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Biochemical protein interactions of Gliotactin at the tricellular junction Que, Jaimmie 2006

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BIOCHEMICAL PROTEIN INTERACTIONS OF GLIOTACTIN AT T H E T R I C E L L U L A R JUNCTION by JAIMMIE QUE B.Sc, University of British Columbia, 1999 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE In THE F A C U L T Y OF G R A D U A T E STUDIES (Zoology) THE UNIVERSITY OF BRITISH C O L U M B I A December 2006 © Jaimmie Que, 2006 Abstract The septate junction is an occluding junction in invertebrates, similar in function to tight junctions, playing a role in epithelial barriers, and in apical/basal polarity. Septate junction interactions are still being characterized as new component proteins are discovered. One septate junction protein, Gliotactin, was discovered in Drosophila to correlate with the mislocalization of characteristic septate junction proteins in a Gliotactin null animal. However, Gliotactin is the only component found exclusively at the level of septate junctions at tricellular junctions in epithelia. The tricellular junctions are the structure at the convergence of three cells and a potential organizing factor of the septate junction. This led to the question, what is Gliotactin's role in the organization of the tricellular and septate junctions? To study this, we looked at Gliotactin interactors, and attempted to elucidate a model of tricellular and septate junction protein interactions. Previous attempts at finding Gliotactin interactors were made using in vitro systems or by using transgenic animals using an over-expressed epitope-tagged Gliotactin, that showed that Gliotactin interacts with known septate junction proteins in a calcium dependent manner. This thesis aims to further explore Gliotactin interactions with the hypothesis that native Gliotactin in Drosophila interacts in a calcium dependant manner with septate junction proteins, Discs Large, and Neurexin IV. Using co-immunoprecipitation and GST pulldowns, on native Gliotactin protein in Drosophila, I have shown that Gliotactin does not interact with Neurexin IV but does interact with Discs Large in a calcium-dependent manner. This is significant in that, to date, there has been no known interactor of Discs Large or of native Gliotactin at the tricellular junction. I also present data on unidentified potential Gliotactin interactors seen in a GST pulldown assay. The data presented in this thesis has contributed to a new working model of the tricellular junction and the role of Gliotactin. i i Table of Contents A b s t r a c t i i T a b l e o f C o n t e n t s i i i L i s t o f T a b l e s v L i s t o f F i g u r e s vi L i s t o f A b b r e v i a t i o n s vii A c k n o w l e d g e m e n t s ix 1. I n t r o d u c t i o n 1 1.1 Cell Junctions and Permeability Barriers 1 1.2 Tight Junctions 2 1.3 Paranodal Junctions 3 1.3.1 Paranodal Junctions - Contactin, Caspr and Neurofascin 4 1.4 Septate Junctions 5 1.4.1 Septate Junction Development 8 1.4.2 Septate Junction Function 8 1.4.3 Septate Junction Components 9 1.4.4 Septate Junction Component Interdependancy 16 1.4.5 Gliotactin 16 1.5 Neuroligins 20 1.6 Specific Aim of the Thesis 21 2. M a t e r i a l s a n d M e t h o d s 24 2.1 Fly Strains Used 24 2.2 Fly Growth Conditions 24 2.3 Membrane Preparations 25 2.4 GST Pulldowns 26 2.5 Immunoprecipitation 27 2.6 Western Transfer and Immunoblotting 28 2.7 Coomassie Staining 29 2.8 Protein Sequencing Preparation 29 3. R e s u l t s 30 3.1 Identifying Gliotactin Protein Interactors From Known Septate Junction Proteins 30 3.1.1 Overexpressed H A Tagged Gliotactin is Unstable in Drosophila in the Long Term 30 3.1.2 Gliotactin Interacts With Discs Large 33 3.1.3 Gliotactin and Neurexin IV Do Not Show a Biochemical Interaction 38 3.2 GST Pulldowns Confirm Immunoprecipitation Results and Validates GST Pulldowns as a Method for Studying Gliotactin Biochemical Interactors 41 ii i 3.3 Identifying Unknown Proteins as Gliotactin Interactors 46 4. Discussion 51 4.1 Gliotactin May Be In A Separate Protein Complex Than Other Currently Known Septate Junction Proteins 51 4.2 Unknown Gliotactin Interactors 59 4.3 Summary and Future Studies 60 4.4 Conclusions 62 References 63 iv List of Tables Table 1: Comparison of N-terminal Sequence Data of Unknown Proteins Seen in a GST Pulldown of GST-Gli-cter List of Figures Figure 1: Septate Junction Transmission Electron Micrograph 7 Figure 2: Model of the Septate Junction 10 Figure 3: Gliotactin and Gliotactin Localization in Epithelial Cells 19 Figure 4: H A Tagged Gliotactin Expression is Unstable in Drosophila 32 Figure 5: Gliotactin Interacts Biochemically With Discs Large 35 Figure 6: Gliotactin Fails to Show an Interaction With Neurexin IV 39 Figure 7: GST Pulldowns Show a Similar Result to Immunoprecipitation Experiments 43 Figure 8: GST Pulldown Using GST Fused to Gliotactin C Terminus Shows the Presence of Unidentified Proteins 47 Figure 9: Amino Acid Sequence of CG31652 50 Figure 10: Model of Protein Interactions at the Tricellular Junction 57 vi List of Abbreviations Ab Antibody aHAJ Apical hemi adherens junction B L A S T Basic Local Alignment Search Tool Band 4.1 see Ferm bHAJ Basal hemi adherens junction CNS Central Nervous System Cor Coracle DE-cad DE cadherin Dig Discs Large DTT Dithiothreitol E C M Extracellumar matrix EDTA Ethylenediaminetetraacetic acid EF hand A helix-turn-helix protein domain which can bind calcium ions EGF Epidermal Growth Factor EGFR Epidermal Growth Factor Receptor FasIII Fasciclin III Ferm Protein domain named for 4.1, Ezrin, Radixin and Moesin GFP Green Fluorescent Protein GJ Gap junction Gli Gliotactin GPI Glycosylphosphatidylinisotol GST Glutathione-S-Transferase G U K guanylate kinase H A Hemaglutinin IF6.3 Mouse monoclonal anti-Gliotactin antibody Ig Immunoglobulin IgG Immunoglobulin G K+ Potassium ion K D a KiloDalton m M MilliMolar M Z Marginal zone Na/K+ Sodium/Potassium ion NAPS Nucleic Acid and Protein Services Nig Neuroligin Nrg Neuroglian NrxIV Neurexin IV PBS Phosphate Buffered Saline PDZ Protein interaction domain, short for PSD-95, Discs Large, ZO-1 PJ Paranodal Junction PMSF Phenylmethylsulphonylfluoride PNS Peripheral Nervous System PSJ Pleated Septate Junction PVDF PolyVinylidine DiFluoride Scrib Scribble SDS Sodium Dodecyl Sulfate SDS- Sodium Dodecyl Sulfate - Polyacrylamide Gel Electrophoresis SH2/3 Src Homology 2/3, binds phosphotyrosine residues SJ (pleated) Septate Junction TBS Tris Buffered Saline TCJ Tricellular Junction TJ Tight Junction T K M Tris, KC1 (potassium chloride), M g C l 2 (magnesium chloride) TRAF TNF (tumor necrosis factor) receptor associated factor, a protein signalling domain uL MicroLitre u M MicroMolar um MicroMeter WD-40 Beta-Transducin Repeats Z A Zonula adherens ZO-1 Zonula Occludens 1 Acknowledgements I would like to thank my supervisor, Dr. Vanessa Auld, for providing me with the opportunity to pursue a graduate degree in her lab at the University of British Columbia. I am truly grateful for all the help and support she provides with her open door policy to all the lab members. This work would not have been possible without her guidance, knowledge and kindness. Her positive support and encouragement have been invaluable. M y committee members, Dr. Linda Matsuuchi and Dr. Cal Roskelley have been a wealth of resources for ideas and suggestions as well as providing me with motivation and encouragement along the way. To Dr. Murry Gilbert and the Auld lab past and present, your friendship, support and of course, ideas and research discussion are well and truly appreciated. To Dr. Jacob Hodgson, who provided me with so much help with protein biochemistry, again, this work would not have been possible without your valuable insight and knowledge in the field. To friends made at UBC, thank you for your help, ideas, assistance and most of all your friendship throughout these years. To the Zoology Department staff, especially our graduate secretary, thank you. Finally, much love and thanks to my family for everything. ix 1. Introduction One of the global questions often addressed in development of organisms is how do the formation and maintenance of separate body cavities and structures arise. Integral to this question is the role of permeability barriers. These structures help to control and regulate the movement of molecules through a layer of cells. Permeability barriers are helped by the presence of occluding junctions located between cells. These junctions help to prevent the diffusion of most molecules across the intercellular space. Study of these occluding junctions is an ongoing task, and we are just beginning to understand how they work. In the invertebrate model organism, Drosophila melanogaster, we have an opportunity to study the structure of the pleated septate junction, a type of occluding junction exclusive to invertebrates, in a living organism. 1.1 Cell Junctions and Permeability Barriers Cell attachment, communication, polarity, and the formation of permeability barriers, are all functions of cell junctions. Occluding junctions are a subset of cell junctions which help maintain the integrity of different body cavities; these junctions create a seal which results in the creation of permeability barriers. These are seen in the blood nerve barrier, epithelial and endothelial barriers, and they also help in creating and maintaining cell polarity (Cereijido et al., 2004; Staddon and Rubin, 1996). Permeability barriers are integral to both vertebrates and invertebrates, though the different organism types also have different methods for creating their permeability barriers. In 1 invertebrates, septate junctions (SJ) function as permeability barriers, while in vertebrate systems, the tight junctions (TJ) are the functionally analogous structures in epithelial tissues. There are many differences between these two junction types, the most obvious is their apicobasal location relative to the adherens junctions, and their components. Septate junctions are located basally to, whereas tight junctions occur apical to adherens junctions (Tepass et al., 2001). 1.2 Tight Junctions Tight junctions are a type of vertebrate occluding junction. Depending on cell type, there is quite a bit of variability in the junction, the similarity is in their structure, which are often described as "kissing points". Tight junctions are varied because of the number of different proteins thought to exist at these junctions. There are scaffold proteins such as the zonula occludins and ZO-1, signalling proteins, such as G proteins, involved in the different aspects of assembly and cell polarity, and the structural proteins such as occludins and the large family of claudins, proteins which are implicated in cell adhesion and tight junction assembly (Anderson et al., 1993; Tsukita et al., 2001). The first tight junction protein discovered, occludin, has four transmembrane domains (Balda and Matter, 2000), and shows homophilic binding in the extracellular space, aiding in TJ strand formation (Anderson, 2001; Fanning et al., 1999). They are known to be differentially phosphorylated, and that overexpression shows an increased number of TJ strands (Denker and Nigam, 1998). 2 Like occludin, claudins are proteins characterized by four transmembrane domains, and have two extracellular loops. Different claudins are expressed based on tissue type and is thought to be directly involved in the formation of TJ strands and in the ability of TJ's to act as permeability barriers (Tsukita et al., 2001; Sonoda et al., 1999; Anderson, 2001). There is however, little sequence homology between the various known claudins (Anderson, 2001), and heterophilic binding between different types of claudins have been observed (Furuse et al., 2001) . Recently, another barrier protein in vertebrates had been identified called tricellulin. Tricllulin appears at the corners where three cells meet (the vertebrate tricellular junction) and has some structural similarity to occludin (Ikenouchi et al., 2005). This protein however does not resemble Gliotactin and does not appear to have a Drosophila homolog. 