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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. The immunological synapse (IS)3 is a specialized contact between T lymphocytes and antigen-presenting cells (APC) into which accumulate T-cell antigen receptor (TCR), coreceptors, and signaling and cytoskeletal components (1-7). Enrichment of proteins in the IS occurs in part by diffusion and retention at the contact region (i.e."mutual co-capping") (8, 9). Furthermore, submembranous flow of the actomyosin cortex contributes to molecular concentration in the IS (10, 11). Recent data indicate that polarized exocytosis from a rapid recycling pathway is particularly important for the enrichment of some synapse components, including TCR (12-16).Rab proteins are major intracellular transport regulators in eukaryotes; they function in vesicle formation, motility, docking, and fusion (17, 18). Over 60 mammalian Rab proteins have been identified, and each is thought to regulate distinct intracellular transport steps through its temporal and spatial association with various interacting proteins. Guanine exchange factors and GTPase-activating proteins (GAPs) control the switch between active GTP-bound and inactive GDP-bound Rab proteins. Although the TBC (Tre/Bub2/Cdc16) domain is a hallmark of RabGAPs (19), few of the more than 50 putative TBC domain-containing proteins present in the human genome have been paired with their target Rab. One recent notable success was identification of EPI64 (EBP50-PDZ interactor of 64kD) as a GAP specific for Rab27a (20).EPI64 is a broadly expressed TBC domain-containing protein first identified in placental microvilli (21). Recent studies have implicated it in regulating microvillus architecture (22). EPI64 has two paralogs in mouse and...

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Regulation of the number ofFurthermore, the effect of EPI64C was dependent upon its GTPase-activating proteins activity. Co-immunoprecipitation studies confirmed an association between KCa2.3 and both Rab35 and RME-1. In contrast to KCa2.3, KCa3.1 was rapidly endocytosed and degraded in an RME-1 and Rab35-independent manner. A series of N-terminal deletions identified a 12-amino acid region, Gly 206 -Pro 217 , as being required for the rapid recycling of KCa2.3. Deletion of Gly 206 -Pro 217 had no effect on the association of KCa2.3 with Rab35 but significantly decreased the association with RME-1. These represent the first studies elucidating the mechanisms by which KCa2.3 is maintained at the plasma membrane.

Vapour cavities in liquid flows have long been associated with cavitation damage to nearby solid surfaces and it is thought that the final stage of collapse, when a highspeed liquid jet threads the cavity, plays a vital role in this process. The present study investigates this aspect of the motion of laser-generated cavities in a quiescent liquid when the distance (or stand-off) of the point of inception from a rigid boundary is between 0.8 and 1.2 times the maximum radius of the cavity. Numerical simulations using a boundary integral method with an incompressible liquid impact model provide a framework for the interpretation of the experimental results. It is observed that, within the given interval of the stand-off parameter, the peak pressures measured on the boundary at the first collapse of a cavity attain a local minimum, while at the same time there is an increase in the duration of the pressure pulse. This contrasts with a monotonic increase in the peak pressures as the stand-off is reduced, when the cavity inception point is outside the stated interval. This phenomenon is shown to be due to a splash effect which follows the impact of the liquid jet. Three cases are chosen to typify the splash interaction with the free surface of the collapsing cavity: (i) surface reconnection around the liquid jet; (ii) splash impact at the base of the liquid jet; (iii) thin film splash. Hydrodynamic pressures generated following splash impact are found to be much greater than those produced by the jet impact. The combination of splash impact and the emission of shock waves, together with the subsequent re-expansion, drives the flow around the toroidal cavity producing a distinctive double pressure peak.