Endocytosis of Cytotoxic Granules Is Essential for Multiple Killing of Target Cells by T Lymphocytes.

CTLs are serial killers that kill multiple target cells via exocytosis of cytotoxic granules (CGs). CG exocytosis is tightly regulated and has been investigated in great detail; however, whether CG proteins are endocytosed following exocytosis and contribute to serial killing remains unknown. By using primary CTLs derived from a knock-in mouse of the CG membrane protein Synaptobrevin2, we show that CGs are endocytosed in a clathrin- and dynamin-dependent manner. Following acidification, endocytosed CGs are recycled through early and late, but not recycling endosomes. CGs are refilled with granzyme B at the late endosome stage and polarize to subsequent synapses formed between the CTL and new target cells. Importantly, inhibiting CG endocytosis in CTLs results in a significant reduction of their cytotoxic activity. Thus, our data demonstrate that continuous endocytosis of CG membrane proteins is a prerequisite for efficient serial killing of CTLs and identify key events in this process.

VAMP8-dependent fusion of recycling endosomes with the plasma membrane facilitates T lymphocyte cytotoxicity.

Cytotoxic T lymphocytes (CTLs) eliminate infected and neoplastic cells through directed release of cytotoxic granule contents. Although multiple SNARE proteins have been implicated in cytotoxic granule exocytosis, the role of vesicular SNARE proteins, i.e., vesicle-associated membrane proteins (VAMPs), remains enigmatic. VAMP8 was posited to represent the cytotoxic granule vesicular SNARE protein mediating exocytosis in mice. In primary human CTLs, however, VAMP8 colocalized with Rab11a-positive recycling endosomes. Upon stimulation, these endosomes rapidly trafficked to and fused with the plasma membrane, preceding fusion of cytotoxic granules. Knockdown of VAMP8 blocked both recycling endosome and cytotoxic granule fusion at immune synapses, without affecting activating signaling. Mechanistically, VAMP8-dependent recycling endosomes deposited syntaxin-11 at immune synapses, facilitating assembly of plasma membrane SNARE complexes for cytotoxic granule fusion. Hence, cytotoxic granule exocytosis is a sequential, multivesicle fusion process requiring VAMP8-mediated recycling endosome fusion before cytotoxic granule fusion. Our findings imply that secretory granule exocytosis pathways in other cell types may also be more complex than previously appreciated.

The Zebrafish GenomeWiki: a crowdsourcing approach to connect the long tail for zebrafish gene annotation.

A large repertoire of gene-centric data has been generated in the field of zebrafish biology. Although the bulk of these data are available in the public domain, most of them are not readily accessible or available in nonstandard formats. One major challenge is to unify and integrate these widely scattered data sources. We tested the hypothesis that active community participation could be a viable option to address this challenge. We present here our approach to create standards for assimilation and sharing of information and a system of open standards for database intercommunication. We have attempted to address this challenge by creating a community-centric solution for zebrafish gene annotation. The Zebrafish GenomeWiki is a 'wiki'-based resource, which aims to provide an altruistic shared environment for collective annotation of the zebrafish genes. The Zebrafish GenomeWiki has features that enable users to comment, annotate, edit and rate this gene-centric information. The credits for contributions can be tracked through a transparent microattribution system. In contrast to other wikis, the Zebrafish GenomeWiki is a 'structured wiki' or rather a 'semantic wiki'. The Zebrafish GenomeWiki implements a semantically linked data structure, which in the future would be amenable to semantic search. Database URL: http://genome.igib.res.in/twiki.

Syntaxin11 serves as a t-SNARE for the fusion of lytic granules in human cytotoxic T lymphocytes.

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.

In the crosshairs: investigating lytic granules by high-resolution microscopy and electrophysiology.

Cytotoxic T lymphocytes (CTLs) form an integral part of the adaptive immune system. Their main function is to eliminate bacteria- and virus-infected target cells by releasing perforin and granzymes (the lethal hit) contained within lytic granules (LGs), at the CTL-target-cell interface [the immunological synapse (IS)]. The formation of the IS as well as the final events at the IS leading to target-cell death are both highly complex and dynamic processes. In this review we highlight and discuss three high-resolution techniques that have proven invaluable in the effort to decipher key features of the mechanism of CTL effector function and in particular lytic granule maturation and fusion. Correlative light and electron microscopy allows the correlation between organelle morphology and localization of particular proteins, while total internal reflection fluorescence microscopy (TIRFM) enables the study of lytic granule dynamics at the IS in real time. The combination of TIRFM with patch-clamp membrane capacitance measurements finally provides a tool to quantify the size of fusing LGs at the IS.

Deciphering dead-end docking of large dense core vesicles in bovine chromaffin cells.

Large dense core vesicle (LDCV) exocytosis in chromaffin cells follows a well characterized process consisting of docking, priming, and fusion. Total internal reflection fluorescence microscopy (TIRFM) studies suggest that some LDCVs, although being able to dock, are resistant to calcium-triggered release. This phenomenon termed dead-end docking has not been investigated until now. We characterized dead-end vesicles using a combination of membrane capacitance measurement and visualization of LDCVs with TIRFM. Stimulation of bovine chromaffin cells for 5 min with 6 µm free intracellular Ca2+ induced strong secretion and a large reduction of the LDCV density at the plasma membrane. Approximately 15% of the LDCVs were visible at the plasma membrane throughout experiments, indicating they were permanently docked dead-end vesicles. Overexpression of Munc18-2 or SNAP-25 reduced the fraction of dead-end vesicles. Conversely, expressing open-syntaxin increased the fraction of dead-end vesicles. These results indicate the existence of the unproductive target soluble N-ethylmaleimide-sensitive factor attachment protein receptor acceptor complex composed of 2:1 syntaxin-SNAP-25 in vivo. More importantly, they define a novel function for this acceptor complex in mediating dead-end docking.