1.3 Paranodal Junctions Another junction in vertebrates, which acts as an occluding junction, is the paranodal junction (PJ). These junctions resemble and show a high degree of conservation to the invertebrate septate junction. They are found at the paranodal region, flanking the nodes of Ranvier in myelinated axons (Scherer and Arroyo, 2002). They are thought to be similar to the invertebrate septate junction because of their morphology and molecular components (Scherer and Arroyo, 2002) . Septate and paranodal junctions have some structural and molecular similarities that have not yet been fully determined. It has been suggested that insect septate junctions took a more specialized role in vertebrates, as the paranodal junction, with tight junctions replacing septate junctions in the epithelia (Hortsch and Margolis, 2003). It is with this idea that we can examine 3 paranodal junctions in vertebrates and compare the structure and function with that of the invertebrate septate junction. 1.3.1 Paranodal Junctions - Contactin, Caspr and Neurofascin Contactin, an axonal protein found at paranodal junctions (PJ), is a GPI anchored protein belonging to the Ig superfamily (Ranscht, 1988). This protein is known to complex with another PJ protein called Caspr (contactin associated protein) (Rios et al., 2000), and its absence causes disrupted paranodal junctions, Caspr expression, and localization of K+ channels, K v l . l and K v l . 2 (Boyle etal., 2001). Caspr is another axonal transmembrane protein required for proper septa formation at the paranodal junction (Poliak et al., 2003). It has a proline rich domain known to bind SH3 domains, and is similar to the Drosophila SJ Protein, Neurexin IV (NrxIV) (Peles et al., 1997). It was found that an intercellular region of Caspr has a Protein 4.1 binding domain that stabilizes it in a complex with Contactin (Gollan et al., 2002). It was also noted that Caspr does not have a PDZ binding domain (Arroyo et al., 1999). Another protein, Caspr 2 on the other hand, does have a PDZ binding domain on the C terminus tail, while also having a protein 4.1 binding domain (Poliak et al., 1999). Caspr 2 was found to also associate with K+ channels (Kvl .2, 1.2 and the beta 2 subunit) which are present at the juxtaparanode (the area before the paranode) (Arroyo etal., 1999). Caspr and Caspr 2, homologues of the septate junction protein Neurexin IV, interact with Protein 4.IB and Protein 4.1R, which are similar to the invertebrate SJ protein, Coracle (Cor), at 4 paranodal junctions (Denisenko-Nehrbass et al., 2003; Einheber et al., 1997; Bellen et al., 1998). As Neurexin IV and Coracle are known to interact in septate junctions (Ward et al., 1998), Caspr2 has been shown to biochemically interact with Protein 4.IB (Denisenko-Nehrbass et al., 2003). As well, mutations in Caspr and Caspr2 show mislocalization of Protein 4. IB (Poliak et al., 2001). Recently, Caspr 3 and 4 have also been discovered in the central nervous system and appear to have the ability to bind PDZ domains (Spiegel and Peles, 2002). Neurofascin, a cell surface glycoprotein, has a variety of splice variants like its invertebrate homolog, Neuroglian (Nrg) (Rathjen et al., 1987; Volkmer et al., 1992; Hortsch and Margolis, 2003). Like Neuroglian, Neurofascin has two isoforms, NF186, a neuronal Neurofascin, and NF155, the form expressed on glial cells (Tait et al., 2000). Neurofascin is implicated in axonal guidance (Rathjen et al., 1992; Shiga and Oppenheim, 1991), is found to colocalize with Caspr (Tait et al., 2000), is found to bind the Caspr/Contactin complex on the extracellular tail (Charles et al., 2002), and has ankyrin binding domains (Zhang et al., 1998). The binding of NF155 to Caspr and Contactin also seems to be important in the proper formation of paranodal junctions (Schafer et al., 2004; Charles et al., 2002). 1.4 Septate Junctions Septate junctions are the invertebrate occluding junction. They are found in epithelial, trachea, gut, and glial cells (Carlson et al., 2000; Tepass et al., 2001). Two different varieties of septate junctions exist, differing in their morphology. Pleated septate junctions arise in tissue originating from ectoderm, and smooth septate junctions arise from tissue originating from the endoderm (Tepass and Ffartenstein, 1994). Physically, pleated septate junctions are visible under 5 electron microscopy, while smooth SJ's are barely, if at all, visible (Tepass and Hartenstein, 1994). In this thesis, SJ will refer to the pleated variety. The invertebrate septate junction is often thought to be analogous to the vertebrate tight junction because of their role as barriers. However, unlike the tight junctions, septate junctions occur basal to adherens junctions. Septate junctions are named because of the ladder-like appearance of septa (Figure 1, panel A and B) which occur in spirals around cells (Tepass et al., 2001). As occluding junctions, septate junctions are involved in permeability (Lord and DiBona, 1976), such as in epithelial barriers, or in the blood-nerve-barrier (Knust and Bossinger, 2002). As well, they are also involved in cell polarity because they can block apical/basal migration of membrane proteins (Knust and Bossinger, 2002). 6 Figure 1: Septate Junction Transmission Electron Micrograph Panel A shows pleated septate junctions show a "ladder-like" morphology at cell junction points, with clusters shown in brackets and single septa shown with arrowheads. Bar = lOOnm. Figure adapted from (Schulte et al., 2003). Panel B shows the septate junction at the tricellular junction (TCJ), with arrows showing the septa between two cells. Bar = 50nm. Figure adapted from (Schulte et al., 2003). Panel C shows the basolateral position o f the septate junction in relation to the adherens junction in invertebrates. 7 1.4.1 Septate Junction Development In Drosophila, septate junction assembly occurs about two thirds through embryogenesis in anticipation of forming permeability barriers later in development. The SJ assembly takes place at approximately stage 14-15 when dorsal closure occurs, although some known SJ components such as discs large and scribble appear earlier, playing what is believed to be an unrelated role in the formation of apical/basal polarity (Tepass et al., 2001; Tepass and Hartenstein, 1994). As SJ components are being continuously discovered, the process of assembly of SJs, on a molecular level, is not yet fully understood. Additionally, at the corners of epithelial cells at the level of SJs, there is a specialized structure called the "tricellular plug", which forms at the convergence of three neighbouring cells to form a permeability barrier (Graf et al., 1982). This is important for the prevention of diffusion between compartments. 1.4.2 Septate Junction Function Because they act as a physical barrier, septate junctions are thought to function in cell adhesion, and have several functions in the formation and maintenance of apicobasal cell polarity (Tepass et al., 2001). Additionally, the tricellular junction (TCJ) is believed to be an organization centre for the SJ strands, since disrupting the TCJ shows a decrease in the number of septa, as well as increased permeability of the SJ, which results in compromised SJ's. (Schulte et al., 2003). 8 1.4.3 Septate Junction Components While new components of SJ's are continuously being discovered, there are several characteristic proteins such as Neurexin IV, Coracle and Discs Large, as well as Scribble, Fasciclin III, Neuroglian, Gliotactin, and Ankyrins which are widely believed to be at the SJ due to known interactions (Hortsch and Margolis, 2003). In addition, there are the more recently discovered components of septate junctions such as the a and (3 subunits of the N a + / K + pump, Lachesin, and the claudins, Megatrachea, Sinuous (Wu et al., 2004; Genova and Fehon, 2003; Llimargas et al., 2004; Behr et al., 2003). How these proteins all interact together is one aspect that is unknown and important for examining the mechanics of the septate junction. Please refer to Figure 2 for diagram. 9 Figure 2: Model of the Septate Junction This shows a model of the possible septate junction components and possible interactions. Grey lines indicate potential interactors and the question mark indicates that the Gliotactin interaction is yet unknown. Note that Ankyrin is has not yet shown to be at the septate junction but is theorized to be present due to known interactors as shown in the figure. 10 Neurexin IV (NrxIV) is a transmembrane protein that has a discoidin domain, EGF domains and Laminin G domains, whose functions are yet to be determined, on the extracellular portion, while it has a PDZ binding domain and a Ferm binding domain on the intercellular portion (Einheber et al, 1997; Bellen et al., 1998). The Ferm binding domain (refer to NrxIV in Figure 2, black dot on intracellular domain) is known to interact with the SJ protein, Coracle (Baumgartner et al, 1996). Additionally, without NrxIV, Cor is mislocalized at the SJ (Baumgartner et al., 1996). Drosophila embryos, which have null mutations in NrxTV, have defective SJ's, leading to a disruption in barrier formation(Baumgartner et al., 1996). This leads to paralysis, and subsequent death of Drosophila embryos due to a leaky blood-nerve-barrier. NrxIV null mutants also show mislocalization of other septate junction proteins such as Coracle and Gliotactin (Baumgartner et al., 1996; Schulte et al., 2003). Coracle, a cytoplasmic protein, is an invertebrate ortholog of Protein 4.1, a structural protein (Fehon et al., 1994). In vertebrates, Protein 4.1 is known to interact with the cytoskeleton, however, Cor seems to lack this function as it is missing the spectrin-actin binding domain present on Protein 4.1 (Ward et al., 2001; Woods et al., 1996a). Mutants of Cor have problems in dorsal closure, perhaps as Cor mutants have a defective septate junction and dorsal closure involves the fusion of epithelial cells (Fehon et al., 1994). As well, Cor mutants also dominantly suppresses ellipse (EGF receptor) mutations in Drosophila eye development (Fehon et al., 1994). Interestingly, a highly conserved domain on the Coracle protein (similar to Protein 4.1), the Ferm (Protein 4.1, Ezrin, Radixin, Moesin) domain, binds NrxTV, and was found to be sufficient for SJ barrier formation (Ward et al, 2001). 