Chika Saegusa

Slp4-a/granuphilin-a regulates dense-core vesicle exocytosis in PC12 cells.

Synaptotagmin-like protein 4-a (Slp4-a)/granuphilin-a was originally identified as a protein specifically associated with insulin-containing vesicles in pancreatic β-cells (Wang, J., Takeuchi, T., Yokota, H., and Izumi, T. (1999)J. Biol. Chem. 274, 28542–28548). Previously, we showed that the N-terminal Slp homology domain of Slp4-a interacts with the GTP-bound form of Rab3A, Rab8, and Rab27A both in vitroand in intact cells (Kuroda, T. S., Fukuda, M., Ariga, H., and Mikoshiba, K. (2002) J. Biol. Chem. 277, 9212–9218). How Slp4-a·Rab complex controls regulated secretion, and which Rab isoforms dominantly interact with Slp4-a in vivo, however, have remained unknown. In this study, we showed by immunocytochemistry and subcellular fractionation that three Rabs, Rab3A, Rab8, and Rab27A, and Slp4-a are endogenously expressed in neuroendocrine PC12 cells and localized on dense-core vesicles, and we discovered that the Slp4-a·Rab8 and Slp4-a·Rab27A complexes, but not Slp4-a·Rab3A complexes, are formed on dense-core vesicles in PC12 cells, although the majority of Rab8 is present in the cell body and is free of Slp4-a. We further showed that expression of Rab27A, but not of Rab8, promotes high KCl-dependent secretion of neuropeptide Y (NPY) in PC12 cells, whereas expression of Slp4-a, but not of an Slp4-a mutant incapable of Rab27A binding, inhibits NPY secretion in PC12 cells. In contrast, expression of Slp3-a, but not of Slp3-b lacking an N-terminal Rab27A-binding domain, promotes NPY secretion. These findings suggest that the Slp family controls regulated dense-core vesicle exocytosis via binding to Rab27A.

Slp1 and Slp2-a Localize to the Plasma Membrane of CTL and Contribute to Secretion from the Immunological Synapse

Rab27a is required for polarized secretion of lysosomes from cytotoxic T lymphocytes (CTLs) at the immunological synapse. A series of Rab27a‐interacting proteins have been identified; however, only Munc13‐4 has been found to be expressed in CTL. In this study, we screened for expression of the synaptotagmin‐like proteins (Slps): Slp1/JFC1, Slp2‐a/exophilin4, Slp3‐a, Slp4/granuphilin, Slp5 and rabphilin in CTL. We found that both Slp1 and Slp2‐a are expressed in CTL. Isoforms of Slp2‐a in CTL showed variation of the linker region but conserved the C2A and C2B and Slp homology (SHD) domains. Both Slp1 and Slp2‐a interact with Rab27a in CTL, and Slp2‐a, but not Slp1, is rapidly degraded when Rab27a is absent. Slp2‐a contains PEST‐like sequences within its linker region, which render it susceptible to degradation. Both Slp1 and Slp2‐a localize predominantly to the plasma membrane of both human and mouse CTLs, and we show that Slp2‐a can focus tightly at the immunological synapse formed with a target cell. Individual knockouts of either Slp2‐a or Slp1 fail to impair CTL‐mediated killing of targets; however, overexpression of a dominant‐negative construct consisting of the SHD of Slp2‐a, which is 56% identical to that of Slp1, reduces target cell death, suggesting that both Slp1 and Slp2‐a contribute to secretory lysosome exocytosis from CTL. These results suggest that both Slp1 and Slp2‐a may form part of a docking complex, capturing secretory lysosomes at the immunological synapse.

Alternative splicing isoforms of synaptotagmin VII in the mouse, rat and human.

Synaptotagmin VII (Syt VII) has been proposed to regulate several different types of Ca2+-dependent exocytosis, but its subcellular localization (lysosome or plasma membrane) and the number of alternative splicing isoforms of Syt VII (single or multiple forms) are matters of controversy. In the present study, we show by reverse transcriptase-PCR analysis that mouse Syt VII has one major isoform (Syt VIIalpha), the original Syt VII, and two minor isoforms (Syt VIIbeta and Syt VIIgamma), which contain unique insertions (of 44 and 116 amino acids respectively) in the spacer domain between the transmembrane and C2 domains of Syt VIIalpha. Similar results were obtained with respect to rat and human Syt VII mRNA expression. An antibody against the N-terminal domain of mouse Syt VII [anti-(Syt VII-N)], which specifically recognized recombinant Syt VII but not other Syt isoforms expressed in COS-7 cells, recognized two major, closely co-migrating bands (p58 and p60) and minor bands of approx. 65 kDa in mouse brain. Immunoaffinity purification of proteins that bind the anti-(Syt VII-N) antibody, and peptide sequence analysis revealed that: (i) the major p58 and p60 bands are identified as adenylate cyclase-associated protein 2; (ii) actin-binding protein is localized at the plasma membrane; and (iii) Syt VIIalpha (65 kDa) is the major Syt VII isoform, but with a much lower expression level than previously thought. It was also shown that FLAG-Syt VII-green-fluorescence-protein fusion protein stably expressed in PC12 cells is localized in the perinuclear region (co-localization with TGN38 protein, even after brefeldin A treatment) and in the tips of neurites (co-localization with Syt I), and not in the plasma membrane.

