Rim2α Determines Docking and Priming States in Insulin Granule Exocytosis

Yasuda, Takahiro; Shibasaki, Tadao; Minami, Kohtaro; Takahashi, Harumi; Mizoguchi, Akira; Uriu, Yoshitsugu; Numata, Tomohiro; Mori, Yasuo; Miyazaki, Jun‐ichi; Miki, Takashi; Seino, Susumu

doi:10.1016/j.cmet.2010.05.017

Cited by 103 publications

(122 citation statements)

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“…4a, b). 'Newcomer SGs' were new SGs appearing de novo under an evanescent field after stimulation and then underwent exocytosis, appearing in two distinct patterns as described previously [4,10,19,23]: 'no-dock' newcomer SGs (shown in white in Fig. 4a, b) were newly recruited by stimulation and immediately fused with PM (docking state of<200 ms, the minimal interval between two consecutive frames); 'short-dock' newcomer SGs (shown in grey in Fig.…”

Section: Resultsmentioning

confidence: 99%

“…We had alternatively postulated that reduced levels of the SM-SNARE complex comprised of Munc18a, Syn-1A, SNAP25 and VAMP2 in beta cells contributed to the defective exocytosis of pre-docked insulin SGs in islets of individuals with type 2 diabetes [29,30]. More recent work showed that newcomer SGs contributed to primary exocytosis [4,5,23], and in fact far exceeded pre-docked SGs in the overall GSIS [5]. Remarkably, these newcomer SGs are recruited to the PM to undergo exocytosis with minimal to no residence time on the PM [4,5].…”

Section: Discussionmentioning

confidence: 99%

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Syntaxin-4 mediates exocytosis of pre-docked and newcomer insulin granules underlying biphasic glucose-stimulated insulin secretion in human pancreatic beta cells

et al. 2015

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Aims/hypothesis Of the four exocytotic syntaxins (Syns), much is now known about the role of Syn-1A (pre-docked secretory granules [SGs]) and Syn-3 (newcomer SGs) in insulin exocytosis. Some work was reported on Syn-4's role in biphasic glucose-stimulated insulin secretion (GSIS), but its precise role in insulin SG exocytosis remains unclear. In this paper we examine this role in human beta cells. Methods Endogenous function of Syn-4 in human islets was assessed by knocking down its expression with lentiviral single hairpin RNA (lenti-shRNA)-RFP. Biphasic GSIS was determined by islet perifusion assay. Single-cell analysis of exocytosis of red fluorescent protein (RFP)-positive beta cells (exhibiting near-total depletion of Syn-4) was by patch clamp capacitance measurements (Cm) and total internal reflection fluorescence microscopy (TIRFM), the latter to further assess single SG behaviour. Co-immunoprecipitations were conducted on INS-1 cells to assess exocytotic complexes. Results Syn-4 knockdown (KD) of 77% in human islets caused a concomitant reduction in cognate Munc18c expression (46%) without affecting expression of other exocytotic proteins; this resulted in reduction of GSIS in the first phase (by 42%) and the second phase (by 40%). Cm of RFP-tagged Syn-4-KD beta cells showed severe inhibition in the readily releasable pool (by 71%) and mobilisation from reserve pools (by 63%). TIRFM showed that Syn-4-KD-induced inhibition of first-phase GSIS was attributed to reduction in exocytosis of both pre-docked and newcomer SGs (which undergo minimal residence or docking time at the plasma membrane before fusion). Second-phase inhibition was attributed to reduction in newcomer SGs. Stx-4 co-immunoprecipitated Munc18c, VAMP2 and VAMP8, suggesting that these exocytotic complexes may be involved in exocytosis of pre-docked and newcomer SGs. Conclusions/interpretation Syn-4 is involved in distinct molecular machineries that influence exocytosis of both predocked and newcomer SGs in a manner functionally redundant to Syn-1A and Syn-3, respectively; this underlies Syn-4's role in mediating portions of first-phase and second-phase GSIS.

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Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Syntaxin-4 mediates exocytosis of pre-docked and newcomer insulin granules underlying biphasic glucose-stimulated insulin secretion in human pancreatic beta cells

et al. 2015

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“…'Newcomer SGs' were new SGs appearing de novo under evanescent field after stimulation and which then underwent exocytosis. Newcomer SGs could be further categorised into two distinct patterns as described previously [5,7,24], designated as 'no-dock' (middle, Fig. 3d) and 'short-dock' (bottom, Fig.…”

