restricts kidney induction to a single site. Dev Cell 6, 709-717 (2004).

Groden, J. et al. Identification and characterization of the familial adenomatous polyposis coli gene. Cell 66, 589-600 (1991).

Grohmann, A., Tanneberger, K., Alzner, A., Schneikert, J. & Behrens, J. AMER1 regulates the distribution of the tumor suppressor APC between microtubules and the plasma membrane. J Cell Sci 120, 3738-3747 (2007).

Guan, H. et al. Neuronal repellent Slit2 inhibits dendritic cell migration and the development of immune responses. J Immunol 171, 6519-6526 (2003).

105

Gutierrez, L., Magee, A.I., Marshall, C.J. & Hancock, J.F. Post-translational processing of p21ras is two-step and involves carboxyl-methylation and carboxy-terminal proteolysis. EMBO J 8, 1093-1098 (1989).

Gyetko, M.R., Todd, R.F., 3rd, Wilkinson, C.C. & Sitrin, R.G. The urokinase receptor is required for human monocyte chemotaxis in vitro. J Clin Invest 93, 1380-1387 (1994).

Hamann, M.J., Lubking, C.M., Luchini, D.N. & Billadeau, D.D. Asef2 functions as a Cdc42 exchange factor and is stimulated by the release of an autoinhibitory module from a concealed C-terminal activation element. Mol Cell Biol 27, 1380-1393 (2007).

Handel, T.M., Johnson, Z., Crown, S.E., Lau, E.K. & Proudfoot, A.E. Regulation of protein function by glycosaminoglycans--as exemplified by chemokines. Annu Rev Biochem 74, 385-410 (2005).

Hao, J.C. et al. C. elegans slit acts in midline, dorsal-ventral, and anterior-posterior guidance via the SAX-3/Robo receptor. Neuron 32, 25-38 (2001).

Hirsch, E. et al. Central role for G protein-coupled phosphoinositide 3-kinase gamma in inflammation. Science 287, 1049-1053 (2000).

Howitt, J.A., Clout, N.J. & Hohenester, E. Binding site for Robo receptors revealed by dissection of the leucine-rich repeat region of Slit. EMBO J 23, 4406-4412 (2004).

Hume, D.A. et al. The mononuclear phagocyte system revisited. J Leukoc Biol 72, 621-627 (2002).

Hwang, D.Y. et al. Mutations of the SLIT2-ROBO2 pathway genes SLIT2 and SRGAP1 confer risk for congenital anomalies of the kidney and urinary tract. Hum Genet 134, 905-916 (2015).

Ikeda, S., Kishida, M., Matsuura, Y., Usui, H. & Kikuchi, A. GSK-3beta-dependent phosphorylation of adenomatous polyposis coli gene product can be modulated by beta-catenin and protein phosphatase 2A complexed with Axin. Oncogene 19, 537-545 (2000).

Imai, T. et al. Identification and molecular characterization of fractalkine receptor CX3CR1, which mediates both leukocyte migration and adhesion. Cell 91, 521-530 (1997).

Ingersoll, M.A., Platt, A.M., Potteaux, S. & Randolph, G.J. Monocyte trafficking in acute and chronic inflammation. Trends Immunol 32, 470-477 (2011).

Ishidate, T., Matsumine, A., Toyoshima, K. & Akiyama, T. The APC-hDLG complex negatively regulates cell cycle progression from the G0/G1 to S phase. Oncogene 19, 365-372 (2000).

Ito, H. et al. Identification of ROBO1 as a novel hepatocellular carcinoma antigen and a potential therapeutic and diagnostic target. Clin Cancer Res 12, 3257-3264 (2006).

106

Itoh, A., Miyabayashi, T., Ohno, M. & Sakano, S. Cloning and expressions of three mammalian homologues of Drosophila slit suggest possible roles for Slit in the formation and maintenance of the nervous system. Brain Res Mol Brain Res 62, 175-186 (1998).

