Recruitment of calcineurin to the TCR positively regulates T cell activation.

Calcineurin is a phosphatase whose primary targets in T cells are NFAT transcription factors, and inhibition of calcineurin activity by treatment with cyclosporin A (CsA) or FK506 is a cornerstone of immunosuppressive therapies. Here we found that calcineurin was recruited to the T cell antigen receptor (TCR) signaling complex, where it reversed inhibitory phosphorylation of the tyrosine kinase Lck on Ser59 (LckS59). Loss of calcineurin activity impaired phosphorylation of Tyr493 of the tyrosine kinase ZAP-70 (ZAP-70Y493), as well as some downstream pathways in a manner consistent with signaling in cells expressing LckS59A (Lck that cannot be phosphorylated) or LckS59E (a phosphomimetic mutant). Notably, CsA inhibited integrin-LFA-1-dependent and NFAT-independent adhesion of T cells to the intercellular adhesion molecule ICAM-1, with little effect on cells expressing mutant Lck. These results provide new understanding of how widely used immunosuppressive drugs interfere with essential processes in the immune response.

Heterogeneity of Signaling Complex Nanostructure in T Cells Activated Via the T Cell Antigen Receptor.

Activation of the T cell antigen receptor (TCR) is a key step in initiating the adaptive immune response. Single-molecule localization techniques have been used to investigate the arrangement of proteins within the signaling complexes formed around activated TCRs, but a clear picture of nanoscale organization in stimulated T cells has not emerged. Here, we have improved the examination of T cell nanostructure by visualizing individual molecules of six different proteins in a single sample of activated Jurkat T cells using the multiplexed antibody-size limited direct stochastic optical reconstruction microscopy (madSTORM) technique. We formally define irregularly shaped regions of interest, compare areas where signaling complexes are concentrated with other areas, and improve the statistical analyses of the locations of molecules. We show that nanoscale organization of proteins is mainly confined to the areas with dense concentrations of TCR-based signaling complexes. However, randomly distributed molecules are also found in some areas containing concentrated signaling complexes. These results are consistent with the view that the proteins within signaling complexes are connected by numerous weak interactions, leading to flexible, dynamic, and mutable structures which produce large variations in the nanostructure found in activated T cells.

Intensity and duration of TCR signaling is limited by p38 phosphorylation of ZAP-70T293 and destabilization of the signalosome.

ZAP-70 is a tyrosine kinase that is essential for initiation of T cell antigen receptor (TCR) signaling. We have found that T cell p38 MAP kinase (MAPK), which is directly phosphorylated and activated by ZAP-70 downstream of the TCR, in turn phosphorylates Thr-293 in the interdomain B region of ZAP-70. Mutant T cells expressing ZAP-70 with an alanine substitution at this residue (ZAP-70T293A) had enhanced TCR proximal signaling and increased effector responses. Lack of ZAP-70T293 phosphorylation increased association of ZAP-70 with the TCR and prolonged the existence of TCR signaling microclusters. These results identify a tight negative feedback loop in which ZAP-70-activated p38 reciprocally phosphorylates ZAP-70 and destabilizes the signaling complex.

Functional nanoscale organization of signaling molecules downstream of the T cell antigen receptor.

Receptor-regulated cellular signaling often is mediated by formation of transient, heterogeneous protein complexes of undefined structure. We used single and two-color photoactivated localization microscopy to study complexes downstream of the T cell antigen receptor (TCR) in single-molecule detail at the plasma membrane of intact T cells. The kinase ZAP-70 distributed completely with the TCRζ chain and both partially mixed with the adaptor LAT in activated cells, thus showing localized activation of LAT by TCR-coupled ZAP-70. In resting and activated cells, LAT primarily resided in nanoscale clusters as small as dimers whose formation depended on protein-protein and protein-lipid interactions. Surprisingly, the adaptor SLP-76 localized to the periphery of LAT clusters. This nanoscale structure depended on polymerized actin and its disruption affected TCR-dependent cell function. These results extend our understanding of the mechanism of T cell activation and the formation and organization of TCR-mediated signaling complexes, findings also relevant to other receptor systems.

Development of nanoscale structure in LAT-based signaling complexes.

