Synaptotagmin 5 Controls SYP132-VAMP721/722 Interaction for Arabidopsis Immunity to Pseudomonas syringae pv tomato DC3000.

Vesicle-associated membrane proteins 721 and 722 (VAMP721/722) are secretory vesicle-localized arginine-conserved soluble N-ethylmaleimide-sensitive factor attachment protein receptors (R-SNAREs) to drive exocytosis in plants. They are involved in diverse physiological processes in plants by interacting with distinct plasma membrane (PM) syntaxins. Here, we show that synaptotagmin 5 (SYT5) is involved in plant defense against Pseudomonas syringae pv tomato (Pst) DC3000 by regulating SYP132-VAMP721/722 interactions. Calcium-dependent stimulation of in vitro SYP132-VAMP722 interaction by SYT5 and reduced in vivo SYP132-VAMP721/722 interaction in syt5 plants suggest that SYT5 regulates the interaction between SYP132 and VAMP721/722. We interestingly found that disease resistance to Pst DC3000 bacterium but not to Erysiphe pisi fungus is compromised in syt5 plants. Since SYP132 plays an immune function to bacteria, elevated growth of surface-inoculated Pst DC3000 in VAMP721/722-deficient plants suggests that SYT5 contributes to plant immunity to Pst DC3000 by promoting the SYP132-VAMP721/722 immune secretory pathway.

Increased urinary exosomal SYT17 levels in chronic active antibody-mediated rejection after kidney transplantation via the IL-6 amplifier.

Chronic active antibody-mediated rejection (CAAMR) is a particular problem in kidney transplantation (KTx), and ~25% of grafts are lost by CAAMR. Further, the pathogenesis remains unclear, and there is no effective cure or marker. We previously found that a hyper NFκB-activating mechanism in non-immune cells, called the IL-6 amplifier, is induced by the co-activation of NFκB and STAT3, and that this activation can develop various chronic inflammatory diseases. Here, we show that synaptotagmin-17 (SYT17) is increased in an exosomal fraction of the urine from CAAMR patients, and that this increase is associated with activation of the IL-6 amplifier. Immunohistochemistry showed that SYT17 protein expression was increased in renal tubule cells of the CAAMR group. While SYT17 protein was not detectable in whole-urine samples by western blotting, urinary exosomal SYT17 levels were significantly elevated in the CAAMR group compared to three other histology groups (normal, interstitial fibrosis and tubular atrophy, and calcineurin inhibitors toxicity) after KTx. On the other hand, current clinical laboratory data could not differentiate the CAAMR group from these groups. These data suggest that urinary exosomal SYT17 is a potential diagnostic marker for CAAMR.

Leishmania promastigotes induce cytokine secretion in macrophages through the degradation of synaptotagmin XI.

Synaptotagmins (Syts) are type-I membrane proteins that regulate vesicle docking and fusion in processes such as exocytosis and phagocytosis. We recently discovered that Syt XI is a recycling endosome- and lysosome-associated protein that negatively regulates the secretion of TNF and IL-6. In this study, we show that Syt XI is directly degraded by the zinc metalloprotease GP63 and excluded from Leishmania parasitophorous vacuoles by the promastigotes surface glycolipid lipophosphoglycan. Infected macrophages were found to release TNF and IL-6 in a GP63-dependent manner. To demonstrate that cytokine release was dependent on GP63-mediated degradation of Syt XI, small interfering RNA-mediated knockdown of Syt XI before infection revealed that the effects of small interfering RNA knockdown and GP63 degradation were not cumulative. In mice, i.p. injection of GP63-expressing parasites led to an increase in TNF and IL-6 secretion and to an augmented influx of neutrophils and inflammatory monocytes to the inoculation site. Both of these cell types have been shown to be infection targets and aid in the establishment of infection. In sum, our data revealed that GP63 induces proinflammatory cytokine release and increases infiltration of inflammatory phagocytes. This study provides new insight on how Leishmania exploits the immune response to establish infection.

[Lambert-Eaton myasthenic syndrome (LEMS)].

Lambert-Eaton myasthenic syndrome (LEMS) is a neuromuscular disorder in which autoantibodies inhibit the presynaptic release of acetylcholine. Autoantibodies against P/Q-type voltage-gated calcium channels (VGCC) are detected in 85% of patients with LEMS. In addition, autoantibodies to synaptotagmin, an M1-type muscarinic acetylcholine receptor and SOX1 are also found in the sera of patients with LEMS. LEMS is closely associated with small cell lung cancer (SCLC) in 50-60% of patients. Patients with SCLC who have anti-VGCC antibodies have been reported to have a favorable prognosis. In contrast to paraneoplatic LEMS, other forms of LEMS may have an autoimmune aspect because of the established association between human leukocyte antigen and a family history of other autoimmune disorders in this condition. The clinical features of LEMS include proximal weakness, areflexia, ptosis, cerebellar ataxia and autonomic dysfunction. The findings of electrophysiological examination show that LEMS is characterized by compound muscle action potential potentials with a low amplitude and increment upon repetitive nerve stimulation at a high rate. Tumor removal is the primary treatment of LEMS. The efficacy of 3,4-diaminopyridine for the treatment of LEMS has also been established. Patients with LEMS require the immunotherapies such as plasma exchange and the administration of high doses of immunoglobulin and prednisolone.

Quantitative analysis of synaptic boutons in Drosophila primary neuronal cultures.