Itoh, T. et al. Dynamin and the actin cytoskeleton cooperatively regulate plasma membrane invagination by BAR and F-BAR proteins. Dev Cell 9, 791-804 (2005).

Jais, P. et al. Absence of somatic alterations of the EB1 gene adenomatous polyposis coli-associated protein in human sporadic colorectal cancers. Br J Cancer 78, 1356-1360 (1998).

Jin, T., Zhang, N., Long, Y., Parent, C.A. & Devreotes, P.N. Localization of the G protein betagamma complex in living cells during chemotaxis. Science 287, 1034-1036 (2000).

Jones, G.E., Allen, W.E. & Ridley, A.J. The Rho GTPases in macrophage motility and chemotaxis. Cell Adhes Commun 6, 237-245 (1998).

Joslyn, G., Richardson, D.S., White, R. & Alber, T. Dimer formation by an N-terminal coiled coil in the APC protein. Proc Natl Acad Sci U S A 90, 11109-11113 (1993).

Jou, T.S., Stewart, D.B., Stappert, J., Nelson, W.J. & Marrs, J.A. Genetic and biochemical dissection of protein linkages in the cadherin-catenin complex. Proc Natl Acad Sci U S A 92, 5067-5071 (1995).

Juanes, M.A. et al. Adenomatous polyposis coli nucleates actin assembly to drive cell migration and microtubule-induced focal adhesion turnover. J Cell Biol 216, 2859-2875 (2017).

Juwana, J.P. et al. EB/RP gene family encodes tubulin binding proteins. Int J Cancer 81, 275-284 (1999).

Kanellis, J. et al. Modulation of inflammation by slit protein in vivo in experimental crescentic glomerulonephritis. Am J Pathol 165, 341-352 (2004).

Kawahara, K. et al. Down-regulation of beta-catenin by the colorectal tumor suppressor APC requires association with Axin and beta-catenin. J Biol Chem 275, 8369-8374 (2000).

Kawasaki, Y., Sato, R. & Akiyama, T. Mutated APC and Asef are involved in the migration of colorectal tumour cells. Nat Cell Biol 5, 211-215 (2003).

Kawasaki, Y. et al. Asef, a link between the tumor suppressor APC and G-protein signaling. Science 289, 1194-1197 (2000).

Kelner, G.S. et al. Lymphotactin: a cytokine that represents a new class of chemokine. Science 266, 1395-1399 (1994).

Kidd, T. et al. Roundabout controls axon crossing of the CNS midline and defines a novel subfamily of evolutionarily conserved guidance receptors. Cell 92, 205-215 (1998).

107

Kintscher, U. et al. Peroxisome proliferator-activated receptor and retinoid X receptor ligands inhibit monocyte chemotactic protein-1-directed migration of monocytes. Eur J Pharmacol 401, 259-270 (2000).

Kinzler, K.W. et al. Identification of FAP locus genes from chromosome 5q21. Science 253, 661-665 (1991).

Kishida, S. et al. Axin, a negative regulator of the wnt signaling pathway, directly interacts with adenomatous polyposis coli and regulates the stabilization of beta-catenin. J Biol Chem 273, 10823- 10826 (1998).

Kjoller, L. & Hall, A. Signaling to Rho GTPases. Exp Cell Res 253, 166-179 (1999).

Kroboth, K. et al. Lack of adenomatous polyposis coli protein correlates with a decrease in cell migration and overall changes in microtubule stability. Mol Biol Cell 18, 910-918 (2007).

Krugmann, S., Hawkins, P.T., Pryer, N. & Braselmann, S. Characterizing the interactions between the two subunits of the p101/p110gamma phosphoinositide 3-kinase and their role in the activation of this enzyme by G beta gamma subunits. J Biol Chem 274, 17152-17158 (1999).

Kurisu, S. & Takenawa, T. The WASP and WAVE family proteins. Genome Biol 10, 226 (2009).

