Computational spatiotemporal analysis identifies WAVE2 and cofilin as joint regulators of costimulation-mediated T cell actin dynamics

Abstract

Fluorescence microscopy is one of the most important tools in cell biology research and 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, methods do not currently exist to assemble these data into three- or four-dimensional maps of protein concentration that can be compared across different cells and conditions. Here, we have developed one such method 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 and we have determined how CD28 modulates actin dynamics. We imaged actin and eight core actin regulators under conditions where CD28 in the context of a strong TCR signal was engaged or blocked to yield over a thousand movies. Our computational analysis identified diminished recruitment of the activator of actin nucleation WAVE2 and the actin severing protein cofilin to F-actin as the dominant difference upon costimulation blockade. Reconstitution of WAVE2 and cofilin activity restored the defect in actin signaling dynamics upon costimulation blockade. Thus we have developed and validated an approach to quantify protein distributions in time and space for analysis of complex regulatory systems.

Micropipette force probe to quantify single-cell force generation: application to T-cell activation

“…This is in agreement with the known effect of CD28 on actin remodeling ( Wülfing, 1998 ). It would thus be particularly interesting to investigate the role of WAVE2 and cofilin, two regulators of CD28-induced actin dynamics ( Roybal et al. , 2016 ), on the pushing forces reported herein.…”

The actin-capping protein alpha-adducin is required for T-cell costimulation

“…CD28 costimulation appears to impact F-actin dynamics in multiple ways. WAVE2 and HS1, which control actin nucleation, and cofilin, which severs F-actin, showed defective accumulation at the synapse upon costimulation blockade (Roybal et al, 2016). Further, the actin nucleation promoting protein WASp has been shown to promote CD28-mediated F-actin accumulation and CD28 endocytosis (Badour et al, 2007).…”

PD-1 suppresses the maintenance of cell couples between cytotoxic T cells and tumor target cells within the tumor

“…In CD4 + T cells differences in the spatiotemporal organization of proximal T cell signaling are tightly linked to differences in actin dynamics (28-30). To similarly gain insight into signaling defects in TILs, which may underpin impaired cell couple maintenance, we investigated key elements of the spatiotemporal organization of Clone 4 CD8 + T cell signaling.…”