The immunological synapse (IS) is a junction between the T cell and antigen-presenting cell and is composed of supramolecular activation clusters (SMACs). No studies have been published on naive T cell IS dynamics. Here, we find that IS formation during antigen recognition comprises cycles of stable IS formation and autonomous naive T cell migration. The migration phase is driven by PKCtheta, which is localized to the F-actin-dependent peripheral (p)SMAC. PKCtheta(-/-) T cells formed hyperstable IS in vitro and in vivo and, like WT cells, displayed fast oscillations in the distal SMAC, but they showed reduced slow oscillations in pSMAC integrity. IS reformation is driven by the Wiscott Aldrich Syndrome protein (WASp). WASp(-/-) T cells displayed normal IS formation but were unable to reform IS after migration unless PKCtheta was inhibited. Thus, opposing effects of PKCtheta and WASp control IS stability through pSMAC symmetry breaking and reformation.

Contact sites of endoplasmic reticulum (ER) and mitochondria locally convey calcium signals between the IP 3 receptors (IP3R) and the mitochondrial calcium uniporter, and are central to cell survival. It remains unclear whether IP3Rs also have a structural role in contact formation and whether the different IP3R isoforms have redundant functions. Using an IP3R-deficient cell model rescued with each of the three IP3R isoforms and an array of super-resolution and ultrastructural approaches we demonstrate that IP3Rs are required for maintaining ER-mitochondrial contacts. This role is independent of calcium fluxes. We also show that, while each isoform can support contacts, type 2 IP3R is the most effective in delivering calcium to the mitochondria. Thus, these studies reveal a non-canonical, structural role for the IP3Rs and direct attention towards the type 2 IP3R that was previously neglected in the context of ER-mitochondrial calcium signaling.

Lymphocyte function-associated antigen-1 (LFA-1) interaction with intercellular adhesion molecules (ICAMs) facilitates T cell antigen receptor (TCR)-mediated killing. To dissect TCR and LFA-1 contributions, we evaluated cytolytic activity and granule release by cytotoxic T lymphocytes (CTL) as well as intracellular granule redistribution and morphology of CTL stimulated with natural TCR ligand in the presence or absence of LFA-1 engagement. Although other adhesion mechanisms, e.g., CD2-CD58 interaction, could substitute for LFA-1 to trigger CTL degranulation, productive LFA-1 ligation was indispensable for effective target cell lysis by the released granules. LFA-1-mediated adhesion to glass-supported bilayers containing intercellular adhesion molecule-1 was characterized by a much larger junction area, marked by LFA-1 segregation, and a more compact cell shape compared with those observed for CD2-mediated adhesion to bilayers containing CD58. A larger contact induced by intercellular adhesion molecule 1 determined a unique positioning of granules near the interface. These data provide evidence that LFA-1 delivers a distinct signal essential for directing released cytolytic granules to the surface of antigenbearing target cells to mediate the effective destruction of these cells by CTL.cytolytic granules ͉ immunological synapse ͉ T cell receptor K illing of virus-infected cells by cytotoxic T lymphocytes (CTL) is triggered by interactions of T cell antigen receptor (TCR) with viral peptides presented by MHC class I proteins on the surface of infected cells and can be mediated by cytotoxic granule exocytosis or FasL-Fas interaction (1, 2). This is a sensitive response often requiring less than a dozen cognate peptide-MHC [complex of antigenic peptide with MHC protein (pMHC)] complexes on the target cell (3, 4). Although productive TCR engagement is necessary and essential to induce CTL cytolytic activity, other accessory and costimulatory molecules are thought to play a role in mediating CTL degranulation and effective cytolytic activity of released granules. For example, both lymphocyte function-associated antigen-1 (LFA-1) and CD2 contribute to CTL adhesion and killing of target cells (5). Ligation of LFA-1 on CTL by high densities of intercellular adhesion molecule-1 (ICAM-1) is sufficient to initiate large-scale molecular segregation and formation of peripheral supramolecular-activating cluster (6), which typically requires antigen for helper T cells (7,8) and CTL precursors (9). In contrast, ligation of CD2 with its natural ligand CD58 (5) mediates formation of a very small adhesion area by CTL (10). However, how specifically LFA-1, CD2, and other adhesion molecules mediate granuleinduced cytotoxicity has not been defined.Here we have investigated the role of productive LFA-1 engagement for antigen-induced granule release and target cell lysis in vitro as well as for granule polarization in CTL exposed to the glasssupported bilayer. We have found that, although blocking of LFA-1-ICAM-1 interaction abrogates ...

