Date of Award

2014

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Graduate Group

Cell & Molecular Biology

First Advisor

Janis K. Burkhardt

Abstract

The efficiency of an immune response critically depends on the ability of T cells to respond to antigens. Upon encountering cognate antigenic peptides on the surface of antigen-presenting cells, T cells form a specialized interface, termed the immunological synapse (IS), which serves as the site of information transfer between the cells. This contact zone is characterized by the enrichment of signaling receptors, kinases and adaptor proteins, and is the site of extensive cytoskeletal remodeling. The versatile nature and spatio-temporal regulation of signaling cascades at the IS has long been recognized but the exact mechanisms that coordinate these processes remain poorly understood. In this work we have investigated the role of cytoskeletal remodeling in propagation of signaling events that lead to T cell activation. Using human T cell lines and primary T cells, we demonstrate that F-actin flow is largely driven by actin polymerization, rather than by myosin IIA contraction. While myosin IIA is able to exert forces on the cytoskeleton, it is dispensable for bulk network flow. Conversely, myosin IIA controls the extent of cell spreading and synaptic symmetry. We have also found that ongoing retrograde flow of F-actin sustains calcium mobilization at the level of release from endoplasmic reticulum stores. This defect is likely due to loss of PLCgamma1 activity at the IS, since the concentration of phosphorylated PLCgamma1 plummets upon F-actin immobilization. Furthermore, we have examined whether F-actin remodeling is required for integrin LFA-1 activation, which in turn strengthens conjugate formation and costimulation. Taking advantage of stimulatory planar lipid bilayers and cell-cell conjugates, we show that F-actin flow drives affinity maturation and spatial organization of LFA-1 at the IS. These observations are in line with a mechanotransduction model, in which F-actin-derived force induces integrin conformational change, thereby modulating binding affinity for ligand. The net inward movement of F-actin also recruits LFA-1 to the interface, thereby increasing its effective concentration. Taken together, these findings indicate that ongoing remodeling of actin cytoskeleton is required to sustain signaling and to choreograph spatio-temporal organization of receptors and their associated complexes at the IS during early phases of T cell activation.

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