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LFA-1 outside-in signalling and actin cytoskeleton reorganization in cytotoxic T lymphocytes MacLeod, Matthew Alexander 2006

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L F A - 1 O U T S I D E - I N S I G N A L L I N G A N D A C T I N C Y T O S K E L E T O N R E O R G A N I Z A T I O N I N C Y T O T O X I C T L Y M P H O C Y T E S by M A T T H E W A L E X A N D E R M A C L E O D B . S c , University of Guelph, 2002 A THESIS S U B M I T T E D I N P A R T I A L F U L F I L L M E N T O F T H E R E Q U I R E M E N T S F O R T H E D E G R E E O F M A S T E R OF S C I E N C E in T H E F A C U L T Y O F G R A D U A T E S T U D I E S (Genetics) T H E U N I V E R S I T Y O F B R I T I S H C O L U M B I A October, 2006 © Matthew Alexander MacLeod, 2006 A B S T R A C T Integrins are heterodimeric cell-surface receptors that mediate the adhesion of cells to the extracellular matrix and to other cells. Leukocyte function-associated molecule-1 ( L F A - 1 ) is the most prevalent integrin expressed on T cells and is known to be crucial for migration, extravasation and immunological synapse formation. In addition to its role as an adhesion molecule, L F A - 1 can induce intracellular signals that influence T cell effector functions, including activation of proteins involved in actin polymerization. However, these studies have used experimental means to activate L F A - 1 , such as phorbol ester treatment or antibody cross-linking. In this study, the effect of L F A - 1 signalling on the activation and redistribution of proteins involved in actin cytoskeleton reorganization was investigated in cytotoxic T lymphocytes (CTLs) . L F A - 1 in C T L s is already in a high avidity form able to bind ligand, and therefore does not require further experimental activation. To elucidate the specific requirement for intercellular adhesion molecule-1 ( I C A M - 1 ) binding in L F A - 1 signalling, C T L s were bound to plastic surfaces coated with either I C A M - 1 or a - L F A - 1 blocking antibody. The binding of C T L s to I C A M - 1 immobilized on polystyrene microspheres induced the site-specific recruitment of actin and W A S P (Wiskott-Aldrich syndrome protein) to the CTL-microsphere interface. This is not due to simple clustering of L F A - 1 , as binding of C T L s to a -LFA-1 coated microspheres did not have the same effect. Recruitment of W A S P to the contact site formed between C T L s and I C A M - 1 coated microspheres was not dependent on an increase in W A S P tyrosine phosphorylation or incorporation of W A S P into Brij 35 insoluble lipid rafts. The guanine nucleotide exchange factor Vav-1 , which has been previously found to influence actin dynamics, was constitutively tyrosine phosphorylated in C T L s . This suggests that Vav-1 , as well as other proteins involved in actin polymerization may already be active in C T L s . The tyrosine kinase Pyk-2 (proline-rich i i kinase 2) has been previously implicated to play a role in LFA-1 signalling. We found that Pyk-2 tyrosine phosphorylation is greatly increased after binding of CTLs to both ICAM-1 and a-LFA-1 coated surfaces. These results suggest that clustering of LFA-1 can induce signalling, however, LFA-1-ICAM-1 binding is necessary to direct site-specific actin reorganization. iii T A B L E O F C O N T E N T S 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 iv L I S T O F F I G U R E S v L I S T O F A B B R E V I A T I O N S v i A C K N O W L E D G E M E N T S ix C H A P T E R 1 INTRODUCTION 1 1.1 L F A - 1 1 1.1.1 L F A - 1 Structure and Role in Immune Responses 1 1.1.2 L F A - 1 Activation: Affinity and Avidi ty 4 LFA-1 Affinity Regulation 5 LFA-lLateral Mobility and Clustering 6 1.1.3 L F A - 1 Mediated Outside-in Signalling 7 1.2 Act in Cytoskeleton 9 1.2.1 Role of the actin cytoskeleton during T cell responses 9 1.2.2 Dyamics of actin polymerization and reorganization during T-cell conjugate formation 9 1.2.3 Vav-1 11 1.2.4 WASP/Arp2 /3 complex 14 1.2.5 Pyk-2 16 1.3 Thesis objectives 18 C H A P T E R 2 M A T E R I A L S AND M E T H O D S 20 2.1 Tissue Culture 20 2.2 Commercial antibodies and reagents 21 2.3 Antibody purification 22 2.4 Purification of soluble I C A M - 1 23 2.5 Immunoprecipitation and Western blotting 23 2.6 Sucrose gradient centrifugation 25 2.7 Confocal microscopy 26 C H A P T E R 3 28 3.1 The role of I C A M - 1 - L F A - 1 interaction in translocation of proteins involved in actin cytoskeleton rearrangement 28 3.2 The role of I C A M - 1 - L F A - 1 interaction in activation of proteins involved in actin cytoskeleton rearrangement 37 C H A P T E R 4 DISCUSSION 42 R E F E R E N C E S 51 iv L I S T O F F I G U R E S Page Fig . 3.1 Act in is enriched at the interface between C T L s and I C A M - 1 but not 29 a -LFA-1 coated beads F ig . 3.2 C T L binding to I C A M - 1 but not a -LFA-1 coated beads results in 30 accumulation of actin at the CTL-bead contact site F ig . 3.3 W A S P is enriched at the interface between C T L s and I C A M - 1 but not 31 a -LFA-1 coated beads F ig . 3.4 Act in and W A S P do not accumulate at the interface of C T L s and a -LFA-1 33 coated beads after 1 or 2 hour time-points F ig . 3.5 W A S P and talin are located exclusively in detergent-soluble high-density 35 sucrose gradient fractions upon C T L binding to I C A M - 1 and a -LFA-1 coated surfaces F ig . 3.6 Pyk-2 is evenly distributed throughout the cytoplasm in C T L s bound to 36 I C A M - 1 and a -LFA-1 coated beads and does not co-localize with actin F ig . 3.7 Tyrosine phosphorylation of W A S P is not induced by C T L binding to 38 I C A M - 1 or a -LFA-1 coated surfaces F ig . 3.8 Tyrosine phosphorylation of Vav is unchanged by C T L binding to I C A M - 1 39 or a -LFA-1 coated surfaces F ig . 3.9 Tyrosine phosphorylation of Pyk-2 is enhanced in C T L s after binding to 41 both I C A M - 1 and a -LFA-1 coated surfaces F ig . 4.1 Proposed model to explain the observed effects of L F A - 1 signalling in this 50 study v L I S T O F A B B R E V I A T I O N S A D A P Adhesion and degranulation-promoting adaptor protein A P Activator protein A P C Antigen presenting cell Arp Actin-related protein A T P adenosine 5'-triphosphate B S A Bovine serum albumin C D Cluster of differentiation Cdc42 Cel l division cycle 42 C H Calponin homology C T L Cytotoxic T lymphocyte D H D b l homology D M E M Dulbecco's modified minimal essential medium E B V Epstein-Barr virus E D T A Ethylene diamine tetraacetate Erk Extracellular signal regulated kinase F A K Focal adhesion kinase F C S Fetal calf serum F E R M band four-point-one/ezrin/radixin/moesin F R E T Fluorescence resonance energy transfer G B D GTPase binding domain G E F Guanine nucleotide exchange factor G M - 1 Ganglioside ceramide tetrahexoside (Cer-Glc-Gal-Sialic acid-GalNac-Gal hDlg human homologue of discs large I C A M Intercellular adhesion molecule I F N Interferon Ig Immunoglobulin IL Interleukin IS Immunological synapse v i I T A M Immunoreceptor tyrosine-based activation motif J A B Jun activation domain-binding protein J A M Junctional adhesion molecule L A D Leukocyte adhesion deficiency L F A - 1 Leukocyte function-associated antigen-1 m A b Monoclonal antibody M A P K Mitogen activated kinase M H C Major histocompatibility complex M L C K Myos in light chain kinase M T O C Microtubule organizing centre N - W A S P Neural Wiskott-Aldrich syndrome protein P H Pleckstrin homology PI3K phosphatidylinositol 3-kinase P K C Protein kinase C P L L Poly-L-Lysine P M A Phorbol 12-myristate 13-acetate PSTPIP Proline, serine, threonine phosphatase-interacting protein PtdIns[4.5]P2 Phosphatidyl 4,5-bisphosphate Pyk2 Proline-rich kinase 2 R O C K Rho-associated kinase S F K Src-family tyrosine kinase SLP-76 Src homology 2 domain-containing leukocyte protein of 76 k D a S M A C Supramolecular activation clusters T H T helper T N F Tumour necrosis factor V C A M Vascular cellular adhesion molecule V L A Very late antigen W A S Wiskott-Aldrich syndrome W A S P Wiskott Aldr ich syndrome protein v i i W A V E W A S P family verprolin homologous W H W A S P homology domain Z A P - 7 0 ^-chain-associated protein of 70 kDa v i n A C K N O W L E D G E M E N T S Firstly, I would like to thank my supervisor, Dr. Fumio Takei, for the opportunity to carry out my graduate studies in his lab and for his patience and guidance throughout the entire process. Thanks to my committee members, Dr. Michael Gold and Dr. Gerry Krystal for their insights, suggestions and critical review of my thesis. I am very grateful to have had the opportunity to work alongside a great bunch of fun and supportive individuals (Linnea Veinotte, Nooshin Tabatabaei, Emi ly Mace, Evette Haddad, Valeria A lcon and Eva Backstrom). I would especially like to express my gratitude to Dr. Erica Wilson, Dr. Reza Marwal i , Dr. Motoi Maeda and Lisa Dreolini for their skillful teachings and explanations in response to my many questions. Lastly, I cannot begin to express how sincerely thankful I am to friends and family for the love and encouragement they have given me while completing this thesis. I am particularly indebted to Michael Bonneau and to my parents, Gai l and A . D . MacLeod for being there to support me along every step of this journey. i x C H A P T E R 1 I N T R O D U C T I O N 1.1 LFA-1 Integrins are heterodimeric cell-surface receptors that are composed of the non-covalent association of a and (3 subunits, both of which are type I transmembrane glycoproteins. They mediate the adhesion of cells to the extracellular matrix and to other cells. A s a result, integrins play a central role in many cellular processes, including adhesion, migration, differentiation, proliferation and programmed cell death. Leukocytes, depending on their particular subtype and maturation state, are able to express at least 12 of the 24 known integrin heterodimers (Hynes, 2002). Four of these are leukocyte-specific P2 integrins (aLP2, aMp2, a xP2 and aDP2), with aLP2 ( C D 1 l a / C D 18), also known as leukocyte function-associated antigen (LFA-1) , being the most prevalent. Recent studies suggest that L F A - 1 , in addition to its role as an adhesion molecule, is also capable of generating intracellular signals that influence T cell activation and differentiation. This research project was therefore initiated to investigate in cytotoxic T lymphocytes (CTLs) the effect of LFA-bind ing to its ligand intracellular adhesion molecule 1 ( I C A M - 1 ) on activation and/or redistribution of proteins involved in actin cytoskeleton reorganization. 