Section: Syn-3 Is a T-snare On Sgsmentioning

confidence: 99%

Syntaxin-3 regulates newcomer insulin granule exocytosis and compound fusion in pancreatic beta cells

et al. 2012

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Aims/hypothesis The molecular basis of the exocytosis of secretory insulin-containing granules (SGs) during biphasic glucose-stimulated insulin secretion (GSIS) from pancreatic beta cells remains unclear. Syntaxin (SYN)-1A and SYN-4 have been shown to mediate insulin exocytosis. The insulinsecretory function of SYN-3, which is particularly abundant in SGs, is unclear. Methods Mouse pancreatic islets and INS-1 cells were treated with adenovirus carrying Syn-3 (also known as Stx3) or small interfering RNA targeting Syn-3 in order to examine insulin secretion by radioimmunoassay. The localisation and distribution of insulin granules were examined by confocal and electron microscopy. Dynamic single-granule fusion events were assessed using total internal reflection fluorescence microscopy (TIRFM). Results Depletion of endogenous SYN-3 inhibited insulin release. TIRFM showed no change in the number or fusion competence of previously docked SGs but, instead, a marked reduction in the recruitment of newcomer SGs and their subsequent exocytotic fusion during biphasic GSIS. Conversely, overexpression of Syn-3 enhanced both phases of GSIS, owing to the increase in newcomer SGs and, remarkably, to increased SG-SG fusion, which was confirmed by electron microscopy. Conclusions/interpretation In insulin secretion, SYN-3 plays a role in the mediation of newcomer SG exocytosis and SG-SG fusion that contributes to biphasic GSIS.

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Section: Identification Of the Rab37-binding Proteinmentioning

confidence: 99%

“…We found 29 different putative Rab37-binding proteins that might be involved in TNF-a secretion from macrophages (Supporting Information Table S1). One, Munc13-1, has a functional interaction with Rab3A-interacting molecule 2 (RIM2) in the final step of exocytosis and is essential for insulin secretion from islet b cells [22].Using immunofluorescence staining, we determined that Munc13-1 colocalized with both EGFP-Rab37 and TNF-a in LPSstimulated RAW264.7 macrophages ( Fig. 5C; 76.375.4% n 5 18 and D; 73.878.3% n 5 18 cells), suggesting that Munc13-1 is associated with Rab37-bound TNF-a-containing vesicles in activated macrophages.…”

mentioning

confidence: 99%

Release of TNF‐α from macrophages is mediated by small GTPase Rab37

et al. 2011

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Activated macrophages at wound sites release many cytokines which positively affect skin wound healing. However, the molecular mechanisms controlling cytokine secretion from macrophages have not been elucidated. In the present study, we performed an RT-PCR analysis and found that 19 small GTPase Rab isoforms were expressed at skin wound sites, with six of them (i.e. Rab3B, Rab27B, Rab30, Rab33A, Rab37, and Rab40C) being upregulated during the inflammation and proliferation/migration phase of skin repair. We also found that gene expression of Rab37 in murine primary and RAW264.7 macrophages was significantly induced after stimulation with LPS. Overexpression of wild type and constitutively active Rab37 in RAW264.7 cells significantly increased TNF-a secretion, whereas knockdown of Rab37 by siRNA significantly decreased it. We also identified 29 putative Rab37-interacting proteins, including the membrane fusion regulating Munc13-1, using liquid chromatography/ linear ion trap mass spectrometry (LC-MS/MS). Immunocytochemical analysis further revealed that TNF-a-containing vesicles were colocalized with both Rab37 and Munc13-1 in activated macrophages. Knockdown of Munc13-1 by siRNA significantly decreased TNF-a secretion. Taken together, these findings demonstrate that Rab37 interacts with Munc13-1 to control TNF-a secretion from activated macrophages.Key words: Macrophage . Munc13-1 . Rab37 . Skin wound healing . TNF-a Supporting Information available online IntroductionIn response to skin tissue damage, inflammatory cells rapidly infiltrate the wound site to protect against infection. In the acute phase of inflammation, neutrophils, the first responder inflammatory cells, migrate toward wound sites to internalize and eliminate pathogens. Subsequently, macrophages preferentially infiltrate to wound sites after neutrophil recruitment, where they release cytokines, chemokines, and growth factors to control skin wound healing by stimulating the activation and proliferation of fibroblasts and keratinocytes, and by promoting wound contraction and angiogenesis [1]. Although this inflammatory response 3230is part of the wound-healing process, wound models in several organisms have shown that the inflammatory response can cause fibrotic diseases (i.e. scarring) in the skin and other organs [2]. Recently, we have shown that osteopontin is secreted by activated fibroblasts in response to platelet-derived growth factor secreted from macrophages, and that silencing of osteopontin at skin wound sites reduces scarring and improves the quality of healing in vivo [3]. Therefore, the pathways controlling inflammatory mediator secretion could provide novel molecular therapeutic targets [4].One such inflammatory mediator, TNF-a, is a pleiotropic proinflammatory cytokine expressed by macrophages and neutrophils at skin wound sites [5,6]. It has been reported that signaling via TNF-a negatively regulates skin wound healing, because TNF-a receptor p55 KO mice showed rapid skin wound healing coincident with reduced inflammatory respons...

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Rim2α Determines Docking and Priming States in Insulin Granule Exocytosis

Cited by 103 publications

References 48 publications

Syntaxin-4 mediates exocytosis of pre-docked and newcomer insulin granules underlying biphasic glucose-stimulated insulin secretion in human pancreatic beta cells

Syntaxin-4 mediates exocytosis of pre-docked and newcomer insulin granules underlying biphasic glucose-stimulated insulin secretion in human pancreatic beta cells

Syntaxin-3 regulates newcomer insulin granule exocytosis and compound fusion in pancreatic beta cells

Release of TNF‐α from macrophages is mediated by small GTPase Rab37

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