No abstract

IFT20, a component of the intraflagellar transport (IFT) system that controls ciliogenesis, regulates immune synapse assembly in the non-ciliated T-cell by promoting T-cell receptor (TCR) recycling. Here, we have addressed the role of Rab8 (for which there are two isoforms Rab8a and Rab8b), a small GTPase implicated in ciliogenesis, in TCR traffic to the immune synapse. We show that Rab8, which colocalizes with IFT20 in Rab11 + endosomes, is required for TCR recycling. Interestingly, as opposed to in IFT20-deficient T-cells, TCR + endosomes polarized normally beneath the immune synapse membrane in the presence of dominant-negative Rab8, but were unable to undergo the final docking or fusion step. This could be accounted for by the inability of the vesicular (v)-SNARE VAMP-3 to cluster at the immune synapse in the absence of functional Rab8, which is responsible for its recruitment. Of note, and similar to in T-cells, VAMP-3 interacts with Rab8 at the base of the cilium in NIH-3T3 cells, where it regulates ciliary growth and targeting of the protein smoothened. The results identify Rab8 as a new player in vesicular traffic to the immune synapse and provide insight into the pathways co-opted by different cell types for immune synapse assembly and ciliogenesis.

Accumulating evidence underscores the T-cell immune synapse (IS) as a site of intense vesicular trafficking, on which productive signaling and cell activation crucially depend. Although the T-cell antigen receptor (TCR) is known to exploit recycling to accumulate to the IS, the specific pathway that controls this process remains to be elucidated. Here we demonstrate that the small GTPase Rab29 is centrally implicated in TCR trafficking and IS assembly. Rab29 colocalized and interacted with Rab8, Rab11 and IFT20, a component of the intraflagellar transport system that regulates ciliogenesis and participates in TCR recycling in the non-ciliated T cell, as assessed by co-immunoprecipitation and immunofluorescence analysis. Rab29 depletion resulted in the inability of TCRs to undergo recycling to the IS, thereby compromizing IS assembly. Under these conditions, recycling TCRs accumulated in Rab11 + endosomes that failed to polarize to the IS due to defective Rab29-dependent recruitment of the dynein microtubule motor. Remarkably, Rab29 participates in a similar pathway in ciliated cells to promote primary cilium growth and ciliary localization of Smoothened. These results provide a function for Rab29 as a regulator of receptor recycling and identify this GTPase as a shared participant in IS and primary cilium assembly. T-cell activation is triggered by T-cell antigen receptor (TCR) engagement by MHC-bound peptide dispayed by an antigen presenting cell (APC). While a substantial proportion of the TCR has long been known to be associated with recycling endosomes, 1 it is only recently that the central function of this pool has emerged with the finding that, on assembly of the specialized interface with the APC, known as the immune synapse (IS), intracellular TCRs are delivered to this location by polarized recycling. 2 This process ensures a steady supply of TCRs at the IS to sustain signaling for T-cell activation 3 and has been co-opted by other receptors, such as the transferrin receptor (TfR) and the chemokine receptor CXCR4, 4,5 as well as membrane-bound signaling mediators, such as the kinase Lck and the adaptor LAT. 6,7 Receptor recycling is orchestrated by the small GTPases Rab4 and Rab11. 8 Cargo specificity is achieved with the assistance of specific regulators and effectors. The TCR recycling pathway has only started to be delineated with the identification of specific mediators, which include Rab35 and its GAP EPI64C 9 and the actin adaptor WASH 10 . Specific combinations of Rabs and SNAREs have been recently associated with recycling endosomes carrying either Lck, or TCRζ and LAT. 11 We have moreover identified IFT20 12 and other components of the intraflagellar transport (IFT) system, which regulates ciliary assembly and function, 13 as unexpected regulators of TCR recycling in the non-ciliated T cell. 14 Recently a Rab GTPase subfamily, which includes Rab29, Rab32 and Rab38, has been implicated in trafficking of the Salmonella-containing vacuole (SCV) in infected epithelial cells. 15,16 Rab32 and Rab38 ...

The primary cilium has gone from being a vestigial organelle to a crucial signaling hub of growing interest given the association between a group of human disorders, collectively known as ciliopathies, and defects in its structure or function. In recent years many ciliogenesis proteins have been observed at extraciliary sites in cells and likely perform cilium-independent functions ranging from regulation of the cytoskeleton to vesicular trafficking. Perhaps the most striking example is the non-ciliated T lymphocyte, in which components of the ciliary machinery are repurposed for the assembly and function of the immunological synapse even in the absence of a primary cilium. Furthermore, the specialization traits described at the immunological synapse are similar to those seen in the primary cilium. Here, we review common regulators and features shared by the immunological synapse and the primary cilium that document the remarkable homology between these structures.