11 Fasciclin III (Fas III), is a transmembrane glycosylated protein, and while it has been a known SJ protein for some time, its function at the SJ is not yet known as null mutants have no discernable SJ phenotype (Patel et al., 1987). It is known that Fas III is a cell adhesion molecule with three Ig domains and that it binds in a homophilic manner (Snow et al., 1989; Patel et al., 1987). However, proteins that bind Fas III are not known, though it was found that Dig and Lachesin are required for proper Fas III localization (Woods et al., 1996b; Llimargas et al., 2004). One of the most recently discovered SJ components is the GPI (glycosylphosphatidylinositol) anchored protein, Lachesin (Llimargas et al., 2004; Karlstrom et al., 1993). Lachesin is first expressed during neurogenesis (Karlstrom et al., 1993), and has three Ig domains plus the GPI anchor, suggesting it may have a role in adhesion due to its similarities to Neuroglian and other cell adhesion molecules. Lachesin was later found to localize to the SJ and has a role in epithelial integrity and tracheal tube size control (Llimargas et al., 2004). An in vitro study showed homophilic properties of this protein, and co-localization experiments showed the mislocalization of known SJ proteins in Lachesin mutants, with Cor, Dig, Scrib, NrxIV, Nrg and Fas III showing basolateral spreading and no longer being tightly localized to the SJ region (Llimargas et al., 2004). Neuroglian is a transmembrane glycosylated protein that belongs to the Ig superfamily, similar to the vertebrate neuronal cell adhesion molecules (Bieber et al., 1989). It has six Ig domains and five fibronectin domains on the extracellular portion (Bieber et al., 1989). Neuroglian mutants have compromised septate junctions and do not survive past embryogenesis (Genova and Fehon, 2003). There are two splice variants of the Neuroglian protein which differ in their 12 cytoplasmic domains, and are either neuronal specific (longer splice variant) or expressed in a wide range of other tissues, including the epithelia (shorter splice variant) (Hortsch et al., 1990). Nrg, found at the SJ, has a highly conserved ankyrin binding site, and the non-neuronal Nrg interacts with echinoid, a cell adhesion molecule involved in EGFR signalling (Hortsch and Margolis, 2003; Bieber et al., 1989; Islam et al., 2003; Dubreuil et al., 1996). Interestingly, neurofascin, a vertebrate homolog of Nrg, does not show ankyrin binding when the highly conserved sequence on the intercellular tail of neurofascin, FIGQY (single aa code), is tyrosine phosphorylated, leading to a decrease in cell-cell interaction (Tuvia et al., 1997). However, this may not be the case for Nrg (Hortsch et al., 1995). Ankyrins are membrane adapter proteins found in the cytoplasm, and are thought to be at the SJ because of known interactors. They contain many ankyrin repeats and have a spectrin binding domain, enabling it to cross link with actin and molecular scaffold proteins (Bennett and Baines, 2001). Ankyrins have the ability to bind to N a + / K + pumps (Nelson and Veshnock, 1987; Zhang et al., 1998), and Neuroglian (Bouley et al., 2000). If ankyrins are at the SJ as suspected, it would indicate that the SJ could also play a role in cell structure as ankyrins would provide the link with the cytoskeleton. One of the more recently discovered septate junction components are the a and P subunits of the sodium/potassium ATP-ase pump; the N a + / K + ATP-ase pump components found at SJ's consists of the ATPa , and the Nervana 2 P subunit (Genova and Fehon, 2003). Null mutants of the N a 7 K + pump result in a disrupted barrier function of the SJ, missing septae, and tracheal tube size defects which are seen in many other mutations of known SJ components (Genova and 13 Fehon, 2003; Paul et al., 2003). The N a + / K + pump is known to interact with ankyrin (Bennett and Chen, 2001), which leads to the speculation that the cytoskeleton is linked to the SJ. Drosophila contactin is another SJ protein, with a lectin domain, six Ig domains, and four fibronectin domains (Hortsch and Margolis, 2003). This extracellular protein, like Lachesin, is covalently anchored to the membrane via a GPI anchor (Walsh and Doherty, 1991). The next two septate junction proteins are unique because they are similar to claudins. These proteins contain four transmembrane domains, characteristic of tight junction claudins, although the septate junctions do not show any TJ-like strands despite these claudin-like proteins. The first claudin-like protein found at SJ's was Megatrachea (Mega), which has the four characteristic transmembrane domains(Behr et al., 2003). As well, Mega has a potential PDZ binding domain on the C terminus (Behr et a l , 2003). Megatrachea mutants have compromised septate junctions (and thus epithelial barriers), as well as mislocalized SJ markers such as NrxIV, Cor, Sinuous, and FasIII, (Wu et al., 2004; Behr et al., 2003). The second claudin-like protein found at SJ's is Sinuous (Sin), also containing the characteristic four transmembrane domains and a potential PDZ binding domain on the C terminus (Wu et al., 2004). Sinuous is also required for proper SJ barrier formation, tracheal development, and localization of known SJ proteins, Cor, NrxIV, ATPoc, and Dig in the epidermis, hindgut and trachea, though strangely, not in the salivary gland (Wu et al., 2004). Which proteins these two claudins interact with have not yet been determined. Discs Large (Dig), was first discovered as a tumor suppressor gene expressed about half way through development (Watson et al., 1994). The protein was discovered to have at least ten 14 different isoforms, and localizes to the septate junction (Woods et a l , 1996a; Woods and Bryant, 1991). Dig is also necessary for proper localization of known SJ proteins, Coracle and Fasciclin III (Woods et al., 1996b). The Dig protein is cytoplasmic, as it lacks a signal sequence, and has guanylate kinase (GUK) and SH3 domains (Woods and Bryant, 1991). Later, PDZ domains, known to be involved in protein-protein interactions, were found on Dig, as well as a binding site for the Protein 4.1 family (Cho et al., 1992; Lue et al., 1994a; Marfatia et al., 1996; Morais Cabral et a l , 1996). Dig was also the first Maguk (membrane associated guanylate kinase homologs) protein found. Maguk proteins are involved in cell junctions and are known to have some importance in cell signalling; they are characterized as possessing G U K , SFI3 and PDZ domains (Anderson, 1996; Caruana, 2002). In 1997 the presence of three types of PDZ domains on Dig, which were termed type 1,2 and 3, were discovered (Hough et a l , 1997). These PDZ binding domains are required for localization to the membrane, and the PDZ type 2 domain is required for SJ localization. The PDZ domains are particularly interesting as it makes Dig a candidate for Gl i binding, especially as Dig appears to be enriched at the tricellular corners (Schulte et al., 2006). Another protein with many PDZ domains, Scribble (Scrib), is also at the septate junction (Hortsch and Margolis, 2003). Scribble is a protein whose absence causes mislocalization of apical proteins, and is also a protein that has multiple PDZ type 1 domains on the carboxy terminus and many leucine rich repeats (also known to be involved in protein-protein interactions) on the amino end (Bilder and Perrimon, 2000). These potential protein interaction domains would seem to suggest that Scrib binds other proteins at the SJ, and while some have been identified (Discs Large, and Lethal Giant Larvae) (Bilder et al., 2000), it could potentially 15 have other as yet unknown interactions. Of course the presence of PDZ domains makes it a potential Gliotactin binding partner. 1.4.4 Septate Junction Component Interdependency When one component of the septate junction is removed or mutated, often, the result is the disruption of other SJ components. For example, Coracle is mislocalized when Neurexin IV is disrupted (Baumgartner et al., 1996), Megatrachea is mislocalized in Cor and NrxTV mutant embryos, (Behr et al., 2003), and the mislocalization of FasIII is observed when Dig and Lachesin are disrupted (Woods et al., 1996a; Llimargas et al., 2004). NrxIV, Cor, Sinuous and FasIII are dependant on Megatrachea (Behr et al., 2003), while Cor, NrxIV, ATPa , and Dig are dependant on Sinouous (Wu et al., 2004). There is also mislocalization of Discs Large, Cor, NrxIV, Scribble, Fasciclin III and Neuroglian in Lachesin mutant embryos (Llimargas et al., 2004). 1.4.5 Gliotactin Gliotactin (Gli), the protein examined in this thesis, is a transmembrane protein present exclusively at the tricellular junction, at the level of septate junctions (see figure 3) (Llimargas et al., 2004; Schulte et al., 2003). As the tricellular junction is thought to possibly organize the septate junction (Schulte et al., 2003), I am interested in understanding the role of Gl i , and specifically address what proteins are bound to Gli . Characterization of Gl i shows a type 1 PDZ recognition domain (Schulte et a l , 2003), a non-functional choline-esterase-like domain on the extracellular portion of the protein, which may serve in cell adhesion, a putative EF hand 16 domain, which may bind calcium ions, a potential TRAF domain, which may be involved in signalling, potential SH2 or SH3 domains, known to be involved in protein-protein interactions, and possible tyrosine phosphorylation sites (V. Auld, personal communication). It was recently found that this intercellular domain of the protein lacks a fixed 3D structure (Zeev-Ben-Mordehai et al., 2003), though the implications of this has yet to be determined. As well, some genetic analysis shows the possibility that Gl i may act as a dimer (Venema et al., 2004). Additionally, Gliotactin mutants show mislocalization of Cor, NrxTV and Dig, septate junction markers that had spread more basally while still showing enrichment apically (Schulte et al., 2003). These mutants have delayed dorsal closure, the trans-epithelial barriers are compromised, and show a reduced number of septa, possible evidence for the tricellular junction as an organizing factor for septate junctions (Schulte et al., 2003). Dig and NrxTV, invertebrate homologs of PSD-95, and Neurexin family member, Caspr (Bellen et al., 1998), have been tested for interactions with Gliotactin. NrxTV, Dig and Cor showed calcium dependant binding to a transgenic Gl i (Schulte, 2002 PhD thesis), while Dig has been shown to complex with, though not directly binding to, Gliotactin (C. MacKinnon, M.Sc. Thesis, 2005). Gliotactin also shows homology (sharing a common developmental origin) to the Neuroligins, and more specifically, to Neuroligin 3 (Nlg3) (Gilbert et al., 2001). Nlg3 is expressed in a range of glia in the CNS and the PNS (Gilbert et al., 2001; Philibert et al., 2000), but interestingly, is not present at the septate-like paranodal junctions (M. Gilbert and A. Khurana, personal communication). Still, Nlg3 can potentially provide clues on molecular binding of its invertebrate homolog, Gliotactin. 17 Other k n o w n interactors o f the Neuro l i g ins are S - S C A M s (synapt ic scaf fo ld ing mo lecu le ) , b i nd ing N i g v i a one o f its f i ve P D Z domains (H i rao et a l . , 1998), and C I P P (channel interact ing P D Z domain ) , w h i c h also contains several P D Z domains (Kurschner et a l . , 1998). The S - S C A M interact ion hints at G l io tac t in b ind ing partners, as a G S T p u l l d o w n showed M a g i , an S - S C A M homologue , potent ia l ly interact ing w i th the C terminus end o f G l i (C . M a c K i n n o n , M S c . thesis, 2005). 18 Figure 3: Gliotactin and Gliotactin Localization in Epithelial Cells Panel A shows a model of Gliotactin with domains of interest labelled. Note that the second phosphotyrosine is a potential SH2 domain. Panel B shows Discs Large, a SJ marker in red (anti-Dig primary antibody with fluorescent secondary antibody) and Gliotactin in green (anti-Gli primary antibody with fluorescent secondary antibody) in Drosophila larval epidermal cells. Note that Gl i is shown to exist at the tricellular junction (appears yellow) and shows the characteristic pattern on Gl i at the SJ in epithelial cells. Sizebar is 15 um. Adapted from Schulte et al., 2006. conserved tyrosine phosphorylation sites P signal sequence non-functional serine-esterase-like domain EFhand PDZ binding domain A B 19 It remains to be seen which other known, or unknown SJ proteins interact with Gl i . Though PDZ containing proteins are likely candidates, Dig, while present in a complex with Gl i , does not appear to directly bind (C. MacKinnon, M.Sc. Thesis, 2005). It remains to be seen if Scribble, which has multiple type 1 domains (Bilder and Perrimon, 2000), binds to the intercellular portion of Gli . 