Lammers, M., Meyer, S., Kuhlmann, D. & Wittinghofer, A. Specificity of interactions between mDia isoforms and Rho proteins. J Biol Chem 283, 35236-35246 (2008).

Lanier, L.M. et al. Mena is required for neurulation and commissure formation. Neuron 22, 313-325 (1999).

Laudanna, C., Kim, J.Y., Constantin, G. & Butcher, E. Rapid leukocyte integrin activation by chemokines. Immunol Rev 186, 37-46 (2002).

Ley, K., Laudanna, C., Cybulsky, M.I. & Nourshargh, S. Getting to the site of inflammation: the leukocyte adhesion cascade updated. Nat Rev Immunol 7, 678-689 (2007).

Li, X. et al. Structural basis of Robo proline-rich motif recognition by the srGAP1 Src homology 3 domain in the Slit-Robo signaling pathway. J Biol Chem 281, 28430-28437 (2006).

Li, Z. et al. Roles of PLC-beta2 and -beta3 and PI3Kgamma in chemoattractant-mediated signal transduction. Science 287, 1046-1049 (2000).

Liang, Y. et al. Mammalian homologues of the Drosophila slit protein are ligands of the heparan sulfate proteoglycan glypican-1 in brain. J Biol Chem 274, 17885-17892 (1999).

Liu, C. et al. Control of beta-catenin phosphorylation/degradation by a dual-kinase mechanism. Cell 108, 837-847 (2002).

108

Liu, Y. Renal fibrosis: New insights into the pathogenesis and therapeutics. Kidney International 69, 213-217 (2006).

Lundstrom, A. et al. Vilse, a conserved Rac/Cdc42 GAP mediating Robo repulsion in tracheal cells and axons. Genes Dev 18, 2161-2171 (2004).

Luster, A.D. Chemokines--chemotactic cytokines that mediate inflammation. N Engl J Med 338, 436- 445 (1998).

Machesky, L.M. & Insall, R.H. Signaling to actin dynamics. J Cell Biol 146, 267-272 (1999).

Marillat, V. et al. Spatiotemporal expression patterns of slit and robo genes in the rat brain. J Comp Neurol 442, 130-155 (2002).

Matsumine, A. et al. Binding of APC to the human homolog of the Drosophila discs large tumor suppressor protein. Science 272, 1020-1023 (1996).

Mattila, P.K. & Lappalainen, P. Filopodia: molecular architecture and cellular functions. Nat Rev Mol Cell Biol 9, 446-454 (2008).

Mellado, M. et al. Chemokine receptor homo- or heterodimerization activates distinct signaling pathways. EMBO J 20, 2497-2507 (2001).

Mimori-Kiyosue, Y., Shiina, N. & Tsukita, S. Adenomatous polyposis coli (APC) protein moves along microtubules and concentrates at their growing ends in epithelial cells. J Cell Biol 148, 505-518 (2000).

Moreau, V., Tatin, F., Varon, C. & Genot, E. Actin can reorganize into podosomes in aortic endothelial cells, a process controlled by Cdc42 and RhoA. Mol Cell Biol 23, 6809-6822 (2003).

Morrison, E.E., Wardleworth, B.N., Askham, J.M., Markham, A.F. & Meredith, D.M. EB1, a protein which interacts with the APC tumour suppressor, is associated with the microtubule cytoskeleton throughout the cell cycle. Oncogene 17, 3471-3477 (1998).

Mukovozov, I. et al. The Neurorepellent Slit2 Inhibits Postadhesion Stabilization of Monocytes Tethered to Vascular Endothelial Cells. J Immunol 195, 3334-3344 (2015).

Muller, W.A. New mechanisms and pathways for monocyte recruitment. J Exp Med 194, F47-51 (2001).

Muller, W.A. Leukocyte-endothelial-cell interactions in leukocyte transmigration and the inflammatory response. Trends Immunol 24, 327-334 (2003).

