Genetic and biochemical studies have identified roles for Itk in PLC-γ phosphorylation, actin accumulation at the T cell–
APC interface, and cytokine secretion. Here, we have identified Itk as a central regulator of the spatiotemporal organization of T cell signaling molecules. Because the patterning of 14 of the 16 sensors that we studied was altered in Itk-deficient cells compared to that in wild-type cells, such regulation by Itk may extend across the entire T cell signaling system. The segregation of signaling intermediates into distinct spatiotemporal clusters was diminished upon loss of Itk; T cell signaling molecules became more homogeneous in time and space. As part of these defects, the accumulation of signaling intermediates at the center of the T cell–
APC interface was widely impaired. The lone exception to this finding was the immediate, but transient, central accumulation of SLP-76 at the interface (fig. S2, S and T
), which suggested that Itk was required for the maintenance, but not establishment, of the central accumulation of signaling molecules. Spatiotemporal segregation in T cell signaling, in particular the formation of the cSMAC signaling complex, is often related to efficient T cell signaling (3
). If such segregation promoted signaling efficiency, then the impaired spatiotemporal organization of T cell signaling molecules observed in Itk-deficient T cells should yield its most pronounced functional effects under conditions in which T cell activation is relatively inefficient. In support of this idea, we found that central TCR clustering and cytokine secretion were more strongly impaired in TH
2-polarized Itk-deficient T cells than in T cells that were not polarized during cell culture (fig. S6, A and B
). Similarly, CD8+
T cells, which have less extensive spatiotemporal organization of signaling molecules than is observed in CD4+
T cells (34
), are particularly sensitive to Itk deficiency (12
). Although the results of our experiments investigating T cell activation by APCs presented here and previously (3
) are consistent with the suggestion that the formation of the cSMAC signaling complex and its regulation by Itk contribute to efficient T cell signaling, other studies involving mathematical modeling and the use of supported lipid bilayers as a substitute for APCs suggest a role for the cSMAC in TCR signal termination, with activating T cell signaling occurring in microclusters at the periphery of the T cell–
APC interface (8
). In addition, strength of TCR engagement, efficacy of proximal signaling, and T cell spatiotemporal organization are linked in a nonlinear fashion (2
). As previously discussed (3
), these data sets can potentially be reconciled. Regardless of the role of the cSMAC, the role of Itk as a regulator of the spatiotemporal organization of T cell signaling is of general interest, because it illustrates how the absence of a single protein can markedly alter the spatiotemporal organization of an entire signaling system. The extent to which the regulation of the T cell spatiotemporal organization by Itk contributes to Itk-dependent cytokine secretion, and whether such regulation depends on the kinase activity of Itk or a potential adaptor function of Itk, needs to be conclusively addressed in future work.
A general challenge in the investigation of the spatiotemporal organization of signaling systems is the need to show that changes in spatiotemporal distributions lead to altered cell function. Because of the general role of Itk in the regulation of spatiotemporal patterning, a conclusive investigation of the relationship between spatiotemporal organization and established Itk effector function requires a systems-scale manipulation of the localization of signaling intermediates, which is a daunting challenge. By addressing the molecular mechanism of Itk-dependent actin regulation, an unresolved critical element of Itk function, we provide here a first case study that suggests that the regulation of spatiotemporal patterning can be required for function. Itk mediated the activation of Cdc42 at the center of the T cell–
APC interface, but the amount of active Cdc42 generated across the entire T cell was not affected by Itk deficiency. These data are in contrast to earlier experiments in which antibody staining of fixed cell couples 10 min after the conjugation of AND T cells and APCs showed that the amount of active Cdc42 at the cell interface was diminished upon Itk deficiency (20
). The discrepancy between that study and the data presented here could be a result of the different TCR trans-genes used in the two studies (3
), because in the case of AND T cells, only 30% of the cell couples showed the accumulation of active Cdc42 at the interface (20
) in comparison to the >80% of cell couples involving DO11.10 T cells presented here (). The impaired central activation of Cdc42 in Itk-deficient DO11.10 T cells proved functionally relevant, because only centrally targeted active Cdc42, and not active Cdc42 targeted to any other part of the cell, restored the diminished amounts of actin at the interface in Itk-deficient DO11.10 T cells. Why was alternately targeted active Cdc42, even at 10-fold higher concentrations than that of centrally targeted active Cdc42, ineffective? When T cells are activated under conditions that yield diminished Cdc42 activation, provision of active Cdc42 can compensate for the limited endogenous Cdc42 activation (22
); however, under conditions of sufficient endogenous Cdc42 activation, Cdc42ca interferes with actin dynamics, likely by competing with endogenous Cdc42 for effectors (22
). In Itk-deficient DO11.10 T cells, the deficiency in Cdc42 activation was spatially restricted to the center of the T cell–
APC interface. Therefore, to reconstitute activity, Cdc42ca had to be provided with comparable spatial restriction. The importance of spatially restricted Cdc42 activity provides initial causal support for the functional relevance of the spatiotemporal organization of T cell signaling.
Signaling occurs in complex networks. The control of actin dynamics, in general and downstream of Itk as demonstrated here, likely involves multiple Rho GTPases and GEFs. In support of this, the accumulation at the interface of three candidate GEFs, Vav1, SLAT, and α-Pix, was consistently impaired upon Itk deficiency. However, distinctions emerged when we considered the spatiotemporal patterning of the GEFs and the regulation thereof by Itk. Although our data illustrate complexity in network-based regulation, spatiotemporal constraints also implied SLAT as a likely prominent element of the Itk-dependent actin regulatory network, which needs to be confirmed by further systems-scale investigations. Thus, our study supports the utility of analyzing spatiotemporal patterning in the investigation of signaling connections in complex networks.
In summary, we found that the absence of a single protein, Itk, altered the spatiotemporal organization of an entire signaling system; control of the central patterning of active Cdc42 rather than its cell-wide activity was functionally most important for Itk-dependent accumulation of actin at the interface, and spatiotemporal analysis provided sensitive access to the complex regulation of T cell actin dynamics. Given the ubiquity of spatiotemporal patterning across various cell types, in particular in the immune system (1
), our findings emphasize the utility of systems-scale spatiotemporal analysis for the comprehensive understanding of signaling systems.