1.1.1 LFA-1 Structure and Role in Immune Responses L F A - 1 mediated leukocyte adhesion is brought about by binding to any of five intercellular adhesion molecules ( I C A M s 1-5). Additionally, L F A - 1 binding to junctional adhesion molecule 1 ( JAM-1) has been described (Ostermann, Weber et al. 2002). I C A M s are members of the immunoglobulin (Ig) superfamily of proteins, as their extracellular domains contain anywhere from two to nine tandem Ig repeats. The 90 kDa I C A M - 1 is the highest affinity, most widely distributed and extensively studied of the L F A - 1 ligands. It contains five Ig domains and is expressed constitutively at low levels on cells of both hematopoeitic and non-1 hematopoetic origin, including leukocytes, endothelial cells, epithelial cells, macrophages, fibroblasts and dendritic cells (Anderson, Schmalsteig et al. 1985; Dustin and Cooper 2000). Expression of ICAM-1 is upregulated in response to various inflammatory stimuli, such as interleukin ( I L ) - l , tumour necrosis factor (TNF)-a and interferon (IFN)-y (Pober, Bevilacqua et al. 1986; Pober, Gimbrone et al. 1986; Dustin and Cooper 2000). Monoclonal antibody (mAb) inhibition of ICAM-1 demonstrates its importance in antigen-independent conjugate formation and antigen-specific cytotoxicity of particular target cells (Makgoba, Sanders et al. 1988). The extracellular domains of integrin a and (3 subunits are relatively large (> 700 residues), while the cytoplasmic domains, with the exception of the 04 subunit, are short (Reviewed in Hynes 2002). The amino-terminal regions of integrin a and P subunits fold to form an extracellular globular 'head-piece' domain that binds ligand. The crystal structure o f the vitronectin (aVP3) extracellular domain shows the head-piece is formed by the interaction o f the P-propeller domain on the a-subunit with the hybrid and I-like domains on the P-subunit (Xiong, Stehle et al. 2002). The P-propeller is composed of seven segments of approximately 60 amino acids each that display weak homology to one another (Springer 1997). Hal f of the a-subunits, including a L of L F A - 1 , contain an inserted (I) domain of approximately 200 amino acids that is inserted between the second and third P-sheets of the P-propeller (Larson, Corbi et al. 1989; Springer 1997). The I-domain is the site of ligand binding for integrins that contain it (Diamond, Garcia-Aguilar et al. 1993; Michishita, Videm et al. 1993; Randi and Hogg 1994). It has been shown to exist in three conformational states that are influenced by the position of the seventh a-helix and which correspond with ligand binding affinities of low, intermediate and high magnitude (Shimaoka, Xiao et al. 2003). The linker peptide that connects the seventh a-helix to the propeller domain binds the I-like domain in the P-subunit. This intramolecular interaction is proposed to cause a downward 'bell-rope' movement o f the seventh a-helix, causing 2 conformational changes in the magnesium-containing ligand binding site and thus facilitating activation (Yang, Shimaoka et al. 2004). Two integrin stalks, comprised of a series of globular domains, serve to connect the head-piece to the cell membrane. The stalk of the a-subunit incorporates a highly flexible 'genu' (knee). This allows the integrin ecotodomain to bend at a 135° angle and assume a folded structure that places the ligand binding site within 5 nm of the membrane. When the genu is straightened, the integrin maintains an extended conformation that positions the ligand binding site 20-25 nm from the membrane (Xiong, Stehle et al. 2001; Takagi, Petre et al. 2002). The importance of P2-integrins in human immune function is evident in patients with Leukocyte Adhesion Deficiency ( L A D ) type I. L A D - 1 is a rare autosomal recessive disorder characterized by recurrent life-threatening bacterial and fungal infections. It is caused by mutations in the P2 (CD 18) gene, resulting in protein that is either undetectable or unable to associate with the a subunits (Kishimoto, Hollander et al. 1987; Kuijpers, Van Lier et al. 1997; Harris, Shigeoka et al. 2001; Ardouin, Bracke et al. 2003; McDowa l l , Inwald et al. 2003; Kinashi , Aker et al. 2004; Stradal, Rottner et al. 2004). Thus, leukocytes in L A D - 1 patients are deficient in all P2-integrins including L F A - 1 . Neutrophils from L A D - 1 patients have defective trafficking to sites of inflammation. Lymphocyte adhesion and migration, however, appear to be normal, which is likely due to redundancy with very late antigen-4 ( V L A - 4 ) (Wehrle-Haller and Imhof 2003). Moreover, L A D - 1 patients display normal delayed-type hypersensitivity responses and have no major problem with severe viral infections (Anderson, Schmalsteig et al. 1985). The importance of L F A - 1 in many diverse T cell immune responses has been well established. During homing to lymph nodes and/or peripheral tissues, L F A - 1 is involved in the firm arrest of lymphocytes to the endothelium (Warnock, Askari et al. 1998). Blocking of the unique L F A - 1 a L chain with monoclonal antibody reduces the in vivo migration of lymphocytes 3 into lymph nodes by 40-60% (Gomez, Hamann et al. 2005). These results were confirmed using lymphocytes from L F A - l " 7 " mice, which display a marked reduction in their ability to traffic to peripheral lymph nodes, and to a lesser extent mesenteric lymph nodes and sites of acute inflammation (Andrew, Spellberg et al. 1998). Homing of these L F A - 1 deficient lymphocytes is not completely abolished due to the compensatory effects of the a4 integrins, a4p7 and a4pi ( V L A - 4 ) , which both bind vascular cellular adhesion molecule ( V C A M ) - l (Berlin-Rufenach, Otto etal . 1999). L F A - 1 plays a vital role in the activation of naive T cells by antigen presenting cells (APCs) and is required for target cell ki l l ing by C T L s . Antibodies against L F A - 1 abrogate T-cell interactions with A P C , thereby disrupting T-cell effector functions (Davignon, Martz et al. 1981). In T cells, the mature immunological synapse (IS) is characterized by a peripheral ring o f adhesive L F A - 1 that surrounds a central cluster of T cell receptor (TCR) /CD3 complexes and signaling molecules (Monks, Freiberg et al. 1998). Formation of the IS is believed to be necessary for full activation of naive T cells. In C T L s , it has been suggested to play a role in the polarization of lytic granules towards target cells (Stinchcombe, Bossi et al. 2001). 1.1.2 LFA-1 Activation: Affinity and Avidity L F A - 1 is expressed constitutively on resting T cells, however, it is in an inactive state that is unable to bind ligand. This ensures that resting lymphocytes remain free to circulate the vasculature by preventing them from binding inappropriately to ligand on endothelium or other lymphocytes. L F A - 1 can be rapidly activated through intracellular signals generated by T C R engagement with M H C bearing appropriate peptide on A P C , or by cytokine or chemokine stimulation. This process is known as 'inside-out' signalling as intracellular activation signals are transduced to the extracellular portion of L F A - 1 capable of binding I C A M - 1 . Alternatively, 4 signals emanating from L F A - 1 - I C A M - 1 binding transduced across the cell membrane and into the cytoplasm are referred to as 'outside-in' (Dustin, Bivona et al. 2004). Activation of L F A - 1 does not require an increase in its cell surface expression, but rather an increase in its avidity (Larson and Springer 1990). Avidi ty is an all-encompassing term that provides a physiological measure of adhesive strength but which cannot be related to the free energy (Dustin, Bivona et al. 2004). Inside-out signals are proposed to influence L F A - 1 avidity by two mechanisms which are not mutually exclusive. The first involves conformational changes in L F A - 1 that enhance monomeric affinity for ligand. The second is by increased L F A - 1 lateral mobility within the cell membrane that allows it to cluster and form multivalent interactions with ligand. LFA-1 Affinity Regulation Integrin affinity can be regulated in a bidirectional manner, both by inside-out signalling and ligand binding (Reviewed in Dustin, Bivona et al. 2004). The 3-D structure of L F A - 1 has not been resolved, however, studies done on the allbp3 integrin have been used to suggest a mechanism by which affinity changes in L F A - 1 are related to global protein conformation. A bent integrin ectodomain is of low affinity, while a straightened conformation corresponds with intermediate or high affinity depending on whether the head-piece is closed or open respectively (Takagi, Petre et al. 2002). The idea that integrin conformation and affinity changes are related is supported by the finding that many monoclonal antibodies bind specifically to the activated form of integrins (Bazzoni and Hemler 1998; Aghazadeh, Lowry et al. 2000). Inside-out signalling is believed to mediate integrin affinity through separation of the cytoplasmic tails. In resting cells, the a and P-subunit cytoplasmic tails are thought to be clasped together by a salt bridge that maintains the integrin in a low affinity state. Deletion of the membrane proximal G F F K R sequence in the a-subunit disrupts this interaction, rendering the 5 integrin constitutively active with a high affinity conformation (Hibbs, X u et al. 1991; Hughes, Diaz-Gonzalez et al. 1996; Rozelle, Machesky et al. 2000). In L F A - 1 , cytoplasmic tail separation has been measured using fluorescence resonance energy transfer (FRET) in response to ligand binding and inside-out signals (Kim, Carman et al. 2003). Experimentally, L F A - 1 can be activated by phorbol esters, stimulatory monoclonal antibodies, or binding of divalent cations such as manganese or magnesium. The physiological dissociation of the clasp is thought to be mediated by binding of intracellular proteins, such as RapL and/or talin (Reviewed in Dustin, Bivona et al. 2004). LFA-1 Lateral Mobility and Clustering L F A - 1 in resting cells is proposed to be made inactive through anchoring interactions with the actin cytoskeleton that prevent it from clustering and forming multivalent interactions with ligand (Reviewed in Cannon and Burkhardt 2002; Dustin, Bivona et al. 2004). After T C R engagement, intracellular signals are generated within T cells that are believed to cause the transient solubilization of the cortical cytoskeleton through proteases like calpain (Stewart, Treiber-Held et al. 1996). This liberates L F A - 1 , allowing it to move laterally in the cell membrane and form multivalent interactions with I C A M - 1 at the site of conjugate formation. Concurrently, other T C R signals serve to activate and recruit proteins involved in actin polymerization to the target cell contact site. After clustered L F A - 1 binds I C A M - 1 , L F A - 1 reassociates with the actin cytoskeleton to form a strong adhesion. The link between L F A - 1 and actin filaments is thought to be bridged by the actin-binding protein talin ( K i m , Carman et al. 2003). Evidence for the above model comes from studies done in Epstein-Barr Virus ( E B V ) transformed B cells. Nanogold beads coated with antibodies to the L F A - 1 I domain were used to 6 perform single molecule tracking experiments (Kucik, Dustin et al. 1996). In resting cells, almost all L F A - 1 molecules were found to be immobile on the cell surface. Treatment of these cells with the phorbol ester phorbol 12-myristate 13-acetate ( P M A ) , which experimentally activates L F A - 1 , increased the mobility of the L F A - 1 molecules by a factor of ten. Additionally, cell adhesion assays were performed in which these same cells were first treated with various concentrations of cytochalasin D , a fungal toxin that disrupts actin filaments and inhibits actin polymerization. After treatment, cells were bound to I C A M - 1 coated surfaces and the percentage of cell binding measured. L o w concentrations of cytochalasin D treatment were found to stimulate cell adhesion and were proposed to facilitate the release o f L F A - 1 from actin constraints, thus allowing it to cluster. A t higher concentrations of cytochalasin treatment, L F A -1 mediated adhesion was suppressed, presumably because clustered L F A - 1 was unable to reassociate with actin filaments in order to create a strong adhesion. 1.1.4 L F A - 1 M e d i a t e d O u t s i d e - i n S i g n a l l i n g L F A - 1 binding to soluble I C A M - 2 has been shown to contribute to naive C D 4 + T cell responses initiated by T C R signalling and CD28 co-stimulation (Perez, Mitchel l et al. 2003). In this study, L F A - 1 engagement, in conjunction with anti-CD3 and anti-CD28 ligation, resulted in a lower threshold of T cell activation than did co-stimulation with C D 3 and CD28 alone. This involved an accelerated mitogenic response, earlier induction o f extracellular signal regulated r kinase (Erk)-l /2 activation and greater IL-2 production. Additionally, a larger number of cells differentiated into the T helper 1 (TH 1) subtype. These effects were dependent on functional Jun activation domain-binding protein ( J A B ) - l and cytohesin-1 responses which occurred following L F A - 1 - I C A M - 2 binding. 