Abstract The SM-protein Munc18 and its binding partner syntaxin are crucial for two distinct steps of regulated exocytosis, docking of secretory granules at the release site, and priming reactions that prepare docked granules for Ca 2+ dependent fusion. Both proteins cluster simultaneously at the docking site seconds after a granule arrives at the plasma membrane. To study the mechanisms of these separate Munc18 functions, we generated a Munc18-1 knockout (1KO) and a double knockout of both Munc18-1 and Munc18-2 (DKO) in insulin-secreting cells, and quantified granule docking, exocytosis, and protein clustering at the docking site by TIRF imaging and capacitance measurements. In the 1KO, priming and exocytosis were lost, but granule docking remained intact. In the DKO, both priming and granule docking were impaired. Expression of either Munc18-1 or -2 rescued these defects, as did expression of mutants with weakened binding to syntaxin (EA and EK mutants). Munc18 clustered at granule docking sites, with apparent affinities decreasing in the order Munc18-1 > Munc18-1EA > Munc18-1EK > Munc18-2. Clustering of syntaxin-1 and − 3 at granules was impaired in both knockout lines. Finally, single molecule and imaging fluorescence recovery after photobleaching (FRAP) revealed that Syntaxin and Munc18 molecules were bound to each other, regardless of whether the proteins were clustered at a granule or not. We conclude that that Munc18-1 and − 2 molecules co-cluster with syntaxin at the release site and support docking and priming, and different affinities of the isoforms for the granule docking site contribute to the observed functional preferences.

Somatostatin secretion from pancreatic δ-cells inhibits nearby α-and β-cells, and tunes the body’s glycemic set-point. The role of δ-cells in diabetes remains unclear, in part due to the difficulty separating intrinsic regulation from intra-islet paracrine effects. Here we compared the function of isolated δ-cells of cadaveric non-diabetic and type-2 diabetic donors, by single cell TIRF-microscopy and electrophysiology. Elevated glucose stimulated exocytosis of somatostatin, which was further amplified by glucagon, exendin-4, or forskolin, independent of diabetic status. GABA enhanced exocytosis and electrical activity, while insulin had no effect. Adrenaline and somatostatin strongly inhibited δ-cell activity, leading to autocrine feedback inhibition of somatostatin exocytosis. In type-2 diabetes, δ-cell inhibition by somatostatin and adrenaline was lost, together with a marked reduction in somatostatin receptor (SSTR2) surface expression. We further show that resistance to somatostatin leads to hyperactive δ-cells in type-2 diabetes, and propose that this mechanism contributes to defective blood glucose control.

Munc13 proteins are essential for regulated exocytosis in neurons and endocrine cells. They consist of an elongated MUN domain that templates SNARE complex formation during priming, flanked by regulatory membrane-associated C1 and C2 domains. Here, we show, using quantitative high-resolution imaging, that priming of insulin granules coincides with recruitment of on average six copies of Munc13 to individual docked granules, similar to estimates of SNARE complexes formed during exocytosis. Intracellular Ca2+- or lipid-signaling accelerates granule priming by promoting C2B-dependent translocation of Munc13 to the plasma membrane, followed by slower (tens of seconds) C2A-domain dependent accumulation at docked granules. Exocytosis in human β-cells also exhibits rapid Ca2+-dependent short-term facilitation that involves Ca2+/C2B-dependent activation of Munc13 but not further accumulation at the release site. Thus, Munc13 controls secretory granule release probability by two separate C2B-dependent mechanisms that affect its recruitment to the release site and its subsequent activation by Ca2+.

ARTICLE HIGHLIGHTS:Vesicle-associated membrane protein 8 (VAMP8) localizes to endosomal vesicles and mediates their exocytosis in pancreatic β-cells. VAMP8-dependent vesicle fusion delivers glucagon-like peptide 1 receptor and GLUT2 to the plasma membrane. VAMP8 overexpression inhibits insulin granule exocytosis. "Newcomer" exocytosis likely involves endosomal compartments, not insulin granules.

Activity recognition in live-cell imaging is labor-intensive and requires significant human effort. Existing automated analysis tools are largely limited in versatility. We present the Intelligent Vesicle Exocytosis Analysis (IVEA) platform, an ImageJ plugin for automated, reliable analysis of fluorescence-labeled vesicle fusion events and other burst-like activity. IVEA includes three specialized modules for detecting: (1) synaptic transmission in neurons, (2) single-vesicle exocytosis in any cell type, and (3) nano-sensor-detected exocytosis. Each module uses distinct techniques, including deep learning, allowing the detection of rare events often missed by humans at a speed estimated to be approximately 60 times faster than manual analysis. IVEA’s versatility can be expanded by refining or training new models via an integrated interface. With its impressive speed and remarkable accuracy, IVEA represents a seminal advancement in exocytosis image analysis and other burst-like fluorescence fluctuations applicable to a wide range of microscope types and fluorescent dyes.

