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, 283 (16), 10716-26

Filamentous Actin Regulates Insulin Exocytosis Through Direct Interaction With Syntaxin 4

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Filamentous Actin Regulates Insulin Exocytosis Through Direct Interaction With Syntaxin 4

Jenna L Jewell et al. J Biol Chem.

Abstract

Glucose-induced insulin exocytosis is coupled to associations between F-actin and SNARE proteins, although the nature and function of these interactions remains unknown. Toward this end we show here that both Syntaxin 1A and Syntaxin 4 associated with F-actin in MIN6 cells and that each interaction was rapidly and transiently diminished by stimulation of cells with d-glucose. Of the two isoforms, only Syntaxin 4 was capable of interacting directly with F-actin in an in vitro sedimentation assay, conferred by the N-terminal 39-112 residues of Syntaxin 4. The 39-112 fragment was capable of selective competitive inhibitory action, disrupting endogenous F-actin-Syntaxin 4 binding in MIN6 cells. Disruption of F-actin-Syntaxin 4 binding correlated with enhanced glucose-stimulated insulin secretion, mediated by increased granule accumulation at the plasma membrane and increased Syntaxin 4 accessibility under basal conditions. However, no increase in basal level Syntaxin 4-VAMP2 association occurred with either latrunculin treatment or expression of the 39-112 fragment. Taken together, these data disclose a new underlying mechanism by which F-actin negatively regulates exocytosis via binding and blocking Syntaxin 4 accessibility, but they also reveal the existence of additional signals and/or steps required to trigger the subsequent docking and fusion steps of exocytosis.

