Title: TNanoscale kinetic segregation of the T cell antigen receptor and glycoproteins in engaged microvilli facilitate early T cell activation
Author: Yair Razvag , Yair Neve-Oz, Julia Sajman, Meital Reches, Eilon Sherman
Abstract:
T cell activation plays a central role in mounting an immune response. However, the molecular mechanisms of early T cell activation in response to cognate antigens remain poorly understood. Here, we combined single molecule localization microscopy (SMLM) techniques in multiple colours to visualize the dynamic nanoscale patterning of TCRs and CD45 glycoproteins at the plasma membrane of live activated T cells. SMLM imaging and second-order statistics showed a phase separation between these molecules in early forming and isolated contacts under a range of TCR-activating and non-activating conditions. This phase separation grew over time under TCR activating conditions, yet was significantly reduced under non-TCR activating conditions. AFM and SMLM further served to identify the early contacts with touching microvilli and to characterize their morphology, rigidity and dynamics. A physical simulation of the cell interface could accurately capture the phase separation between these molecules and its dynamics under the experimental conditions. TCR triggering, highlighted by pTCRz, negatively correlated with the physical separation between TCRs and CD45. Our results indicate a refined view of the kinetic segregation model where it occurs within seconds from TCR activation and that TCRs should be segregated, yet not removed too far, from CD45 for their optimal activation within clusters, thus shedding new light on this critical process of T cell activation. Moreover, our imaging techniques and computational tools are relevant to the study of the dynamics and function of a wide range of membrane proteins in various cell types.