FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W4323344605> ?p ?o ?g. }

• W4323344605 abstract "Article Figures and data Abstract Editor's evaluation Introduction Results Discussion Materials and methods Data availability References Decision letter Author response Article and author information Metrics Abstract The Rab27 effectors are known to play versatile roles in regulated exocytosis. In pancreatic beta cells, exophilin-8 anchors granules in the peripheral actin cortex, whereas granuphilin and melanophilin mediate granule fusion with and without stable docking to the plasma membrane, respectively. However, it is unknown whether these coexisting effectors function in parallel or in sequence to support the whole insulin secretory process. Here, we investigate their functional relationships by comparing the exocytic phenotypes in mouse beta cells simultaneously lacking two effectors with those lacking just one of them. Analyses of prefusion profiles by total internal reflection fluorescence microscopy suggest that melanophilin exclusively functions downstream of exophilin-8 to mobilize granules for fusion from the actin network to the plasma membrane after stimulation. The two effectors are physically linked via the exocyst complex. Downregulation of the exocyst component affects granule exocytosis only in the presence of exophilin-8. The exocyst and exophilin-8 also promote fusion of granules residing beneath the plasma membrane prior to stimulation, although they differentially act on freely diffusible granules and those stably docked to the plasma membrane by granuphilin, respectively. This is the first study to diagram the multiple intracellular pathways of granule exocytosis and the functional hierarchy among different Rab27 effectors within the same cell. Editor's evaluation This study examines how Rab27 and its different effectors regulate insulin secretory granule exocytosis. Using single and double knockouts and rescue experiments, the work presents convincing data to characterize the relative hierarchy of the Rab27 effectors, their action with the exocyst, and their roles in distinct types of exocytosis. Overall, this is a valuable study that sheds new light on the regulation of distinct forms of secretory granule exocytosis. https://doi.org/10.7554/eLife.82821.sa0 Decision letter Reviews on Sciety eLife's review process Introduction Synaptic vesicles must be docked and primed on the plasma membrane prior to stimulation for successful neuronal transmission to occur within 1 ms after electrical stimulation (Südhof, 2013). In contrast, secretory granules in exocrine and endocrine cells initiate exocytosis at a timepoint that is 1000-fold later than this, even when the Ca2+ concentration is abruptly upregulated by caged-Ca2+ compounds (Kasai, 1999). Their physiological release of bioactive substances likewise occurs much later. For example, digestive enzymes and insulin are secreted from pancreas cells continuously over a range of minutes to hours during food intake and subsequent hyperglycemia. To support such slow and consecutive exocytosis, the exocytic pathway can be neither single nor linear, and the requisite steps for recruiting granules from the cell interior to the cell limits are therefore critical. In fact, total internal reflection fluorescence (TIRF) microscopy monitoring of prefusion behavior in living cells has revealed that insulin granules residing beneath the plasma membrane prior to stimulation, and those recruited from the cell interior after stimulation, fuse in parallel, with some variability in their ratios, during physiological glucose-stimulated insulin secretion (GSIS) (Kasai et al., 2008; Ohara-Imaizumi et al., 2004; Shibasaki et al., 2007). In many secretory cells, a greater number of granules are clustered in the actin cortex at the cell periphery and/or along the plasma membrane compared with other cytoplasmic areas. It has traditionally been considered that granules docked to the plasma membrane form a readily releasable pool, whereas those accumulated within the actin cortex form a reserve pool. However, this may be an oversimplification, considering that there is a gating system to prevent spontaneous or unlimited vesicle fusion in regulated