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Rajat Varma

Publications and source records attributed to Rajat Varma.

8 recordsLinked to original sources
PubMed2006

T cell-dendritic cell immunological synapses.

Dendritic cells (DCs) are myeloid lineage cells that are imprinted by their environment and that mature in response to microbial products. A crucial role of the DC is to impart this context-specific information to T cells as well as to present self and foreign MHC-peptide complexes through formation of an immunological synapse. The structure of the T cell-DC immunological synapse departs from the canonical structure formed with B cells or with supported planar bilayers in that it has multiple foci of T-cell receptor interactions rather than a central focus. Recent studies on model systems provide insight into the mechanisms and biological consequences of the unique T cell-DC synaptic patterns.

Animals↗
PubMed2006

T cell receptor-proximal signals are sustained in peripheral microclusters and terminated in the central supramolecular activation cluster.

T cell receptor (TCR) signaling is initiated and sustained in microclusters; however, it's not known whether signaling also occurs in the TCR-rich central supramolecular activation cluster (cSMAC). We showed that the cSMAC formed by fusion of microclusters contained more CD45 than microclusters and is a site enriched in lysobisphosphatidic acid, a lipid involved in sorting ubiquitinated membrane proteins for degradation. Calcium signaling via TCR was blocked within 2 min by anti-MHCp treatment and 1 min by latrunculin-A treatment. TCR-MHCp interactions in the cSMAC survived these perturbations for 10 min and hence were not sufficient to sustain signaling. TCR microclusters were also resistant to disruption by anti-MHCp and latrunculin-A treatments. We propose that TCR signaling is sustained by stabilized microclusters and is terminated in the cSMAC, a structure from which TCR are sorted for degradation. Our studies reveal a role for F-actin in TCR signaling beyond microcluster formation.

Actins↗
PubMed2005

Actin and agonist MHC-peptide complex-dependent T cell receptor microclusters as scaffolds for signaling.

T cell receptor (TCR) microclusters form within seconds of T cell contact with supported planar bilayers containing intercellular adhesion molecule-1 and agonist major histocompatibility complex (MHC)-peptide complexes, and elevation of cytoplasmic Ca2+ is observed within seconds of the first detectable microclusters. At 0-30 s after contact, TCR microclusters are colocalized with activated forms of Lck, ZAP-70, and the linker for activation of T cells. By 2 min, activated kinases are reduced in the older central microclusters, but are abundant in younger peripheral microclusters. By 5 min, TCR in the central supramolecular activation cluster have reduced activated kinases, whereas faint peripheral TCR microclusters efficiently generated activated Lck and ZAP-70. TCR microcluster formation is resistant to inhibition by Src family kinase inhibitor PP2, but is abrogated by actin polymerization inhibitor latrunculin A. We propose that Src kinase-independent formation of TCR microclusters in response to agonist MHC-peptide provides an actin-dependent scaffold for signal amplification.

Actins↗
PubMed2005

A molecular dissection of lymphocyte unresponsiveness induced by sustained calcium signalling.

In lymphocytes, integration of Ca2+ and other signalling pathways results in productive activation, while unopposed Ca2+ signalling leads to decreased responsiveness to subsequent stimulation (anergy). The Ca(2+)-regulated transcription factor NFAT has an integral role in both aspects of lymphocyte function. NFAT cooperates with the transcription factor AP-1 (Fos/Jun) to up-regulate genes involved in productive activation of lymphocytes. However, in the absence of AP-1, NFAT imposes an opposing genetic programme that leads to lymphocyte anergy. Anergy is implemented at least partly through proteolytic degradation of the key signalling proteins PKCtheta and PLCgamma1. Sustained Ca(2+)-calcineurin signalling increases mRNA and protein levels of the E3 ubiquitin ligases Itch, CblB and Grail and induces expression of Tsg1O1, the ubiquitin-binding component of the ESCRT1 endosomal sorting complex. Subsequent stimulation or homotypic cell adhesion promotes membrane translocation of Itch and the related protein Nedd4, resulting in PKCtheta and PLCgamma1 degradation. T cells from Itch- and CblB-deficient mice are resistant to anergy induction. Anergic T cells show impaired calcium mobilization after TCR triggering and are unable to maintain a mature immunological synapse. Thus Ca(2+)-calcineurin-NFAT signalling links gene transcription to a multi-step programme that leads to impaired signal transduction in anergic T cells.

Calcium Signaling↗
PubMed2004

Nanoscale organization of multiple GPI-anchored proteins in living cell membranes.

Cholesterol and sphingolipid-enriched "rafts" have long been proposed as platforms for the sorting of specific membrane components including glycosyl-phosphatidylinositol-anchored proteins (GPI-APs), however, their existence and physical properties have been controversial. Here, we investigate the size of lipid-dependent organization of GPI-APs in live cells, using homo and hetero-FRET-based experiments, combined with theoretical modeling. These studies reveal an unexpected organization wherein cell surface GPI-APs are present as monomers and a smaller fraction (20%-40%) as nanoscale (<5 nm) cholesterol-sensitive clusters. These clusters are composed of at most four molecules and accommodate diverse GPI-AP species; crosslinking GPI-APs segregates them from preexisting GPI-AP clusters and prevents endocytosis of the crosslinked species via a GPI-AP-selective pinocytic pathway. In conjunction with an analysis of the statistical distribution of the clusters, these observations suggest a mechanism for functional lipid-dependent clustering of GPI-APs.

Animals↗
PubMed2004

Calcineurin imposes T cell unresponsiveness through targeted proteolysis of signaling proteins.

Sustained calcium signaling induces a state of anergy or antigen unresponsiveness in T cells, mediated through calcineurin and the transcription factor NFAT. We show here that Ca(2+)-induced anergy is a multistep program that is implemented at least partly through proteolytic degradation of specific signaling proteins. Calcineurin increased mRNA and protein of the E3 ubiquitin ligases Itch, Cbl-b and GRAIL and induced expression of Tsg101, the ubiquitin-binding component of the ESCRT-1 endosomal sorting complex. Subsequent stimulation or homotypic cell adhesion promoted membrane translocation of Itch and the related protein Nedd4, resulting in degradation of two key signaling proteins, PKC-theta and PLC-gamma1. T cells from Itch- and Cbl-b-deficient mice were resistant to anergy induction. Anergic T cells showed impaired calcium mobilization after TCR triggering and were unable to maintain a mature immunological synapse, instead showing late disorganization of the outer ring containing lymphocyte function-associated antigen 1. Our results define a complex molecular program that links gene transcription induced by calcium and calcineurin to a paradoxical impairment of signal transduction in anergic T cells.

Animals↗
PubMed2003

The immunological synapse balances T cell receptor signaling and degradation.

The immunological synapse is a specialized cell-cell junction between T cell and antigen-presenting cell surfaces. It is characterized by a central cluster of antigen receptors, a ring of integrin family adhesion molecules, and temporal stability over hours. The role of this specific organization in signaling for T cell activation has been controversial. We use in vitro and in silico experiments to determine that the immunological synapse acts as a type of adaptive controller that both boosts T cell receptor triggering and attenuates strong signals.

Adaptor Proteins, Signal Transducing↗