G protein-coupled receptors (GPCRs) are integral membrane proteins which closely interact with their plasma membrane lipid microenvironment. Cholesterol is a lipid enriched at the plasma membrane with pivotal roles in the control of membrane fluidity and maintenance of membrane microarchitecture, directly impacting on GPCR stability, dynamics, and function. Cholesterol extraction from pancreatic beta cells has previously been shown to disrupt the internalisation, clustering, and cAMP responses of the glucagon-like peptide-1 receptor (GLP-1R), a class B1 GPCR with key roles in the control of blood glucose levels via the potentiation of insulin secretion in beta cells and weight reduction via the modulation of brain appetite control centres. Here, we unveil the detrimental effect of a high cholesterol diet on GLP-1R-dependent glucoregulation in vivo, and the improvement in GLP-1R function that a reduction in cholesterol synthesis using simvastatin exerts in pancreatic islets. We next identify and map sites of cholesterol high occupancy and residence time on active vs inactive GLP-1Rs using coarse-grained molecular dynamics (cgMD) simulations, followed by a screen of key residues selected from these sites and detailed analyses of the effects of mutating one of these, Val229, to alanine on GLP-1R-cholesterol interactions, plasma membrane behaviours, clustering, trafficking and signalling in INS-1 832/3 rat pancreatic beta cells and primary mouse islets, unveiling an improved insulin secretion profile for the V229A mutant receptor. This study (1) highlights the role of cholesterol in regulating GLP-1R responses in vivo; (2) provides a detailed map of GLP-1R - cholesterol binding sites in model membranes; (3) validates their functional relevance in beta cells; and (4) highlights their potential as locations for the rational design of novel allosteric modulators with the capacity to fine-tune GLP-1R responses.

AIMS/HYPOTHESIS:Regulatory factor X 6 (RFX6) is crucial for pancreatic endocrine development and differentiation. The RFX6 variant p.His293LeufsTer7 is significantly enriched in the Finnish population, with almost 1:250 individuals as a carrier. Importantly, the FinnGen study indicates a high predisposition for heterozygous carriers to develop type 2 and gestational diabetes. However, the precise mechanism of this predisposition remains unknown. METHODS:To understand the role of this variant in beta cell development and function, we used CRISPR technology to generate allelic series of pluripotent stem cells. We created two isogenic stem cell models: a human embryonic stem cell model; and a patient-derived stem cell model. Both were differentiated into pancreatic islet lineages (stem-cell-derived islets, SC-islets), followed by implantation in immunocompromised NOD-SCID-Gamma mice. RESULTS:Stem cell models of the homozygous variant RFX6-/- predictably failed to generate insulin-secreting pancreatic beta cells, mirroring the phenotype observed in Mitchell-Riley syndrome. Notably, at the pancreatic endocrine stage, there was an upregulation of precursor markers NEUROG3 and SOX9, accompanied by increased apoptosis. Intriguingly, heterozygous RFX6+/- SC-islets exhibited RFX6 haploinsufficiency (54.2% reduction in protein expression), associated with reduced beta cell maturation markers, altered calcium signalling and impaired insulin secretion (62% and 54% reduction in basal and high glucose conditions, respectively). However, RFX6 haploinsufficiency did not have an impact on beta cell number or insulin content. The reduced insulin secretion persisted after in vivo implantation in mice, aligning with the increased risk of variant carriers to develop diabetes. CONCLUSIONS/INTERPRETATION:Our allelic series isogenic SC-islet models represent a powerful tool to elucidate specific aetiologies of diabetes in humans, enabling the sensitive detection of aberrations in both beta cell development and function. We highlight the critical role of RFX6 in augmenting and maintaining the pancreatic progenitor pool, with an endocrine roadblock and increased cell death upon its loss. We demonstrate that RFX6 haploinsufficiency does not affect beta cell number or insulin content but does impair function, predisposing heterozygous carriers of loss-of-function variants to diabetes. DATA AVAILABILITY:Ultra-deep bulk RNA-seq data for pancreatic differentiation stages 3, 5 and 7 of H1 RFX6 genotypes are deposited in the Gene Expression Omnibus database with accession code GSE234289. Original western blot images are deposited at Mendeley ( https://data.mendeley.com/datasets/g75drr3mgw/2 ).