exocytosis. In fact, multiple Rab27 effectors that are involved in intracellular granule trafficking show complex and differential effects on exocytosis (Izumi, 2021). For example, granuphilin (also known as exophilin-2 and Slp4) mediates stable granule docking to the plasma membrane but simultaneously prevents their spontaneous fusion by interacting with a fusion-incompetent, closed form of syntaxins (Gomi et al., 2005; Torii et al., 2002). Another effector, exophilin-8 (also known as MyRIP and Slac2-c), anchors secretory granules within the actin cortex (Bierings et al., 2012; Desnos et al., 2003; Fan et al., 2017; Huet et al., 2012; Mizuno et al., 2011; Nightingale et al., 2009), which is considered to have dual roles in accumulating granules at the cell periphery and in preventing their access to the plasma membrane. Although another effector, melanophilin (also known as exophilin-3 and Slac2-a), similarly captures melanosomes within the peripheral actin network in skin melanocytes (Hammer and Sellers, 2012), it mediates stimulus-induced granule mobilization and immediate fusion to the plasma membrane in pancreatic beta cells (Wang et al., 2020). It is unknown, however, just how the different Rab27 effectors coexisting in the same cell function in a coordinated manner to support the entire exocytic processes. Neither is it known whether multiple secretory pathways and/or rate-limiting steps exist in the final fusion stages of regulated granule exocytosis. To answer these questions, we must first seek to determine whether each effector functions in sequence or in parallel. In this study, we compared the exocytic profiles in beta cells lacking the two effectors with those in cells deficient in each single effector and in wild-type (WT) cells. This yielded valuable insights into the functional hierarchy and relationship among the exocytic steps in which individual effectors are involved. We also found that the exocyst, which universally functions in constitutive exocytosis (Wu and Guo, 2015), plays roles in the physical and functional connections between different Rab27 effectors. Results Melanophilin exclusively functions downstream of exophilin-8 to mediate the exocytosis of granules recruited from the actin cortex to the plasma membrane after stimulation In monolayer mouse pancreatic beta cells, insulin granules were unevenly distributed with accumulation in the actin cortex (Figure 1—figure supplement 1A). Because the Rab27 effectors, melanophilin and exophilin-8, are known to show affinities to the actin motors, myosin-Va and/or -VIIa (Hammer and Sellers, 2012, Izumi, 2021), these effectors are presumed to function on granules within this peripheral actin network. In fact, they showed a similar uneven distribution and were colocalized especially at the cell periphery (Figure 1—figure supplement 1B). To examine the functional relationship between these effectors, we generated melanophilin/exophilin-8 double-knockout (ME8DKO) mice by crossing exophilin-8-knockout (Exo8KO) mice (Fan et al., 2017) with melanophilin-knockout (MlphKO) mice (see ‘Materials and methods’). The doubly deficient beta cells displayed even distribution of insulin granules in the cytoplasm without accumulation at the cell periphery, in contrast to WT and MlphKO cells, but in a manner similar to that in Exo8KO cells (Figure 1A). Expression of exophilin-8, but not that of melanophilin, in ME8DKO cells at the endogenous level in WT cells redistributed them to the cell periphery (Figure 1B). This is consistent with the previous findings obtained from cells deficient in each single effector, which revealed that exophilin-8 is essential for granule accumulation within the actin cortex, whereas melanophilin is not (Fan et al., 2017; Wang et al., 2020). Figure 1 with 1 supplement see all Download asset Open asset Exophilin-8, but not melanophilin, accumulates insulin granules in the action cortex. (A) WT, MlphKO, Exo8KO, and ME8DKO beta cells were immunostained with anti-insulin antibody. A peripheral accumulation of insulin immunosignals was quantified under confocal microscopy: clustering of insulin immunosignals at least in one corner (upper) or along the plasma membrane (lower) were counted as positive. More than 