Significance HIV-1 infection and the associated disease AIDS are a major cause of human death worldwide with no vaccine or cure available. The assembly of virus particles involves numerous host and viral proteins that are potential therapeutic targets. We used high-resolution microscopy techniques to investigate how the virus hijacks cellular proteins to enable the release of virions from an infected cell. We show with high temporal and spatial resolution that components of the host endosomal sorting complex required for transport (ESCRT) machinery are recruited to the neck of the assembling virus to facilitate scission of the link between the virus and the cell.

Spatial organization of signaling complexes is a defining characteristic of the immunological synapse (IS), but its impact on cell communication is unclear. In T cell-APC pairs, more IL-2 is produced when CD28 clusters are segregated from central supramolecular activation cluster (cSMAC)-localized CD3 and into the IS periphery. However, it is not clear in these cellular experiments whether the increased IL-2 is driven by the pattern itself or by upstream events that precipitate the patterns. In this article, we recapitulate key features of physiological synapses using planar costimulation arrays containing antibodies against CD3 and CD28, surrounded by ICAM-1, created by combining multiple rounds of microcontact printing on a single surface. Naïve T cells traverse these arrays, stopping at features of anti-CD3 antibodies and forming a stable synapse. We directly demonstrate that presenting anti-CD28 in the cell periphery, surrounding an anti-CD3 feature, enhances IL-2 secretion by naïve CD4 ؉ T cells compared with having these signals combined in the center of the IS. This increased cytokine production correlates with NF-B translocation and requires PKB/Akt signaling. The ability to arbitrarily and independently control the locations of anti-CD3 and anti-CD28 offered the opportunity to examine patterns not precisely attainable in cell-cell interfaces. With these patterns, we show that the peripheral presentation of CD28 has a larger impact on IL-2 secretion than CD3 colocalization/ segregation.costimulation ͉ immunology ͉ naïve T cells ͉ microarrays C ommunication between T cells and antigen-presenting cells (APCs) forms a key regulatory point of the immune system, mediated in large part by interactions between cell-surface proteins in a small (Ϸ75 m 2 ) intercellular contact area termed the immunological synapse (IS). It is hypothesized that the spatial organization of receptor/ligand complexes within the IS is an important part of the language of T cell-APC communication (1, 2). The archetypical organization of mature synapses consists of a central supramolecular activation cluster (cSMAC) containing TCR/MHC complexes and surrounded by a peripheral (pSMAC) region rich in ICAM-1/LFA-1 clusters; the function of this bull's-eye pattern, as well as alternative geometries, in modulating T cell activation is of much contemporary interest (3-7). Recent studies have also suggested a functional role of microscale organization of CD28 ligation, a classic costimulatory signal, in modulating T cell response. Andres et al. (8) showed that during the initial minutes of T cell/APC interaction, CD28 and TCR comigrate, leading to cSMAC localization of both complexes. At later time points, Tseng and colleagues (9) showed that CD28/CD80 complexes segregate from TCR/MHC into either the cSMAC periphery or pSMAC. Surprisingly, truncation of the CD80 cytosolic domain, which reduces T cell activation (10), promoted colocalization of TCR/MHC and CD28/CD80 complexes in the cSMAC (9). Because the modifications to CD80 were in the APC, ...