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Imaging T cell actin dynamics
T cells must receive signals through the T cell receptor (TCR) and the costimulatory receptor CD28 to become fully activated. Critical to this process is the reorganization of plasma membrane actin at the immunological synapse, the interface between a T cell and an antigen-presenting cell. Roybal et al. imaged actin and fluorescently tagged actin regulatory proteins in T cells activated through the TCR in the absence or presence of CD28 signaling. Computational image processing to normalize differences in cell shape enabled tracking of the fluorescent proteins. The regulatory proteins WAVE2 and cofilin were efficiently recruited to the immunological synapse only when both TCR and CD28 signaled. Constitutive activation of either protein in TCR-stimulated T cells enabled normal actin reorganization even when CD28 signaling was blocked. This combination of imaging and computational analysis could be applied to other systems to determine the spatiotemporal dynamics of signaling molecules.
Fluorescence microscopy is one of the most important tools in cell biology research because it provides spatial and temporal information to investigate regulatory systems inside cells. This technique can generate data in the form of signal intensities at thousands of positions resolved inside individual live cells. However, given extensive cell-to-cell variation, these data cannot be readily assembled into three- or four-dimensional maps of protein concentration that can be compared across different cells and conditions. We have developed a method to enable comparison of imaging data from many cells and applied it to investigate actin dynamics in T cell activation. Antigen recognition in T cells by the T cell receptor (TCR) is amplified by engagement of the costimulatory receptor CD28. We imaged actin and eight core actin regulators to generate over a thousand movies of T cells under conditions in which CD28 was either engaged or blocked in the context of a strong TCR signal. Our computational analysis showed that the primary effect of costimulation blockade was to decrease recruitment of the activator of actin nucleation WAVE2 (Wiskott-Aldrich syndrome protein family verprolin-homologous protein 2) and the actin-severing protein cofilin to F-actin. Reconstitution of WAVE2 and cofilin activity restored the defect in actin signaling dynamics caused by costimulation blockade. Thus, we have developed and validated an approach to quantify protein distributions in time and space for the analysis of complex regulatory systems.