100 cells were inspected in each of three independent experiments. Note that Exo8KO and ME8DKO cells do not show the peripheral accumulation of insulin granules in contrast to WT and MlphKO cells. (B) ME8DKO cells were infected by adenovirus expressing either HA-exophilin-8 or HA-melanophilin at the endogenous protein levels found in WT cells. LacZ was expressed in WT and ME8DKO cells as controls. The cell extracts were immunoblotted with the indicated antibodies (left). The ME8DKO cells expressing LacZ (upper), HA-exophilin-8 (middle), and HA-melanophilin (lower) were immunostained with anti-insulin and anti-HA antibodies (center), and a peripheral accumulation of insulin was quantified as in (A) (right). Insets represent higher magnification photomicrographs of a cell within the region outlined by frames. Note that expression of HA-exophilin-8, but not HA-melanophilin, rescues the peripheral granule accumulation in ME8DKO cells. Bars, 10 μm. ###p<0.001 by one-way ANOVA. Figure 1—source data 1 Uncropped blot images of Figure 1B. https://cdn.elifesciences.org/articles/82821/elife-82821-fig1-data1-v2.zip Download elife-82821-fig1-data1-v2.zip ME8DKO mice show glucose intolerance without changes in body weight or insulin sensitivity, as found in each singly deficient mice (Figure 2—figure supplement 1A). The cells deficient in melanophilin and/or exophilin-8 all showed decreases in GSIS compared with WT cells in both islet batch and perifusion assays, although the decreases in ME8DKO cells tended to be larger than those in MlphKO cells (Figure 2A and B, Figure 2—figure supplement 1B and C). We next monitored insulin granule exocytosis directly by TIRF microscopy in living cells expressing insulin fused with enhanced green fluorescent protein (EGFP). The numbers of visible granules under TIRF microscopy were not different among the four genotypes of cells (Figure 2C). Lack of the change in Exo8KO cells suggests that granules residing beneath the plasma membrane are not necessarily derived or supplied from granules anchored within the actin cortex. The amount of insulin released in the medium correlated well with a reduction in the total number of fusion events detected as a flash followed by diffusion of insulin-EGFP fluorescence during glucose stimulation in each of the mutant cells (Figure 2D, Figure 2—figure supplement 1D). We previously categorized fused insulin granules into three types depending on their prefusion behaviors (Kasai et al., 2008): those having been visible prior to stimulation, ‘residents’; those becoming visible during stimulation, ‘visitors’; and those invisible until fusion, ‘passengers.’ MlphKO cells with the genetic background of C57BL/6N mice showed a specific decrease in the passenger-type exocytosis, particularly in a later phase, compared with WT cells (Figure 2D and E), which is consistent with previous findings in parental leaden cells with the genetic background of C57BR/cdJ mice (Wang et al., 2020). In contrast, both Exo8KO and ME8DKO cells with the same C57BL/6N genetic background displayed decreases in both the resident and passenger types of exocytosis, while changes in the markedly less frequent visitor type were difficult to compare among the cells. Because there are no significant differences in exocytic phenotypes between Exo8KO and ME8DKO cells, melanophilin is thought to function downstream of exophilin-8. Considering the previously identified role of each effector in beta cells (Fan et al., 2017; Wang et al., 2020), the passenger-type exocytosis mediated by melanophilin via interactions with myosin-Va and syntaxin-4 appears to be derived from granules anchored in the actin cortex by exophilin-8. Figure 2 with 1 supplement see all Download asset Open asset Insulin secretory defects of beta cells doubly deficient in melanophilin and exophilin-8 are indistinguishable from those singly deficient in exophilin-8. (A) Islets isolated from WT, MlphKO, Exo8KO, and ME8DKO mice at 12–18 weeks of age were preincubated in 2.8 mmol/L low glucose (LG)-containing KRB buffer at 37℃ for 1 hr. They were then incubated in new LG buffer for 1 hr followed by 25 mmol/L high glucose (HG) buffer for 1 hr. The ratios of insulin secreted in the media to that left in the cell