7 L F A - 1 engagement in T cells has been shown to cause cytoskeletal rearrangements and cell migration. Following binding of Mg 2 +-activated L F A - 1 to I C A M - 1 , T cells become polarized and begin to migrate randomly (Smith, Bracke et al. 2003). Coritical F-actin also increases by approximately 30%, predominantly at the leading edge of the migrating T cell where it can support lamellipodial extension (Porter, Bracke et al. 2002). L F A - 1 mediated migration on I C A M - 1 is dependent on myosin light chain kinase ( M L C K ) and Rho-associated kinase ( R O C K ) , which control forward propulsion and rear detachment respectively (Smith, Bracke et al. 2003). L F A - 1 signals also modify the microtubule cytoskeleton. Upon antibody cross-linking of L F A - 1 , protein kinase C (PKC) pi and 5 are targeted to the microtubule organizing centre ( M T O C ) and microtubules (Volkov, Long et al. 1998). T cell clones deficient for P K C P are unable to polarize their microtubule cytoskeleton or crawl in response to L F A - 1 cross-linking (Volkov, Long et al. 2001). Magnesium stimulated L F A - 1 on T lymphoblasts transduces signals causing the redistribution of focal adhesion kinase ( F A K ) and proline-rich kinase 2 (Pyk-2) to the M T O C following I C A M - 1 binding (Rodriguez-Fernandez, Gomez et al. 1999). Electron microscopy studies of aVp3 and allbp3 integrins demonstrate conformational changes following ligand binding (Takagi, Petre et al. 2002). Separation of L F A - 1 a and p cytoplasmic tails has also been measured using F R E T following engagement with soluble I C A M - 1 ( K i m , Carman et al. 2003). L F A - 1 , in addition to mediating intracellular binding is also capable o f transmitting signals that promote T cell activation and differentiation. It remains unclear, however, how ligand induced conformational changes in cytoplasmic tails, which themselves lack enzymatic activity, act to induce intracellular signals. 8 1.2 Actin Cytoskeleton 1.2.1 Role of the actin cytoskeleton during T cell responses The coordinated regulation of the actin cytoskeleton is crucial during nearly every stage of T cell function (Reviewed in Sanchez-Martin, Sanchez-Sanchez et al. 2004). This includes migration during thymic development, homing in lymphoid organs and extravasation into target tissues. Reorganization of the actin cytoskeleton also occurs in cell division, adhesion, apoptosis, phagocytosis and T cell activation. During T cell conjugate formation, cytoskeletal changes induce morphological transformations in the T cell allowing it to encompass the A P C or target cell. The actin cytoskeleton orchestrates the clustering of receptors and signalling molecules into highly organized supramolecular activation clusters ( S M A C ) . This structure, also referred to as the immunological synapse, is believed to be important for the sustained transduction of T C R signals necessary for full T cell activation. 1.2.2 Dynamics of actin polymerization and reorganization during T-cell conjugate Formation The actin cytoskeleton in a highly flexible system that is able to instantaneously restructure based on the momentary needs of the cell (Reviewed in Huff, Muller et al. 2001). In order for T cells to be capable of undergoing large scale actin polymerization within seconds of chemotactic stimuli or antigen recognition, they must maintain large pools of monomeric or globular (G)-actin. In leukocytes, this is mediated by the sequestering capabilities of thymosin-P4, which when bound to monomeric actin prevents it from becoming incorporated into actin filaments. Additionally, capping proteins, such as gelsolin, bind to the fast growing "barbed end" of actin filaments to prevent the attachment of further actin monomers (Sun, Yamamoto et al. 1999). Thymosin-p4 competes for adenosine 5'-triphosphate (ATP)-actin monomer binding with profilin. Act in bound to profilin can be incorporated onto the barbed ends of actin filaments, 9 although the complex is incapable of spontaneous actin nucleation (the formation of new actin filaments created from actin monomers) (Pollard and Cooper 1984; Pring, Weber et al. 1992). Act in nucleation requires additional help from the association of Wiskott Aldr ich syndrome protein ( W A S P ) with the actin-related protein (Arp)2/3 complex. WASP/Arp2 /3 complexes bind to profilin-bound monomeric actin molecules to promote nucleation (Pollard, Blanchoin et al. 2000). Triggering of the T C R / C D 3 complex alone is sufficient to induce polymerization of F-actin at the site of T C R engagement (Lowin-Kropf, Shapiro et al. 1998). Proper actin dynamics, however, necessary for the formation of S M A C s and stabilization of the IS are thought to involve costimulation. The actin binding proteins cofilin and L-plastin, that act to sever/depolymerize and bundle F-actin respectively, only become activated via costimulation through receptors such as C D 2 or CD28 (Henning, Meuer et al. 1994; Samstag, Eckerskorn et al . 1994; Lopez-Lago, Lee et al. 2000). A biphasic model has been proposed to explain actin cytoskeletal remodelling following T C R engagement (Reviewed in Fuller, Braciale et al. 2003). After antigen recognition, downstream signals cause the transient solubilization of the cortical cytoskeleton through proteases, while concurrently initiating actin polymerization at the site of conjugate formation. A s mentioned previously, the dissolution of actin filaments is believed to liberate L F A - 1 from its cytoskeletal restraints. L F A - 1 then moves laterally in the cell membrane towards the site of target cell binding where it forms multivalent interactions with I C A M - 1 . Concomitant with this, polymerization of new actin filaments is initiated when the immunoreceptor tyrosine-based activation motifs ( ITAMs) of CD3£, y, 8 and s proteins become phosphorylated following T C R clustering. Phosphorylated tyrosine residues in I T A M s allow binding via their SH2 domains to the Src family kinases Lck and Fyn, which in turn become activated and phosphorylate further 10 I T A M residues. Fully phosphorylated I T A M s can recruit Z A P - 7 0 (^-chain-associated protein of 70 kDa) through its tandem Src homology domains, leading to its phosphorylation and activation by Fyn. These initial signalling events cause the activation and redistribution of a number of additional proteins that promote localized actin polymerization at the site of target cell binding. This provides the framework to direct the location of receptors and signalling molecules into supramolecular activation clusters ( S M A C s ) necessary for T cell activation. Additionally, actin reassociates with clustered L F A - 1 , presumably through the actin-binding protein talin, to establish a strong adhesion between the T cell and A P C or target cell. Our lab has previously shown that actin accumulates in the IS formed between C T L s and I C A M - 1 transfected fibroblasts in an antigen independent manner (Marwali , 2004). It is therefore possible that initial interactions between L F A - 1 on C T L s and I C A M - 1 on targets cells act in a costimulatory manner to induce actin polymerization at the site of conjugate formation. This initial actin assembly could then serve to direct the localization of further actin polymerization by T C R signals, assuming recognition of appropriate M H C (major histocompatibility complex) plus peptide. 1 . 2 . 3 Vav-1 One of the proteins rapidly tyrosine phosphorylated by T C R signalling is the 95 kDa protein Vav-1 . It has been shown to play a key role in many T cell processes, acting to bridge downstream T C R signals with actin dynamics and IS formation (Reviewed in Hornstein, Alcover et al. 2004). Three members of the Vav family of proteins have been identified (Vav-1, Vav-2 and Vav-3). Vav-1 is predominantly restricted to cells of hematopoietic origin, whereas Vav-2 and Vav-3 are more ubiquitously expressed (Katzav, Martin-Zanca et al. 1989; Schuebel, Bustelo et al. 1996; Mov i l l a and Bustelo 1999). Vav family members are guanine nucleotide exchange factors (GEFs). They serve to catalyze the removal of G D P from Rho family GTPases 11 and hence allow for their activation by G T P binding. Rho GTPases are well established mediators of cytoskeletal regulation in many cell types, including leukocytes. Vav-1 has been shown to act specifically on R a c l , Rac2 and RhoG. Additionally, it has also been suggested that R h o A and Cdc42 are candidates for Vav-1 G E F activity, although this is controversial (Crespo, Schuebel et al. 1997; Han, Das et al. 1997). Vav-1 acts as a key transducer of downstream T C R signals that serve to regulate the actin cytoskeleton. Vav-1 7 " T cells have diminished actin cap formation following T C R engagement (Fischer, Kong et al. 1998; Holsinger, Graef et al. 1998). Additionally, there is evidence to support a role for Vav-1 in T C R and lipid raft clustering to the c S M A C , as these are impaired in Vav-1 deficient T cells (Wulfing, Bauch et al. 2000; Vil la lba, B i et al. 2001). A s V a v - l ^ T cells are inefficient at forming conjugates with A P C s , it has been suggested that Vav-1 mediates inside-out signals that activate L F A - 1 , although there is no direct evidence to support this (Krawczyk, Oliveira-dos-Santos et al. 2002; Ardouin, Bracke et al. 2003). Recruitment of W A S P is unaffected in Vav-1 deficient T cells, although its activation as well as the accumulation of GTP-bound Cdc42 is greatly diminished (Zeng, Cannon et al. 2003). Cdc42 is known to be necessary for M T O C polarization towards the A P C upon antigen recognition (Stowers, Yelon et al. 1995). Accordingly, Vav _ /" thymocytes also have impaired microtubule polarization following T C R engagement (Ardouin, Bracke et al. 2003). Vav proteins contain a Db l homology (DH) region, typical of GEFs , that is flanked by many regulatory domains (Adams, Houston et al. 1992). To the amino-terminal side of the D H domain are the calponin homology (CH) domain and acidic domain. The acidic domain contains at least three different tyrosine residues (Y142, Y160 and Y174), phosphorylation of which result in increased Vav-1 G E F activity (Crespo, Schuebel et al. 1997; Lopez-Lago, Lee et al. 2000; Huang, Shimaoka et al. 2006). Unphosphorylated Y174 is capable of binding the D H 12 domain and inhibiting its G E F activity, while phosphorylation of this tyrosine residue relieves this autoinhibition (Aghazadeh, Lowry et al. 2000). The C H domain has also been shown to inhibit D H domain activity, possibly through an intramolecular interaction that blocks the D H domain, thus preventing its association with GTPases (Zugaza, Lopez-Lago et al. 2002). To the carboxy-terminal side of the D H domain are the pleckstrin homology (PH) and C1 domains, and at the carboxy-terminal end of Vav-1 are an SH2 and two SH3 domains that serve to regulate protein-protein interactions. Costimulation with both C D 3 and CD28 is required for full Vav-1 activation in T cells, although either pathway can independently cause Vav-1 phosphorylation (Michel and Acuto 2002). The specific kinase(s) responsible for V a v l phosphorylation is controversial. A multi-step process for Vav-1 activation has been proposed (Reviewed in Valensin, Paccani et al. 2002). During the first wave, Vav-1 phosphorylation is controlled by Fyn. This initial activation lasts for about 5 minutes, takes place before Z A P - 7 0 activation, is l ipid raft independent and is thought to promote l ipid raft clustering at the IS. The second wave of Vav-1 activation occurs at 15 minutes post T C R engagement, after lipid raft coalescence has occurred. During this phase, Vav-1 is recruited to the T ce l l /APC interface through an association with SLP-76 (Src homology 2 domain-containing leukocyte protein of 76 kDa) and ZAP-70 . Vav-1 phosphorylation has also been reported to occur independently of T C R engagement following clustering of L F A - 1 molecules in the HSB-2 T cell line (Sanchez-Martin, Sanchez-Sanchez et al. 2004). Whether the L F A - 1 - I C A M - 1 interaction is sufficient to induce Vav-1 phosphorylation was not tested. Using constitutively active and dominant-negative Vav-1 mutants, Vav-1 was shown to regulate the transient Rac-1 activation phase. Transient Rac-1 activation was required for T cell polarization and migration on I C A M - 1 coated surfaces in H S B - 2 T cells that first underwent L F A - 1 activation with stimulatory antibody (Sanchez-Martin, 13 Sanchez-Sanchez et al. 2004). It is not known whether L F A - 1 can induce outside-in signals that phosphorylate Vav-1 in tissue derived T cells, as compared with the cell line used in this experiment. Additionally, L F A - 1 was activated with stimulatory antibody or antibody cross-linking which may induce signalling cascades that are physiologically irrelevant. Because L F A -1 in C T L s is already in a high avidity form able to bind I C A M - 1 , further experimental activation o f L F A - 1 is unnecessary. 