No abstract

Cytotoxic T cells (CTLs) are the main cellular mediators of the adaptive immune defenses against intracellular pathogens and malignant cells. Upon recognition of specific antigen on their cellular target, CTLs assemble an immunological synapse where they mobilise their killing machinery that is released into the synaptic cleft to orchestrate the demise of their cell target. The arsenal of CTLs is stored in lysosome-like organelles that undergo exocytosis in response to signals triggered by the T cell antigen receptor following antigen recognition. These organelles include lytic granules carrying a cargo of cytotoxic proteins packed on a proteoglycan scaffold, multivesicular bodies carrying the death receptor ligand FasL, and the recently discovered supramolecular attack particles that carry a core of cytotoxic proteins encased in a non-membranous glycoprotein shell. Here we will briefly review the main features of these killing entities and discuss their interrelationship and interplay in CTL-mediated killing.

Macroautophagy/autophagy has emerged as a central process in lymphocyte homeostasis, activation and differentiation. Based on our finding that the p66 isoform of SHC1 (p66SHC) pro-apoptotic ROS-elevating SHC family adaptor inhibits MTOR signaling in these cells, here we investigated the role of p66SHC in B-cell autophagy. We show that p66SHC disrupts mitochondrial function through its CYCS (cytochrome c, somatic) binding domain, thereby impairing ATP production, which results in AMPK activation and enhanced autophagic flux. While p66SHC binding to CYCS is sufficient for triggering apoptosis, p66SHC-mediated autophagy additionally depends on its ability to interact with membrane-associated LC3-II through a specific binding motif within its N terminus. Importantly, p66SHC also has an impact on mitochondria homeostasis by inducing mitochondrial depolarization, protein ubiquitination at the outer mitochondrial membrane, and local recruitment of active AMPK. These events initiate mitophagy, whose full execution relies on the role of p66SHC as an LC3-II receptor which brings phagophore membranes to mitochondria. Importantly, p66SHC also promotes hypoxia-induced mitophagy in B cells. Moreover, p66SHC deficiency enhances B cell differentiation to plasma cells, which is controlled by intracellular ROS levels and the hypoxic germinal center environment. The results identify mitochondrial p66SHC as a novel regulator of autophagy and mitophagy in B cells and implicate p66SHC-mediated coordination of autophagy and apoptosis in B cell survival and differentiation. Abbreviations: ACTB: actin beta; AMPK: AMP-activated protein kinase; ATP: adenosine triphosphate; ATG: autophagy-related; CYCS: cytochrome c, somatic; CLQ: chloroquine; COX: cyclooxygenase; CTR: control; GFP: green fluorescent protein; HIFIA/Hif alpha: hypoxia inducible factor 1 subunit alpha; IMS: intermembrane space; LIR: LC3 interacting region; MAP1LC3B/LC3B: microtubule associated protein 1 light chain 3 beta; MTOR/mTOR: mechanistic target of rapamycin kinase; OA: oligomycin and antimycin A; OMM: outer mitochondrial membrane; PHB: prohibitin; PBS: phosphate-buffered saline; PINK1: PTEN induced putative kinase 1; RFP: red fluorescent protein; ROS: reactive oxygen species; SHC: src Homology 2 domain-containing transforming protein; TMRM: tetramethylrhodamine, methyl ester; TOMM: translocase of outer mitochondrial membrane; ULK1: unc-51 like autophagy activating kinase 1; WT: wild-type.

Components of the intraflagellar transport (IFT) system that regulates the assembly of the primary cilium are co-opted by the non-ciliated T cell to orchestrate polarized endosome recycling and to sustain signaling during immune synapse formation. Here we have investigated the potential role of BBS1, an essential core component of the Bardet-Biedl syndrome complex that cooperates with the IFT system in ciliary protein trafficking, in the assembly of the T cell synapse. We demonstrate that BBS1 allows for centrosome polarization towards the immune synapse. This function is achieved through the clearance of centrosomal F-actin and its positive regulator WASH, a process that we demonstrate to be dependent on the proteasome. We show that BBS1 regulates this process by coupling the 19S proteasome regulatory subunit to the microtubule motor dynein for its transport to the centrosome. Our data identify the ciliopathy-related protein BBS1 as a new player in T cell synapse assembly that acts upstream of the IFT system to set the stage for polarized vesicular trafficking and sustained signaling.