The adapter molecule linker for activation of T cells (LAT) plays a crucial role in forming signaling complexes induced by stimulation of the T cell receptor (TCR). These multi-molecular complexes are dynamic structures that activate highly regulated signaling pathways. Previously, we have demonstrated nanoscale structure in LAT-based complexes where the adapter SLP-76 (also known as LCP2) localizes to the periphery of LAT clusters. In this study, we show that initially LAT and SLP-76 are randomly dispersed throughout the clusters that form upon TCR engagement. The segregation of LAT and SLP-76 develops near the end of the spreading process. The local concentration of LAT also increases at the same time. Both changes require TCR activation and an intact actin cytoskeleton. These results demonstrate that the nanoscale organization of LAT-based signaling complexes is dynamic and indicates that different kinds of LAT-based complexes appear at different times during T cell activation.

Super-resolution characterization of TCR-dependent signaling clusters.

Multi-molecular signaling complexes drive the earliest events of immune cell activation via immunoreceptors with unexplained specificity and speed. Fluorescence microscopy has shown that these complexes form microclusters at the plasma membrane of activated T cells upon engagement of their antigen receptors (TCRs). Although crucial for cell function, much remains to be learned about the molecular content, fine structure, formation mechanisms, and function of these microclusters. Recent advancements in super-resolution microscopy have enabled the study of signaling microclusters at the single molecule level with resolution down to approximately 20 nm. These techniques have now helped to characterize the size distributions of signaling clusters at the plasma membrane of intact cells and to shed light on the formation mechanisms that govern their assembly. Surprisingly, dynamic and functional nanostructures have been identified within the signaling clusters. We expect that these novel methodologies, combined with older techniques, will shed new light on the nature of signaling clusters and their critical role in T-cell activation.

The linker for activation of T cells (LAT) signaling hub: from signaling complexes to microclusters.

Since the cloning of the critical adapter, LAT (linker for activation of T cells), more than 15 years ago, a combination of multiple scientific approaches and techniques continues to provide valuable insights into the formation, composition, regulation, dynamics, and function of LAT-based signaling complexes. In this review, we will summarize current views on the assembly of signaling complexes nucleated by LAT. LAT forms numerous interactions with other signaling molecules, leading to cooperativity in the system. Furthermore, oligomerization of LAT by adapter complexes enhances intracellular signaling and is physiologically relevant. These results will be related to data from super-resolution microscopy studies that have revealed the smallest LAT-based signaling units and nanostructure.

Cutting edge: cell surface linker for activation of T cells is recruited to microclusters and is active in signaling.

A controversy has recently emerged regarding the location of the cellular pool of the adapter linker for activation of T cells (LAT) that participates in propagation of signals downstream of the TCR. In one model phosphorylation and direct recruitment of cell surface LAT to activation-induced microclusters is critical for T cell activation, whereas in the other model vesicular, but not surface, LAT participates in these processes. By using a chimeric version of LAT that can be tracked via an extracellular domain, we provide evidence that LAT located at the cell surface can be recruited efficiently to activation-induced microclusters within seconds of TCR engagement. Importantly, we also demonstrate that this pool of LAT at the plasma membrane is rapidly phosphorylated. Our results provide support for the model in which the cell utilizes LAT from the cell surface for rapid responses to TCR stimulation.

In vivo functional mapping of the conserved protein domains within murine Themis1.

Thymocyte development requires the coordinated input of signals that originate from numerous cell surface molecules. Although the majority of thymocyte signal-initiating receptors are lineage-specific, most trigger 'ubiquitous' downstream signaling pathways. T-lineage-specific receptors are coupled to these signaling pathways by lymphocyte-restricted adapter molecules. We and others recently identified a new putative adapter protein, Themis1, whose expression is largely restricted to the T lineage. Mice lacking Themis1 exhibit a severe block in thymocyte development and a striking paucity of mature T cells revealing a critical role for Themis1 in T-cell maturation. Themis1 orthologs contain three conserved domains: a proline-rich region (PRR) that binds to the ubiquitous cytosolic adapter Grb2, a nuclear localization sequence (NLS), and two copies of a novel cysteine-containing globular (CABIT) domain. In the present study, we evaluated the functional importance of each of these motifs by retroviral reconstitution of Themis1(-/-) progenitor cells. The results demonstrate an essential requirement for the PRR and NLS motifs but not the conserved CABIT cysteines for Themis1 function.

