Planar Immunological Synapses Formed by Human CD4 T Cells as In Vitro System to Study Actin and Tubulin Dynamics Through TIRF Microscopy.

The rapid and transient rearrangement of the actin cytoskeleton and the microtubule network toward the antigen contact site is essential for immunological synapse (IS) formation in T cells. This process can be reproduced in vitro using activating antibodies against the T-cell receptor and costimulatory molecules. During the IS formation, the actin cytoskeleton is remodeled into a dense peripheral F-actin ring, accompanied by polarization of the centrosome, which acts as the major microtubule-organizing center at the synapse. Both processes rely on tightly regulated cycles of polymerization and depolymerization of cytoskeletal filaments. In this chapter, we present a live-cell, super-resolution microscopy approach based on Total Internal Reflection Fluorescence (TIRF), using planar IS as an in vitro model to investigate actin and tubulin dynamics in primary human CD4 T cells.