Munemitsu, S., Albert, I., Souza, B., Rubinfeld, B. & Polakis, P. Regulation of intracellular beta-catenin levels by the adenomatous polyposis coli (APC) tumor-suppressor protein. Proc Natl Acad Sci U S A 92, 3046-3050 (1995).

109

Munemitsu, S. et al. The APC gene product associates with microtubules in vivo and promotes their assembly in vitro. Cancer Res 54, 3676-3681 (1994).

Nassar, N., Hoffman, G.R., Manor, D., Clardy, J.C. & Cerione, R.A. Structures of Cdc42 bound to the active and catalytically compromised forms of Cdc42GAP. Nat Struct Biol 5, 1047-1052 (1998).

Nathke, I. Cancer Biology: APC Delivers Kiss of Death to Focal Adhesions. Curr Biol 27, R805-R807 (2017).

Nathke, I.S., Adams, C.L., Polakis, P., Sellin, J.H. & Nelson, W.J. The adenomatous polyposis coli tumor suppressor protein localizes to plasma membrane sites involved in active cell migration. J Cell Biol 134, 165-179 (1996).

Neish, A.S. et al. Endothelial interferon regulatory factor 1 cooperates with NF-kappa B as a transcriptional activator of vascular cell adhesion molecule 1. Mol Cell Biol 15, 2558-2569 (1995).

Neufeld, K. & White, R. Nuclear and cytoplasmic localization of the adenomatous polyposis coli protein. Proc. Natl. Acad. Sci. 94 (1997).

Nguyen, D.H. et al. Myosin light chain kinase functions downstream of Ras/ERK to promote migration of urokinase-type plasminogen activator-stimulated cells in an integrin-selective manner. J Cell Biol 146, 149-164 (1999).

Nicholson-Dykstra, S., Higgs, H.N. & Harris, E.S. Actin dynamics: growth from dendritic branches. Curr Biol 15, R346-357 (2005).

Nobes, C.D. & Hall, A. Rho, rac, and cdc42 GTPases regulate the assembly of multimolecular focal complexes associated with actin stress fibers, lamellipodia, and filopodia. Cell 81, 53-62 (1995).

Noordermeer, J., Klingensmith, J., Perrimon, N. & Nusse, R. dishevelled and armadillo act in the wingless signalling pathway in Drosophila. Nature 367, 80-83 (1994).

Noritake, J., Watanabe, T., Sato, K., Wang, S. & Kaibuchi, K. IQGAP1: a key regulator of adhesion and migration. J Cell Sci 118, 2085-2092 (2005).

Olofsson, B. Rho guanine dissociation inhibitors: pivotal molecules in cellular signalling. Cell Signal 11, 545-554 (1999).

Ozawa, M., Baribault, H. & Kemler, R. The cytoplasmic domain of the cell adhesion molecule uvomorulin associates with three independent proteins structurally related in different species. EMBO J 8, 1711-1717 (1989).

Ozawa, M. & Kemler, R. Correct proteolytic cleavage is required for the cell adhesive function of uvomorulin. J Cell Biol 111, 1645-1650 (1990).

Patel, N.R. et al. Cell elasticity determines macrophage function. PLoS One 7, e41024 (2012).

110

Pilling, D., Zheng, Z., Vakil, V. & Gomer, R.H. Fibroblasts secrete Slit2 to inhibit fibrocyte differentiation and fibrosis. Proc Natl Acad Sci U S A 111, 18291-18296 (2014).

Prasad, A., Qamri, Z., Wu, J. & Ganju, R.K. Slit-2/Robo-1 modulates the CXCL12/CXCR4-induced chemotaxis of T cells. J Leukoc Biol 82, 465-476 (2007).

Prelich, G. Gene overexpression: uses, mechanisms, and interpretation. Genetics 190, 841-854 (2012).