15 kDa granulysin causes differentiation of monocytes to dendritic cells but lacks cytotoxic activity.

Granulysin is expressed as two isoforms by human cytotoxic cells: a single mRNA gives rise to 15 kDa granulysin, a portion of which is cleaved to a 9 kDa protein. Studies with recombinant 9 kDa granulysin have demonstrated its cytolytic and proinflammatory properties, but much less is known about the biologic function of the 15 kDa isoform. In this study, we show that the subcellular localization and functions of 9 and 15 kDa granulysin are largely distinct. Nine kilodalton granulysin is confined to cytolytic granules that are directionally released following target cell recognition. In contrast, 15 kDa granulysin is located in distinct granules that lack perforin and granzyme B and that are released by activated cytolytic cells. Although recombinant 9 kDa granulysin is cytolytic against a variety of tumors and microbes, recombinant 15 kDa granulysin is not. The 15 kDa isoform is a potent inducer of monocytic differentiation to dendritic cells, but the 9 kDa isoform is not. In vivo, mice expressing granulysin show markedly improved antitumor responses, with increased numbers of activated dendritic cells and cytokine-producing T cells. Thus, the distinct functions of granulysin isoforms have major implications for diagnosis and potential new therapies for human disease.

Interchangeability of Themis1 and Themis2 in thymocyte development reveals two related proteins with conserved molecular function.

Themis1, a recently identified T cell protein, has a critical function in the generation of mature CD4(+)CD8(-) and CD4(-)CD8(+) (CD4 and CD8 single-positive [SP]) thymocytes and T cells. Although Themis1 has been shown to bind to the adaptor proteins LAT and Grb2, previous studies have yielded conflicting results regarding whether thymocytes from Themis1(-/-) mice exhibit TCR-mediated signaling defects. In this study, we demonstrate that, in the absence of Themis1, TCR-mediated signaling is selectively impaired in CD4 SP and CD8 SP thymocytes but is not affected in CD4(+)CD8(+) double-positive thymocytes despite high expression of Themis1 in double-positive thymocytes. Like Themis1, Themis2, a related member of the Themis family, which is expressed in B cells and macrophages, contains two conserved cysteine-based domains, a proline-rich region, and a nuclear localization signal. To determine whether Themis1 and Themis2 can perform similar functions in vivo, we analyzed T cell development and TCR-mediated signaling in Themis1(-/-) mice reconstituted with either Themis1 or Themis2 transgenes. Notably, Themis1 and Themis2 exhibited the same potential to restore T cell development and TCR-mediated signaling in Themis1(-/-) mice. Both proteins were tyrosine phosphorylated and were recruited within Grb2 signaling complexes to LAT following TCR engagement. These results suggest that conserved molecular features of the Themis1 and Themis2 proteins are important for their biological activity and predict that Themis1 and Themis2 may perform similar functions in T and B cells, respectively.

Resolving multi-molecular protein interactions by photoactivated localization microscopy.

Multi-molecular protein complexes are critical to many cellular functions, including signaling, DNA transcription and enzymatic reactions. In spite of their importance, current research techniques such as biochemistry and diffraction-limited microscopy cannot resolve the heterogeneity and nanoscale organization of protein complexes in intact cells. Here we describe a technique that enables the study of multi-molecular protein complexes at the single molecule level in intact cells. The technique uses photoactivated localization microscopy (PALM) to resolve individual proteins with a resolution down to 20nm in intact cells, and second-order statistics to study the spatial interactions of the proteins. We demonstrate the feasibility of this technique by studying signaling complexes that form in activated T cells. We first use single color PALM imaging and univariate second-order statistics to resolve the clustering of Linker for Activation of T cells (LAT) at the plasma membrane (PM) of the cells. We then use two color PALM and bivariate second-order statistics to resolve the interaction of LAT with key interacting proteins. We discuss potential caveats in studying molecular clustering and the robustness of the technique to study bimolecular interactions. Our proposed technique, combined with older techniques, could help shed new light on the nature of multimolecular protein complexes and their significance to cell function.