1.5 Neuroligins In vertebrates, the Neuroligins are a family of transmembrane cell adhesion proteins that are most closely related to the invertebrate septate junction protein, Gliotactin (Gilbert et al., 2001). They bind some a-Neurexins and f}-Neurexins, neuronal cell surface proteins which play a role in adhesion and signalling, in a calcium dependant manner (Ichtchenko et al., 1995; Ichtchenko et al., 1996; Nguyen and Sudhof, 1997; Boucard et al., 2005). They also bind PSD-95, a homolog of Drosophila protein Discs Large, at a PDZ binding domain (Irie et al., 1997; Nguyen and Sudhof, 1997). Neuroligin 1 (Nlgl) was specifically found to form synaptic junctions through interactions with P-Neurexins (Song et al., 1999). In a significant finding, Nig 1 and 2 with their receptor, 6-Neurexin, were found to initiate the formation of presynaptic structures showing that neuroligins are able to initiate and are required for synaptic development (Scheiffele et al., 2000). At first, the Neuroligins (1-3) were thought to occur only in the CMS (Ichtchenko et al., 1995; Ichtchenko et al., 1996). However, this concept was revised when the discovery of a novel human Neuroligin, Nlg4, was found in many different tissue types, including the heart, liver, 20 pancreas, skeletal muscle, but only at low levels in the brain (Bolliger et al., 2001). Furthermore, Nlg3 was later found in glia both in the CNS and PNS (Gilbert et al., 2001). Neuroligin 3 was first believed to be at the paranodal junction, but was found to not be present (M. Gilbert, unpublished data). However, this does not exclude the other Neuroligins such as 2 and 4, which could be at the paranodal junction. Although there are obvious differences in the vertebrate paranodal junctions when compared to the invertebrate septate junctions, it isn't unreasonable to believe that analogous components can provide clues to the interactions in both systems. Recently, Nlg3 and Nlg4 have been implicated in a human disease, autism and Asperger syndrome (Jamain et al., 2003). The families analyzed either had a shortened Nlg4, or a point mutation in Nlg3 which converted an arginine to a cysteine in a section of the non-functional serine-esterase-like domain which is thought to show calcium binding properties (Jamain et al., 2003). This is interesting as the Drosophila septate junction counterpart, Gliotactin has shown calcium dependent binding to NrxIV and Coracle (Schulte, 2002 PhD thesis). 1.6 Specific A im of the Thesis Gliotactin is currently the only known protein present at in septate junctions that localizes exclusively to the tricellular junction. We know that when Gliotactin is disrupted, other septate junction components become mislocalized, and the barrier function of the SJ becomes compromised. Loss of Gliotactin leads to a mislocalization of Neurexin IV, Coracle and Discs Large, as well as a reduced number of septa, and septa spread down the basolateral edge 21 (Schulte et al., 2003). This indicates that Gliotactin may have an important role in either organization or structure of the pS J, and specifically the TCJ by perhaps organizing or stabilizing the core SJ complex (Schulte et al., 2003). Additionally, an overexpressed H A tagged Gliotactin was shown to biochemically interact with known SJ proteins, Cor, NrxIV and Dig in a calcium dependant manner (Schulte et al., 2003). It is the aim of this thesis is to examine if Gliotactin interacts with other septate junction components, and to discover other potential Gliotactin protein interactors in an effort to elucidate further, the role of Gliotactin at the T C J . Hypothesis: Gliotactin interacts in a calcium dependent manner with other septate junction proteins. Objectives of the thesis: 1. Look for Gliotactin protein interactors by testing potential known septate junction protein candidates. 2. Look for unknown Gliotactin protein interactors. Methods to achieve thesis objectives: 1. Use immunoprecipitation of membrane protein extracts of Drosophila melanogaster to purify the protein of interest and then probe the resulting blots with various antibodies to known septate junction proteins. 22 2. Use glutathione-agarose beads bound to a protein construct of GST fused to the C terminus of Gliotactin in a GST pulldown using membrane protein extracts of Drosophila melanogaster. Probing of the resulting blots with various antibodies to known septate junction proteins will give supportive in vitro results of co-immunoprecipitation experiments. 3. Use glutathione-agarose beads bound to a protein construct of GST fused to the C terminus of Gliotactin in a GST pulldown using membrane protein extracts of Drosophila melanogaster. Resulting blots would be stained and prominent proteins that had bound to the C terminus of Gliotactin would be sent for sequencing to attempt to identify unknown proteins capable of binding Gliotactin. These methods for identifying Gliotactin interactors have the limitations of a biochemical system. Immunoprecipitations show us entire organism protein interactions of Gl i , not solely the Gli interactors in the TCJ, and GST pulldowns show only potential interactors of the C terminus. However these biochemical experiments are intended to complement other current research on Gliotactin. Additionally, the use of membrane protein extracts from the collection of adult and embryo Drosophila should help show native interactions occurring in the animal itself, with less risk of showing artifacts of a purely in vitro system of identifying protein-protein interactions. 23 2. Materials and Methods 2.1 Fly Strains Used Oregon R - a wild type lab strain (OrgR) 1119 w - Drosophila lacking eye pigment, making the eye appear to be white in colour. Used here as a pseudo wild type strain, lab stock #107 (w 1 1 1 8 P { P T T - G A J N r g 0 0 0 3 0 5 [Nrg G 3 0 5]) - expresses GFP tagged Neuroglian (NrgGFP), lab stock # 138, Bloomington Stock # 6844. Drosophila strain from: (Morin et al., 2001). w; U A S - g / i P D Z A # 1 " H A ; da.G32 - expressing HA-tagged gliotactin with the last three amino acids truncated to exclude the last PDZ domain (FLA-gliAPDZ). Drosophila strain from: Schulte et al., 2003. w; UAS-g//™13"1^; ga.G32 - expressing FLA tagged gliotactin (FLA-gli). Drosophila strain from: Schulte et al., 2003. 2.2 Fly Growth Conditions Flies were raised at 22°C, on standard fly food media (12.6% agar, 31 m M potassium sodium tartrate, 5 m M calcium chloride, 350 m M dextrose, 175 m M sucrose, 3.2% w/v yeast, 7.6% w/v cornmeal, 2.5% ethanol, 480 m M tegosept, 0.5% ampicillin, 0.15% tetracycline). Adult collection involved exposing flies to carbon dioxide and collecting unconscious adults into eppendorf tubes. Embryo collection involved growth of flies in a population chamber on apple juice plates: apple juice agar media (2.6% agar, 25% apple juice, 73 m M sucrose, 10 m M Methyl 4-hydroxybenzoate) in large (Falcon, #351058, Franklin Lakes, NJ, USA) Petri dishes. Flies laid eggs on apple juice media, and apple juice plates were collected after 12 hours. Embryos were collected by dechorination through applying a 50% bleach solution to apple juice plates, which were allowed to sit for five minutes. Collection proceeded by filtering the media through fine nylon mesh, then washing off remaining embryos on apple juice plates with 0.05% 24 Triton X-100 solution. Embryos were then suspended in a small amount of 0.05% Triton X-100 solution and collected with a Pasteur pipette into eppendorf tubes. 2.3 Membrane Preparations Membrane preparations were made from the following strains: w; U A S - g / / ^ " 1 ^ ; ga.G32 adults, w; U A S - g / / P D Z / s * 1 " H A ; da.G32 adults, w 1 1 1 8 adults, OregonR embryos, andNrgGFP adults. Membrane extracts were prepared using the method described by Zhang and Hsieh (2000). This method involved the extraction of membrane and membrane bound proteins by centrifugation through a density gradient, and all steps were performed on ice or at 4°C. Approximately 4-6 mL of Drosophila adults or embryos were homogenized using a handheld 15 mL glass homogenizer with 2-4 mL of detergent-free lysis buffer (50 m M Tris-Cl pH 7.5,150 m M KC1, 5 m M MgCl 2,0.25 M Sucrose, 0.1 m M DTT, 1 m M PMSF, 2 ug/mL leupeptin, 2 ug/mL pepstatin), chosen because it would not dissociate integral proteins from cell membranes. Centrifugation at 1000G followed, to sediment particulates. The resulting supernatant was mixed with 15.2 mL of 2.5 M Sucrose in T K M (50 m M Trizma Base, 150 m M potassium chloride, 5 m M magnesium chloride, pH 7.5), and subsequent layers of 12.5 mL 2.0 M and 7.0 mL 0.5 M Sucrose solutions (of the 2.5 M Sucrose in T K M diluted with lysis buffer) were layered on top. A 100,000 G spin for 2.5 hours followed and the result was isolated membranes appearing at the interface of the 0.5 M and 2.0 M layers. This was collected using a Pasteur pipette and mixed with 2 volumes of lysis buffer. A final centrifugation to wash the isolated membranes was performed at 30,000 G for 30 minutes. The membrane pellet was resuspended in 200 uL of lysis buffer and total protein concentration was then determined using the Bradford dye-binding assay. 25 The Bradford assay is based on coomassie dye binding of proteins in a solution. More protein present results in a stronger blue colour, and thus protein concentration is tested against a standard curve of absorbance at 595 nm against known protein concentrations (usually ranging from 0-100 ug/100 uL). In this case, Bradford reagent was prepared using Bio-Rad Protein Assay Reagent (#500-0006, Hercules, C A , USA) by dilution of the concentrate in double distilled water at 1 in 5, and then filtered to remove particulates. Five standards of 0, 20, 60, 80 and 100 ug/uL bovine serum albumin were prepared and 5 mL of Bradford reagent was added. Similarly, the sample(s) tested were usually diluted as appropriate, typically 1 in 10, with 5 mL Bradford reagent added. Standards and samples were mixed by brief vortexing, and the colour was allowed to develop for 5 minutes. Following colour development, absorbance was measured at 595 nm, and a standard curve of absorbance versus protein concentration was generated to compare absorbance of the sample(s) and determine the protein concentration. The membrane extract was then flash frozen with liquid nitrogen and stored at -80°C. Membrane extracts were thawed on ice immediately prior to use. 2.4 GST Pulldowns Glutathione agarose beads, (Sigma, cat #G4510, St. Louis, M O , USA), 30 uL in a 50:50 slurry in PBS (1.86 m M N a H 2 P 0 4 * H 2 0 , 8.41 m M Na 2 HPO, 174.9 m M NaCl, pH 7.2-7.6), were prebound overnight with 75 ug GST-Gli-cter (a construct designed by C. MacKinnon containing the intracellular portion of Gliotactin fused to GST - C. MacKinnon, 2005, MSc. Thesis) were incubated in 250 uL lysis buffer (10 m M Tris-HCl, 4 m M EDTA, 1 m M EGTA, 150 m M NaCl, 0.5% Triton X-100,1 m M PMSF, 2 ug/mL leupeptin, 2 ug/mL pepstatin) with varying calcium concentrations of either 0.0,0.05,0.1 or 0.2 uM. Calcium concentration was determined by the 26 on-line chelator calculator which was located at: http://www.stanford.edu/~cpatton/webmaxCS.htm. After the overnight incubation, beads were washed three times with lysis buffer, then incubated with 150 ug w l 118 adult or OregonR embryo membrane preparations for 3 hours at 4°C with gentle agitation. Following incubation, beads were washed 3-5 times with lysis buffer. After the final wash, 30uL of Laemmli loading buffer (2% SDS, 100 m M DTT, 50 m M Tris-Cl pH 6.8, 10% glycerol, 0.05% Bromophenol blue, 2% 2-mercaptoethanol) was added, and the beads heated for 15 minutes at 65°C. The loading buffer/protein mixtures were then loaded onto 8% 0.75 mm SDS-PAGE gels and run at 80-110V with Laemmli running buffer (10X running buffer: 250 m M Trizma base, 1.92 M glycine, 1%SDS) in a C.B.S. Scientific Co. mini gel box (#M6V-202, Del Mar, C A , USA). 