Prosser, J. et al. APC mutation analysis by chemical cleavage of mismatch and a protein truncation assay in familial adenomatous polyposis. Br J Cancer 70, 841-846 (1994).

Randolph, G.J., Beaulieu, S., Lebecque, S., Steinman, R.M. & Muller, W.A. Differentiation of monocytes into dendritic cells in a model of transendothelial trafficking. Science 282, 480-483 (1998).

Rao, Y., Wong, K., Ward, M., Jurgensen, C. & Wu, J.Y. Neuronal migration and molecular conservation with leukocyte chemotaxis. Genes Dev 16, 2973-2984 (2002).

Ridley, A.J. Rho GTPases and cell migration. J Cell Sci 114, 2713-2722 (2001).

Ronca, F., Andersen, J.S., Paech, V. & Margolis, R.U. Characterization of Slit protein interactions with glypican-1. J Biol Chem 276, 29141-29147 (2001).

Rossi, D. & Zlotnik, A. The biology of chemokines and their receptors. Annu Rev Immunol 18, 217-242 (2000).

Rossman, K.L., Der, C.J. & Sondek, J. GEF means go: turning on RHO GTPases with guanine nucleotide-exchange factors. Nat Rev Mol Cell Biol 6, 167-180 (2005).

Rothberg, J.M. & Artavanis-Tsakonas, S. Modularity of the slit protein. Characterization of a conserved carboxy-terminal sequence in secreted proteins and a motif implicated in extracellular protein interactions. J Mol Biol 227, 367-370 (1992).

Rothberg, J.M., Hartley, D.A., Walther, Z. & Artavanis-Tsakonas, S. slit: an EGF-homologous locus of D. melanogaster involved in the development of the embryonic central nervous system. Cell 55, 1047- 1059 (1988).

Rothberg, J.M., Jacobs, J.R., Goodman, C.S. & Artavanis-Tsakonas, S. slit: an extracellular protein necessary for development of midline glia and commissural axon pathways contains both EGF and LRR domains. Genes Dev 4, 2169-2187 (1990).

Rothlein, R., Dustin, M.L., Marlin, S.D. & Springer, T.A. A human intercellular adhesion molecule (ICAM-1) distinct from LFA-1. J Immunol 137, 1270-1274 (1986).

Roux, K.J., Kim, D.I., Raida, M. & Burke, B. A promiscuous biotin ligase fusion protein identifies proximal and interacting proteins in mammalian cells. J Cell Biol 196, 801-810 (2012).

111

Rubinfeld, B. et al. Association of the APC gene product with beta-catenin. Science 262, 1731-1734 (1993).

Sabatier, C. et al. The divergent Robo family protein rig-1/Robo3 is a negative regulator of slit responsiveness required for midline crossing by commissural axons. Cell 117, 157-169 (2004).

Salmon, J.A. & Higgs, G.A. Prostaglandins and leukotrienes as inflammatory mediators. Br Med Bull 43, 285-296 (1987).

Sampath, R., Gallagher, P.J. & Pavalko, F.M. Cytoskeletal interactions with the leukocyte integrin beta2 cytoplasmic tail. Activation-dependent regulation of associations with talin and alpha-actinin. J Biol Chem 273, 33588-33594 (1998).

Sarkar, K., Kruhlak, M.J., Erlandsen, S.L. & Shaw, S. Selective inhibition by rottlerin of macropinocytosis in monocyte-derived dendritic cells. Immunology 116, 513-524 (2005).

Sasaki, T. et al. Function of PI3Kgamma in thymocyte development, T cell activation, and neutrophil migration. Science 287, 1040-1046 (2000).

Seiradake, E. et al. Structure and functional relevance of the Slit2 homodimerization domain. EMBO Rep 10, 736-741 (2009).

Seki, M. et al. Human ROBO1 is cleaved by metalloproteinases and gamma-secretase and migrates to the nucleus in cancer cells. FEBS Lett 584, 2909-2915 (2010).