lysates (Figure 2—figure supplement 1B) are shown (left; n = 9 from three mice each). (B) Islets from age-matched mice (19- to 25-week-old) were perfused with 16.7 mmol/L glucose buffer for 30 min, and the ratios of insulin secreted in the media to that left in the cell lysates are plotted as in Figure 2—figure supplement 1C. The area under the curve (AUC) is shown (n = 9 from three mice each). (C–E) A monolayer of islet cells from the above four kinds of mice were infected with adenovirus encoding insulin-EGFP and were observed by total internal reflection fluorescence (TIRF) microscopy (C, left). The numbers of visible granules were manually counted for WT (n = 10 cells from three mice), MlphKO (n = 9 from three mice), Exo8KO (n = 5 cells from three mice), and ME8DKO (n = 8 cells from three mice) cells (C, right). Insulin granule fusion events in response to 25 mmol/L glucose for 20 min were counted as in Figure 2—figure supplement 1D for WT (n = 21 cells from five mice), MlphKO (n = 10 from three mice), Exo8KO (n = 12 cells from three mice), and ME8DKO (n = 12 cells from three mice) cells. The observed fusion events were categorized into three types: residents, visitors, and passengers (D). Summary of the three modes of fusion events (E) in the first phase (left, 0–5 min) and the second phase (right, 5–20 min). Note that the decrease in insulin exocytosis in ME8DKO cells is greater than that in MlphKO cells, specifically due to the decrease in the resident-type exocytosis. Bar, 10 μm. #p<0.05, ##p<0.01, ###p<0.001 by one-way ANOVA. Exophilin-8 and melanophilin form a complex via the exocyst To investigate the molecular basis by which exophilin-8 and melanophilin sequentially promote the passenger-type exocytosis, we first examined the expression level of each effector in cells lacking the other effector. Although the expression of exophilin-8 was not affected in MlphKO cells, that of melanophilin was decreased to 59.8 ± 9.0% (n = 4) in Exo8KO cells compared with WT cells (Figure 3A). However, this decrease should not affect GSIS in Exo8KO cells because a similar level of expression (65.4 ± 4.7%) in heterozygous MlphKO cells did not reduce it (Figure 3—figure supplement 1). We further found that the exophilin-8 and melanophilin expressed in the pancreatic beta cell line MIN6 form a complex (Figure 3B). These findings suggest that the protein stability of melanophilin partially depends on its interaction with exophilin-8, which is consistent with the model that melanophilin functions downstream of exophilin-8. However, they do not interact when expressed in HEK293A cells (Figure 3—figure supplement 2A), suggesting that they do not interact directly. The two effectors have been shown to interact with different proteins, except for Rab27a, in beta cells (Fan et al., 2017; Wang et al., 2020). The exophilin-8 mutant that loses its binding activity to RIM-BP2, and the melanophilin mutants that lose their binding activity to Rab27a, myosin-Va, or actin, all preserved their binding activity to the other WT effector in MIN6 cells (Figure 3—figure supplement 2B). To identify unknown intermediate proteins, we individually expressed Myc-TEV-FLAG (MEF)-tagged exophilin-8 and melanophilin in MIN6 cells, and we analyzed the proteins in each of the anti-FLAG immunoprecipitates using a liquid chromatography-tandem mass spectrometry (LC-MS/MS) (Figure 3—figure supplement 3). As a result, we identified the exocyst complex components, SEC8, SEC10, and EXO70, in both immunoprecipitates, which is consistent with the previous finding that exophilin-8 interacts with SEC6 and SEC8 in INS-1 832/13 cells (Goehring et al., 2007). We confirmed the endogenous interactions among melanophilin, exophilin-8, SEC6, and SEC10 in MIN6 cells (Figure 3C). The exocyst forms an evolutionarily conserved heterooctameric protein complex (Wu and Guo, 2015). To identify the components responsible for the interaction with two Rab27 effectors, we performed a visible immunoprecipitation (VIP) assay, which permits examination of large numbers of protein combinations and complicated one-to-many or many-to-many protein interactions simultaneously (Katoh et al., 2015). As a result, we found that exophilin-8 and melanophilin immediately, if