1.2.4 W A S P / A r p 2 / 3 Complex W A S P facilitates actin polymerization through concomitant binding of actin monomers and the Arp2/3 complex. W A S P is the gene product of the Wiskott-Aldrich syndrome ( W A S ) gene, a defect of which was first identified in patients with a recessive X- l inked immunodeficiency disease (Deny, Ochs et al. 1994). The syndrome is characterized by micro-thrombocytopenia (a decreased number of small platelets) and eczema. Patients also experience a progressive decline in T cell numbers and function, resulting from defective T cell proliferation and impaired delayed-type hypersensitivity responses (Krawczyk, Oliveira-dos-Santos et al. 2002). The W A S P family of proteins consists of 5 members in mammals: W A S P , neural W A S P ( N - W A S P ) , and three W A S P family verprolin homologous ( W A V E ) proteins (also known as suppressor of c A M P receptor, or S C A R ) . N - W A S P and W A V E 2 are expressed ubiquitously in various tissues, whereas W A V E 1 and W A V E 3 are expressed primarily in the brain and W A S P is hematopoietic lineage-restricted (Deny, Ochs et al. 1994; M i k i , Miura et al. 1996; Stewart, Treiber-Held et al. 1996; Suetsugu, M i k i et al. 1999). The carboxy-terminal region of W A S P consists of three independent domains: the verprolin homology (V), central basic connecting (C) and the acidic (A) regions. Collectively these domains form the V C A module that alone is capable o f activating Arp2/3-dependent actin 14 polymerization in vitro (Hufner, Higgs et al. 2001). Act in monomers bind the V region, while the Arp2/3 binds the C and A regions (Machesky and Insall 1998; Rohatgi, M a et al. 1999; Marchand, Kaiser et al. 2001; Panchal, Kaiser et al. 2003). Upstream of the V C A module is a proline-rich region capable of binding SH3 domain containing proteins, the central GTPase binding domain (GBD) , the basic-rich region and the NH 2 -terminal W A S P homology domain 1 (WH1) (Reviewed in Badour, Zhang et al. 2004). Regulation o f W A S P activity is a complex process that has not been completely elucidated. Multiple W A S P binding partners establish a partly overlapping multi-level system of W A S P regulation brought about by changes in W A S P protein conformation, subcellular distribution and phosphorylation. In its inactive state, W A S P is autoinhibited by an intramolecular interaction between the G B D and V C A modules or via direct binding of the basic-rich region to V C A bound Arp2/3 (Prehoda, Scott et al. 2000). In either case, W A S P autoinhibition prevents Arp2/3 activation. Some dispute exists in the literature concerning the manner by which W A S P is relieved of its autoinhibition. The first theory involves the cooperative binding of active, GTP-bound Cdc42 and phosphatidyl 4,5-bisphosphate (PtdIns[4,5]P2) to the G B D and the basic-rich region respectively (Higgs and Pollard 2000; K i m , Carman et al. 2003). After relief of autoinhibition though Cdc42 binding, tyrosine residue Y291 within the G B D region is able to become phosphorylated and is predicted to stabilize the open conformation of W A S P , thereby enhancing its activity (Bompard and Caron 2004). Alternatively, Y291 is in such close proximity to the G B D region that its phosphorylation in inactive W A S P may serve to disrupt the autohibited conformation and uncover the V C A module. Support for the later argument comes from a study done in Jurkat T cells in which Fyn phosphorylation of the Y291 residue following T C R engagement is unaffected by changes in Cdc42 activity or disruption o f the Cdc42-WASP interaction (Badour, Zhang et al. 2004). In this 15 instance, Y291 phosphorylation is not merely a stabilizing modification for the open conformation of W A S P , but instead essential for W A S P induced actin polymerization following T C R engagement. Whether L F A - 1 - I C A M - 1 binding induces signalling events influencing the tyrosine phosphorylation status of W A S P has not been studied. W A S P is also capable o f interacting with several proteins that optimize its Arp2/3 binding capacity or direct its cellular localization following T C R engagement. Constitutive phosphorylation of serines 483 and 484 within the V C A module by caseine kinase 2 strongly increases the affinity of the V C A module for the Arp2/3 complex in vitro and is required for optimal actin polymerization by full-length W A S P (Cory, Cramer et al. 2003). Binding of the W A S P proline-rich region to the SH3 domain of proline, serine, threonine phosphatase interacting protein (PSTPIP)l adaptor targets W A S P towards the T ce l l -APC interface where it directs the localized polymerization of actin required for IS formation (Badour, Zhang et al. 2003). Fol lowing T C R / C D 2 8 stimulation, a small fraction of W A S P is recruited to l ipid rafts. W A S P is required for upregulation of the lipid raft marker G M 1 to the cell membrane during T cell activation and for clustering of lipid rafts at the site of IS formation (Dupre, Aiu t i et al. 2002). Whether L F A - 1 - I C A M - 1 binding affects the cellular localization of W A S P or its incorporation into l ipid rafts is unknown. 1.2.5 Pyk-2 Pyk-2 is a 116 kDa member of the focal adhesion kinase ( F A K ) family of nonreceptor tyrosine kinases. Unlike the ubiquitous expression of F A K , Pyk2 distribution is primarily limited to cells of neural, epithelial and hematopoetic origin (Karnovsky, Unanue et al. 1972; Avraham, London et al. 1995). Both F A K and Pyk-2 are unusual because they do not contain the Src homology 2 (SH2) or SH3 domains typically found in most nonreceptor tyrosine kinases 16 (Avraham, London et al. 1995). Pyk-2 does, however, contain a number of potential binding domains for SH2 and SH3 containing proteins. The two proline-rich regions in the C-terminal tail are also capable of binding various proteins. Pyk-2 has four sites of tyrosine phosphorylation that are conserved with F A K , including the Tyr 402 autophosphorylation site known to bind the SH2 domain of Src and other Src-family tyrosine kinases (SFK) (Dikic, Tokiwa et al. 1996; Avraham, Park et al. 2000; Park, Avraham et al. 2004). S F K binding is believed to facilitate the phosphorylation of the other Pyk2 tyrosine residues, Tyr 579, 580 and 881, resulting in increased kinase activity and subsequent recruitment of further SH2 domain containing proteins (Ostergaard and Lysechko 2005). L ip id rafts become enriched for S F K upon T C R activation, however, despite a known association between Pyk-2 and S F K , Pyk-2 is not recruited into l ipid rafts after stimulation of Jurkat T cells with anti-CD3/CD28-coated beads (Sancho, Montoya et al. 2002). In C T L s , minimal amounts of Pyk-2 have been reported in l ipid rafts isolated after combined fibronectin and antigen stimulation (Doucey, Legler et al. 2003). Whether Pyk-2 becomes enriched in lipid rafts in C T L s following L F A - 1 - I C A M - 1 binding has not been studied. The function o f Pyk-2 has not been fully elucidated. B-cells and macrophages from Pyk-2 knockout mice have an impaired response to chemokines due to a decreased migratory capacity. In macrophages this appears to be the result of inappropriate F-actin accumulation in a direction that is not facing the chemokine gradient (Guinamard, Okigaki et al. 2000; Okigaki , Davis et al. 2003). Expression of a dominant negative form of Pyk-2 in Jurkat T cells inhibits the production of IL-2 by about 50% (Katagiri, Takahashi et al. 2000). Pyk-2 becomes tyrosine phosphorylated in T cells in response to various stimuli, including 03 integrin stimulation, T C R stimulation, chemokine stimulation and with various cell surface molecules including C D 2 , CD44 and CD45 (Ostergaard and Lysechko 2005). Additionally, L F A - 1 stimulates Pyk-2 activation and redistribution to the M T O C in human T lymphoblasts pretreated with the L F A - 1 17 activating m A b KIM-127 and plated on I C A M - 1 plates (Rodriguez-Fernandez, Gomez et al. 1999). Pyk-2 in T cells may therefore act downstream of L F A - 1 signalling to control actin cytoskeleton dynamics. 1.3 Thesis Objectives There are many discrepancies between the currently accepted model of L F A - 1 regulation by lateral mobility and clustering. Recent studies in our laboratory suggest L F A - 1 in unchallenged primary and cytotoxic T lymphocytes, unlike some T cell lines, does not seem to be constitutively anchored to the actin cytoskeleton. Accumulation of actin at the immunological synapse of C T L s , however, does occur in an antigen independent manner. Whether this specifically requires L F A - 1 - I C A M - 1 binding has not been determined. L F A - 1 , in addition to its role as an adhesion molecule can also generate intracellular signals. Many of the studies examining L F A - 1 signalling, however, have been done in cell lines or using systems were L F A - 1 must be experimentally activated using antibody cross-linking, activating a -LFA-1 antibodies or phorbol esters. The objective of this thesis was to test the following hypothesis: binding of L F A - 1 on C T L s to I C A M - 1 , but not simple clustering of L F A - 1 , induces outside-in signalling events causing the activation and/or redistribution of proteins involved in cytoskeletal reorganization. To test this, I use microspheres coated with soluble I C A M - 1 (s ICAM-1) or those coated with a -LFA-1 mAb as controls. The rationale for this approach is that L F A - 1 - I C A M - 1 binding likely induces a conformational change needed to induce a signalling cascade, while binding of microspheres coated with blocking a -LFA-1 antibody induces clustering of L F A - 1 but not conformational change. To test this hypothesis, the cellular distribution of actin, W A S P and Pyk-2 is determined after C T L binding to I C A M or a -LFA-1 coated polystyrene microspheres. The activation of Vav-1 , W A S P and Pyk-2 is tested at various time-points by 18 assessing their tyrosine phosphorylation status post binding of L F A - 1 on C T L s to immobilized I C A M or a - L F A - 1 . Clustering of lipid rafts in C T L s at the immunological synapse occurs in an antigen independent manner. Therefore, incorporation o f proteins involved in actin polymerization/redistribution into sucrose gradient lipid fractions was examined after C T L binding to I C A M - 1 or a -LFA-1 coated surfaces. 19 C H A P T E R 2 Materials and Methods 2.1 Tissue Culture 2.1.1 Mice C 5 7 B L / 6 mice were obtained from Jackson Laboratories (Bar Harbor, M E ) and H Y T C R transgenic Rag 2~'~ C57BL/10 mice were obtained from Taconic Farms (Tarrytown, N Y ) . These mice were bred in the Joint Animal Facility and A R C of the B C Cancer Research Centre. 2.1.2 Murine splenocyte extraction Splenocytes were isolated from three to six month old C 5 7 B L / 6 mice. Single cell suspensions were prepared in a sterile environment by crushing spleens on a 100 mm Petri dish using the blunt end of a 3 ml syringe. Cells were recovered, resuspended in D M E M 5% F C S and passed through a #21 G needle. Cells were centrifuged for 5 minutes at 1450 rpm and the pellet was resuspended in 0.8% ammonium chloride/0.1 m M E D T A to lyse red blood cells. Splenocytes were washed and resuspended in medium. In order to enrich for T cell , splenocytes were passed through a nylon wool column (Polysciences, Inc., Warrington, P A ) at 37°C according to the manufacturer's protocols. 