2.5 Immunoprecipitation Calbiochem Protein G agarose beads (cat #539207, San Diego, C A , USA), 30 uL in a 50:50 slurry in PBS (1.86 m M N a H 2 P 0 4 * H 2 0 , 8.41 m M Na 2 HPO, 174.9 m M NaCl, pH 7.2-7.6), were prebound overnight with 715 ug of 1F6.3 (mouse monoclonal anti-Gliotactin, hydridoma cell line rederived by ImmunoPrecise Antibodies Ltd., Victoria, B C , Canada) antibody in 250 uL PBS (10X PBS is: 13.7 m M sodium chloride, 2.7 m M potassium chloride, 4.3 m M sodium phosphate, 1.4 m M potassium phosphate). Negative control was 20 ug mouse IgG (Jackson Immunoresearch #015-000-001) in 250 uL PBS. Follwing the overnight incubation, bead-Ab complexes were incubated in 250 uL lysis buffer (10 m M Tris-HCl, 4 m M EDTA, 1 m M EGTA, 150 m M NaCl or 300 m M NaCl where specified, 0.5% Triton X-100, ImM PMSF, 2 ug/mL leupeptin, 2 ug/mL pepstatin) with a final calcium concentration of 0.0, 0.05,0.1 or 0.2 uM and 150 ug w l l l 8 adult, Nrg-GFP, or OregonR embryo membrane preparations for 3 hours 27 at 4°C with gentle agitation. Following incubation, beads were washed 3-4 times with appropriate lysis buffer. Laemmli loading buffer was added, and the beads heated for 15 minutes at 65°C. Loading buffer with immunoprecipitated proteins were then loaded onto 8% 0.75 mm SDS-PAGE gels and run at 80-110 volts with Laemmli running buffer in a C.B.S. Scientific Co. mini gel box. 2.6 Western Transfer and Immunoblotting SDS-PAGE gels were transferred to nitrocellulose (Amersham Biosciences Hybond #RPN303D) or PVDF (Amersham Biosciences Hybond-P #RPN1416F, Piscataway, NJ, USA) using semi-dry transfer. SDS-PAGE gel, membrane and thick blotting paper (Bio-Rad Protean X L #1703969, Hercules, C A , USA) were soaked for 5 minutes in transfer buffer (25 m M Trizma base, 150 m M glycine) before being arranged on the Trans-Blot SD semi-dry transfer cell (Bio-Rad #170-3940, Hercules, CA, USA), as per manufacturer's directions, and then allowed to seperate at 10V for 15 minutes followed by 15V for 15 minutes. Blots were then blocked overnight with 5% skim milk in TBS (10X TBS is: 200 m M Trizma base, 1.37 M NaCl, HCI to pH 8.0) solution. Primary antibodies were allowed to bind for 2 hours at 4°C with gentle agitation. Primary antibodies of rabbit anti-Neurexin IV (gift from M . Bhat) and 4F3 (from University of Iowa Hybridoma Bank), mouse anti-Discs Large were used at a 1 in 500 dilution, while 1F6.3, mouse anti-Gli was used at 1 in 100 dilution, HA. 11 mouse anti-HA (Covance Research #MMS-101R, Princeton, NJ, USA) was used at 1 in 1000 dilution, and BP104 (from University of Iowa Hybridoma Bank), mouse anti-Nrg, was used at 1 in 10 dilution. After incubation, blots were washed three times in TBS for 15 minutes. Membranes were then incubated for an hour with secondary antibodies Peroxidase-conjugated AffiniPure Goat Anti-28 Mouse IgG (Jackson Immunoresearch #115-035-003, West Grove, PA, USA) or Peroxidase-conjugated AffiniPure Goat Anti-Rabbit IgG (Jackson Immunoresearch #111-035-045, West Grove, PA, USA) at an end dilution of 1 in 5000. Membranes were again washed three times with TBS for 15 minutes, then washed twice for 5 minutes, first in TBS with 0.5% Tween-20, then again with TBS before developing with Western Lighting Chemiluminescence Reagent Plus (Perkin Elmer, cat #NEL105, Wellesley, M A , USA) according to manufacturer's directions. 2.7 Coomassie Staining Proteins transferred to PVDF membrane from SDS-PAGE gels were coomassie stained using Gel Code Blue (Pierce, cat #24590, Rockford, IL, USA) according to manufacturer's directions. 2.8 Protein Sequencing Preparation Proteins from w l 118 adults or OregonR embryos were prepared by GST pulldown as described above, then transferred to PVDF membrane and stained with Gel Code Blue as described above. Bands of interest were indicated and the blot sent to NAPS (Nucleic Acid and Protein Sequencing) at the University of British Columbia for N-terminal protein sequencing based on the Edman degradation method. Alternatively, protein samples were also prepared by collecting 6-12 of the same protein bands from identical GST pulldowns, using a new razor blade (VWR Industrial Razor Blades #55411-050, West Chester, PA, USA) to cut out bands from PVDF membrane blots, and these bands were then sent to University of Victoria - Genome B C Proteomics Centre for N-terminal sequencing based on the Edman degradation method. 29 3. Results 3.1 Identifying Gliotactin Protein Interactors from Known Septate Junction Proteins. Gliotactin is the only identified protein exclusively found at the TCJ at the level of septate junctions. Determining Gli interactors may help elucidate its role and function at the TCJ. A biochemical approach was used to determine which proteins interact with Gl i , although this approach shows all Gl i interactions in the animal. Previous preliminary immunoprecipitation work by Joost Schulte (2002, PhD. Thesis) with modified Gli proteins (HA-gli and H A -gliAPDZ) had shown possible interactions of Gl i with Discs Large, Neurexin IV and Neuroglian. Both immunoprecipitation and GST pulldowns were used as a test for biochemical protein interactions. 3.1.1 Overexpressed H A Tagged Gliotactin is Unstable in Drosophila in the Long Term Previously, no reliable anti-Gliotactin antibody existed, therefore an epitope tagged Gliotactin was the only possible method for studying proteins interactions in Drosophila using immunoprecipitation. The constructed transgenic flies expressed HA-tagged, full length Gliotactin and Gliotactin with the PDZ recognition motif deleted (the last 3 amino acids were deleted) were initially used to test biochemical interactions. These lines expressing H A tagged versions of Gliotactin and a Gl i mutant lacking the PDZ domain were generated by Joost Schulte (Schulte et al., 2003). The constructs were expressed in a wild type (wl 118) background using the UAS/Gal4 system (Brand and Perrimon, 1993), using the ubiquitous daughterless-Gal4 driver. The UAS/Gal4 system is a yeast derived expression system, where the Gal-4 30 transcription factor causes expression of genes through binding with the upstream activating sequence (UAS). This system allows for strong ubiquitous expression of HA-tagged Gl i , facilitating detection and biochemical work, though expressing the protein at higher than normal levels in the animal. However, the expression of HA-tagged Gliotactin was found to be unstable when continually expressed, as HA-tagged Gl i rapidly disappeared from the transgenic Drosophila lines in a span of as little as two months as seen in figure 4. The HA-tagged Gl i APDZ also proved to be unstable in the months following the disappearance of the HA-tagged Gl i , though lasting longer in Drosophila than HA-tagged Gl i , showing extensive degradation of the protein in the Drosophila lines (figure 4 does not yet show this degradation as it only became apparent in March-April of 2004). These Drosophila lines with the HA-tagged versions of Gl i were not comparable to wild type in growth and reproduction as the transgenic animals had leg defects and did not grow at the same rate as wild type Drosophila (J. Schulte, PhD. thesis, 2003; D. Venema, PhD. thesis, 2003). It is thought that because of the disadvantages of carrying the transgene, overexpression of Gl i may have created a selective pressure for the elimination of the transgenic protein. Transient expression may have helped to avoid this problem, however, it would have been an inconvenient system to use as large quantities of protein are required to perform biochemical studies. As well, transient expression would have required individual scoring of thousands of Drosophila. It is for these reasons that endogenous Gl i was examined instead, despite the need to collect larger quantities of Drosophila adults and embryos to obtain enough Gl i protein to study. 31 Figure 4: H A Tagged Gliotactin Expression is Unstable in Drosophila. Whole adult extracts from strains expressing H A tagged G l i and H A tagged G l i A P D Z , driven by the daughterless-GAL4 line, probed with mouse anti-HA. Lane 1 shows the presence of H A tagged G l i while lane 2 shows the disappearance of H A tagged G l i from the same fly strain just two months later. Lane 3 and 4 shows H A tagged G l i A P D Z , not yet degraded. Lane 5 is the control lane, with whole adult extract from w l 118 Drosophila, which does not show any H A -tagged proteins. Arrow indicates HA-tagged G l i and H A tagged G l i A P D Z (both approximately the same molecular weight), and size markers in kDa are shown at left. 8^ o as o 5 199 j 133< 87-40-I* ff 4> O 8> O <B CN N CN Q 1 ^ 1 probe: anti-HA 32 3.1.2 Gliotactin Interacts with Discs Large In previous work, it was shown that over-expressed HA-tagged Gliotactin interacts with Discs Large in a calcium dependent manner in immunoprecipitation experiments (Schulte, PhD thesis, 2003). M y aim was to test i f this was true with native Gl i , using a rederived monoclonal antibody to Gli . To test i f Gliotactin and Discs Large are in the same protein complex, immunoprecipitation of membrane preparations from wild type Drosophila adults and embryos were carried out using a newly rederived monoclonal antibody raised to the C terminus of Gliotactin; the original antibody was first used by Auld et al. (1995). These immunoprecipitations were carried out in the presence or absence of calcium due to the calcium dependant interaction seen in previous experiments using the HA-tagged Gli (Schulte, PhD thesis, 2003). The resulting immunoprecipitations using the anti-Gli antibody was then probed with a monoclonal anti-Discs Large antibody, and shows Dig in the same protein complex as Gl i in both adult and embryo Drosophila (figure 5, panel B - adult, D - embryo). This was seen to occur in a calcium dependant manner (n=5 for adult, figure 5, panel B). Dig occurs in multiple isoforms in Drosophila, with at least 10 known versions (Woods et a l , 1996a). As expected, many distinct protein bands appear in the crude membrane protein lane ranging from about 90-133 kDa (figure 5, panel B, D). Multiple Dig isoforms are able to complex with Gl i , in particular proteins of 120, 110 and 97 kDa (Figure 5b, arrows). With increasing free calcium in the assay, we observe increasing amounts of Dig, seen in figure 5, panel B. The free calcium concentrations of 0.1 and 0.2 u M were used. At 0.1 u M free 33 calcium, we observe the presence of Dig brought down by Gl i , and this intensifies as free calcium increases to 0.2 uM (figure 5, panel B, compare lanes 5 and 6). This pattern is reflected in a probe of the same immunoprecipitation using anti-Gli antibody, showing increasing amounts of Gl i with increasing calcium concentration from 0.1 u M to 0.2 u M (figure 5, panel C, compare lanes 5 and 6). We see the calcium dependant immunoprecipitation of Gl i in the w l 118 adult Drosophila. Immunoprecipitations were repeated with membrane proteins isolated from wild type embryos (n=4), and similarly, Dig was found in Gli immunoprecipitated complexes (figure 5, panel D). However, the embryo immunoprecipitation seems to be less calcium dependent (see figure 5, compare panel B and D), as anti-Gli probed embryo immunoprecipitations also did not appear to be calcium dependant. In the converse experiment, the immunoprecipitation of Discs Large failed to show the presence of Gliotactin in the subsequent immunoblots even with calcium. 34 Figure 5: Gliotactin Interacts Biochemically with Discs Large. Panel A shows a predicted model of the immunoprecipitation, with the antibody binding the C terminus of Gl i , and Gli then binding to an unknown intermediary (support for an indirect Gl i and Dig interaction is seen in C. MacKinnon's M.Sc. thesis, 2005), which then binds to Dig. Panel B shows the immunoprecipitation of Gl i from w l 118 adult Drosophila membrane preparations, probed for Discs Large. Lane 1 shows crude membrane preparation protein, showing the presence of multiple isoforms of Dig in the membrane preparation. Lane 2 is the negative control, an immunoprecipitation performed with mouse IgG and 0.05 u M free calcium (later replicates contained 0.1 uM free calcium in the negative control). Lanes 3 and 4 shows an experimental lane with 0.0 and 0.05 u M free calcium, respectively, while lanes 5 and 6 show experimental lanes with 0.1 and 0.2 u M free calcium, respectively. Note that Dig shows interaction with Gl i in lanes 5 and 6, with possible dependency on free calcium concentration. Bands seen across the bottom at approximately 50 kDa are thought to be the antibody heavy chain, the band seen below the 50 kDa bands correspond with the dye front and are unknown. Arrows indicate known Dig sizes of 120,110 and 97 kDa, with size markers in kDa shown at left. Five replicates of this gel were performed and showed consistent results. Panel C shows the same blot as in 2B, the immunoprecipitation of Gl i from w l 118 adult Drosophila membrane preparations, probed with anti-Gliotactin. Lane 1 shows crude membrane preparation protein, though no Gli protein is seen, this is likely due to insufficient loading of crude membrame prep. Lane 2 is the negative control, an immunoprecipitation performed with mouse IgG and 0.05 uM free calcium. Lanes 3 and 4 shows an experimental lane with 0.0 and 35 0.05 u M free calcium, respectively, while lanes 5 and 6 show experimental lanes with 0.1 and 0.2 u M free calcium, respectively. Note the presence of Gl i in lanes 5 and 6. Bands seen across the bottom at approximately 50 kDa (variation may be due to difference in IgG types) are thought to be the antibody heavy chain, the band seen below the 50 kDa bands correspond with the dye front and are unknown. Arrow indicates Gl i at 110 kDa, with size markers in kDa shown at left. Panel D shows the immunoprecipitation of Gl i from Oregon R embryo membrane preparations, probed for Discs Large. Lane 1 shows 20 ug of crude membrane preparation protein, showing the presence of multiple isoforms of Dig in the membrane preparation. Lane 2 is the negative control, an immunoprecipitation performed with mouse IgG and 0.1 u M free calcium. Lane 3 shows an experimental lane with 0.0 u M free calcium, while lanes 4 and 5 show experimental lanes with 0.1 and 0.2 u M free calcium, respectively. Note that Dig shows interaction with Gl i in lanes 3-5. Bands seen across the bottom at approximately 50 kDa are thought to be the antibody heavy chain. Bands seen at 75 - 85 kDa (arrows) are possible Dig isoforms. Size markers in kDa are shown at left. In contrast with the immunoprecipitation performed on adult membrane preparation, there appears to be less or no calcium dependency on the Gli-Dlg interaction in embryos. Four replicates of this gel were performed and shod consistent results. 