Figures

FIGURE 1.
FIGURE 1.
Latrunculin potentiates insulin secretion in human islets: conservation of function with rodent islets and cultured beta cells. A, mouse islets were handpicked for a 2-h preincubation in KRB with 2.8 mm glucose ± latrunculin (LAT;10 μm) and then 2 h more under basal or stimulated conditions (20 mm glucose (Gluc)) with 10 islets/condition performed in duplicate. Three independent sets of mouse islets were combined; data are expressed as insulin secretion normalized to insulin content. *, p < 0.05, versus vehicle (VEH)-basal condition. B, human islets were isolated from cadaver pancreas at the ICR center and shipped for receipt within 48 h of isolation. Islets were allowed to recover for 3 h and then handpicked for preincubation for 1 h in KRB containing 2 mm glucose ± latrunculin (10 μm LAT) followed by 1 h of incubation under basal (2.8 mm glucose) or stimulated conditions (20 mm glucose). Data represent the average of three independent batches of human islets normalized for insulin content and expressed as percent vehicle-basal secretion set equal to 100% (*, p < 0.05, versus vehicle-basal condition). C, MIN6 cells were preincubated in glucose-free MKRBB containing vehicle or LAT for 2 h and then stimulated with 20 mm glucose for 5 min. Data represent the average of six independent sets of MIN6 cells normalized for protein content and expressed as percent vehicle-basal secretion; *, p < 0.02, versus vehicle-basal condition.
FIGURE 2.
FIGURE 2.
Latrunculin causes increased granule accumulation at the plasma membrane in the absence of glucose. A, transmission electron microscopy of MIN6 cells treated with vehicle (VEH) or latrunculin (LAT). Bar = 500 nm. B, latrunculin treatment mobilizes granules toward the plasma membrane compartment from the storage granule pool under basal conditions. MIN6 cells were left unstimulated, stimulated with 20 mm glucose for 5 min, or treated with 10 μm latrunculin for 2 h and then fixed and permeabilized for anti-insulin immunofluorescent (IF) confocal microscopy. Bar = 5 μm. C, MIN6 cells were treated with latrunculin or the DMSO vehicle for 2 h and then subfractionated, and 20 μg of protein from the PM and SG fractions were resolved on 12% SDS-PAGE for immunoblotting. Ponceau S staining was used to verify equal loading; syntaxin immunoblotting (IB) was used to validate PM fraction integrity in each fractionation. Data are representative of two independent experiments. D, optical density scanning quantitation (each set of fractions normalized to VEH control = 100%) of VAMP2; data shown are the average ± S.E. of three independent sets of fractions. Ponceau S staining was used to verify loading in SG and PM fractions each experiment.
FIGURE 3.
FIGURE 3.
Glucose-induced dissociation of F-actin from Syntaxin 1A and Syntaxin 4. MIN6 cells were preincubated in MKRBB for 2 h and stimulated with 20 mm glucose (Gluc) for 10, 30, and 60 min. Detergent cell lysates were prepared by harvesting in lysis buffer containing 1% Nonidet P-40 and n-octylglucoside. Cleared detergent cell lysates (2 mg of protein) were immunoprecipitated (IP) with mouse anti-Syntaxin 1 (A) or rabbit anti-Syntaxin 4 (B) for 2 h at 4 °C. Immunoprecipitates were resolved on 12% SDS-PAGE, and proteins were transferred to PVDF for immunoblotting (IB) with mouse anti-Syntaxin 1A or Syntaxin 4, anti-SNAP-25, and rabbit anti-actin. C, lysates prepared from cells pretreated for 2 h with vehicle (DMSO, VEH) or latrunculin (LAT) were used in immunoprecipitation reactions with anti-Syn4 antibody for subsequent immunoblotting for co-immunoprecipitation of actin. Parallel reactions including IgG immunoprecipitation reactions were included to control for nonspecific binding as demonstrated previously (Ref. ; not shown here). Data are representative of at least five independent co-immunoprecipitation experiments. Optical density scanning quantitation of actin association with syntaxin is shown directly below each of the three immunoprecipitation studies (each set of lysates normalized to basal = 1). Bars represent the average ± S.E. of 3-5 independent experiments.
FIGURE 4.
FIGURE 4.
Syntaxin 4 but not Syntaxin 1A binds directly to F-actin in vitro. Recombinant purified proteins were tested for direct F-actin binding in an in vitro sedimentation assay. Following incubation and centrifugation, F-actin and associated binding proteins are recovered in the pellet (P) fraction, and non-F-actin-binding proteins remain in the supernatant (S). Proteins were resolved on 10% SDS-PAGE and detected by Coomassie Blue staining. This reaction was determined to be at saturation for binding (data not shown). Data are representative of six independent experiments using four different preparations of proteins.
FIGURE 5.
FIGURE 5.