2.1.3 Generation of C T L s Dendritic cells were isolated from the spleens of two to six month old male C 5 7 B L / 6 mice by preparing a single cell suspension (as per 2.1.2). Red blood cells were lysed with 0.8% ammonium chloride/0. I m M E D T A and Fc receptors were blocked with 2.4G2 hybridoma ( A T C C ) supernatant for 10 minutes at room temperature. Dendritic cells were labelled with 0.65 ug/ml phycoerythrin (PE)-conjugated anti-CD 1 l c antibody ( B D Pharmingen) for 15 minutes at room temperature and then separated using the Easy Sep™ P E selection kit (StemCell Technologies Inc., Vancouver, Canada) according to the manufacturer's instructions. 20 Splenocytes from the spleens of 6-12 week old female H Y T C R transgenic Rag 2"" C57BL/10 mice were stimulated with dendritic cells at a 20:1 ratio in the presence of 20 U / m l IL-2 in R P M I media supplemented with 10% fetal calf serum, 5 x 10"5 M p-mercaptoethanol and penicillin/streptomycin. The cell mixture was added to 12 well culture plates at a concentration of 3 x 10 6 cells/ml (2.5 ml/well). C T L s were harvested for experiments on day 5-6. 2.2 Commercial antibodies and reagents 2.2.1 Primary Antibodies Antibody Company Catalogue Number Species raised in Monoclonal or Polyclonal? Dilution for Western Blotting A n t i - V a v l (D-7) Santa Cruz Biotechnology (Santa Cruz, C A ) sc-8039 Mouse Monoclonal 1:500 A n t i - W A S P Santa Cruz sc-13139 Mouse Monoclonal 1:500 Anti-Pyk2 (N-19) Santa Cruz sc-1514 Goat Polyclonal 1:500 Anti-Tal in (C-20) Santa Cruz sc-7534 Goat Polyclonal 1:500 4G10 Ant i -Phosphotyrosine Upstate Cel l Signaling Solutions (Charlottesville, V A ) 05-321 Mouse Monoclonal 1.5 pg/ml An t i -CD3 s chain B D Pharmingen (San Diego, C A ) 557306 Armenian Hamster Monoclonal Ant i -CD28 B D Pharmingen 553294 Syrian Hamster Monoclonal 21 2.2.2 Secondary Antibodies Antibody Company Catalogue Number Conjugated to Dilution Anti-mouse IgG Jackson ImmunoResearch Laboratories, Inc. (West Grove, P A ) 715-036-150 Horseradish peroxidase (HRP) 1:10 000 Anti-rat IgG Jackson 112-036-062 H R P 1:5 000 Anti-goat IgG Santa Cruz sc-2352 H R P 1:5 000 Anti-mouse IgG Molecular Probes(Eugene, OR) A11029 Alexa Fluor 488 10 ng/ml (Confocal Microscopy) Anti-goat IgG Molecular Probes A11078 Alexa Fluor 488 10 ug/ml (Confocal Microscopy) Anti-mouse IgG Molecular Probes A11031 Alexa Fluor 568 10 ng/ml (Confocal Microscopy) 2.2.3 Reagents Bovine serum albumin ( B S A ) fragment V, Brij 35, protease inhibitors (leupeptin, phenyl-methyl-sulfonyl fluoride, aprotinin and pepstatin A), NaV04, NaMo04 and saponin were from Sigma Aldrich (St. Louis, M O ) . 2.3 Antibody purification TIB213 (anti-LFA-1) and TIB218 (anti-CD 18) rat hybridomas were from A T C C . They were grown in D M E M / 5 % F C S for 10-14 days. Cultures were cleared by centrifugation followed by filtration through a 0.22 pm bottle top filter (Falcon). In order to purify monoclonal TIB213 antibody, the resultant TIB213 hybridoma supernatant was passed through a 5 ml H i Trap affinity protein G column (Amersham Biosciences, Piscatway, NJ) at a flow rate of 1 ml/minute. The column was washed with 20 ml equilibrium buffer (20 m M NaP04, p H 7.0) and the protein eluted with five column volumes of elution buffer (0.1 M citric acid, p H 2.7). Eluted 22 antibody was neutralized with 0.2 M sodium bicarbonate (pH 8.0) and dialyzed into P B S . Quantitation was achieved by running purified samples alongside B S A standards on an S D S -P A G E gel using the Mini-Protean 3 electrophoresis system (BioRad, Hercules, C A ) . Staining of gels with Coomassie Blue allowed for identification of bands and quantitation. 2.4 Purification of soluble ICAM-1 Soluble I C A M - 1 was produced by Lisa Dreolini by growing NS-1 cells transfected with c D N A encoding soluble recombinant I C A M - 1 in D M E M + 5% F C S for 10 days (Welder, Lee et al. 1993). The culture was cleared by centrifugation and filtration through a 0.22 um bottle top filter (Falcon). The resultant supernatant was purified by passing it through a column containing Affigel-10 Active Ester Agarose beads coupled to anti-ICAM-1 m A b (YN1/1.7.4). The column was washed with 100 ml 10 m M Tris (pH 4.5) + 0.15 M N a C l and the protein eluted with 0.1 M glycine-HCl (pH 2.8) + 0.15 M N a C l . Twenty 1 ml fractions were collected and neutralized with 1.5 M Tris buffer. Quantitation of eluded fractions was achieved by running purified samples alongside B S A standards on an S D S - P A G E gel, followed by staining with Coomassie Blue. 2.5 Immunoprecipitation and Western blotting 2.5.1 Polystyrene Petri dish preparation Polystyrene 60 x 15 mm Petri dishes ( B D Falcon) were coated with 10 pg/ml I C A M - 1 or anti-LFA-1 antibody (TIB213, 2.3) in Adhesion Buffer (20 m M Tr i s -HCl , 150 m M N a C l p H 8.2) overnight at 4°C. Poly-L-Lysine (PLL) coated plates were used as a negative control and prepared by incubating polystyrene Petri Dishes with 0.01% P L L (Sigma-Aldrich) for 1 hour at room temperature. I C A M - 1 and TIB213 coated plates were blocked with Adhesion Buffer 23 containing 1% B S A for 1 hour at room temperature. Coated plates were washed three times with P B S before use. 2.5.2 Vav-1 and WASP C T L s were harvested, centrifuged and resuspended in 37°C R P M I + 10% F B S at 4 and 8 mil l ion cells/ml for W A S P and Vav-1 experiments respectively. One millilitre of cell suspension was added to each coated plate. Plates were incubated at 37°C for various time-points, washed twice with 1 ml of 37°C R P M I and cells were lysed with 1 ml of ice cold lysis buffer (25 m M T r i s - H C l , p H 7.8, 1 m M E D T A p H 8.0, 0.5% B S A , 10% glycerol, 1% Triton X-100, 10 m M NaF, 1 m M Na3V04, 1 m M P M S F , 5 ng/ml aprotinin, 10 ng/ml leupeptin and 10 ng/ml pepstatin). For time zero time-points, 8 million C T L s were lysed in a microfuge tube. 2.5.3 Pyk-2 C T L s were harvested, centrifuged and resuspended in 37°C R P M I + 10% F B S at 10 mil l ion cells/ml. One millilitre (10 mill ion cells) of cell suspension was added to each coated plate. Plates were incubated at 37°C for various time-points, washed twice with 1 ml of 37°C R P M I and cells were lysed with 1 ml of ice cold lysis buffer (10 m M Tr i s -HCl , p H 8.0, 150 m M KC1, 2 m M E D T A p H 8.0, 1% Triton X-100, 0.5% B S A , 1 m M N a 3 V 0 4 , 0.2 m M N a M o 0 4 , 1 m M P M S F , 5 ng/ml aprotinin, 10 ng/ml leupeptin and 10 ng/ml pepstatin). For time zero time-points, 10 mill ion C T L s were lysed in a microfuge tube. 2.5.4 Immunoprecipitation of proteins Supernatants were added to microfuge tubes and cells were sheared with two passages through a # 26 G needle followed by incubation on ice for 10 minutes. After clarification at 24 13,000 rpm (Heraeus Instruments) for 20 minutes at 4°C, supernatants were taken as cell lysates. Immunoprecipitations were performed by incubating lysates with primary antibodies (1.5 pg ant i -WASP, 1.5 pg anti-Vav-1, or 1 pg anti-Pyk-2) for 1 hour at 4°C with constant agitation. Protein G beads (Amersham Biosciences) were added and lystates were agitated for an additional hour at 4°C. Protein G beads were washed three times with Wash Buffer (lysis buffer without B S A ) and the immunoprecipitated proteins eluted with S D S - P A G E sample buffer and boiling for 5 minutes. Samples for each time-point (8 million cell equivalent for W A S P , 10 mill ion cell equivalent for Pyk-2 and 16 mill ion cell equivalent for Vav-1) were divided in two equal portions, with one half used as a loading control and the other half used to look at tyrosine phosphorylation. 2.5.5 Western blotting of samples Samples were loaded on a 10% S D S - P A G E gel (WASP) , 7% S D S - P A G E gel ( L F A - 1 , Vav-1 and Pyk-2) or 5% S D S - P A G E gel (talin). S D S - P A G E gels were run using the M i n i -Protean 3 system (BioRad) and transferred to polyvinylidene fluoride ( P V D F ; Pal l , Pensacola, F L ) membranes at 80 volts for 1.5 hours using a Hoefer T E 22 (Amersham Biosciences) transfer apparatus. Blots were visualized by incubation with antibodies specific for talin, L F A - 1 (TIB218), Vav-1 , W A S P , Pyk-2 or tyrosine phosphorylation (4G10) and the appropriate H R P -conjugated anti-IgG secondary antibody followed by chemiluminescence using an E C L system (Amersham Biosciences, Piscatway, NJ) according to the manufacturer's protocol. 2.6 Sucrose gradient centrifugation C T L s (5 x 107) were harvested, washed twice and resuspended in 37°C R P M I + 10% F B S at 12.5 mill ion cells/ml. One millilitre (12.5 mill ion cells) of cell suspension was added to each 25 of four I C A M - 1 or TIB213 coated polystyrene plates (2.5.1) and incubated at 37°C for 30 minutes. Polystyrene plates were washed twice with 1 ml of 37°C R P M I and cells were lysed with 1 ml ice-cold lysis buffer ( l O m M Tris, p H 8.0, 150 m M KC1, 1% Brij 35, 2 m M E D T A , p H 8.0, 0.25 m M N a 3 V 0 4 , 0.2 m M N a M o 0 4 , 0.2 m M P M S F , 1 ng/ml aprotinin, 1 ng/ml leupeptin and 1 ng/ml pepstatin). The cell lysates were sheared by five successive passages through a #26 gauge hypodermic needles, mixed with an equal volume of 80% sucrose (w/v) in ice-cold lysis buffer without detergent and then transferred to SW41 centrifuge tubes. The samples were overlaid with 6 ml of 30% sucrose and 3.5 ml of 5% sucrose and then centrifuged at 200,000 x g for 18 hours at 4°C (Beckman, Palo Al to , C A ) . Following centrifugation, 8 fractions of 1.5 ml each were collected starting from the top of the gradient. Fractions 2-3 are referred to as the low-density, l ipid raft fractions while fractions 7-8 are the high-density, non-lipid raft fractions (Marwali , 2004). The materials at the bottom of the tube were referred to as the pellet. Aliquots of each fraction were boiled in S D S - P A G E sample buffer (non-reducing conditions for L F A - 1 only) and run on S D S - P A G E gels (2.5.5) 2.7 Confocal microscopy 2.7.1 Slide preparation Polystyrene beads (Polybead® polystyrene 10 micron microspheres, Polysciences Inc., Warrington P A ) were mixed at a 1:1 ratio with 40 ng/ml solutions of TIB213 or I C A M - 1 in Adhesion Buffer (20 m M Tr i s -HCl , 150 m M N a C l , p H 8.2) for 1 hour at room temperature with occasional agitation to prevent settling. Coated beads were blocked with Adhesion Buffer containing 10 mg/ml B S A for 1 hour at room temperature, followed by three washes with R P M I 10% F B S . C T L s were harvested, centrifuged and resuspended in R P M I + 10% F B S . C T L s and coated beads were mixed at a 1:2 ratio in a microfuge tube and incubated at 37°C for 5 minutes. 26 The cell-bead mixture was gently resuspended and plated onto a Poly-L-lysine coated coverslip, followed by incubation at 37°C for various time-points. Cells were fixed with 4% paraformaldehyde for at least 15 minutes, blocked with P B S + 1% B S A for 30 minutes and permeablized by washing with Hank's Saponin solution (HBSS, 2% F B S , 5 m M E D T A , 0.5% Saponin) for 5 minutes at room temperature. Act in was detected by incubating coverslips with a 1:40 solution of rhodamine-phalloidin in Hank's Saponin solution for 20 minutes at room temperature. W A S P and Pyk-2 were detected by incubating coverslips with a 1:40 solution of the corresponding primary antibody in Hank's Saponin for 1 hour at room temperature followed by incubation with 10 ug/ml Alex Fluor 488- or Alexa Fluor 568-conjugated goat anti-mouse IgG antibody for W A S P , or Alex Fluor 488-conjugated rabbit anti-goat IgG antibody for Pyk-2. Coverslips were washed 3 times with Hank's Saponin and 2 times with H B S S and mounted on slides using Vectashield Hard Set Antifade Mounting Medium (Vector Laboratories, Inc., Burlingame, C A ) . 