Enhanced T-cell signaling in cells bearing linker for activation of T-cell (LAT) molecules resistant to ubiquitylation.

Linker for activation of T cells (LAT) plays a central role in T-cell activation by nucleating signaling complexes that are critical for the propagation of T-cell signals from the plasma membrane to the cellular interior. The role of phosphorylation and palmitoylation in LAT function has been well studied, but not much is known about other strategies by which the cell modulates LAT activity. We have focused on LAT ubiquitylation and have mapped the sites on which LAT is ubiquitylated. To elucidate the biological role of this process, we substituted LAT lysines with arginines. This resulted in a dramatic decrease in overall LAT ubiquitylation. Ubiquitylation-resistant mutants of LAT were internalized at rates comparable to wild-type LAT in a mechanism that required Cbl family proteins. However, these mutants displayed a defect in protein turnover rates. T-cell signaling was elevated in cells reconstituted with LAT mutants resistant to ubiquitylation, indicating that inhibition of LAT ubiquitylation enhances T-cell potency. These results support LAT ubiquitylation as a molecular checkpoint for attenuation of T-cell signaling.

GTP-binding protein-like domain of AGAP1 is protein binding site that allosterically regulates ArfGAP protein catalytic activity.

AGAPs are a subtype of Arf GTPase-activating proteins (GAPs) with 11 members in humans. In addition to the Arf GAP domain, the proteins contain a G-protein-like domain (GLD) with homology to Ras superfamily proteins and a PH domain. AGAPs bind to clathrin adaptors, function in post Golgi membrane traffic, and have been implicated in glioblastoma. The regulation of AGAPs is largely unexplored. Other enzymes containing GTP binding domains are regulated by nucleotide binding. However, nucleotide binding to AGAPs has not been detected. Here, we found that neither nucleotides nor deleting the GLD of AGAP1 affected catalysis, which led us to hypothesize that the GLD is a protein binding site that regulates GAP activity. Two-hybrid screens identified RhoA, Rac1, and Cdc42 as potential binding partners. Coimmunoprecipitation confirmed that AGAP1 and AGAP2 can bind to RhoA. Binding was mediated by the C terminus of RhoA and was independent of nucleotide. RhoA and the C-terminal peptide from RhoA increased GAP activity specifically for the substrate Arf1. In contrast, a C-terminal peptide from Cdc42 neither bound nor activated AGAP1. Based on these results, we propose that AGAPs are allosterically regulated through protein binding to the GLD domain.

Endocytic events in TCR signaling: focus on adapters in microclusters.

Although the critical role of T-cell receptor (TCR) microclusters in T-cell activation is now widely accepted, the mechanisms of regulation of these TCR-rich structures, which also contain enzymes, adapters, and effectors, remain poorly defined. Soon after microcluster formation, several signaling proteins rapidly dissociate from the TCR. Recent studies from our laboratory demonstrated that the movement of the adapters linker for activation of T cells (LAT) and Src homology 2 domain-containing leukocyte protein of 76 kDa (SLP-76) away from initial microcluster formation sites represents endocytic events. Ubiquitylation, Cbl proteins, and multiple endocytic pathways are involved in the internalization events that disassemble signaling microclusters. Several recent studies have indicated that microcluster movement and centralization plays an important role in signal termination. We suggest that microcluster movement is directly linked to endocytic events, thus implicating endocytosis of microclusters as a means to regulate signaling output of the T cell.

Formation of STIM and Orai complexes: puncta and distal caps.

In the last few years, great progress has been made in understanding how stromal interacting molecule 1 (STIM1), a protein containing a calcium sensor that is located in the endoplasmic reticulum, and Orai1, a protein that forms a calcium channel in the plasma membrane, interact and give rise to store-operated calcium entry. Pharmacological depletion of calcium stores leads to the formation of clusters containing STIM and Orai that appear to be sites for calcium influx. Similar puncta are also produced in response to physiological stimuli in immune cells. In T cells engaged with antigen-presenting cells, clusters containing STIM and Orai accumulate at the immunological synapse. We recently discovered that in activated T cells, STIM1 and Orai1 also accumulate in cap-like structures opposite the immune synapse at the distal pole of the cell. Both caps and puncta are long-lived stable structures containing STIM1 and Orai1 in close proximity. The function of puncta as sites of calcium influx is clear. We speculate that the caps may provide a secondary site of calcium entry. Alternatively, they may serve as a source of preformed channel complexes that move to new immune synapses as T cells repeatedly engage antigen-presenting cells.

