Rab35 and its GAP EPI64C in T cells regulate receptor recycling and immunological synapse formation
ABSTRACT Upon antigen recognition, T-cell receptor (TCR/CD3) and other signaling molecules become enriched in a specialized contact site between the T cell and antigen-presenting cell, i.e. the immunological synapse (IS). Enrichment occurs via mechanisms that include polarized secretion from recycling endosomes, but the Rabs and RabGAPs that regulate this are unknown. EPI64C (TBC1D10C) is an uncharacterized candidate RabGAP we identified by mass spectrometry as abundant in human peripheral blood T cells that is preferentially expressed in hematopoietic cells. EPI64C is a Rab35-GAP based both on in vitro Rab35-specific GAP activity and findings in transfection assays. EPI64C and Rab35 dominant negative (DN) constructs each impaired transferrin export from a recycling pathway in Jurkat T-cells and induced large vacuoles marked by transferrin receptor, TCR, and SNAREs implicated in TCR-polarized secretion. Rab35 localized to the plasma membrane and to intracellular vesicles where it substantially colocalized with TfR and with TCR. Rab35 was strongly recruited to the IS. Conjugate formation was impaired by transfection with Rab35-DN or EPI64C and by EPI64C knock down. TCR enrichment at the IS was impaired by Rab35-DN. Thus, EPI64C and Rab35 regulate a recycling pathway in T cells and contribute to IS formation, most likely by participating in TCR transport to the IS.
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ABSTRACT: T cell activation requires sustained signaling at the immune synapse (IS), a specialized interface with the APC that assembles following TCR engagement by MHC-bound peptide. Central to sustained signaling is the continuous IS recruitment of TCRs which are partly mobilized from an endosomal pool by polarized recycling. We have identified IFT20, a component of the intraflagellar transport (IFT) system that controls ciliogenesis, as a central regulator of TCR recycling to the IS. Here we have investigated the interplay of IFT20 with the Rab GTPase network that orchestrates recycling. We found that IFT20 forms a complex with Rab5 and the TCR on early endosomes. IFT20 knockdown resulted in a block of recycling TCRs in Rab5(+) endosomes. Recycling of the transferrin receptor, but not of CXCR4, was disrupted by IFT20 deficiency. The IFT components, IFT52 and IFT57, were found to act in concert with IFT20 in regulating TCR and TfR recycling. The results provide novel insights into the mechanisms that control TCR recycling and IS assembly and underscore the trafficking-related function of the IFT system beyond ciliogenesis.Journal of Cell Science 02/2014; 127(9). DOI:10.1242/jcs.139337 · 5.33 Impact Factor
Frontiers in Cell and Developmental Biology 09/2014; 2:52. DOI:10.3389/fcell.2014.00052
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ABSTRACT: The signals that orchestrate the process of T cell activation are coordinated at the specialized interface that forms upon contact with an antigen presenting cell displaying specific MHC-associated peptide ligand, known as the immune synapse. The central role of vesicular traffic in the assembly of the immune synapse has emerged only in recent years with the finding that sustained TCR signaling involves delivery of TCR/CD3 complexes from an intracellular pool associated with recycling endosomes. A number of receptors as well as membrane-associated signaling mediators have since been demonstrated to exploit this process to localize to the immune synapse. Here we will review our current understanding of the mechanisms responsible for TCR recycling, with a focus on the intraflagellar transport system, a multimolecular complex that is responsible for the assembly and function of the primary cilium which we have recently implicated in polarized endosome recycling to the immune synapse.Traffic 11/2014; 16(3). DOI:10.1111/tra.12241 · 4.71 Impact Factor