2.7.2 A n a l y s i s o f samp les CTL-bead conjugates were analyzed using a BioRad Radiance 2000 Multiphoton microscope (Hercules, C A ) with a 60x objective lens. The lasers used were K r and M a i Tai T i Sapphire. Alexa Fluor 488 was excited at 488 nm and the emission filter was H Q 515/30. Alexa Fluor 568 and rhodamine were excited at 568 nm and the emission filter was H Q 600/50. Stacks were collected using 0.15 um z steps and reconstructed using Volocity software (Improvision, Lexington, M A ) . The CTL-bead interface was cropped and rotated to obtain a view from the C T L side. The fluorescence intensity and area of actin staining were measured using Voloci ty software on conjugates collected under identical confocal conditions. The "Integrated Intensity" was determined to be the total fluorescence within an encircled selection divided by the area. 27 CHAPTER 3 RESULTS 3.1 The role of I C A M - l - L F A -1 interaction in translocation of proteins involved in actin cytoskeletal rearrangement Actin accumulates at the interface of CTLs and ICAM-1 but not a-LFA-1 coated beads In order to study the effect of signals generated by LFA-1-ICAM-1 binding on distribution of actin, CTLs were bound to 10 pm polystyrene beads coated with either ICAM-1 or control beads coated with TIB213 a-LFA-1 antibody. Confocal microscopy was used to examine conjugates that were bound to only one bead and to no other cells. Distinct morphological differences were observed between CTLs bound to ICAM-1 versus a-LFA-1 coated beads. CTLs bound to ICAM-1 beads were flatter with extensions that wrapped around the bead, while cells bound to a-LFA-1 were rounder and displayed only minor cupping of beads. Actin accumulated at the contact site between CTLs and ICAM-1 coated beads in 92% of conjugates analyzed (n=37; Fig. 3.1 top panel), while in CTLs bound to a-LFA-1 coated beads actin accumulated at the contact site in only 12.5% of conjugates (n=40; Fig. 3.1 bottom panel). The distribution of actin was further analyzed by 3-D reconstruction of the confocal images. To quantitatively determine the effect of LFA-1-ICAM-1 binding, the intensity (Fig. 3.2A) and area (Fig. 3.2B) of actin staining at the CTL-bead contact-site were measured using Volocity software. Because intensity is influenced by the power of the confocal microscope laser, these conjugates were collected at the same time using identical laser settings. Both the fluorescence intensity and area of actin staining were significantly larger in CTLs bound to ICAM versus a-LFA-1 coated beads. In order to account for the greater contact-site area of CTLs bound to ICAM-1 coated beads, the "Integrated Intensity" was calculated by dividing the fluorescence intensity by the encircled area. Even after adjusting for area differences, the amount of actin at the CTL-bead interface was still significantly greater in CTLs bound to ICAM-1-coated versus 28 a-LFA-1-coated beads (Fig. 3.2C). To further analyze actin enrichment at the CTL-bead interface, the ratio between the integrated intensity at the contact-site to that at the non-contact-site was calculated (Fig. 3.2D). The mean ratio for CTLs bound to ICAM-1 coated beads was significantly greater than one, suggesting actin enrichment is occurring at the CTL-bead contact site. The mean ratio for CTLs bound to a-LFA-1 coated beads was significantly less than one, suggesting that actin enrichment is occurring at the non-contact site. Consistent with this, actin was found to accumulate randomly at a site other than the CTL-bead interface in 32.5% of CTLs bound to a-LFA-1 coated beads (see Fig. 3.6). Brightfield Actin Merged _ ro < ~o O 2 — ro o o -o • <D < -o - i 1 JB D nj O o 1 i %•* • I % * • • Figure 3.1: Actin is enriched at the interface between C T L s and ICAM-1 but not a-LFA-1 coated beads. CTLs were allowed to bind to polystyrene beads coated with 20 ug/ml ICAM-1 or 20 ng/ml a-LFA-1 for 30 minutes at 37°C. CTL-bead conjugates were fixed with 4%> paraformaldehyde, permeabilized with 0.5% saponin and then stained for F-actin with rhodamine-phalloidin (red). Mid-section images of CTLs bound to ICAM-1 coated beads (upper panel) and a-LFA-1 coated beads (lower panel) were collected with confocal microscopy and analysis performed with Volocity software. Images are representative of 37 conjugates formed with ICAM-1 coated beads and 40 conjugates formed with a-LFA-1 coated beads. The asterisk denotes the position of the bead in the CTL-bead conjugate. 29 350000-300000->» 250000-nsit 200000-JZ 150000-100000-50000-0-ICAM-1 coated beads (n=23) a-LFA-1 coated beads (n=18) ICAM-1 coated beads (n=23) a-LFA-1 coated beads (n=18) 20000-1 £ 10000-1 0) ICAM-1 coated beads (n=23) a-LFA-1 coated beads (n=18) ICAM-1 coated beads (n=37) a-LFA-1 coated beads (n=40) Figure 3.2: C T L binding to ICAM-1 but not a-LFA-1 coated beads results in accumulation of actin at the CTL-bead contact site. The intensity (A) and area (B) of actin staining was measured at the contact site of conjugates prepared as in Figure 3.1. Integrated intensity (C) was determined to be the total fluorescence within an encircled selection divided by the area. For (D), the integrated intensity of actin staining at the CTL-bead contact site and non-contact site was determined and the ratio between the two calculated. Results for all graphs represent the mean ± S E M . The asterisk represents statistically significant differences between C T L conjugates formed with ICAM-1-coated and a-LFA-1-coated beads, *p < 0.05. 30 WASP accumulates at the interface of CTLs and ICAM-1 but not a-LFA-1 coated beads Since W A S P is thought to be important for actin polymerization in T cells, I next examined the distribution of W A S P in C T L s following binding to I C A M - 1 or a -LFA-1 coated beads. Very similar patterns of staining were observed for W A S P distribution as was seen with actin. W A S P accumulated at the interface in 100% of C T L s bound to I C A M - 1 coated beads (n=25, Fig . 3.3 bottom panel) versus 15% of C T L s bound to a -LFA-1 coated beads (n=20, F ig . 3.3 top panel). _A B Brightfield WASP CTL-bead interface Figure 3.3: WASP is enriched at the interface between CTLs and ICAM-1 but not a-LFA-1 coated beads. C T L s were allowed to bind to polystyrene beads coated with 20 ng/ml I C A M - 1 or 20 ug/ml a -LFA-1 for 30 minutes at 37°C. CTL-bead conjugates were fixed with 4% paraformaldehyde, permeabilized with 0.5% saponin and then stained for W A S P with anti-WASP primary m A b and Alexa Fluor anti-mouse 568 (red). Z-sections of CTL-bead conjugates were collected with confocal microscopy and then 3-D reconstructions done with Volocity software, (a) Mid-section images o f C T L s bound to I C A M - 1 coated beads (lower panel) and a -LFA-1 coated beads (upper panel), (b) 3-D reconstruction of CTL-bead interface viewed from the C T L side. Images are representative of 25 conjugates formed with I C A M - 1 coated beads and 20 conjugates formed with a -LFA-1 coated beads. 31 Actin and WASP fail to accumulate at the interface of CTLs and a-LFA-1 coated beads even after longer timepoints In order to rule out the possibility that clustering of L F A - 1 with a -LFA-1 takes longer than 30 minutes to initiate actin and W A S P accumulation at the cell-bead interface, C T L s were bound to a -LFA-1 coated beads for 60 and 120 minutes and analyzed with confocal microscopy. Distribution of actin and W A S P for these longer time-points was observed to be similar to that of the 30 minute time-point. After 60 minutes of binding, actin and W A S P accumulated at the CTL-bead contact site in 10% (n=21) and 8% (n=25) of conjugates respectively (Fig. 3.4, top panel). After 120 minutes o f binding, actin and W A S P accumulated at the bead contact site in 10%o (n=20) and 8% (n=24) of conjugates respectively (Fig. 3.4, bottom panel). 32 Actin WASP Merged 1 HR. 2 HR. Figure 3.4: Actin and WASP do not accumulate at the interface of C T L s and a-LFA-1 coated beads after 1 or 2 hour time-points. C T L s were allowed to bind to polystyrene beads coated with 20 ug/ml a -LFA-1 for 60 minutes (upper panel) or 120 minutes (lower panel) at 37°C. CTL-bead conjugates were fixed with 4% paraformaldehyde, permeabilized with 0.5% saponin and then stained for F-actin with rhodamine-phalloidin (red) and for W A S P with anti-W A S P primary A b and Alexa Fluor anti-mouse 488 (green) secondary A b . Mid-sections of CTL-bead conjugates were collected with confocal microscopy and analysis performed with Voloci ty software. Images are representative of 20-25 conjugates. The asterisk denotes the position of the bead in the CTL-bead conjugate. 33 Accumulation of WASP and Talin at the interface of CTLs and ICAM-1 coated beads is not dependent on recruitment into 1% Brij 35 extracted lipid rafts After T C R / C D 2 8 stimulation in untransformed human T cells, W A S P has been found to be both recruited into l ipid rafts and required for clustering of G M 1 lipid rafts (Dupre, Aiu t i et al. 2002). Additionally, G M l - r i c h lipid rafts are enriched in the C T L immunological synapse in an antigen-independent manner (Marwali , MacLeod et al. 2004). To determine i f the recruitment o f W A S P into lipid rafts is responsible for its accumulation in C T L s at the interface between I C A M - 1 coated beads, C T L s bound to either I C A M - 1 or a -LFA-1 coated Petri dishes were solubilized with 1% Brij 35 on ice and fractionated by sucrose gradient centrifugation at 4°C. L F A - 1 was used as a protein marker for lipid raft isolation, as it has previous been found in C T L s to be present in both the detergent-insoluble low-density l ipid raft fractions (fractions 2-3) and the high-density non-raft fractions (fractions 7-8) (Marwali, 2004). In C T L s bound to both I C A M - 1 and a -LFA-1 coated plates, W A S P was found to be distributed exclusively in the high-density, non-lipid raft fractions (Fig. 3.5). Tal in distribution in sucrose gradient fractions was also analyzed. Talin has been implicated as the link between L F A - 1 and the actin cytoskeleton. Talin, like actin, accumulates at the contact-site between C T L s and I C A M - 1 , but not a -LFA-1 coated beads (Dreolini, 2005). Ligand-specific binding by L F A - 1 may therefore be needed to induce a conformational change in the L F A - 1 cytoplasmic tails that then allows L F A - 1 to bind talin. Despite these findings, talin and L F A - 1 l ipid raft distribution are dissimilar. Talin, like W A S P , is found exclusively in the high-density, non-lipid rafts fractions in C T L s bound to both I C A M - 1 and a -LFA-1 coated plates (Fig. 3.5). 34 We also determined the distribution of Vav-1 and Pyk-2 in sucrose gradient fractions. Both Vav-1 and Pyk-2 were found exclusively in the high-density, non-lipid rafts fractions in C T L s bound to both I C A M - 1 and a-LFA-1 coated plates (Fig. 3.5). B 1 2 3 4 5 6 7 8 P 1 2 3 4 5 6 7 8 P WASP Talin-LFA-1-Vav-1-Pyk-2-Hi ^ w * l i t Figure 3.5: WASP and talin are located exclusively in detergent-soluble high-density sucrose gradient fractions upon CTL binding to ICAM-1 and a-LFA-1 coated surfaces. C T L s were allowed to bind to polystyrene Petri dishes coated with 10 pg/ml a -LFA-1 (A) or 10pg/ I C A M - 1 (B) for 30 minutes at 37°C, solubilized with ice cold 1% Brij 35 and subjected to sucrose gradient centrifugation for 18 hr at 4°C. Eight fractions were collected from top to bottom. Fractions 2-3 are the detergent resistant light density lipid raft fractions, fractions 7-8 are the detergent soluble high-density fractions and the material at the bottom was referred to as the pellet (P). Indicated molecules in each fraction were detected by Western blot using ant i -WASP A b , anti-talin A b , anti-CD 18 A b , anti-Vav-1 A b and anti-Pyk-2 A b with anti-mouse IgG secondary, anti-goat IgG secondary or anti-rat IgG secondary Abs conjugated with horseradish peroxidase and the E C L chemiluminescence. 