Little information is currently available for structural and quantitative aspects of Drosophila central synapses due to difficulties in accessing those synapses in the tiny fly brain. Here, we developed a new approach to quantitatively analyze central synapses using Drosophila primary neuronal cultures. Two different markers were used to identify synaptic boutons: GFP marking with a synaptotagmin (Syt)::eGFP transgene and anti-Syt antibody. These markers clearly recognized puncta-like synaptic boutons and both signals were well overlapped. In addition, these puncta signals were completely absent in neuronal cultures derived from a Syt null mutant Syt(AD4), firmly demonstrating that anti-Syt(+) puncta are presynaptic terminals. Since anti-Syt signals were stronger and extensive, it was chosen to quantify synaptic boutons in the neuronal culture. Using an image analysis software Image J, synaptic boutons were quantified on the basis of the size and intensity of anti-Syt(+) signals. The number of synaptic boutons in wild type neurons increased by 27% between 3 and 9 days in culture. This increase was much greater (142%) in neuronal cultures derived from a FasII(e86) mutant known to show alterations in synapse growth and stabilization. A parallel increase in neurite length was also observed in both wild type and FasII(e86) neurons. Interestingly, the number of GABAergic synaptic boutons did not increase during this time, indicating distinctive mechanisms underlying development and maintenance of specific types of boutons. Our results successfully showed that Drosophila synaptic boutons can be quantified and thus we can examine genes and signaling pathways regulating structural properties of central synapses in Drosophila.

Differential role of the Ca(2+) sensor synaptotagmin VII in macrophages and dendritic cells.

Synaptotagmin VII (Syt VII) is a Ca(2+) sensing molecule that regulates lysosomal exocytosis in several cell types. In macrophages (MØ), Syt VII is required for efficient uptake of large particle loads, by promoting the delivery of lysosomal membrane to phagocytic cups. Here we compare the phagocytic capacity of bone marrow-derived MØs and dendritic cells (DC), and show that the requirement for Syt VII correlates with the unique ability of MØs for continuous phagocytosis. In contrast to MØs, Syt VII(+/+) and Syt VII(-/-) immature DCs show similar levels of initial phagocytosis, followed by a marked decrease in particle uptake. [Ca(2+)](i) chelation and PI-3 kinase inhibition reduce particle uptake by MØs, but are markedly less inhibitory in DCs. Thus, immature DCs appear to lack the Syt VII, Ca(2+) and PI-3 kinase-dependent forms of phagocytosis that are present in MØs. Interestingly, expression of Syt VII is up-regulated during LPS-induced DC maturation, a stimulus that also induces Syt VII translocation from intracellular compartments to the plasma membrane. Syt VII(-/-) DCs show a delayed translocation of MHC class II to the cell surface during maturation, consistent with the possibility that Syt VII facilitates exocytosis and/or surface retention of molecules critical for antigen presentation.

The exocytosis regulator synaptotagmin V controls phagocytosis in macrophages.

Synaptotagmins (Syts) play a key role in the regulation of Ca(2+)-triggered exocytosis and membrane fusion events, two crucial events associated to the phagocytic process. In the present study, we investigated the role of Syt V, a regulator of focal exocytosis, in phagocytosis. In macrophages, Syt V is localized on recycling endosomes and on filopodia-like structures and is recruited to the nascent phagosomes independently of the phagocytic receptor engaged. Silencing of Syt V by RNA interference revealed a role for this protein for phagocytosis, particularly under conditions of high membrane demand. In contrast, silencing of Syt V had no effect on the recruitment of the lysosomal marker LAMP1 to phagosomes, indicating that phagosome maturation is not regulated by Syt V. Collectively, these results illustrate the importance of Syt V in the regulation of an important innate function of macrophages. Furthermore, our results are consistent with the concept that focal exocytosis of endocytic organelles is a key event in phagocytosis and suggest that Syt V regulates this process.

Expression and function of synaptotagmin VII in CTLs.

The Ca(2+) sensor synaptotagmin (Syt) VII regulates the exocytosis of conventional lysosomes in several cell types. In CTLs, the Ca(2+)-regulated exocytosis of lytic granules/secretory lysosomes is responsible for the perforin/granzyme-mediated lysis of target cells. To investigate the role of Syt VII in CTL effector function, the expression and function of Syt VII were examined in wild-type and Syt VII-deficient mice. In comparison with Syt VII(+/+) controls, Syt VII(-/-) animals were impaired in their ability to clear an infection with the intracellular pathogen Listeria monocytogenes. When isolated CTLs were examined, we found that Syt VII is expressed upon CTL activation and localizes to granzyme A-containing lytic granules. Syt VII-deficient CTLs have no defects in proliferation and cytokine production, and their lytic granules contain normal amounts of perforin and granzyme A and polarize normally at the immunological synapse. However, despite normal conjugate formation with target cells, CTLs from Syt VII(-/-) mice exhibit reduced effector activity, when compared with controls. Treatment of Syt VII(+/+) or Syt VII(-/-) CTLs with an inhibitor of the perforin-mediated lytic pathway resulted in comparable levels of cytotoxic activity, suggesting that Syt VII regulates perforin-mediated cytolytic CTL responses.

Membrane resealing: synaptotagmin VII keeps running the show.

Ca2+ influx, an immediate consequence of plasma membrane disruption, triggers a resealing mechanism involving exocytosis. Although this has been known for about a decade, a better understanding of the organelles involved and of the molecular machinery controlling membrane repair has been slower to emerge. Recent studies have changed this picture, by identifying lysosomes as exocytotic vesicles involved in membrane resealing and the Ca2+-binding protein synaptotagmin VII as a regulator of this process. New evidence reinforces the role of the C2A and C2B domains of synaptotagmin VII in plasma membrane repair, highlighting the importance of this molecule as a powerful tool for future studies.