Vesicle exocytosis is a fundamental component of intercellular communication, in all organisms. It has been studied for decades, using various imaging tools. Nevertheless, exocytosis research is still limited by the lack of reliable automated analysis procedures. To address this, we developed the Intelligent Vesicle Exocytosis Analysis Platform (IVEA), a nearly universal solution for analyzing exocytosis acquired with live cell imaging. IVEA is applicable to a wide variety of experimental model systems, microscopes and reporter fluorophores. IVEA combines state-of-the-art deep-learning and computer vision regimes to enable fully automated analysis of large data. IVEA runs as a FIJI plugin and does not require prior training or human intervention. IVEA is 60 times faster than manual analysis and is able to detect rare events often missed by the human eye. Overall, IVEA represents a breakthrough in the analysis of cellular secretory mechanisms and has a transformative potential for the exocytosis imaging field. ### Competing Interest Statement The authors have declared no competing interest.

Defects in insulin processing and granule maturation are linked to pancreatic beta-cell failure during type 2 diabetes (T2D). Phosphatidylinositol transfer protein alpha (PITPNA) stimulates activity of phosphatidylinositol (PtdIns) 4-OH kinase to produce sufficient PtdIns-4-phosphate (PtdIns-4-P) in the trans-Golgi network to promote insulin granule maturation. PITPNA in beta-cells of T2D human subjects is markedly reduced suggesting its depletion accompanies beta-cell dysfunction. Conditional deletion of Pitpna in the beta-cells of Ins-Cre, Pitpnaflox/flox mice leads to hyperglycemia resulting from decreasing glucose-stimulated insulin secretion (GSIS) and reducing pancreatic beta-cell mass. Furthermore, PITPNA silencing in human islets confirms its role in PtdIns-4-P synthesis and leads to impaired insulin granule maturation and docking, GSIS, and proinsulin processing with evidence of ER stress. Restoration of PITPNA in islets of T2D human subjects reverses these beta-cell defects and identify PITPNA as a critical target linked to beta-cell failure in T2D.

Phosphatidylinositol(4,5)bisphosphate (PI(4,5)P2) is an important signaling phospholipid that is required for regulated exocytosis and some forms of endocytosis. The two processes share a topologically similar pore structure that connects the vesicle lumen with the outside. Widening of the fusion pore during exocytosis leads to cargo release, while its closure initiates kiss&run or cavicapture endocytosis. We show here, using live-cell total internal reflection fluorescence (TIRF) microscopy of insulin granule exocytosis, that transient accumulation of PI(4,5)P2 at the release site recruits components of the endocytic fission machinery and stalls the late fusion pore expansion that is required for peptide release. The absence of clathrin differentiates this mechanism from clathrin-mediated endocytosis. Knockdown of phosphatidylinositol-phosphate-5-ki-nase-1c or optogenetic recruitment of 5-phosphatase reduces PI(4,5)P2 transients and accelerates fusion pore expansion, suggesting that acute PI(4,5)P2 synthesis is involved. Thus, local phospholipid signaling in-hibits fusion pore expansion and peptide release through an unconventional endocytic mechanism.

Hormones and neurotransmitters are released from (neuro)endocrine cells by regulated exocytosis of secretory granules. During exocytosis, the granule membrane fuses with the plasma membrane, which allows release of the stored content into the bloodstream or the surrounding tissue. Here, we give a detailed description of two complementary methods to observe and quantify exocytosis in single cells: high-resolution TIRF microscopy and patch-clamp capacitance recordings. Precise stimulation of exocytosis is achieved by local pressure application or voltage-clamp depolarizations. While the chapter is focused on insulin-secreting cells as an accessible and disease-relevant model system, the methodology is applicable to a wide variety of secretory cells including chromaffin and PC12 cells.

Protein function can be modulated by phase transitions in their material properties, which can range from liquid- to solid-like; yet, the mechanisms that drive these transitions and whether they are important for physiology are still unknown. In the model plant Arabidopsis, we show that developmental robustness is reinforced by phase transitions of the plasma membrane-bound lipid-binding protein SEC14-like. Using imaging, genetics, and in vitro reconstitution experiments, we show that SEC14-like undergoes liquid-like phase separation in the root stem cells. Outside the stem cell niche, SEC14-like associates with the caspase-like protease separase and conserved microtubule motors at unique polar plasma membrane interfaces. In these interfaces, SEC14-like undergoes processing by separase, which promotes its liquid-to-solid transition. This transition is important for root development, as lines expressing an uncleavable SEC14-like variant or mutants of separase and associated microtubule motors show similar developmental phenotypes. Furthermore, the processed and solidified but not the liquid form of SEC14-like interacts with and regulates the polarity of the auxin efflux carrier PINFORMED2. This work demonstrates that robust development can involve liquid-to-solid transitions mediated by proteolysis at unique plasma membrane interfaces.