35 Pyk-2 is dispersed throughout the cytoplasm of CTLs bound to ICAM-1 and a-LFA-1 coated beads Since Pyk-2 activity and distribution are thought to be influenced by L F A - 1 signalling, I next examined the localization of Pyk-2 in C T L s following binding to I C A M - 1 or a -LFA-1 coated beads for 30 minutes. Because Pyk-2 staining was weak, the power of the confocal laser had to be increased to a level at which the microspheres were observed to autofluorescence in some conjugates. The staining for Pyk-2, however, was specific as fluorescence of unstained C T L s was not detected using the same laser power. Pyk-2 was observed to have a diffuse distribution in C T L s bound to either I C A M - 1 or a -LFA-1 coated beads and did not co-localize with actin (Fig. 3.6). Actin Pyk-2 Merged ICAM-1 coated beads a-LFA-1 coated beads * * * * * » Figure 3.6: Pyk-2 is evenly distributed throughout the cytoplasm in CTLs bound to ICAM-1 and a-LFA-1 coated beads and does not co-localize with actin. C T L s were allowed to bind to polystyrene beads coated with 20 ug/ I C A M - 1 (upper panel) or 20 ug/ a-L F A - 1 (lower panel) for 30 minutes at 37°C. CTL-bead conjugates were fixed with 4% paraformaldehyde, permeabilized with 0.5% saponin and then stained for F-actin with rhodamine-phalloidin (red) and for Pyk-2 with anti-Pyk-2 primary A b and Alexa Fluor anti goat 488 (green) secondary A b . Mid-sections of CTL-bead conjugates were collected with confocal microscopy. Images are representative of 20 conjugates. The asterisk denotes the position of the bead in the CTL-bead conjugate. 36 3.2 The role of I C A M - l - L F A - 1 interaction in activation of proteins involved in cytoskeletal rearrangement WASP is not tyrosine phosphorylated upon binding of CTLs to ICAM-1 or a-LFA-1 coated surfaces Activation of W A S P has been shown to be mediated by tyrosine phosphorylation of Y291 in mouse thymocytes after T C R engagement. Tyrosine phosphorylation is required for W A S P contribution to actin polymerization (Badour, Zhang et al. 2004). We immunoprecipitated W A S P from C T L s post binding to I C A M - 1 or a -LFA-1 coated surfaces and used the 4G10 anti-phosphotyrosine antibody to investigate differences in W A S P activation. A s a control for equal gel loading, half of the immunoprecipitates were probed for W A S P . A s a positive control for W A S P phosphorylation, resting mouse splenocytes, enriched for T cells, were bound to polystyrene plates coated with anti-CD3e and anti-CD28. A s a negative control, resting T cells were bound to poly-L-lysine coated plates. W A S P was found not to be phosphorylated in either resting splenocytes or C T L s . After 10 minutes of T C R stimulation, W A S P from primary splenocytes was tyrosine phosphorylated. W A S P from C T L s bound to either I C A M - 1 or a -LFA-1 coated plates did not become tyrosine phosphorylated at any of the time-points tested. These results suggest that L F A-1 outside-in signalling events are apparently unable to induce tyrosine phosphorylation of W A S P . 37 Figure 3.7: Tyrosine phosphorylation of WASP is not induced by C T L binding to ICAM-1 or a-LFA-1 coated surfaces. C T L s (8 x 106) were allowed to bind to polystyrene Petri dishes coated with 10 ug/ml a-CD3e + 5 ug/ml a-CD28 (A), 10 ug/ml a - L F A - 1 (B) or 10 ug/ml I C A M - 1 (C) for the indicated times (in minutes) at 37°C and then solubilized with ice cold 1% Triton X-100 lysis buffer. Time zero samples were lysed in microfuge tubes with ice cold 1% Triton X-100 lysis buffer. W A S P was immunoprecipitated as described in Materials and Methods. Immunoprecipitated samples were divided in half and analyzed for W A S P (loading control) and tyrosine phosphorylation with ant i -WASP and 4G10 primary Abs respectively followed by anti-mouse IgG secondary Abs conjugated with horseradish peroxidase and the E C L chemiluminescence. Each timepoint is representative of at least two independent experiments. 38 Vav-1 in resting CTLs is already tyrosine phosphorylated and does not become further tyrosine phosphorylated upon binding to ICAM-1 or a-LFA-1 coated surfaces We immunoprecipitated Vav-1 from C T L s post binding to I C A M - 1 or a -LFA-1 coated surfaces and probed with the 4G10 anti-phosphotyrosine antibody to investigate differences in Vav-1 activation. A s a control for equal gel loading, half of the immunoprecipitates were probed for Vav-1 . A s a negative control for phosphorylation, Vav-1 was immunoprecipitated from C T L s lysed in a microfuge tube (represents time zero). We found that in C T L s , Vav-1 was tyrosine phosphorylated without stimulation at time zero. The level of Vav-1 tyrosine phosphorylation was unaltered by binding of C T L s to either I C A M - 1 or a -LFA-1 coated surfaces at any of the time-points tested (Fig. 3 . 8 ) . ICAM-1 coated plates a-LFA-1 coated plates p-Tyr Vav-1 0 5 10 30 mm m* *** 5 10 30 Figure 3.8: Tyrosine phosphorylation of Vav is unchanged by CTL binding to ICAM-1 or a-LFA-1 coated surfaces. C T L s (16 x 106) were allowed to bind to polystyrene Petri dishes coated with 10 pg/ml I C A M - 1 or 10 pg/ml a -LFA-1 for the indicated times (in minutes) at 37°C and then solubilized with ice cold 1% Triton X-100 lysis buffer. Time zero samples were lysed in microfuge tubes with ice cold 1% Triton X-100 lysis buffer. Vav-1 was immunoprecipitated as described in Materials and Methods. Immunoprecipitated samples were divided in half and analyzed for Vav-1 (loading control) and tyrosine phosphorylation with anti-Vav-1 and 4G10 primary Abs respectively followed by anti-mouse IgG secondary Abs conjugated with horseradish peroxidase and the E C L chemiluminescence. Each timepoint is representative of at least two independent experiments. 39 Pyk-2 is tyrosine phosphorylated upon binding of CTLs to both ICAM-1 and a-LFA-1 coated surfaces Pyk-2 activity has been shown to increase in T lymphoblasts treated with activating L F A -1 antibody and plated on I C A M - 1 coated plates (Rodriguez-Fernandez, Gomez et al. 1999) . This increase in Pyk-2 activity was dependent on cytoskeletal integrity and reached a maximum 30 minutes after stimulation with activating L F A - 1 antibody, while decreasing at longer times. L F A - 1 in C T L s , however, is already in a high avidity state able to bind I C A M - 1 , and therefore does not need to be stimulated experimentally. To study the dynamics of Pyk-2 activation in C T L s , cells were bound to I C A M - 1 or a -LFA-1 coated plates for various time-points, after which they were solubilized with ice-cold 1% Triton X-100 lysis buffer and Pyk-2 was immunoprecipitated as described in Materials and Methods. C T L s were plated at a confluency that limited cell-cell contact, and therefore L F A - 1 - I C A M - 1 interactions between C T L s . A t time zero, there is a small amount o f basal Pyk-2 tyrosine phosphorylation that is dramatically increased after 5 minutes in C T L s bound to both I C A M - 1 and a -LFA-1 coated plates. This tyrosine phosphorylation persisted for at least 60 minutes in both conditions, demonstrating that in addition to L F A - 1 - I C A M - 1 binding, clustering of L F A - 1 with a -LFA-1 is capable of initiating signalling events. 40 ICAM-1 coated plates a-LFA-1 coated plates 0 5 10 30 60 5 10 30 60 Figure 3.9: Tyrosine phosphorylation of Pyk-2 is enhanced in CTLs after binding to both ICAM-1 and a-LFA-1 coated surfaces. C T L s (10 x 106) were allowed to bind to polystyrene Petri dishes coated with 10 ug/ml I C A M - 1 or 10 ug/ml a - L F A - 1 for the indicated times (in minutes) at 37°C and then solubilized with ice cold 1% Triton X-100 lysis buffer. Time zero samples were lysed in microfuge with ice cold 1% Triton X-100 lysis buffer. Ce l l lysates were immunoprecipitated as described in Materials and Methods. Immunoprecipitated samples were divided in half and analyzed for Pyk-2 (loading control) and tyrosine phosphorylation with anti-Pyk-2 and 4G10 primary Abs respectively followed by anti-goat IgG and anti-mouse IgG secondary Abs conjugated with horseradish peroxidase and the E C L chemiluminescence. Each timepoint is representative of at two independent experiments. 41 CHAPTER 4 DISCUSSION We have investigated the effect of L F A - 1 outside-in signalling on the activation and redistribution of proteins involved in actin cytoskeleton reorganization. Herein we show that binding of C T L s to I C A M - 1 immobilized on plastic surfaces induces the reorganization of the actin cytoskeleton and W A S P in a consistent manner. This is not due to simple clustering of L F A - 1 , as binding of C T L s to a -LFA-1 coated surfaces does not have the same effect. A n increase in Pyk2 tyrosine phosphorylation after C T L binding to a -LFA-1 coated surfaces indicates that clustering of L F A - 1 can induce signalling, however it is not sufficient to cause site-specific actin reorganization. The significance of these findings is that future studies must take into consideration the biological relevance of systems that activate L F A - 1 using antibody cross-linking, as this methodology is limited in the scope of L F A - 1 mediate signals that can be generated. Act in reorganization accompanies many processes necessary for proper T cell function. Changes in actin dynamics permit flexibility in cell shape needed during migration and extravasation, while disruption of the actin cytoskeleton by cytochalasins prevents the formation of T ce l l /APC contacts (Delon, Bercovici et al. 1998). T cells deficient for proteins involved in actin polymerization, such as Vav-1 and W A S P , have impaired immunological synapse formation and are poorly activated (Fischer, Kong et al. 1998; Holsinger, Graef et al. 1998; Badour, Zhang et al. 2003). In C T L s , immunological synapse formation is initiated by L F A - 1 , independent of recognition of antigen/MHC by the T C R (Marwali, MacLeod et al. 2004). We demonstrate that actin accumulates at the contact-site formed between C T L s and microspheres coated with I C A M - 1 but not a - L F A - 1 . Even when taking into consideration the increased area of contact-sites formed between C T L s and I C A M - 1 coated beads, actin staining at the CTL-bead interface was on average approximately 60% greater in these conjugates than in 42 ones formed with a -LFA-1 coated beads. Furthermore, the ratio of integrated intensity at the contact-site versus the non-contact site in C T L s bound to I C A M - 1 coated beads was significantly greater than one, while in C T L s bound to a -LFA-1 coated beads this ratio was significantly less than one. This suggests that while L F A - 1 - I C A M - 1 binding induces site-specific actin reorganization towards the CTL-bead interface, clustering o f L F A - 1 with m A b may be capable of causing actin accumulation in areas of the cell other than the contact-site. Consistent with this, actin was found to cluster at sites other than the CTL-bead interface in approximately 30% of conjugates formed between C T L s and a -LFA-1 beads. This coincides with an experiment done in our lab in which L F A - 1 in C T L s was capped by antibody crosslinking and stained for both L F A - 1 and actin. Capping of L F A - 1 was not found to induce co-capping of actin, although it was able to cause clustering of actin at different sites (Marwali, 2004). H o w actin reorganization is induced by I C A M - 1 binding to L F A - 1 is still unknown. Separation of L F A - 1 cytoplasmic tails has been measured using F R E T following ligand engagement ( K i m , Carman et al. 2003). Both I C A M - 1 and a -LFA-1 binding presumably cluster L F A - 1 at the site of bead binding, however, only I C A M - 1 binding would cause separation of L F A - 1 cytoplasmic tails which could then serve as a docking site in the recruitment of cytoskeletal proteins. Consistent with this is the finding that talin, which has long been implicated as the link between integrins and the cytoskeleton, was previously found in our lab to accumulate at the contact-site formed between C T L s and I C A M - 1 , but not a -LFA-1 coated microspheres (Dreolini, 2005). This is most likely because a talin binding site on L F A - 1 is exposed only after separation of L F A - 1 cytoplasmic tails. Talin bound to L F A - 1 could then serve to direct the recruitment of actin through one of its actin binding sites (Fig. 4.1C). Upon T C R mediated signalling, D N A M - 1 (CD226) becomes serine phosphorylated at residue 329, allowing it to interact with L F A - 1 (Shirakawa, Shibuya et al. 2005). Because some 43 proteins, such as Vav-1 , appear to be already phosphorylated in C T L s , it is possible that this L F A - l - D N A M - 1 interaction is constitutive in C T L s , or that L F A - 1 - I C A M - 1 binding facilitates the interaction of L F A - 1 with serine phosphorylated-DNAM-1. D N A M - 1 is involved in a ternary complex along with