The localization of receptors, signaling intermediates, and cytoskeletal components at the T cell/APC interface is thought to be a major determinant of efficient T cell activation. However, important questions remain open. What are the dynamics of the T cell cytoskeleton as a potential mediator of such localization? How are they regulated by the TCR and costimulatory receptors? Do they actually mediate receptor localization? In this study, we have addressed these questions. Even under limiting T cell activation conditions, actin accumulated immediately and transiently at the T cell/APC interface, the microtubule organizing center reoriented toward it. In contrast, sustained (>5 min) actin accumulation in highly dynamic patterns depended on an optimal T cell stimulus: high concentrations of the strong TCR ligand agonist peptide/MHC and engagement of the costimulatory receptors CD28 and LFA-1 were required in an overlapping, yet distinct, fashion. Intact sustained actin dynamics were required for interface accumulation of TCR/MHC in a central pattern and for efficient T cell proliferation, as established using a novel approach to selectively block only the sustained actin dynamics. These data suggest that control of specific elements of actin dynamics by TCR and costimulatory receptors is a mechanism to regulate the efficiency of T cell activation.

T cell polarization toward and within the cellular interface with an APC is critical for effective T cell activation. The Rho family GTPase Cdc42 is a central regulator of cellular polarization. Using live-cell imaging, we characterized the spatiotemporal patterns of Cdc42 activity and their physiological regulation. Using three independent means of experimental manipulation of Cdc42 activity, we established that Cdc42 is a critical regulator of T cell actin dynamics, TCR clustering, and cell cycle entry. Using quantification of three-dimensional data, we could relate distinct spatiotemporal patterns of Cdc42 activity to specific elements of T cell activation. This result suggests that Cdc42 activity in specific locations at specific times is most critical for its function in T cell activation.

Cellular interaction is vital to the activation of most lymphocytes. At the interface between the lymphocyte and the cell that activates it, multiple receptor/ligand pairs accumulate in distinct patterns. This accumulation is intriguing, as it is likely to shape the quality of receptor signaling and thereby lymphocyte behavior. Here we address such receptor/ligand accumulation with an emphasis on T and natural killer (NK) cells. First, we discuss the strengths and limitations of commonly used approaches to visualize receptor/ligand accumulation. Second, we discuss two principal mechanisms of receptor and ligand translocation, diffusion and cytoskeletal transport, as understanding these mechanisms can be invaluable in the determination of the significance of receptor/ligand accumulation. We show that the extent of receptor/ligand accumulation at the T cell/antigen presenting cell interface is dominated by diffusion for all but the lowest affinity interactions, while patterning of these receptors/ligands within the interface is strongly influenced by cytoskeletal transport. Third, we discuss two specific issues in lymphocyte receptor/ligand accumulation. We review the abundant but frequently controversial data on T cell receptor (TCR)/major histocompatibility complex (MHC) accumulation and suggest that central TCR/MHC accumulation is a mediator of efficient T cell activation. In the investigation of NK cell/target cell interactions, we characterize the often tentative NK cell/target cell couple maintenance, as it creates a major obstacle in studying receptor/ligand accumulation.

The Rho family GTPase Cdc42 is a critical regulator of cellular polarization from yeast to man. An analysis of its function in T cell activation is therefore of interest. This analysis poses two substantial challenges, similar to the analysis of many other critical T cell signaling intermediates. First, Cdc42 is required for development and cell survival, necessitating short-term manipulation of its activity. Second, Cdc42 is likely involved in multiple signaling pathways, requiring approaches to distinguish multiple roles. To address these challenges, we first determined and quantified spatio-temporal patterns of Cdc42 activity using live cell video fluorescence microscopy. This generates hypotheses at which times and locations Cdc42 might play possibly distinct roles. Second and as the focus of this manuscript, we employed protein transduction to manipulate Cdc42 activity for the generation of causality. Protein transduction allows such manipulation to be short-term, quantitative, and with multiple reagents. Here, we characterize uptake, retention, and subcellular distribution of protein transduction reagents. We describe how a more quantitative single cell analysis of Cdc42 activity provides superior distinction between experimental conditions. And we show how we have used dose responses of the protein transduction reagents to minimize side effects while retaining efficacy. We suggest that our strategy is an important complement to more established techniques to study protein function in primary T cells, in particular in the investigation of signaling intermediates that are essential for cell survival and regulate multiple aspects of T cell activation.