The primary cilium is an organelle present in most adult mammalian cells that is considered as an antenna for sensing the local microenvironment. Here, we use intact mouse pancreatic islets of Langerhans to investigate signaling properties of the primary cilium in insulin-secreting β-cells. We find that GABAB1 receptors are strongly enriched at the base of the cilium, but are mobilized to more distal locations upon agonist binding. Using cilia-targeted Ca2+ indicators, we find that activation of GABAB1 receptors induces selective Ca2+ influx into primary cilia through a mechanism that requires voltage-dependent Ca2+ channel activation. Islet β-cells utilize cytosolic Ca2+ increases as the main trigger for insulin secretion, yet we find that increases in cytosolic Ca2+ fail to propagate into the cilium, and that this isolation is largely due to enhanced Ca2+ extrusion in the cilium. Our work reveals local GABA action on primary cilia that involves Ca2+ influx and depends on restricted Ca2+ diffusion between the cilium and cytosol.

Regulatory factor X 6 (RFX6) is indispensable for pancreatic endocrine development and differentiation. The RFX6 protein-truncating variant p.His293LeufsTer7 is significantly enriched in the Finnish population with almost 1:250 individuals as a carrier. Importantly, the FinnGen study indicates a high predisposition for heterozygous carriers to develop type 2 diabetes (T2D) and gestational diabetes. To understand the role of this variant in β-cell development and function, we generated allelic series of isogenic pluripotent stem cell models and directed them into pancreatic islet lineages (SC-islets). Expectedly, in-vitro models of the homozygous RFX6−/− variant failed to generate pancreatic endocrine cells, recapitulating the phenotype in Mitchell-Riley syndrome. Notably, heterozygous RFX6+/− derived SC-islets showed reduced β-cell maturation markers and calcium oscillations, resulting in defective insulin secretion, without affecting β-cell number or insulin content. The reduced insulin secretion is sustained during in-vivo implantation studies, consistent with the susceptibility of the carriers to develop diabetes. Teaser Modeling RFX6-assocciated neonatal and type-2 diabetes using allelic series stem cell-derived islets in-vitro and in-vivo.

Adiponectin is a peptide hormone abundantly released from adipocytes, and its reduced circulating levels have a central role in obesity-related diseases, such as type 2 diabetes, insulin resistance and cardiovascular disease. Adiponectin is secreted by regulated exocytosis of vesicles belonging to a pre-formed vesicle pool, in response to the appropriate stimulatory signal. Traditional molecular biology and imaging techniques lack spatial and temporal resolution to visualize and quantify the exocytosis dynamics of adiponectin-containing vesicles. Here we generate 3T3-L1 adipocytes stably expressing mCherry fused to human adiponectin and we employ total internal reflection fluorescence (TIRF) microscopy in real-time to visualise and quantify exocytosis of the fusion protein. The TIRF recordings show that an intracellular elevation of either cAMP or Ca 2+ , mediators known to induce adiponectin exocytosis, lead to rapid disappearance of fluorescent vesicles and we interpreted this as exocytosis events. Secretion measurements confirm that the human adiponectin-mCherry fusion protein is secreted to the surrounding solution upon stimulation with agents that elevate intracellular cAMP or Ca 2+ . We conclude that the fusion protein appear to be directed to vesicles belonging to the endogenous adiponectin vesicle population and that the targeted vesicles are secreted by signals known to stimulate adiponectin exocytosis. Thus, the mCherry-adiponectin-expressing cells are useful for investigations of the adiponectin vesicle exocytosis process in adipocytes, at a level of detail that has not been possible previously.

Transplantation of pancreatic islet cells derived from human pluripotent stem cells is a promising treatment for diabetes. Despite progress in the generation of stem-cell-derived islets (SC-islets), no detailed characterization of their functional properties has been conducted. Here, we generated functionally mature SC-islets using an optimized protocol and benchmarked them comprehensively against primary adult islets. Biphasic glucose-stimulated insulin secretion developed during in vitro maturation, associated with cytoarchitectural reorganization and the increasing presence of alpha cells. Electrophysiology, signaling and exocytosis of SC-islets were similar to those of adult islets. Glucose-responsive insulin secretion was achieved despite differences in glycolytic and mitochondrial glucose metabolism. Single-cell transcriptomics of SC-islets in vitro and throughout 6 months of engraftment in mice revealed a continuous maturation trajectory culminating in a transcriptional landscape closely resembling that of primary islets. Our thorough evaluation of SC-islet maturation highlights their advanced degree of functionality and supports their use in further efforts to understand and combat diabetes.

Significance This project describes the existence of previously unknown non–GPI-anchored CD59 isoforms required for insulin secretion, named CD59–IRIS-1 and CD59–IRIS-2, and finds reduced expression of CD59-IRIS isoforms in human diabetic islets, showing a link between dysregulation of IRIS isoforms and defects in insulin secretion in diabetic patients. These data open a path for future studies into CD59-IRIS expression and function in additional cell types capable of regulated secretion. Identification of additional specific CD59-IRIS binding partners within the cell could provide therapeutic targets for enhancement of insulin secretion in T2D.