Different Munc13 isoforms function as priming factors in lytic granule release from murine cytotoxic T lymphocytes.

In order to fuse lytic granules (LGs) with the plasma membrane at the immunological synapse, cytotoxic T lymphocytes (CTLs) have to render these LGs fusion-competent through the priming process. In secretory tissues such as brain and neuroendocrine glands, this process is mediated by members of the Munc13 protein family. In human CTLs, mutations in the Munc13-4 gene cause a severe loss in killing efficiency, resulting in familial hemophagocytic lymphohistiocytosis type 3, suggesting a similar role of other Munc13 isoforms in the immune system. Here, we investigate the contribution of different Munc13 isoforms to the priming process of murine CTLs at both the mRNA and protein level. We demonstrate that Munc13-1 and Munc13-4 are the only Munc13 isoforms present in mouse CTLs. Both isoforms rescue the drastical secretion defect of CTLs derived from Munc13-4-deficient Jinx mice. Mobility studies using total internal reflection fluorescence microscopy indicate that Munc13-4 and Munc13-1 are responsible for the priming process of LGs. Furthermore, the domains of the Munc13 protein, which is responsible for functional fusion, could be identified. We conclude from these data that both isoforms of the Munc13 family, Munc13-1 and Munc13-4, are functionally redundant in murine CTLs.

Synaptobrevin2 is the v-SNARE required for cytotoxic T-lymphocyte lytic granule fusion.

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.

Identification of novel targets for miR-29a using miRNA proteomics.

MicroRNAs (miRNAs) are short regulatory RNA molecules that interfere with the expression of target mRNA by binding to complementary sequences. Currently, the most common method for identification of targets of miRNAs is computational prediction based on free energy change calculations, target site accessibility and conservation. Such algorithms predict hundreds of targets for each miRNA, necessitating tedious experimentation to identify the few functional targets. Here we explore the utility of miRNA-proteomics as an approach to identifying functional miRNA targets. We used Stable Isotope Labeling by amino acids in cell culture (SILAC) based proteomics to detect differences in protein expression induced by the over-expression of miR-34a and miR-29a. Over-expression of miR-29a, a miRNA expressed in the brain and in cells of the blood lineage, resulted in the differential expression of a set of proteins. Gene Ontology based classification showed that a significant sub-set of these targets, including Voltage Dependent Anion Channel 1 and 2 (VDAC1 and VDAC2) and ATP synthetase, were mitochondrial proteins involved in apoptosis. Using reporter assays, we established that miR-29a targets the 3' Untranslated Regions (3' UTR) of VDAC1 and VDAC2. However, due to the limited number of proteins identified using this approach and the inability to differentiate between primary and secondary effects we conclude that miRNA-proteomics is of limited utility as a high-throughput alternative for sensitive and unbiased miRNA target identification. However, this approach was valuable for rapid assessment of the impact of the miRNAs on the cellular proteome and its biological role in apoptosis.

SNARE protein expression and localization in human cytotoxic T lymphocytes.

The major function of cytotoxic T lymphocytes (CTLs) is to eliminate pathogen-infected and tumorigenic cells. This is mediated mainly through the exocytosis of lytic granules (LGs) containing cytotoxic components, such as perforin and granzymes at the immunological synapse (IS). The soluble NSF attachment receptor (SNARE) protein isoforms are well known to be required for vesicle exocytosis in neuronal synapses, but their potential function in CTLs is only partly understood. Here, we examined the expression of SNARE proteins before and after the activation of primary human CD8(+) T cells and determined their co-localization with LGs and CD3 after IS formation with target cells. We found that several key SNARE proteins in neuronal cells were not expressed in CTLs, such as syntaxin1B2 and SNAP-25. Vti1b, Stx8 and Stx16 had the highest degrees of co-localization with LGs while Stx3, Stx4, Stx6, Stx7, Stx8, Stx13, Vti1b, VAMP3 and VAMP4 co-localized with CD3. Our data provide the first complete expression profile and localization of SNAREs in primary human CD8(+) T cells, laying the groundwork for further understanding their potential role in T-cell function.

Dendritic effect of ligand-coated nanoparticles: enhanced apoptotic activity of silica-berberine nanoconjugates.

We describe the synthesis and biological characterization of a novel prototype, namely, silica nanoconjugates bearing a covalently linked berberine, a plant alkaloid known to have antiproliferative activity. The effect of synthesized nanoconjugates on cell proliferation, the cell cycle profile, and apoptosis in the human cervical carcinoma cell line (HeLa), human hepatocellular liver carcinoma cell line (HepG2), and human embryonic kidney (HEK) 293T cell line has been studied and compared with the results obtained for free berberine. Our results show that all the nanoconjugates display higher antiproliferative activity than free berberine. The ability of these nanoconjugates to inhibit cellular proliferation is mediated by the cell cycle arrest at the G1 phase. Moreover, silica nanoconugates caused selective apoptotic arrest with a higher efficiency than free berberine followed by apoptotic cell death as shown by quantitative fluorescence-activated cell sorting analyses. Efficiency of the nanoconjugates increases upon an increase in the linker chain length, demonstrating the distinct role of the spacer chain that conjugates nanoparticles and ligands. The actual reason to show enhanced efficiency by the nanoconjugates has not been elucidated in the present study; however, we hypothesize that an increase in local concentration due to the confinement of a ligand on the nanosurface ("dendritic" effect) might have led to the observed effect.

MicroRNA-mediated up-regulation of an alternatively polyadenylated variant of the mouse cytoplasmic {beta}-actin gene.

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 beta-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.