the guanylate kinase hDlg (human homologue o f discs large) and 4.1G (Ralston, Hi rd et al. 2004). The 4.1 superfamily are characterized by a F E R M (band four-point-one/ezrin/radixin/moesin) domain toward the amino terminus and an F actin-binding segment at the carboxyl terminus (Hamada, Shimizu-et al. 2003). This complex, in addition to talin, may serve as an additional means by which actin is recruited in C T L s to L F A - 1 clusters (Fig. 4 . IB) . W A S P is a well established mediator of actin polymerization through its association with the Arp2/3 complex, but its role in L F A - 1 signalling has not been established. We have shown that W A S P , like actin, accumulates at the contact site formed between C T L s and microspheres coated with I C A M - 1 but not a - L F A - 1 . It is unclear, however, what drives this W A S P recruitment. T C R induced activation and translocation of W A S P are thought to occur by two distinct pathways (Zeng, Cannon et al. 2003). Following T C R engagement, Z A P - 7 0 is phosphorylated which in turn phosphorylates SLP-76. This creates binding sites for both Nek and Vav-1 on SLP-76. Nek functions to recruit W A S P to the site o f T ce l l -APC interaction via binding of its C-terminal SH3 domain with the proline-rich region of W A S P . Parallel with this, Vav-1 acts to induce the localized activation of Cdc42 which then serves to relieve W A S P from autoinhibition (Zeng, Cannon et al. 2003). It is not known whether Nek is also involved in L F A -1 induced W A S P recruitment. The adhesion and degranulation-promoting adaptor protein ( A D A P ) binds SLP-76 and has been shown to colocalize with L F A - 1 in the p S M A C (da Silva, L i et al. 1997; Musc i , Hendricks-Taylor et al. 1997; Shirakawa, Wang et al. 2006). In C T L s , the immunological synapse is formed in an antigen independent manner (Marwali , 2004). L F A - 1 -44 I C A M - 1 binding may therefore cause A D A P recruitment to the CTL-bead interface, which could then facilitate W A S P recruitment and activation via Nek and Cdc42 respectively (Fig. 4. ID). In preliminary confocal experiments done in our lab, N c k l staining was not sensitive enough to be detected above background fluorescence (data not shown). Previous experiments examining the role of Nek in W A S P recruitment were done in Jurkat T cells, which may have significantly higher levels of Nek expression. Mouse strains lacking N c k l , Nck2 , or both Nek genes have been generated (Bladt, Aippersbach et al. 2003). Only concomitant loss of both N c k l and Nck2 resulted in early embryonic lethality, suggesting that there is a redundancy in N c k l and Nck2 activities. Fibroblast cell lines derived from the Nckl" 7 " Nck2 _ /" embryos had significantly impaired motility as a result of altered lamellipodium formation. To address the role of N c k l and Nck2 in L F A - 1 mediated W A S P recruitment, T cells could be derived from Nckl" 7 " Nck2 - / " embryos. Interestingly, T C R / C D 2 8 engagement has been shown to induce the formation of endogenous Lck-hDlg-Zap70-WASP complexes, with hDlg acting to facilitate interactions of L c k with Zap70 and W A S P (Round, Tomassian et al. 2005). The authors observed that hDlg promoted antigen-induced actin polymerization, l ipid raft and T C R clustering, which have all been observed previously to occur in an antigen independent manner following L F A - 1 - I C A M - 1 binding (Marwali , MacLeod et al. 2004). A s mentioned earlier, D N A M - 1 binds both L F A - 1 and hDlg . Phosphorylation of D N A M - 1 at residue 329 is critical for its interaction with L F A - 1 and is also required for its recruitment into lipid rafts (Shibuya, Lanier et al. 1999; Shirakawa, Shibuya et al. 2005). Since lipid raft recruitment to the immunological synapse in C T L s occurs in an antigen independent manner (Marwali, MacLeod et al. 2004), it is possible that L F A - 1 -I C A M - 1 binding facilitates the incorporation of D N A M - 1 into l ipid rafts by allowing L F A - 1 to bind serine phosphorylated D N A M - 1 . D N A M - 1 could then recruit W A S P via its interaction 45 with hDlg, which could explain the observed accumulation of W A S P in C T L s binding I C A M - 1 coated beads in this study. Immunological synapse formation is dependent on the clustering of l ipid rafts. In untransformed human T cell lines, W A S P has been shown to play a key role in l ipid raft movement. Following T C R / C D 2 8 engagement, a fraction of W A S P is immediately incorporated into sucrose gradient l ipid raft fractions. This recruitment is necessary for l ipid raft clustering, as W A S patients have impaired capacities to cluster the l ipid raft marker G M 1 after T C R / C D 2 8 activation (Dupre, Aiu t i et al. 2002). Results generated in our lab suggest the immunological synapse in C T L s is formed independent of antigen binding. This includes recruitment of L F A - 1 , C D 3 and G M 1 to the site of target cell binding (Marwali, MacLeod et al. 2004). However, W A S P was not found in Brij 35 insoluble lipid raft fractions following C T L binding to either s I C A M - 1 or a -LFA-1 coated dishes. It is possible that W A S P recruitment into l ipid rafts is dependent on T C R / C D 2 8 activation and is not required for L F A - 1 induced recruitment of W A S P . Conversely, the amount of W A S P incorporated into l ipid rafts may have returned to basal levels at the 30 minute time-point tested. It is also possible that solubilization of C T L s caused the dissociation of W A S P from lipid rafts. Since W A S P is not a transmembrane protein, its localization to l ipid rafts is presumably dependent on protein-protein interactions which may become disrupted following treatment with certain detergents. Although both actin and W A S P accumulate at the contact site formed between C T L s and microspheres coated with s I C A M - 1 , it cannot be assumed that W A S P is responsible for this localized polymerization of actin. The role of W A S P in T cell activation has been until recently related to its regulation of actin dynamics. However, T cells from WASP" 7 " mice polymerize actin normally at the interface formed with antigen-pulsed B cells and in response to T C R capping (Sedwick, Morgan et al. 1999; Krawczyk, Oliveira-dos-Santos et al. 2002). Additional 46 pathways controlling actin polymerization in T cells may therefore exist. Recent findings suggest the W A S P family member W A V E 2 may be the primary mediator of actin reorganization at the immunological synapse during T cell activation (Nolz, Gomez et al. 2006; Zipfel , Bunnell et al. 2006). WAVE2-suppressed Jurkat T cells displayed impaired T C R induced actin polymerization and conjugate formation with antigen-pulsed B cells, whereas suppression of W A S P had no affect. W A V E proteins are activated indirectly by the Rho family GTPase R a c l . In human T lymphoblasts, Rac-1 becomes activated after cells adhere to I C A M - 1 coated surfaces (Sanchez-Martin, Sanchez-Sanchez et al. 2004). It is therefore worth investigating i f W A V E 2 is involved in L F A - 1 outside-in signalling pathways leading to actin reorganization in C T L s . Direct phosphorylation of tyrosine residue Y291 in W A S P has been shown to induce actin polymerization following T C R engagement (Badour, Zhang et al. 2004). The data presented here show no increase in W A S P phosphorylation following C T L binding to I C A M - 1 or a - L F A - 1 coated plates. This suggests that either W A S P is not responsible for the observed accumulation of actin following L F A - 1 engagement or that tyrosine phosphorylation of W A S P is not a requirement for W A S P activation in C T L s . A s W A S P is required for proper T cell activation, it would be difficult to generate C T L s from WASP" 7 " mice to directly test its role in L F A - 1 induced actin dynamics. Antibodies specific to the open conformation of W A S P could provide further insight into whether W A S P is activated following L F A - 1 - I C A M - 1 binding. We found that in C T L s , the G E F Vav-1 was basally tyrosine phosphorylated, and that the level of phosphorylation was not increased by binding of cells to I C A M - 1 or a -LFA-1 coated plates. It is therefore possible that other proteins involved in actin cytoskeleton rearrangement, such as Cdc42 and Rac-1, are already activated in C T L s . For this reason, L F A - 1 ligand engagement may be more important in influencing the cytoplasmic localization of these proteins than in activating them further. 47 The tyrosine kinase Pyk-2 has been shown to become activated in response to integrin engagement and has also been suggested to modulate cytoskeletal reorganization and changes in cell morphology (Rodriguez-Fernandez, Gomez et al. 1999; Ostergaard and Lysechko 2005). Our findings indicate that Pyk-2 is tyrosine phosphorylated after 5 minutes of C T L binding to either I C A M - 1 or a -LFA-1 coated plates, and was sustained at this level for at least 60 minutes. These results contrast slightly with a report describing the activation of Pyk-2 in human T lymphoblasts (Rodriguez-Fernandez, Gomez et al. 1999). In this instance, L F A - 1 was activated with stimulatory antibody and cells were plated on I C A M - 1 surfaces for 15 to 60 minutes. Pyk-2 activity increased after 15 minutes, reached a maximum after 30 minutes and then decreased at longer time-points. The phosphorylation of Pyk-2 in this system required cytoskeletal integrity, as drugs that disrupt the actin and microtubule cytoskeletons completely blocked any L F A - 1 mediated increase in Pyk-2 phosphorylation. It should be noted that this inhibition of Pyk-2 phosphorylation could also be due to indirect effects caused by cytoskeletal disruption. The phosphorylation of Pyk-2 in this system was concurrent with its redistribution from a diffuse cytoplasmic distribution to a location near the M T O C (Rodriguez-Fernandez, Gomez et al. 1999). We demonstrated that Pyk-2 in C T L s bound to I C A M - 1 or a -LFA-1 coated beads remains distributed evenly throughout the cytoplasm. It is possible that tyrosine phosphorylated Pyk-2 represents only a fraction of the total cellular Pyk-2. Using antibodies that specifically recognize the Pyk-2 Y402 autophosphorylation site may be better able to distinguish differences in activated Pyk-2 subcellular distribution as a result of I C A M - 1 versus a -LFA-1 binding. L F A - 1 has been shown to induce the transient activation of PI3K (phosphatidylinositol 3-kinase) in human T lymphoblasts following M n 2 + activated L F A - 1 binding to I C A M - 1 coated surfaces (Sanchez-Martin, Sanchez-Sanchez et al. 2004). Additionally, crosslinking of L F A - 1 with the specific a-CD18 Lia3/2.1 mAb also induces PI3K activation (Sanchez-Martin, Sanchez-48 Sanchez et al. 2004). In C T L s , TCR-stimulated PI3K activates Erk leading to paxillin tyrosine phosphorylation (Robertson, Mireau et al. 2005). Tyrosine phosphorylated paxill in is then able to bind the SH2 domain of the Src family kinase L c k (Ostergaard, L o u et al. 1998). In murine C T L clones, paxill in is constitutively associated with Pyk-2 in a manner that is not altered by the phosphorylation of either protein (Ostergaard, Lou et al. 1998). Binding of Lck , or another Src family kinase such as Fyn, to paxillin would therefore place it in close proximity to Pyk-2. Since Src family kinases are known to phosphorylate Pyk-2 tyrosine residues (Qian, Lev et al. 1997), this could explain how Pyk-2 becomes tyrosine phosphorylated in C T L s following both I C A M - 1 and a -LFA-1 binding (Fig. 4.1 A ) . 49 Src family kinase (i.e. Fyn) I Relief of WASP autoinhibition WASP-Arp2/3 binding leading to actin polymerization Figure 4.1: Proposed model to explain the observed effects of LFA-1 signalling in this study. (A) Tyrosine phosphorylation of Pyk-2 by Src family kinases. (B) Recruitment of actin to the CTL-bead interface by the ternary complex D N A M - 1 -hDlg-4.1G which interacts with L F A - 1 via a phosphorylated serine residue on D N A M - 1 . 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