UBC Theses and Dissertations
LFA-1 outside-in signalling and actin cytoskeleton reorganization in cytotoxic T lymphocytes MacLeod, Matthew Alexander
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 (LFA-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, LFA-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 LFA-1 , such as phorbol ester treatment or antibody crosslinking. In this study, the effect of LFA-1 signalling on the activation and redistribution of proteins involved in actin cytoskeleton reorganization was investigated in cytotoxic T lymphocytes (CTLs). LFA-1 in CTLs 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 (ICAM-1) binding in LFA-1 signalling, CTLs were bound to plastic surfaces coated with either ICAM-1 or a-LFA-1 blocking antibody. The binding of CTLs to ICAM-1 immobilized on polystyrene microspheres induced the site-specific recruitment of actin and WASP (Wiskott-Aldrich syndrome protein) to the CTL-microsphere interface. This is not due to simple clustering of LFA-1 , as binding of CTLs to a-LFA-1 coated microspheres did not have the same effect. Recruitment of WASP to the contact site formed between CTLs and ICAM-1 coated microspheres was not dependent on an increase in WASP tyrosine phosphorylation or incorporation of WASP 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 CTLs. This suggests that Vav-1, as well as other proteins involved in actin polymerization may already be active in CTLs. The tyrosine kinase Pyk-2 (proline-rich 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.
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