From actin to Zap70: the outcomes of the interaction of the actin cytoskeleton with T-cell receptors
- Author(s): Smoligovets, Alexander Alexandrovich
- Advisor(s): Groves, Jay T
- Isacoff, Ehud
- et al.
The actin cytoskeleton has for decades been known to be important in the process of T-cell recognition of antigen. Upon receiving stimulus from T-cell receptors (TCRs), the T-cell cytoskeleton adopts a pattern of radially symmetric, centripetal flow that imposes multi-micrometer scale organization on the protein complement of a T-cell membrane. Many of the details of the interactions between actin and cell surface receptors remain unknown; of particular interest are the connections to TCRs themselves. The actin cytoskeleton appears to modulate TCR-mediated signaling, and thus T-cell behaviors, on a number of levels. On the level of single TCR molecules and oligomers, the presence of actin affects TCR-antigen recognition by altering binding kinetics through mechanisms that are still unclear. On a larger spatial and longer temporal level, actin drives the formation and centripetal movement of small TCR assemblies whose radial distribution correlates with their signaling state. Little is known about the local organization and dynamics of the actin cytoskeleton around these assemblies. A more complete understanding of the process of T-cell activation depends on the ability to characterize the involvement of actin in detail.
This thesis describes how the T-cell actin cytoskeleton locally reacts to TCR assemblies, how it modulates the kinetics of the TCR-antigen interaction, and what role myosin IIA plays in organization of and signaling by TCRs. Direct imaging of actin reveals that the cytoskeletal network transiently slows and compresses at sites of mobility-limited T-cell receptors. Actin disruption during direct observation of antigen peptide binding to TCRs shows that the presence of a functional cytoskeleton increases the rate of antigen unbinding from these receptors. Tracking and disruption of labeled myosin IIA indicates that the motor protein helps but is not required for proper establishment of T-cell membrane organization, but it is necessary for appropriate calcium influx into the T-cell cytoplasm during activation. For all of these observations and others, new experimental and analytical methods are developed or applied.