not directly, interact with SEC8 and EXO70, respectively (Figure 3D). Although the exocyst components were found in both immunoprecipitates of melanophilin and exophilin-8, other interacting proteins such as RIM-BP2, RIM2, myosin-VIIa, and myosin-Va were identified in only one of the immunoprecipitates (Figure 3E), suggesting that the majority of each effector form a distinct complex in cells. In addition, although we previously reported that exophilin-8 primarily interacts with myosin-Vlla displaying a molecular mass of ~170 kDa in INS-1 832/13 rat cells (Fan et al., 2017), it interacted with mysosin-VIIa with an authentic mass of ~260 kDa in MIN6 mouse cells, which may reflect a species difference between these beta cell lines. Figure 3 with 3 supplements see all Download asset Open asset Exophilin-8 and melanophilin interact through the exocyst in beta cells. (A) Islet extracts (40 μg) from WT, MlphKO, Exo8KO, and ME8DKO mice were immunoblotted with antibodies toward the indicated proteins (left). Protein expression levels were quantified by densitometric analyses from 3 to 5 independent preparations (right). (B) MIN6 cells were infected with adenoviruses encoding control LacZ, mCherry-exophilin-8, and/or FLAG-melanophilin. After 2 days, the cell extracts underwent immunoprecipitation with mCherry nanobody. The immunoprecipitates (IP), as well as the 1% extracts (Input), were immunoblotted with anti-FLAG and anti-red fluorescent protein (RFP) antibodies. (C) MIN6 cell extracts were immunoprecipitated with rabbit anti-melanophilin antibody or control IgG, and the immunoprecipitates were immunoblotted with antibodies toward the indicated proteins. (D) HEK293A cells cultured in 10 cm dishes were transfected with mCherry-fused, exophilin-8 (left) or melanophilin (right) with the indicated EGFP-fused exocyst components. After 48 hr, the cell lysates were subjected to immunoprecipitation with mCherry-nanobody-bound glutathione-Sepharose beads. EGFP and mCherry fluorescence on the precipitated beads was observed by confocal microscopy. (E) MIN6 cells expressing FLAG-tagged, melanophilin or exophlin-8 were immunoprecipitated with anti-FLAG antibody, and the immunoprecipitates were immunoblotted with antibodies toward the indicated proteins. Note that the exocyst complex components exist in both immunoprecipitates, whereas RIM-BP2, RIM2, myosin-VIIa, and myosin-Va largely exist only in one of them. Bars, 100 μm. ###p<0.001 by one-way ANOVA. Figure 3—source data 1 Uncropped blot images of Figure 3A, B, C and E. https://cdn.elifesciences.org/articles/82821/elife-82821-fig3-data1-v2.zip Download elife-82821-fig3-data1-v2.zip The exocyst functions only in the presence of exophilin-8 In mammalian cells, the exocyst complex is assembled after separate formation of the subcomplex 1 (SEC3, SEC5, SEC6, SEC8) and the subcomplex 2 (SEC10, SEC15, EXO70, EXO84) (Ahmed et al., 2018). Because exophilin-8 and melanophilin immediately bind SEC8 and EXO70, respectively (Figure 3D), it is conceivable that the holo-exocyst links the two effectors. Previous VIP assays detected interactions between SEC8 or SEC3 in the subcomplex 1 and SEC10 in the subcomplex 2 (Katoh et al., 2015). Therefore, silencing of SEC10 is expected to efficiently disrupt the formation of the holo-exocyst. However, SEC10 knockdown by specific siRNAs did not affect the granule localization of exophilin-8 or melanophilin (Figure 4—figure supplement 1). Furthermore, there was no effect on the peripheral accumulation of insulin granules in contrast to the case in exophilin-8-deficient cells (Figure 1A). However, SEC10 knockdown significantly decreased the colocalization ratio of melanophilin, but not that of exophilin-8, with SEC6, another exocyst component that is colocalized with insulin granules in MIN6 cells (Tsuboi et al., 2005; Figure 4A). Considering that the two effectors specifically interact with the component of different subcomplexes (Figure 3D), it is reasonable that silencing of SEC10 that disrupts the subcomplex 2 dissociates melanophilin from exophilin-8 associated with the subcomplex 1. To obtain further evidence that the two effectors associate through the exocyst, we downregulated SEC8 that is thought to interact with