36 1 2 3 4 5 1 2 3 4 5 6 + + + probe: anti-Gli probe: anti-Dig 3.1.3 Gliotactin and Neurexin IV Do Not Show a Biochemical Interaction In previous work, it was shown that HA-tagged Gliotactin interacts with Neurexin IV in a calcium dependant manner in immunoprecipitation experiments (Schulte, 2003, PhD thesis). However, in these experiments, my aim was to reproduce this with native Gl i , which is only expressed at the tricellular junction. This would avoid any potential artifacts of an overexpression of the Gl i transgene which causes the protein to spread throughout the SJ domain. Immunoprecipitation of Gl i from either adult or embryonic membrane preparations were carried out under the same conditions used for the Dig immunoprecipitations, and was unable to show an interaction with Neurexin IV in membrane preparations of both adult and embryo Drosophila (n=4 for adult, n=2 for embryo). For both adult and embryo immunoprecipitation experiments, the resulting immunoblot, probed with rabbit anti-NrxIV, failed to show any NrxTV in the immunoprecipitation lanes (figure 6), while the crude membrane preparation protein lane shows a band corresponding to the expected range at approximately 155-160 kDa for NrxTV (figure 6, panel A , lane 1 and panel B , lane 1). 38 Figure 6: Gliotactin Fails to Show an Interaction with Neurexin IV. Panel A shows the immunoprecipitation of Gl i from w l 118 adult Drosophila membrane preparations, probed with anti-NrxIV. Lane 1 shows crude membrane preparation protein, showing the presence of NrxIV in the membrane preparation at approximately 155-160 kDa (arrow). Lane 2 is the negative control, an immunoprecipitation performed with mouse IgG and 0.1 u M free calcium. Lanes 3,4 and 5 show a experimental lanes with 0.0, 0.1 and 0.2 uM free calcium, respectively. Size markers in kDa are indicated at left. Note that we fail to see NrxIV interaction with Gl i in any of the experimental lanes. Panel B shows the immunoprecipitation of Gl i from Oregon R membrane preparations, probed with anti-NrxIV. Lane 1 shows crude membrane preparation protein at pproximately 155-160 kDa (arrow), showing the presence of NrxIV in the membrane preparation. Lane 2 is the negative control, an immunoprecipitation performed with mouse IgG and 0.1 u M free calcium. Lanes 3,4 and 5 show a experimental lanes with 0.0, 0.1 and 0.2 uM free calcium, respectively. Size markers in kDa are indicated at left. Note that we fail to see NrxIV interaction with Gl i in any of the experimental lanes. 39 Figure 6 Q. E c • S S 3 P O I 3 4 5 + + + CM CM CM CC CO CO O O O 3 3 O t— CM o o O 25d probe: anti-NrxIV Adult B 250b 50ft 00b 75 501ft Q. E 2 u o . o + CN CO O 3 O d + CM CO O 3 probe: anti-NrxIV Embryo In summary, Gliotactin immunoprecipitation experiments were able to show interaction with Discs Large in both Drosophila adult and embryo membrane protein preparations, but were not able to show interaction with Neurexin IV. 3.2 GST Pulldowns Confirm Immunoprecipitation Results and Validates GST Pulldowns as a Method for Studying Gliotactin Biochemical Interactors To support the results seen from immunoprecipitation experiements, another biochemical approach using a GST pulldown was used to study the interaction of Gliotactin with Discs Large and Neurexin IV. The purpose of the GST pulldowns was two-fold, to attempt to replicate the results of the inimunoprecipitation experiments, and to validate the GST pulldown as a method to study protein interactors of Gliotactin, to look for unknown Gli interactors. GST-pulldowns were performed using GST-Gli-Cter, a construct comprised of GST fused to the C terminus of Gli, the section of the protein following the transmembrane domain. This does differ from immunoprecipitatons in that the GST pulldowns would only reveal proteins that interact with the C terminus of Gliotactin while immunoprecipitations using the anti-Gli antibody would reveal proteins interacting with the entire protein. A diagram of the predicted mechanism of the GST pulldown with GST-Gli-Cter is shown in figure 7, panel A. In figure 7, panel D a probe of the GST pulldown using anti-Gli is shown. This shows a protein (arrow) that corresponds to native Gliotactin that appars to be non-Calcium dependent, and is the basis for the predicted GST pulldown mechanism which shows GST-Gli-Cter binding to native Gli (figure 7, panel A). It was thought that the GST-Gli-Cter might interact with the protein complex containing Discs Large but not directly to Dig as Gli and Dig have been shown to not directly interact (C. MacKinnon, M.Sc. thesis, 2005). Alternatively, it is possible that GST-Gli-Cter would interact 41 with native Gl i and pull out any proteins bound to native Gl i because the over abundance of GST-Gli-Cter would outcompete the native Gli . The GST-Gli-Cter/Glutathione agarose complex was allowed to incubate with the membrane preparations from adults and the resulting immunoblots reflected the results shown with the co-immunoprecipitation experiments. When probed with anti-Dig, the immunoblots (n=2) showed similar results to the Gl i immunoprecipitatios, showing a wide range of Dig isoforms interacting with GST-Gli-Cter (compare figure 7 panel B, with figure 5, panel B). We see similar multiple Dig isoforms in the experimental lanes, with increasing Dig signal in 0.2 u M free calcium compared to the 0.1 u M free calcium lane (see figure 7, panel B , compare lanes 4 and 5). When GST pulldowns with GST-Gli-Cter were probed with rabbit anu-NrxiV, the immunoblots (n=2) again showed similar results to the Gl i immunoprecipitation probed with rabbit anti-NrxIV; we failed to detect any NrxIV lanes in the experimental lanes of the immunoblot (compare figure 7, panel C, with figure 5, panel C). 42 Figure 7: GST Pulldowns Show a Similar Result to Immunoprecipitation Experiments. Panel A shows a predicted model of the GST-pulldown, with the GST-Gli-cter binding Gl i , and Gl i then binding to an unknown intermediary, which then binds to Dig. Panel B shows GST pulldowns performed using GST fused to the C terminus of Gliotactin, bound to glutathione agarose beads, and w l 118 adult Drosophila membrane preparations. This blot was probed for Discs Large. Lane 1 shows 20 ug crude membrane preparation protein, showing the multiple isoforms of Dig present in the membrane preparation. Lanes 2-5 includes 150 ug of crude membrane preparation input into the GST pulldowns. Lane 2 is a negative control using GST in place of GST-Gli-cter with 0.1 uM free calcium. Lanes 3-5 show experimental lanes with 0.0, 0.1 and 0.2uM free calcium concentrations, respectively. Note that Dig shows an interaction with GST-Gli-cter in lanes 4-5 with possible dependency on free calcium concentration. Arrows indicate known Dig sizes of 120, 110 and 97 kDa, with size markers in kDa shown at left. Faint bands seen in lnaes 4 and 5 at 50 - 75 kDa are thought to be possible Dig isoforms. Panel C shows GST pulldowns performed using GST fused to the C terminus of Gliotactin, bound to glutathione agarose beads, and w l 118 adult Drosophila membrane preparations. This was probed with anti-NrxIV. Lane 1 shows 20 ug of crude membrane preparation protein, showing the presence of NrxIV in the membrane preparation (arrow at approximately 155-160 kDa, the predicted NrxIV size). Bands seen from 75 to 100 kDa in lane 1 are unknown. Lanes 2-5 includes 150 ug of crude membrane preparation input into the GST pulldowns. Lane 2 is a negative control using GST in place of GST-Gli-cter with 0.1 u M free calcium. Lanes 3-5 show 43 experimental lanes with 0.0, 0.1 and 0.2 uM free calcium concentrations, respectively. Size markers in kDa are shown at left. Note that we fail to see NrxIV interaction with GST-Gli-cter in any of the experimental lanes. Panel D shows the same blot as in panel B, showing GST pulldowns performed using GST fused to the C terminus of Gliotactin, bound to glutathione agarose beads, and incubated with w l 118 adult Drosophila membrane preparations. This was probed with anti-Gli. Lane 1 shows 20ug of crude membrane preparation protein, showing the presence of Gl i in the membrane preparation. Lanes 2-5 includes 150 ug of crude membrane preparation input into the GST pulldowns. Lane 2 is a negative control using GST in place of GST-Glicter with 0.1 uM free calcium. Lanes 3-5 show experimental lanes with 0.0, 0.1 and 0.2uM free calcium concentrations, respectively. Note arrow indicates Gl i in lanes 1, 3-5 at approximately 1 lOkDa, with size markers in kDa shown at left. 44 F igure 7 cter B C L E CD "D ~ 3 O > 3 4 5 + + + CM CM CN re CO CD o o o 2 2 3 3 3 O *— CN — e i — O — © probe: anti-Dig Q-£ C 3 O 250 150 100 CD > 3 4 5 + + + CM C\J 05 CD CD O o u 2 2 2 D 3 O CNJ d H o Q-E ^•S § ,2 CN 3 CM =3 CN 22 5: Q co •<- co CN re o O dO dOprobe: anti-NrxIV probe: anti-Gli These results show us that GST pulldowns using GST-Gli-Cter can be used as a system to study Gliotactin protein interactions because it is consistent with results from Gl i immunoprecipitation experiments (GST-Gli-Cter showing an interaction with Discs Large and not with Neurexin IV). In addition, this also supports the observation that Gl i is able to interact with Dig. 3.3 Identifying Unknown Proteins as Gliotactin Interactors. Previous results had indicated that GST pulldown was a valid method for finding Gliotactin interactors, as GST pulldowns reflected the result of co-immunoprecipitations for Discs Large and Neurexin IV. It was hypothesized that unknown proteins could also be brought down in this assay, and thus this method might be employed to discover unknown proteins complexed with the C terminal tail of Gl i . GST pulldowns using the GST-Gli-cter fusion were carried out using membrane preparations from w l 118 adults (n=3) or Oregon R embryos (n=l). Following SDS-PAGE protein separation, the protein bands were transferred to PVDF membrane. Gel Code Blue (coomassie based dye) staining of blotted PVDF membranes revealed several unknown proteins, one prominent band at approximately 75 kDa, and a fainter doublet at approximately 250 kDa, seen in figure 9, lanes 4-6. There are multiple bands at 50 kDa and below in the GST-Gli-Cter pulldown (figure 8, lanes 4-6), and they are most likely background bands with the most prominent band at 50 kDa corresponding to GST-Gli-Cter, and the smaller bands likely degradation of the fusion protein. A negative control containing just GST protein, without the C terminal tail of Gliotactin was run as a negative control (figure 8, lane 3), and did show a background band at 45 kDa. Interestingly, the proteins seen on the Gel Code Blue stained PVDF membranes did not appear to be calcium dependent as they appeared in all lanes equally in an attempt (not shown) that included a free calcium concentration range of 0.0 uM to 0.2 uM. 46 Figure 8: GST Pulldown Using GST Fused to Gliotactin C Terminus Shows the Presence of Unidentified Proteins. GST pulldowns were performed using GST fused to the C terminus of Gliotactin, bound to glutathione agarose beads, and embryo OregonR membrane preparations. Lane 1 shows size markers with corresponding sizes in kDa indicated. Lane 2 shows crude membrane preparation protein, showing all detectable proteins in the membrane preparation. Lane 3 is a negative control using GST in place of GST-Gli-cter with 0.0 uM free calcium, note that GST protein alone is approximately 26 kDa and was mostly run off the gel. Lanes 4-6 show experimental lanes with 0.0 uM free calcium concentrations. Note the doublet bands seen at approximately 250 kDa, and the robust band at approximately 75 kDa, seen in the experimental lanes. 