Inherited ARPC5 mutations cause an actinopathy impairing cell motility and disrupting cytokine signaling.

We describe the first cases of germline biallelic null mutations in ARPC5, part of the Arp2/3 actin nucleator complex, in two unrelated patients presenting with recurrent and severe infections, early-onset autoimmunity, inflammation, and dysmorphisms. This defect compromises multiple cell lineages and functions, and when protein expression is reestablished in-vitro, the Arp2/3 complex conformation and functions are rescued. As part of the pathophysiological evaluation, we also show that interleukin (IL)-6 signaling is distinctively impacted in this syndrome. Disruption of IL-6 classical but not trans-signaling highlights their differential roles in the disease and offers perspectives for therapeutic molecular targets.

Bam32: a novel mediator of Erk activation in T cells.

Bam32 (B lymphocyte adapter molecule of 32 kDa) is an adapter protein expressed in some hematopoietic cells including B and T lymphocytes. It was previously shown that Bam32-deficient mice have defects in various aspects of B cell activation including B cell receptor (BCR)-induced Erk activation, BCR-induced proliferation and T-independent antibody responses. In this study, we have examined the role of Bam32 in T cell activation using Bam32-deficient mice. By comparing CD4(+) T cells from lymph nodes of wild-type and Bam32-deficient mice, we found that Bam32 was required for optimal TCR-induced Erk activation, cytokine production, proliferation and actin-mediated spreading of CD4(+) T cells. These results indicate a novel pathway to Erk activation in T cells involving the adapter protein Bam32.

T cell receptor ligation induces the formation of dynamically regulated signaling assemblies.

Tcell antigen receptor (TCR) ligation initiates tyrosine kinase activation, signaling complex assembly, and immune synapse formation. Here, we studied the kinetics and mechanics of signaling complex formation in live Jurkat leukemic T cells using signaling proteins fluorescently tagged with variants of enhanced GFP (EGFP). Within seconds of contacting coverslips coated with stimulatory antibodies, T cells developed small, dynamically regulated clusters which were enriched in the TCR, phosphotyrosine, ZAP-70, LAT, Grb2, Gads, and SLP-76, excluded the lipid raft marker enhanced yellow fluorescent protein-GPI, and were competent to induce calcium elevations. LAT, Grb2, and Gads were transiently associated with the TCR. Although ZAP-70-containing clusters persisted for more than 20 min, photobleaching studies revealed that ZAP-70 continuously dissociated from and returned to these complexes. Strikingly, SLP-76 translocated to a perinuclear structure after clustering with the TCR. Our results emphasize the dynamically changing composition of signaling complexes and indicate that these complexes can form within seconds of TCR engagement, in the absence of either lipid raft aggregation or the formation of a central TCR-rich cluster.

Persistence of cooperatively stabilized signaling clusters drives T-cell activation.

Antigen recognition triggers the recruitment of the critical adaptor protein SLP-76 to small macromolecular clusters nucleated by the T-cell receptor (TCR). These structures develop rapidly, in parallel with TCR-induced increases in tyrosine phosphorylation and cytosolic calcium, and are likely to contribute to TCR-proximal signaling. Previously, we demonstrated that these SLP-76-containing clusters segregate from the TCR and move towards the center of the contact interface. Neither the function of these clusters nor the structural requirements governing their persistence have been examined extensively. Here we demonstrate that defects in cluster assembly and persistence are associated with defects in T-cell activation in the absence of Lck, ZAP-70, or LAT. Clusters persist normally in the absence of phospholipase C-gamma1, indicating that in the absence of a critical effector, these structures are insufficient to drive T-cell activation. Furthermore, we show that the critical adaptors LAT and Gads localize with SLP-76 in persistent clusters. Mutational analyses of LAT, Gads, and SLP-76 indicated that multiple domains within each of these proteins contribute to cluster persistence. These data indicate that multivalent cooperative interactions stabilize these persistent signaling clusters, which may correspond to the functional complexes predicted by kinetic proofreading models of T-cell activation.