Decreased basal mucus secretion by Slp2-a-deficient gastric surface mucous cells

Synaptotagmin‐like protein (Slp) 2‐a is a putative Rab27A/B‐effector protein and is implicated in intracellular membrane transport. However, the precise tissue distribution of Slp2‐a protein and its functions remain largely unknown. In this study we used a specific anti‐Slp2‐a antibody to investigate the tissue distribution of Slp2‐a in mice and found that Slp2‐a is most abundantly expressed in mouse stomach. Co‐immunoprecipitation experiments indicated that Slp2‐a interacts with Rab27A/B in vivo. We also discovered that Slp2‐a and Rab27A/B are predominantly localized at the apical region of gastric‐surface mucous cells, where mucus granules are accumulated. Analysis of Slp2‐a mutant mice generated by homologous recombination showed a reduced number of mucus granules, a deficiency of granule docking with the apical plasma membrane in the gastric‐surface mucous cells and reduction of mucus secretion by Slp2‐a‐deficient gastric primary cells. Based on these results, we propose that Slp2‐a is part of the mucin secretory machinery in surface mucous cells of mouse stomach.

Nerve Growth Factor-dependent Sorting of Synaptotagmin IV Protein to Mature Dense-core Vesicles That Undergo Calcium-dependent Exocytosis in PC12 Cells

Synaptotagmin IV (Syt IV) is a fourth member of the Syt family and has been shown to regulate some forms of memory and learning by analysis of Syt IV null mutant mice (Ferguson, G. D., Anagnostaras, S. G., Silva, A. J., and Herschman, H. R. (2000) Proc. Natl. Acad. Sci. U. S. A. 97, 5598–5603). However, the involvement of Syt IV protein in vesicular trafficking and even its localization in secretory vesicles are still matters of controversy. Here we present several lines of evidence showing that the Syt IV protein in PC12 cells is normally localized in the Golgi or immature vesicles at the cell periphery and is sorted to fusion-competent mature dense-core vesicles in response to short nerve growth factor (NGF) stimulation. (i) In undifferentiated PC12 cells, Syt IV protein is mainly localized in the Golgi and small amounts are also present at the cell periphery, but according to the results of an immunocytochemical analysis, they do not colocalize with conventional secretory vesicle markers (Syt I, Syt IX, Rab3A, Rab27A, vesicle-associated membrane protein 2, and synaptophysin) at all. By contrast, limited colocalization of Syt IV protein with dense-core vesicle markers is found in the distal parts of the neurites of NGF-differentiated PC12 cells. (ii) Immunoelectron microscopy with highly specific anti-Syt IV antibody revealed that the Syt IV protein in undifferentiated PC12 cells is mainly present on the Golgi membranes and immature secretory vesicles, whereas after NGF stimulation Syt IV protein is also present on the mature dense-core vesicles. (iii) An N-terminal antibody-uptake experiment indicated that Syt IV-containing vesicles in the neurites of NGF-differentiated PC12 cells undergo Ca2+-dependent exocytosis, whereas no uptake of the anti-Syt IV-N antibody was observed in undifferentiated PC12 cells. Our results suggest that Syt IV is a stimulus (e.g. NGF)-dependent regulator for exocytosis of dense-core vesicles.

Rab27 effector Slp2-a transports the apical signaling molecule podocalyxin to the apical surface of MDCK II cells and regulates claudin-2 expression

Slp2-a is required for targeting of the signaling molecule podocalyxin to the apical membrane in MDCK II cells in a Rab27A-dependent manner. Apical membrane localization of podocalyxin is required for expression of the tight junction protein claudin-2 through modulation of intracellular signals, including MAPK signals.

Expression of Rab27B-binding protein Slp1 in pancreatic acinar cells and its involvement in amylase secretion.

Slp1 is a putative Rab27 effector protein and implicated in intracellular membrane transport; however, the precise tissue distribution and function of Slp1 protein remain largely unknown. In this study we investigated the tissue distribution of Slp1 in mice and found that Slp1 is abundantly expressed in the pancreas, especially in the apical region of pancreatic acinar cells. Slp1 interacted with Rab27B in vivo and both proteins were co-localized on zymogen granules. Morphological analysis of fasted Slp1 knockout mice showed an increased number of zymogen granules in the pancreatic acinar cells, indicating that Slp1 is part of the machinery of amylase secretion by the exocrine pancreas.

The C2A Domain of Double C2 Protein γ Contains a Functional Nuclear Localization Signal

The C2 domain was originally defined as a homologous domain to the C2 regulatory region of Ca2+-dependent protein kinase C and has been identified in more than 50 different signaling molecules. The original C2 domain of protein kinase Cα functions as a Ca2+ binding module, and the Ca2+ binding to the C2 domain allows translocation of proteins to phospholipid membranes. By contrast, however, some C2 domains do not exhibit Ca2+ binding activity because of amino acid substitutions at Ca2+-binding sites, and their physiological meanings remain largely unknown. In this study, we discovered an unexpected function of the Ca2+-independent C2A domain of double C2 protein γ (Doc2γ) in nuclear localization. Deletion and mutation analyses revealed that the putative Ca2+ binding loop 3 of Doc2γ contains six Arg residues (177RLRRRRR183) and that this basic cluster is both necessary and sufficient for nuclear localization of Doc2γ. Because of the presence of the basic cluster, the C2A domain of Doc2γ did not show Ca2+-dependent phospholipid binding activity. Our findings indicate that by changing the nature of the putative Ca2+ binding loops the C2 domain has more diversified function in cellular signaling than a simple Ca2+ binding motif.