1 2 3 4 5 6 47 Attempts at sequencing the unknown 75 kDa band have yielded unsatisfactory results. Samples from GST pulldowns using the w l 118 adult membrane preparations were sent for protein sequencing to the NAPS protein sequencing facility at U B C . Two attempts yielded two different amino acid sequences. A third attempt was made from samples from GST pulldowns using OregonR embryo membrane preparations, sent to the protein sequencing facility at the University of Victoria. This yielded yet another amino acid sequence appearing unrelated to the previous sequencing attempts (see table 1). Sequencing of the 250 kDa doublet from embryonic tissue, yielded more interesting results as both bands of the doublet yielded similar sequences (see table 1). After performing a short-sequence amino acid B L A S T search using the sequence E N E N D X X X D N (single amino acid code), a potential gene (CG31652) and gene product were identified for this 250 kDa doublet protein. The candidate contains the matching short amino acid sequence 17 amino acids into the predicted 2148 amino acid sequence, placing the candidate's potential molecular weight at a predicted 248 kDa (http://www.ebi.ac.uk/servicestmp/saps-20060523-14280096995539.html). 48 Table 1: Comparison of N-terminal Sequence Data of Unknown Proteins Seen in a GST Pulldown of GST-Gli-cter. Sequence comparison of four sequencing attempts of the unknown protein seen at 75 kDa which associated with GST-Gli-cter, seen in rows 1-4 Sequence comparison of the 250 kDa protein doublet (named A and B), which are associated with GST-Gli-cter seen in rows 5-6. Sequence sample N-terminal peptide sequence U B C NAPS 75kDa adult unknown - trial 1 S/T/?, P/Q, E/N/I, V / P / M , D/T/?, Q/V/I, F, T/A, N/I, E U B C NAPS 75kDa adult unknown - trial 2 H/G/?, Q/K/L/?, I/V, I/Q, G, Q/P/I/V, D, T/L/K, G, T, T /M/A UVic Protein Sequencing 75kDa adult unknown P/W/I, K , I, I, A , I, H , L , L , L UVic Protein Sequencing 75kDa embryo unknown D/G, L, A , I, D, D, S UVic Protein Sequencing 250kDa A E/D/N, N , E, N , D, ?, ?, ?, D/S/?, N UVic Protein Sequencing 250kDa B E/D/N, ?, D/N/?, E/S/?, N/S/?, E, D/S/? 49 Figure 9: Amino Acid Sequence of CG31652 Amino acid sequence of potential Gliotactin interactor, CG31652 with matching N-terminal sequence of unknown 250 kDa protein underlined. MDTTQESDLLGEEEELENENDVSFPRDIVSVEHSFGYDCKKLFNLVLVDSDTLVFASGNF LNYFSISRQEISFQETVYGCGVGFITKNEHPDYTNLFTVGENGARPTVFIYEYPSLNVRV KLLNAAQNCFTAGSYNKTGELFASQAGYPDFIITIWRWENAEVVLRAKSFQSDILFVHFS EHNPILLCSSGLSHIKFWKMANTFTGLKLKGDLGRFGKTDFSDIYAMYMLQDENVISGSD WGNMLLWQAGLIKFEICRKGRKPCHTKPITRITMKNGEVTTVGMDGYVRVWYWETVDLAD PPEDDLFVEIDPIYEFKIADVELRCMQKIHPFDESDFTHYAQDGNGGIWFCDINTYDVPQ KPRKLYSCIGGKVLAAQMSPVSPHFLCMSESGKLFVYQYDEQRLILEKEFPAEGVDVIWL DTNISVKGTELVAAFKDGILRQMYLDLSNGERPKMTRVRAFKAHTSPITALTVSRNSSLL LTGSADKSIFIYQLSRDEHQMVDMRPLGFVQFAAIPNCFYWHETEPTVVLVGCKSGDLYE YNIRTEVTDHETYLSYNITENSRMRHTKFASIKSRIRRDIHRENVKKRKERKRERKMNEV EKLKKANPGLQIDIESALADSEPDEEEEPLHIPTVPNAIIWLRYTLRDTIWVSMAGYDAG YIYELEFNAAEPTRATIIAEADDIEIHSYCIIDEFIIFGLHNGRIRINRINPDNFTDLSE FRIYPMHDGLKGTIPKIDISYSGEHLLTIGYDGNVFLHKWNGPKIVRNKVRDKLPPLPEG VTQIPDIEPEACSLEQEKINAELRRQQEAADAHDRDVLSKIGALQNVYFDIIRINEELPP GLRAKDTDLLLDDRITRQIRNELQAELDDVREDLAYDLEFAQVGHTKLYNHFLKELVQVP FTVAPLRANVPGVSTFRLQALGEEHAQIKQDIEDRLKREHDMGLHDYVAPERSDESIGEP PPESFFFGRDPKSIEPRFSKKMMRLLTRYRKRQIFEVRRIFDWDKLERQKPDPNRNHPDD DAQIEDAKRNLGDFKLKIGSDYEPKSSETLTQKYIEVVECREQYFAMVDAFNQMVLDLRE RKGELDQFITAKRNRLAVIHNYLPEPDRQPLDPIQEIDMDLEYPELNLIEHYTPGCGVEI DDILTLEHSVDQVIARMAPMRSTANVSLFSLHGLDVPELHEHTAFTMLQISKLNRSLPNA ELDEELLKLPPKSDPAYFELNDDGQVPYMMETRHRWLRQLLVEQNAIRTEVDEEVRQFDK ALEKLQVRRFHVKMDAEFMTAFMNSLNQELYILRDSEEIETQLLLNAKTAMSTRNDLQMV INSTNRQLDELRRNIDKLGEQIVALQVLFATTVKGHKFFDFLRRIFKKKWRPPKVSRGDD ESSSSSSSSSSSSEDEQADNKSLDSLDMTTIRLDEATCPTGLDRSLYDLAFSMRSDRHDL ERNVRELTREVENKRKEIAEMQIKMKFHEEVYQREKNALLQFRRNRQQEVNKVHISAILR MDQLQHFYDGDDYRDLSKAILFDADMLVDLRRRADQLSEETLATKRWHRINFIHLRRMNT DIKFMRFEITRLEEEIRQAMMKKFGIIVNLDELEEEVLRRYIFDLETNAEDELMALEKEL LEKQKELARCEEELVLETQNNTEKVNIMTVLREENNILRTLLDIQNKNYAECWANPNALNL SYDIEKLRGIEKSLLDQIECLEREICALRLKSKPLQINYEFEVDPNAQPQVVTSEIMPND DAMVCPAILNVDAYMLPPMPDEFIMDRVQTIVQKSFNRFFGKSTTPENVRKFAQRSSLYL CQAAYSFQGRYTDRIAECITEHLQTFVPKKYFIHISNEELRKLFNEVVAVFDYERSDVNT EELISGIFDHAKDSLCAHVHMGDSEVVNRTHFIVAHMFKELIEVLPLEEFQADETLRMIV DVLEREPMVDPRAIAVEQLIDSTLQHARENLIDGITAVPIRSLGSSIQRDLIKRRQYKQP SCESPIRDKMSKPSKAGPPSEGSAPAYDYRHSGDDMAMRAPPPTYEESQRNGGTGAGAYG YPQGGGAPPSQNQYYGMISHQQQQMPFSGQPNYGMTHGSGFGLAGPSTSAGAAAAQVLHL DSRAEVRTNSNGNVVIPPPPPGCLPTPAQWAAMQGQPVVLKQKKRSFF 50 4. Discussion 4.1 Gliotactin May Be In A Separate Protein Complex Than Other Currently Known Septate Junction Proteins It has been previously seen that the majority of known SJ proteins are interdependent and a loss of one protein results in the mislocalization of the other SJ proteins (Hortsch and Margolis, 2003; Genova and Fehon, 2003; Llimargas et al., 2004). It has also been seen that a loss of Gliotactin leads to a mislocalization of Neurexin IV, Coracle and Discs Large (Schulte et al., 2003). In this case, characteristic SJ septa are still seen, though spread down the basolateral edge, suggesting that perhaps Gl i plays an integral role in organizing or stabilizing the core SJ complex just under the adherens junction (Schulte et al., 2003). When overexpressed, an H A -tagged version of Gl i spread throughout the SJ domain and was able to biochemically interact with Discs Large, Neurexin IV and Coracle (Schulte et al., 2003). In an effort to study other potential Gl i protein interactors, an attempt was first made to replicate Schulte's (Ph.D. thesis, 2003) immunoprecipitation experiments, which showed the biochemical interaction of H A -tagged Gl i with NrxIV, Cor and Dig. This was done to verify i f I was able to reproduce the same results, demonstrating an ability to successfully immunoprecipitate HA-tagged Gl i . When it was discovered that the Drosophila strains used were unstable and had lost the HA-tagged Gl i , we looked to other methods of studying Gli protein interactions including immunoprecipitation and GST pulldown of essentially wild type Drosophila membrane preparations. This approach also allowed the study of endogenous protein complexes, rather than those formed with overexpressed protein in transgenic flies, thus, reducing the potential for artifacts due to the modification of the protein sequence and expression, 51 Through both co-immunoprecipitation experiments and GST pulldown experiments, it has been shown that Gliotactin, a protein localized to the septate junctions at the tricellular junction, does not appear to interact with Neurexin IV (figure 6) in both adult and embryo membrane preparations. However, Gliotactin does appear to be in a complex with Discs Large in both adult and embryo membrane preparations. The only difference seen between embryo and adult preparations being that there seems to be no calcium dependence in the binding in the embryos. This is possibly due to changes in the cell and protein interactions during embryo to adult development. As identical detergent conditions were used to in immunoprecipitations from both adult and embryo membrane preparations, it is possible that the detergent conditions would extract different proteins in adult and embryo membrane preparations. To date, Dig is the only known SJ protein with which Gl i interacts. Interestingly, this interaction in adults is only seen in the presence of calcium at 0.1 or 0.2 uM. It is possible that the Gl i epitope recognized by the anti-Gli antibody requires calcium to be properly recognized, as we see in the calcium dependency of Gl i immunoprecipitation (figure 5, panel C). Alternatively, this may indicate that Gl i requires calcium for proper protein interactions, protein conformation, or as a factor in dimer stabilization. Gl i genetically appears to act as a dimer due to some combinations of different Gl i mutations being able to show rescue of the mutation (Venema et al., 2004). In addition, the candidate Gl i vertebrate homologs, the Neuroligins are known to bind Neurexins in a calcium dependent manner (Ichtchenko et al., 1995; Ichtchenko et al., 1996; Nguyen and Sudhof, 1997), possibly through a putative EF-hand domain on Neuroligin (Gilbert and Auld, 2005). This may point to a similarity in the function of calcium in the Gli-Dlg interaction, and this is supported by the possibility of a calcium binding EF-hand domain on the 52 extracellular portion of Gl i (Gilbert and Auld, 2005). Alternatively, the immunoprecipitation of Gliotactin itself may be calcium dependant (figure 5, panel C), i f the Gl i epitope requires calcium for proper binding of the anti-Gli antibody, a property also seen in the antibody binding to CD29 and to leukocyte function-associated molecule-1 (Levesque et al., 2002; van Kooyk et al., 1991). The data showing Discs Large as the only protein seen to interact with Gliotactin, had led to the idea that perhaps Gli may be independent of other known septate junction proteins, existing in a unique complex at the tricellular junction (Schulte et al., 2006), illustrated in figure 10. Discs Large is known as a SJ protein, occurring around the entire cell and additionally, showing enrichment at the TCJ (Woods and Bryant, 1991). Gl i is known to only occur at the TCJ (Schulte et al., 2003; Woods and Bryant, 1991). As there are many known Dig isoforms (Mendoza et al., 2003), perhaps Gli interacts with different isoforms present at the TCJ, which are different from those in the known septate junction protein complex that occur around the cell (Hortsch and Margolis, 2003). Which isoforms of Dig interact with Gli is a more difficult question to answer as the anti-Dig antibody recognizes the PDZ 2 domain of Dig, and this domain occurs on many different Dig isoforms. It would be interesting to have seen i f indeed different Dig isoforms associate with Gli as with the SJ by performing immunohistochemistry. It is interesting to note that the interaction of Gl i and Dig does not appear to be PDZ dependant in in GST pulldowns using a Gl i construct with a truncated PDZ binding domain (Schulte et al., 2006). Interestingly, human Discs Large (hDlg) also has several different isoforms (so far all contain PDZ domains 1 to 3), and 5 have been identified (McLaughlin et al., 2002). They have a wide 53 variety of locations and functions including cell-cell contacts interacting with other known structural proteins human Scribble, and the vertebrate ortholog of Coracle, Protein 4.1 (Lue et al., 1994b). Recent cell biological work has pointed to a possible link between Gliotactin and Discs Large. In 3 r d instar larve columnar epithelial cells of the imaginal wing disc and the peripodial cells, Dig shows enrichment at the TCJ, as well as Gl i and Dig showing strong colocalization at the TCJ. As well, the overexpression of Gl i and its subsequent spreading of Gl i into the entire SJ domain, appears to correlate with the down regulation of Dig in the immaginal discs of 3 r d instar larvae (Schulte et al., 2006; Charish, personal communication). This would make sense i f Gl i was indeed somehow bound to Dig, perhaps the overexpression of Gl i provides competition for the usual Dig ligand at the septate junction. However, there is the possibility that Gliotactin and Discs Large do not work through a direct interaction, but an indirect one, as supported by work by C. MacKinnon (2005, M.Sc. thesis), which included experiments involving GST pulldowns of GST-Gli-Cter expressed in E. coli, with in vitro translated Dig. Her work showed no evidence of direct physical interaction between the two proteins, though the experiments did not involve testing the effect of free calcium (C. MacKinnon, 2005, M.Sc. thesis). As we see some calcium dependence of the Gl i -Dlg interaction in immunoprecipitations and GST pulldowns, this could be taken as Gl i needing calcium to show binding to Dig, that Gl i itself is calcium dependant or that the TCJ as a whole is somehow calcium dependant. 