The region containing the HA-HB domains of Syntaxin 4 is necessary and sufficient to confer direct binding to F-actin. A, four C-terminal truncations and one N-terminal truncation of Syntaxin 4 were generated as GST fusion proteins for expression in E. coli and purification on glutathione-Sepharose. B, Coomassie Blue staining showed the expression of GST fusion Syntaxin 4 truncation proteins. C, thrombin-cleaved Syntaxin 4 N-terminal and C-terminal truncated fragments 1-194, 1-112, and 39-273 were prepared at a concentration of 1 mg/ml for use in the in vitro actin sedimentation assay. Both the supernatants (S) and pellets (P) were subjected to 15% SDS-PAGE for Coomassie Blue staining. Syn4 and Syn1A reactions were used as positive and negative controls, respectively, in each experiment (see Fig. 4). D, purified Syntaxin 4 C-terminal truncations 1-112 and 1-70 were prepared and tested in the actin sedimentation assay. Both the supernatants and the pellets were subjected to 18% SDS-PAGE followed by transfer to PVDF membranes for immunoblotting with rabbit anti-Syntaxin 4. Data are representative of 2-3 independent sets of proteins.
FIGURE 6.
FIGURE 6.
Expression of Syntaxin 4 residues 39-112 in MIN6 cells disrupted the endogenous F-actin-Syn4 association and increased glucose-stimulated secretion. A, the Ha-Hb region of Syntaxin 4 was fused to the C terminus of EGFP for expression in mammalian cells. This is a schematic representation of EGFP and EGFP-(39-112) proteins. B, MIN6 lysates expressing the ∼37-kDa EGFP-(39-112) protein were used for immunoprecipitation (IP) with Syntaxin 4 antibody and immunoblotted (IB) for co-precipitation of actin. EGFP (29 kDa) in lysates served as control; the actin/Syn4 ratio in EGFP-expressing lysates was set equal to 1 for normalization in each of four immunoprecipitation experiments (*, p < 0.05). C, MIN6 cells were co-transfected with EGFP or EGFP-(39-112) with human proinsulin to report effects upon glucose-stimulated human C-peptide secretion only from transfected cells. MIN6 cells were preincubated in glucose-free MKRBB followed by stimulation with 20 mm glucose for 30 min. Data represent the mean ± S.E. from five independent experiments normalized for total protein content. Data were normalized to the unstimulated level for each construct to obtain stimulation index (*, p < 0.05).
FIGURE 7.
FIGURE 7.
Like latrunculin treatment, expression of EGFP-(39-112) mobilizes granules into the PM compartment from the storage granule pool under basal conditions. MIN6 cells were transfected with EGFP or EGFP-(39-112) DNA and 48 h later were subfractionated, and 20 μg of protein each from the PM and SG fractions was resolved on 12% SDS-PAGE for immunoblotting (IB). Optical density scanning quantitation (each set of fractions normalized to EGFP control = 100%) of VAMP2. Data are shown as the average ± S.E. of four independent sets of fractions.β-Catenin immunodetection was used to verify PM fraction integrity and equal protein loading in each set of PM fractions; Ponceau S staining was used to verify loading in SG and PM fractions in each experiment.
FIGURE 8.
FIGURE 8.
EGFP-Syntaxin4 (39-112) expression increases Syntaxin 4 accessibility but not VAMP2 docking/fusion at Syntaxin 4 sites. A, MIN6 cells were treated with vehicle (VEH) or latrunculin (LAT) for 2 h in glucose-free MKRBB for preparation of cleared detergent lysates for use in GST-VAMP2 (soluble form) interaction assays. Lysate protein (2-3 mg) was combined with GST-VAMP2 linked to beads, and precipitated proteins were resolved on 10% SDS-PAGE for Syntaxin 4 immunoblotting (IB). Data are representative of four independent sets of cell lysates. B, MIN6 cells were left untransfected or were transfected with pEGFP or pEGFP-(39-112) DNA. Forty-eight h later, cells were preincubated in glucose-free MKRBB with or without DMSO or LAT added for 2 h and harvested for preparation of cleared detergent lysates. Lysate protein (2 mg/reaction) was incubated with anti-Syntaxin 4, and co-immunoprecipitated proteins (IP) were resolved on 12% SDS-PAGE for immunoblotting with anti-Syntaxin 4, anti-VAMP2, and anti-actin antibodies. Data are representative of 3-6 independent experiments.
FIGURE 9.
FIGURE 9.
Model depicting potential mechanism of regulation of insulin exocytosis by F-actin-Syntaxin 4 complexes under basal and glucose-stimulated conditions. Left panel, under basal conditions, F-actin binds to residues 39-112 of Syntaxin 4 (Ha-Hb domains), decreasing Syntaxin 4 accessibility to VAMP2 (consistent with Syntaxin 4 being in the closed conformation) and also restricting the flow of storage pool granules into the readily releasable pool at the plasma membrane. However, some Syntaxin 4 sites are occupied by granules, consistent with the requirement for Syntaxin 4 in first-phase insulin release (25). Right panel, glucose stimulation results in the localized reorganization of actin, which allows the storage pool of insulin granules to traffic to the plasma membrane and increases accessibility of VAMP2 to Syntaxin 4 sites (open conformation) to facilitate granule fusion and insulin release. An additional step triggered by glucose to “activate” VAMP2 docking is suggested (see question mark).

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