Serre, L., Stoppin-Mellet, V. & Arnal, I. Adenomatous Polyposis Coli as a Scaffold for Microtubule End-Binding Proteins. J Mol Biol 431, 1993-2005 (2019).

Servant, G. et al. Polarization of chemoattractant receptor signaling during neutrophil chemotaxis. Science 287, 1037-1040 (2000).

Shamri, R. et al. Lymphocyte arrest requires instantaneous induction of an extended LFA-1 conformation mediated by endothelium-bound chemokines. Nat Immunol 6, 497-506 (2005).

Sheetz, M.P., Felsenfeld, D., Galbraith, C.G. & Choquet, D. Cell migration as a five-step cycle. Biochem Soc Symp 65, 233-243 (1999).

Shimonaka, M. et al. Rap1 translates chemokine signals to integrin activation, cell polarization, and motility across vascular endothelium under flow. J Cell Biol 161, 417-427 (2003).

Singer, S.J. & Kupfer, A. The directed migration of eukaryotic cells. Annu Rev Cell Biol 2, 337-365 (1986).

Sit, S.T. & Manser, E. Rho GTPases and their role in organizing the actin cytoskeleton. J Cell Sci 124, 679-683 (2011).

112

Smalley, M.J. et al. Interaction of axin and Dvl-2 proteins regulates Dvl-2-stimulated TCF-dependent transcription. EMBO J 18, 2823-2835 (1999).

Smits, R. et al. Apc1638T: a mouse model delineating critical domains of the adenomatous polyposis coli protein involved in tumorigenesis and development. Genes Dev 13, 1309-1321 (1999).

Solomon, E. et al. Chromosome 5 allele loss in human colorectal carcinomas. Nature 328, 616-619 (1987).

Stamos, J.L. & Weis, W.I. The beta-catenin destruction complex. Cold Spring Harb Perspect Biol 5, a007898 (2013).

Su, L.K. et al. APC binds to the novel protein EB1. Cancer Res 55, 2972-2977 (1995).

Su, L.K. et al. Association between wild type and mutant APC gene products. Cancer Res 53, 2728- 2731 (1993).

Svensson, K.J. et al. A Secreted Slit2 Fragment Regulates Adipose Tissue Thermogenesis and Metabolic Function. Cell Metab 23, 454-466 (2016).

Tcherkezian, J. & Lamarche-Vane, N. Current knowledge of the large RhoGAP family of proteins. Biol Cell 99, 67-86 (2007).

Thorpe, L.M., Yuzugullu, H. & Zhao, J.J. PI3K in cancer: divergent roles of isoforms, modes of activation and therapeutic targeting. Nat Rev Cancer 15, 7-24 (2015).

Tole, S. et al. The axonal repellent, Slit2, inhibits directional migration of circulating neutrophils. J Leukoc Biol 86, 1403-1415 (2009).

Tsujita, K. et al. Coordination between the actin cytoskeleton and membrane deformation by a novel membrane tubulation domain of PCH proteins is involved in endocytosis. J Cell Biol 172, 269-279 (2006). van Furth, R. & Cohn, Z.A. The origin and kinetics of mononuclear phagocytes. J Exp Med 128, 415- 435 (1968). van Furth, R., Nibbering, P.H., van Dissel, J.T. & Diesselhoff-den Dulk, M.M. The characterization, origin, and kinetics of skin macrophages during inflammation. J Invest Dermatol 85, 398-402 (1985). van Gils, J.M. et al. Endothelial expression of guidance cues in vessel wall homeostasis dysregulation under proatherosclerotic conditions. Arterioscler Thromb Vasc Biol 33, 911-919 (2013).

Vazquez, F. & Devreotes, P. Regulation of PTEN function as a PIP3 gatekeeper through membrane interaction. Cell Cycle 5, 1523-1527 (2006).

