Actin Regulatory Dynamics Required for T Cell Activation: A Quantitative and Systems-Level Perspective
T cell activation occurs through interaction with an antigen-presenting cell (APC). Upon activation, signaling ensues with the coordination of dozens of diverse signaling molecules in space and time, a feature of cell signaling we call ‘spatiotemporal patterning’. We performed a systems-scale analysis of the spatiotemporal patterning of T cell signaling and have found that it is highly diverse. Over 50 signaling sensors were imaged in live primary T cells activated with APCs under various physiological stimulation conditions, and no two signaling intermediates showed the same dynamic localization. The activation environment controlled spatiotemporal features of T cell signaling and specific spatiotemporal features correlated with efficient T cell activation. To identify underlying cell biological mechanisms controlling spatiotemporal organization of signaling, we complimented our live cell imaging with microscopy across multiple scales and identified a dense transient F-actin network that extends from a highly interdigitated T cell:APC interface several micrometers deep into the T cell lamellum. Systems-scale imaging revealed a large network of proximal T cell signaling intermediates that localized to the lamellal actin network and shared the spatial, temporal, and mobility features of F-actin. Interference with lamellal actin dynamics modulated the activity of the associated proteins and impaired IL-2 production. These data strongly suggest that the transient deep F-actin network by controlling lamellal localization modulates the activity of a substantial part of the T cell signal transduction system. As a next step in understanding how spatiotemporal dynamics of signaling controls T cell activation, we have developed a quantitative 4D analysis approach for signaling networks and coupled it with traditional cell biological techniques to uncover higher order mechanisms of the control of actin dynamics by CD28 co-stimulation during T cell activation. A group of nine actin regulatory proteins that mediate actin polymerization, capping, and severing were assessed and CD28 co-stimulation was required for their sustained activity at the T cell:APC interface. WAVE2 and Cofilin were especially sensitive to blockade of CD28 signaling. Functional relevance of the loss of WAVE2 and Cofilin enrichment was shown by the treatment of T cells with constitutively active Rac1 and Cofilin, which bypassed the requirement of co-stimulation for normal actin dynamics and AKT activation. This study highlights how a systems analysis of actin regulation could identify mechanisms that are inaccessible to more traditional single protein/gene approaches.
Actins, Signal Transduction, T-Lymphocytes
University of Texas Southwestern Medical Center
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