Cytotoxic T lymphocytes kill virus-infected and tumorigenic target cells through the release of perforin and granzymes via fusion of lytic granules at the contact site, the immunological synapse. It has been postulated that this fusion process is mediated by non-neuronal members of the soluble N-ethylmaleimide-sensitive factor attachment protein receptor complex protein family. Here, using a synaptobrevin2-monomeric red fluorescence protein knock-in mouse we demonstrate that, surprisingly, the major neuronal v-SNARE synaptobrevin2 is expressed in cytotoxic T lymphocytes and exclusively localized on granzyme B-containing lytic granules. Cleavage of synaptobrevin2 by tetanus toxin or ablation of the synaptobrevin2 gene leads to a complete block of lytic granule exocytosis while leaving upstream events unaffected, identifying synaptobrevin2 as the v-SNARE responsible for the fusion of lytic granules at the immunological synapse.

Actin is a major cytoskeletal protein in eukaryotes. Recent studies suggest more diverse functional roles for this protein. Actin mRNA is known to be localized to neuronal synapses and undergoes rapid deadenylation during early developmental stages. However, its 3′-untranslated region (UTR) is not characterized and there are no experimentally determined polyadenylation (polyA) sites in actin mRNA. We have found that the cytoplasmic β-actin (Actb) gene generates two alternative transcripts terminated at tandem polyA sites. We used 3′-RACE, EST end analysis and in situ hybridization to unambiguously establish the existence of two 3′-UTRs of varying length in Actb transcript in mouse neuronal cells. Further analyses showed that these two tandem polyA sites are used in a tissue-specific manner. Although the longer 3′-UTR was expressed at a relatively lower level, it conferred higher translational efficiency to the transcript. The longer transcript harbours a conserved mmu-miR-34a/34b-5p target site. Sequence-specific anti-miRNA molecule, mutations of the miRNA target region in the 3′-UTR resulted in reduced expression. The expression was restored by a mutant miRNA complementary to the mutated target region implying that miR-34 binding to Actb 3′-UTR up-regulates target gene expression. Heterogeneity of the Actb 3′-UTR could shed light on the mechanism of miRNA-mediated regulation of messages in neuronal cells.

VAMP8 is associated with the recycling endosome compartment rather than with cytotoxic granules and is required for a fusion step between recycling endosomes and the plasma membrane that brings syntaxin-11 to the immune synapse for cytotoxic granule exocytosis.

CTLs kill target cells via fusion of lytic granules (LGs) at the immunological synapse (IS).Soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) function as executors of exocytosis. The importance of SNAREs in CTL function is evident in the form of familial hemophagocytic lymphohistiocytosis type 4 that is caused by mutations in Syntaxin11 (Stx11), a Qa-SNARE protein. Here, we investigate the molecular mechanism of Stx11 function in primary human effector CTLs with high temporal and spatial resolution. Downregulation of endogenous Stx11 resulted in a complete inhibition of LG fusion that was paralleled by a reduction in LG dwell time at the IS. Dual color evanescent wave imaging suggested a sequential process, in which first Stx11 is transported to the IS through a subpopulation of recycling endosomes. The resulting Stx11 clusters at the IS then serve as a platform to mediate fusion of arriving LGs. We conclude that Stx11 functions as a t-SNARE for the final fusion of LG at the IS, explaining the severe phenotype of familial hemophagocytic lymphohistiocytosis type 4 on a molecular level.Keywords: Cytotoxic T lymphocytes r FHL r Lytic granule fusion r Syntaxin11 clusters r TIRFM Additional supporting information may be found in the online version of this article at the publisher's web-site IntroductionCTLs kill their target cells through the polarized fusion of lytic granules (LGs) at the immunological synapse (IS). The IS is initiated when the TCRs recognize specific MHC peptide complexes on the target cell. The recognition results in the clustering of molecules that leads to the formation of supramolecular activation clusters (SMACs) [1]. TCRs and associated signaling proteins accumulate to form a central SMAC while adhesion proteins Correspondence: Prof. Jens Rettig e-mail: jrettig@uks.eu needed for stable target cell contact form the peripheral SMAC. After IS formation, LGs polarize, dock, prime, and finally fuse to release their contents into the cleft between CTL and target cell [2].Soluble N-ethylmaleimide-sensitive factor attachment receptor (SNARE) proteins are known executors of almost all intracellular fusion events [3]. The SNARE proteins that execute synaptic vesicle fusion in neurons and neuroendocrine cells have been well characterized [4,5]. Vesicle fusion is initiated by the formation of a four helical bundle that consists of one R-SNARE (v-SNARE) that resides on one membrane (mostly vesicle membranes) and three Q-SNAREs (Qa, Qb, Qc; t-SNARE) that reside on another membrane (mostly the plasma membrane) of the cell [6]. paired Student t-test). (B) Cell lysates from naïve CTLs (N), CTLs stimulated for 3 days by anti-CD3/anti-CD28 antibody-coated beads (S) andCTLs overexpressing TFP-Stx11 (OE) were assessed for Stx11 expression by Western blot using a polyclonal anti-Stx11 antibody. (C) Lysates from CTLs transfected with either control or three different Stx11 siRNAs (#1, #2, and #6, respectively) were blotted for Stx11 (top) and GAPDH (bottom) as loading control. (D) Qua...