113

Vetter, I.R. & Wittinghofer, A. The guanine nucleotide-binding switch in three dimensions. Science 294, 1299-1304 (2001).

Vielkind, S., Gallagher-Gambarelli, M., Gomez, M., Hinton, H.J. & Cantrell, D.A. Integrin regulation by RhoA in thymocytes. J Immunol 175, 350-357 (2005).

Wang, K.H. et al. Biochemical purification of a mammalian slit protein as a positive regulator of sensory axon elongation and branching. Cell 96, 771-784 (1999).

Watanabe, T. et al. Interaction with IQGAP1 links APC to Rac1, Cdc42, and actin filaments during cell polarization and migration. Dev Cell 7, 871-883 (2004).

Wen, Y. et al. EB1 and APC bind to mDia to stabilize microtubules downstream of Rho and promote cell migration. Nat Cell Biol 6, 820-830 (2004).

Wiktor-Jedrzejczak, W. & Gordon, S. Cytokine regulation of the macrophage (M phi) system studied using the colony stimulating factor-1-deficient op/op mouse. Physiol Rev 76, 927-947 (1996).

Wills, Z., Marr, L., Zinn, K., Goodman, C.S. & Van Vactor, D. Profilin and the Abl tyrosine kinase are required for motor axon outgrowth in the Drosophila embryo. Neuron 22, 291-299 (1999).

Wojciak-Stothard, B., Williams, L. & Ridley, A.J. Monocyte adhesion and spreading on human endothelial cells is dependent on Rho-regulated receptor clustering. J Cell Biol 145, 1293-1307 (1999).

Wong, K. et al. Signal transduction in neuronal migration: roles of GTPase activating proteins and the small GTPase Cdc42 in the Slit-Robo pathway. Cell 107, 209-221 (2001).

Worthylake, R.A. & Burridge, K. Leukocyte transendothelial migration: orchestrating the underlying molecular machinery. Curr Opin Cell Biol 13, 569-577 (2001).

Wu, J.Y. et al. The neuronal repellent Slit inhibits leukocyte chemotaxis induced by chemotactic factors. Nature 410, 948-952 (2001).

Yamazaki, D., Itoh, T., Miki, H. & Takenawa, T. srGAP1 regulates lamellipodial dynamics and cell migratory behavior by modulating Rac1 activity. Mol Biol Cell 24, 3393-3405 (2013).

Yang, J., Zhang, L., Yu, C., Yang, X.F. & Wang, H. Monocyte and macrophage differentiation: circulation inflammatory monocyte as biomarker for inflammatory diseases. Biomark Res 2, 1 (2014).

Yang, Y.H., Manning Fox, J.E., Zhang, K.L., MacDonald, P.E. & Johnson, J.D. Intraislet SLIT-ROBO signaling is required for beta-cell survival and potentiates insulin secretion. Proc Natl Acad Sci U S A 110, 16480-16485 (2013).

Yuan, W. et al. The mouse SLIT family: secreted ligands for ROBO expressed in patterns that suggest a role in morphogenesis and axon guidance. Dev Biol 212, 290-306 (1999).

114

Zallen, J.A., Yi, B.A. & Bargmann, C.I. The conserved immunoglobulin superfamily member SAX- 3/Robo directs multiple aspects of axon guidance in C. elegans. Cell 92, 217-227 (1998).

Zelina, P. et al. Signaling switch of the axon guidance receptor Robo3 during vertebrate evolution. Neuron 84, 1258-1272 (2014).

Zhou, W. et al. The role of SLIT-ROBO signaling in proliferative diabetic retinopathy and retinal pigment epithelial cells. Mol Vis 17, 1526-1536 (2011).

Ziegler-Heitbrock, L. et al. Nomenclature of monocytes and dendritic cells in blood. Blood 116, e74-80 (2010).

Zigmond, S.H. Mechanisms of sensing chemical gradients by polymorphonuclear leukocytes. Nature 249, 450-452 (1974).

115