exophilin-8 (Figure 4—figure supplement 1) and found that SEC8 knockdown markedly disrupted the complex formation (Figure 4B). These findings indicate that the exocyst physically connects the two effectors on the same granule. Figure 4 with 1 supplement see all Download asset Open asset Knockdown of the exocyst component disrupts the interaction between melanophilin and exophilin-8. (A) HA-exophilin-8 and HA-melanophilin were expressed in Exo8KO and MlphKO monolayer beta cells, respectively, at the endogenous levels in WT cells as described in Figure 1B. They were then transfected with control siRNA or siRNA against SEC10 #11 or #12, as shown in Figure 4—figure supplement 1A. After fixation, the cells were coimmunostained with anti-HA and anti-SEC6 antibodies and were observed by confocal microscopy (left). Fluorescent intensity profiles along the indicated line of SEC6 and either HA-exophilin-8 or HA-melanophilin are shown (center). Colocalization was quantified by Pearson’s correlation coefficient (right, n = 18–26 cells from two mice each). Note that SEC10 knockdown (KD) induces dissociation of melanophilin, but not exophilin-8, from SEC6 (black arrowheads). (B) MIN6 cells were transfected with control siRNA or siRNA against SEC8 #12, as shown in Figure 4—figure supplement 1A, and the cell extracts were subjected to immunoprecipitation with control IgG or anti-melanophilin antibody. The immunoprecipitates (IP), as well as the 1% extracts (Input), were immunoblotted with anti-exophilin-8 and anti-melanophilin antibodies. Bars, 10 μm. ###p<0.001 by one-way ANOVA. Figure 4—source data 1 Uncropped blot images of Figure 4B. https://cdn.elifesciences.org/articles/82821/elife-82821-fig4-data1-v2.zip Download elife-82821-fig4-data1-v2.zip SEC10 knockdown markedly decreased GSIS in WT cells, but induced no further decrease of GSIS in Exo8KO cells, which was already decreased by half compared with WT cells (Figure 5A, Figure 5—figure supplement 1). These findings suggest that the exocyst functions only in the presence of exophilin-8. As shown in Figure 2C, TIRF microscopy of Exo8KO cells expressing insulin-EGFP revealed no decrease in the number of visible granules (Figure 5B). Similarly, SEC10 knockdown does not affect the number of visible granules in either WT or Exo8KO cells. Consistent with insulin secretion assays (Figure 5A), it induces a marked decrease in glucose-stimulated fusion events in WT cells, but no additional decrease in Exo8KO cells (Figure 5C). Categorization by prefusion behavior revealed decreases in both resident and passenger types of exocytosis in WT cells, which phenocopied Exo8KO cells without SEC10 knockdown (Figure 2D). Again, SEC10 knockdown had no effects on either type of exocytosis in Exo8KO cells. The holo-exocyst appears to function with exophilin-8 in the same pathway because SEC10 knockdown is expected to preserve the interaction between exophilin-8 and the subcomplex 1, as shown in Figure 4A. Taken together, it seems that exophilin-8 and the exocyst mediate the passenger-type exocytosis when they interact with melanophilin on the same granule. Figure 5 with 1 supplement see all Download asset Open asset The exocyst affects insulin granule exocytosis only in the presence of exophilin-8. (A–C) WT or Exo8KO mouse islet cells were twice transfected with control siRNA or siRNA against SEC10 #11 or #12, as shown in Figure 4—figure supplement 1A, and were plated in a 24-well plate (A) or glass base dish (B, C). (A) The transfected monolayer cells were incubated for 1 hr in KRB buffer containing 2.8 mmol/L glucose and were then stimulated for 1 hr in the same buffer or in buffer containing 25 mmol/L glucose. Insulin levels secreted in the media and left in the cell lysates were measured (Figure 5—figure supplement 1), and their ratios are shown (n = 3 from three mice each). (B) The control or SEC10 knockdown (KD) cells (control siRNA-treated WT cells, n = 10 from three mice; SEC10 siRNA#11 or #12-treated WT cells, n = 11 from three mice; control siRNA-treated Exo8KO cells, n = 12 from three mice; SEC10 siRNA#11 or #12-treated Exo8KO cells, n = 12 from three mice) were infected with adenovirus encoding insulin-EGFP and were observed by total internal reflection fluorescence (TIRF) microscopy (left). The numbers of visible granules were manually counted (right). (C) The transfected cells (control siRNA-treated WT cells, n = 22 from three mice; SEC10 siRNA#11-treated WT cells, n = 8 from three mice; SEC10 siRNA#12-treated WT cells, n = 10 from three mice; control siRNA-treated Exo8KO cells, n = 15 from three mice; SEC10 siRNA#11-treated Exo8KO cells, n = 9 from three mice; SEC10 siRNA#12-treated Exo8KO cells, n = 7 from three mice) were infected with adenovirus encoding insulin-EGFP, and fusion events in response to 25 mmol/L glucose for 20 min were counted and categorized under TIRF microscopy as described in Figure 2D. Bar, 10 μm. #p<0.05, ##p<0.01, ###p<0.001 by one-way ANOVA versus control siRNA-treated WT cells. Exophilin-8 promotes the exocytosis of granules residing beneath the plasma membrane only in the presence of granuphilin As shown in Figure 2D, exophilin-8 deficiency also affects the resident-type exocytosis. Another Rab27 effector, granuphilin, is thought to be deeply involved in this type of exocytosis because beta cells lacking granuphilin display very few granules directly attached to the plasma membrane under electron microscopy (Gomi et al., 2005). Despite this docking defect, these cells display a marked increase in granule exocytosis, possibly because granuphilin interacts with and stabilizes syntaxins in a fusion-incompetent, closed form (Torii et al., 2002). To explore the functional relationship between the two effectors in this type of exocytosis, we generated granuphilin/exophilin-8 double-knockout (GE8DKO) mice. The GE8DKO cells also exhibited an increase in GSIS compared with Exo8KO cells (Figure 6A, Figure 6—figure supplement 1A), which indicates that a significant number of granules fuse efficiently without exophilin-8 and granuphilin, thus without prior capture in the actin cortex and stable docking to the plasma membrane. TIRF microscopy of these cells expressing insulin-EGFP revealed that, although the number of visible granules was markedly decreased in granuphilin-knockout (GrphKO) cells compared with WT cells, as expected, it was not further decreased in GE8DKO cells compared with GrphKO cells (Figure 6B). The number of fusion events during glucose stimulation under TIRF microscopy was correlated with the amount of insulin released in the medium in each mutant cell (Figure 6C). All the types of exocytosis were increased in GrphKO cells, suggesting that the absence of granuphilin facilitates granule easier access to the fusion-competent machinery on the plasma membrane. Simultaneous absence of exophilin-8 induced strikingly differential effects on each type of exocytosis in GE8DKO cells: the increases in the passenger and visitor types were reduced to the level found in Exo8KO cells, whereas the increase in the resident type was completely unaffected. The former finding is consistent with the view that the passenger and visitor types of exocytosis are derived from granules captured in the actin cortex by exophilin-8. However, the latter finding indicates that, at least in the absence of granuphilin, exophilin-8 is dispensable for the exocytosis of granules already residing beneath the plasma membrane prior to stimulation. Figure 6 with 1 supplement see all Download asset Open asset Exophilin-8 deficiency strongly inhibits the resident-type exocytosis from granuphilin-positive, stably docked granules. (A) Islets isolated from WT, Exo8KO, GrphKO, and GE8DKO mice at 12–17 weeks of ages were preincubated in low glucose (LG) KRB buffer for 1 hr. They were then incubated in another LG buffer for 30 min followed by high glucose (HG) buffer for 30 min. The ratios of insulin secreted in the media of that left in the cell lysates (Figure 6—figure supplement 1A) are shown as in Figure 2A (n = 12 from four mice each). (B) A monolayer of islet cells from the above four kinds of mice were infected with adenovirus encoding insulin-EGFP and were observed by total internal reflection fluorescence (TIRF) microscopy (left). The numbers of visible granules were manually counted for WT (n = 15 cells from four mice), Exo8KO (n = 11 cells from three mice), GrphKO (n = 14 cells from four mice), and GE8DKO (n = 17 cells from four mi" @default.
