Mediating signaling response to actin‐mediated forces: Cas‐L is causal in the T‐cell response to forces triggered by antigen presentation

Intracellular signaling was once considered primarily as a sequence of chemical reactions, and the biochemical tools for studying signaling did not accommodate the context of these reactions inside the cell. It is now appreciated that intracellular signals do not occur in amorphous slush, but in ever-changing microcosms defined by membrane composition and the cytoskeleton. Elucidating the constraints and forces that control molecular movement to coordinate intracellular signals has begun to transform biology. The immunological synapse of a T cell represents one such microcosm of signaling, regulating signals at the interface with the antigen-presenting cell to dictate T-cell fate and to provide a conduit for T-cell-mediated cytotoxicity. In this issue of ICB, Santos et al.1 show how initial T-cell receptor (TCR) signals control actomyosin contractility to mediate molecular trafficking in the immunological synapse. The back-and-forth between chemical and physical signals described here provides a fascinating glimpse of complex biophysical events that might well be prevalent throughout biology. Driven by previous findings that cytoskeletal flow alters the activity of TCR-associated molecules, the authors here explore the role of Cas-L (Crk-associated substrate) in mediating the link between force and signaling events. Cas-L (also known as Nedd9 or HEF1) is a member of a family of force-sensing proteins, and is specifically expressed in lymphocytes. Cas-L has been implicated in T-cell responses to antigen, and the authors have previously shown that the Cas-family proteins can unfold in response to actin-dependent stretch, exposing substrates of the Src-family kinases.2 Here, they demonstrate that Cas-L mediates the response to TCR-induced F-actin polymerization, presumably through a similar unfolding process. TCR clusters form perfectly well in Cas-L−/− T cells, but these microclusters do not effectively move toward the center of the immunological synapse. The reduced migration of the microclusters seems to relate to a reduced adhesion owing to defective inside-out activation of the integrin, LFA1. In support of a direct role for Cas-L in the microclusters, the authors found that Cas-L is transiently recruited to the TCR microclusters and activated by actin polymerization. Similar to previous findings in epithelial cells, the phosphorylation of Cas-L depends upon a Src-family kinase, Lck, presumably again acting on epitopes of Cas-L that are exposed following actin-dependent stretch. Together, these observations make a compeling case for Cas-L as a bridge between the mechanical cues triggered by initial adhesion and subsequent signaling events. Thus, proximal TCR signals, including Lck activation and actin polymerization, lead to stretch-induced Cas-L activation (Figure 1). Cas-family proteins have long been known to regulate morphological changes,3 and their role in the TCR response seems to be no exception. An immediate effect of Cas-L knockout is slowing of the trafficking of TCR microclusters. The direct effect of this process on downstream TCR signaling is not easy to dissect from alternative, indirect effects (for instance, TCR-independent calcium signaling was altered in the Cas-L cells), but several lines of evidence suggest that Cas-L activation influences Ca2+ signaling and inside-out integrin activation. The effects of Cas-L on integrin activation are particularly interesting in light of previous studies showing non-TCR-related effects of Cas-L on inside-out integrin signaling4 and earlier studies showing integrin-mediated activation of Cas-L.5 The potential for complex feedback loops in which signaling from both TCR and adhesion receptors regulate actin and Cas-L, which in turn regulate adhesion, means that the complex model proposed by the authors is probably only the beginning of an extremely sophisticated regulatory network. The findings by Santos et al.1 were obtained using total internal reflection fluorescence (TIRF) microscopy on lipid bilayers. This technology is widely used, and the combination of high resolution and precise control of TCR signaling has proven very powerful. It is clear that the morphological rearrangements observed in response to lipid bilayers will be very different from the response to an antigen-presenting cell, where each cell exerts counter forces on the other and remodels accordingly.6 If Cas-L senses forces triggered by F-actin polymerization, how would those forces differ in interactions with an antigen-presenting cell rather than a glass slide? With new super-resolution imaging technologies emerging7 that will preempt the need for lipid bilayers, we will hopefully soon see whether and how actin polymerization in a cell that is rapidly remodeling in response to morphological changes in the antigen-presenting cell can activate Cas-L.