54 There may also be other proteins in the Gli-Dlg complex, that act as intermediate proteins, linking Gl i and Dig, perhaps transiently because a Dig immunoprecipitation fails to show Gl i . Since there is an abundance of Dig in Drosophila, and only a small subset of Dig might be bound to Gl i at any one time, perhaps we were unable to detect what would be very small amounts of Gl i in a Dig immunoprecipitation. As Gl i does not bind to NrxIV, it is likely that this Gli-Dlg complex is separate from the known septate junction complex. From the data gathered so far, we can arrive at a model of the workings of the tricellular junction at the level of septate junctions. We believe that the TCJ does indeed act as an organizing centre for the septate junction strands, and that the tricellular plug is where SJ strands are anchored at the TCJ by the Gliotactin-Discs Large complex. Gl i , a transmembrane protein, might be responsible for linking the tricelular plug, an extracellular structure, to the cells at the TCJ via Dig, an intracellular protein. This concept would support the observation that the loss of Gl i , and thus the destabilization of this TCJ complex, leads to the basolateral diffusion of the septate junction strands -the tricellular plug is no longer being anchored to the TCJ. Recently, a new vertebrate protein had been identified, Tricellulin, a protein with some structural similarity to occludin and which exists in the area of tight junction strands at tricellular contacts in vertebrate cells (Ikenouchi et a l , 2005). However, there does not appear to be any homology between Tricellulin and Gliotactin though they appear to both be required for epithelial barrier integrity (Ikenouchi et al., 2005; Schulte et al., 2003). In fact, there is no homology between Tricellulin and any Drosophila protein, nor are there any known Tricellulin interactors to date, including PDZ containing proteins as Tricellulin does not appear to have a PDZ binding domain. Though the functional similarities between Tricellulin and Gliotactin are 55 intriguing, the similar functions demonstrated by both proteins may simply be an example of convergent evolution. 56 Figure 10: Model of Protein Interactions at the Tricellular Junction. The tricellular plug (blue), a structure comprised of unknown components, is shown with Gliotactin, at the tricellular junction, as a component of a unique TCJ protein complex. Shown with Gl i are unknown protein(s) (yellow) and Discs Large (red). It is believed that the Gliotactin complex is helping to organize the septate junction and anchor the tricellular plug. 57 Gliotactin is a possible homolog of a class of vertebrate proteins called the Neuroligins, with the closest relation being to Nlg3 (Gilbert et al., 2001). Neuroligin 1 binds to a and (5 Neurexin in the vertebrate system, only in the presence of calcium (Gilbert et al., 2001; Nguyen and Sudhof, 1997). Additionally, neuroligins are thought to function as dimers (Dean et al., 2003) and have been found to bind to the third PDZ domain of PSD-95, a vertebrate Dig homolog (Bolliger et al., 2001; Irie et al., 1997; Willott et al., 1993). Perhaps the calcium dependence observed in the binding of Gl i and Discs Large in Drosophila reflects the binding activity of its vertebrate homologs, since Neuroligins also bind to the Neurexins in a calcium dependant manner (Ichtchenko et al., 1995; Ichtchenko et al., 1996; Nguyen and Sudhof, 1997; Boucard et al., 2005). More recently, further links between neuroligins and PSD-95 have been determined. It had been found that PSD-95 has some role in the clusetering of Nlg2 and Nlg3 (Levinson et al., 2005), and there is suggestion that Nig can regulate PSD-95 (Iida et al., 2004). There has been no evidence of interaction between Neuroligin 3 and Caspr (Gilbert and Khurana, unpublished data), a class of proteins thought to be vertebrate homologs of Neurexin IV (Peles et al., 1997). Again, this reflects our biochemical results in the Drosophila system, where we fail to observe an interaction between Gliotactin and NrxIV. Such vertebrate proteins with invertebrate septate junction homologs as Caspr, Neurofascin and Contactin are found at the paranodes of myelinated nerves, often believed to have septate-like junctions (Hortsch and Margolis, 2003; Bhat et al., 2001). Surprisingly, Neuroligin 3, the closest homolog to Gliotactin, does not appear to be present in the paranode (Gilbert, personal communication). This may be consistent with the idea that Gliotactin is in a separate TCJ protein complex, and thus its vertebrate homolog, are in separate complexes from the septate and septate-like protein complexes, respectively. 58 4.2 Unknown Gliotactin Interactors The next step in searching for potential candidates for Gliotactin binding partners was to use a biochemical screen. This approach isolated membrane preparations and looked for proteins able to bind to a fusion protein: GST fused with the C terminal domain of Gl i , in a GST pulldown. Since GST pulldowns had replicated the results seen in immunoprecipitations for Gliotactin and Dig, as well as Gl i and NrxIV, it was surmised that GST pulldowns would be a valid method of locating other Gl i interactors. Using this GST pulldown method from both adult and embryonic membrane preparations, we are able to observe several unknown proteins, a doublet appearing at about 250 kDa, a faint band at about 150 kDa and a prominent band at 75 kDa. The intensities may be an indication of the prevalence of the Gliotactin-unknown protein interaction. A l l bands appeared to be independent of calcium concentration. As the most prominent, the 75 kDa unknown protein was sequenced using N-terminal degradation. The first two attempts at sequencing the 75 kDa protein resulted in two completely different sequences. The third attempt at sequencing the 75 kDa protein again resulted in a different sequence. It is suspected that the 75 kDa band may be a non-specific cleavage or degradation product as there was a lack of an N-terminus methionine, or a collection of different proteins of similar molecular weight. Other work had since been done on GST pulldowns of adult and embryo membrane preparations using GST-Gli-Cter, as well as GST-Gli-CterAPDZ, a similar construct though missing the last PDZ domain on the C terminus of Gliotactin. It was found that in the presence of high salt (300 m M NaCl), the 75 kDa band persisted, as well, it was also present in pulldowns performed with 59 GST-GlicterAPDZ (Alan Gillet, unpublished data). This shows that the binding of the unknown 75 kDa protein to the C terminus of G l i is a non-PDZ interaction. The 250 kDa doublet, alternatively, resulted in two similar sequences, an indication that they may be isoforms of the same protein. B L A S T searches revealed the most likely candidate for this 250 kDa band is as yet, an unknown and uncharacterized gene sequenced from cDNA, CG31652. This gene appears to be highly conserved across various species, including the human gene AAI01583. This gene product contains potential WD-40 repeats, also known as beta-transducin repeats, which are associated with a variety of functions including signal transduction and transcription regulation to cell cycle control and apoptosis (information from The European Bioinformatics Institute, http://www.ebi.ac.uk/Information/). However, all proteins with WD-40 repeats are associated with the coordination of multi-protein complexes as the WD-40 domains act as a scaffold (European Bioinformatics Institute, electronic communication). As well, WD-40 domains bind to phosphoserine residues, which are potentially present on Gliotactin. A Drosophila line with an insertion of a transposable element in the candidate gene has been examined, though it failed to show any mislocalization of Gl i in the embryo (Julia Wierzchwski, personal communication). 4.3 Summary and Future Studies Curently, there is some evidence that the Gliotactin and Discs Large interaction is somehow dependant on calcium. We also see some unknown proteins which appear to biochemically interact with the C terminus of Gl i . The question remains, what other proteins are present in this unique TCJ complex? 60 It would be interesting to see if any of the unknown proteins discovered in the GST pulldown experiments may potentially be the missing link between Gliotactin and Discs Large, as they are proteins that interact with the cytosolic domain of Gl i . We can begin to examine this by looking to see if any SJ defects, and mislocalization of Dig are present in the Drosophila line (flybase id: FBgn0051652) with an insertion in the gene of the 250 kDa protein candidate. We already do not see a mislocalization of Gl i , which would indicate that perhaps the unknown protein is not required for Gl i localization at the TCJ, or that it does not play a role at the TCJ, or that the insertion into the gene did not cause a mutation. It is of course, equally likely that they may be interacting in other ways or in another location. We can pursue this candidate protein in the future by intentionally making a deletion mutation in this gene, in case the transposon insertion did not cause a mutation. This could be done by injecting embryos with the transposase gene, which might cause an inaccurate excision of the insert, and screening for mutant phenotypes. The easier route to look for an interaction with Gliotactin would be to simply take the CG31652 gene and in vitro translate it to perform a GST pulldown with Gl i . If this proves fruitful, it would be possible to raise an antibody to the CG31652 gene product and attempt to do antibody staining of the gene product and Gli to look for colocalization and subsequently, perform immunoprecipitation experiments looking for the CG31652 gene product with Gliotactin. To further examine Gliotactin interactions, we would in the future, need to be able to verify potential candidates both biochemically and through co-localization studies in vivo in Drosophila as outlined above. If potential candidates co-localize in vivo, and also interact in immunoprecipitation or pulldown assays, then they would be suspected of interacting with Gl i . Additional support would arise if mutations in potential candidates had a visible effect on Gl i , and vice versa in both cell biological and biochemical studies. It would also be intriguing to see 61 if any Gli interactors discovered in the future would interact with other known SJ components (for example, through immunoprecipitation), as this may provide a link between the TCJ and the SJ. 4.4 Conclusions This thesis has shown the first known Gliotactin protein interaction with another known septate junction protein, Discs Large through biochemical assays including immunoprecipitation and GST-pulldowns. Additionally, this interaction appears to be independent of the currently known pSJ complex, which contains Neurexin IV as an integral part of the complex. This observation points to a new, unique pleated SJ complex, perhaps existing at the tricellular junction. Additionally, new unknown Gli interactors have been found, which may help in future endeavours in locating other proteins of this potential new TCJ complex. 62 References Al Awar,0., Radhakrishna,H., Powell,N.N., and Donaldson,J.G. (2000). 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