• W4323344605 created "2023-03-08" @default.
• W4323344605 creator A5059670850 @default.
• W4323344605 creator A5089594621 @default.
• W4323344605 date "2022-09-18" @default.
• W4323344605 modified "2023-09-26" @default.
• W4323344605 title "Decision letter: Functional hierarchy among different Rab27 effectors involved in secretory granule exocytosis" @default.
• W4323344605 doi "https://doi.org/10.7554/elife.82821.sa1" @default.
• W4323344605 hasPublicationYear "2022" @default.
• W4323344605 type Work @default.
• W4323344605 citedByCount "0" @default.
• W4323344605 crossrefType "peer-review" @default.
• W4323344605 hasAuthorship W4323344605A5059670850 @default.
• W4323344605 hasAuthorship W4323344605A5089594621 @default.
• W4323344605 hasBestOaLocation W43233446051 @default.
• W4323344605 hasConcept C151730666 @default.
• W4323344605 hasConcept C162324750 @default.
• W4323344605 hasConcept C175969161 @default.
• W4323344605 hasConcept C185592680 @default.
• W4323344605 hasConcept C31170391 @default.
• W4323344605 hasConcept C34447519 @default.
• W4323344605 hasConcept C49039625 @default.
• W4323344605 hasConcept C49760210 @default.
• W4323344605 hasConcept C51785407 @default.
• W4323344605 hasConcept C55493867 @default.
• W4323344605 hasConcept C86803240 @default.
• W4323344605 hasConcept C95444343 @default.
• W4323344605 hasConceptScore W4323344605C151730666 @default.
• W4323344605 hasConceptScore W4323344605C162324750 @default.
• W4323344605 hasConceptScore W4323344605C175969161 @default.
• W4323344605 hasConceptScore W4323344605C185592680 @default.
• W4323344605 hasConceptScore W4323344605C31170391 @default.
• W4323344605 hasConceptScore W4323344605C34447519 @default.
• W4323344605 hasConceptScore W4323344605C49039625 @default.
• W4323344605 hasConceptScore W4323344605C49760210 @default.
• W4323344605 hasConceptScore W4323344605C51785407 @default.
• W4323344605 hasConceptScore W4323344605C55493867 @default.
• W4323344605 hasConceptScore W4323344605C86803240 @default.
• W4323344605 hasConceptScore W4323344605C95444343 @default.
• W4323344605 hasLocation W43233446051 @default.
• W4323344605 hasOpenAccess W4323344605 @default.
• W4323344605 hasPrimaryLocation W43233446051 @default.
• W4323344605 hasRelatedWork W1570158828 @default.
• W4323344605 hasRelatedWork W2056572393 @default.
• W4323344605 hasRelatedWork W2205678869 @default.
• W4323344605 hasRelatedWork W2286694113 @default.
• W4323344605 hasRelatedWork W2584098703 @default.
• W4323344605 hasRelatedWork W3028785900 @default.
• W4323344605 hasRelatedWork W3111888726 @default.
• W4323344605 hasRelatedWork W3135817955 @default.
• W4323344605 hasRelatedWork W4235264942 @default.
• W4323344605 hasRelatedWork W1586809413 @default.
• W4323344605 isParatext "false" @default.
• W4323344605 isRetracted "false" @default.
• W4323344605 workType "peer-review" @default.