Preparation of Whole-mount Mouse Islets on Vascular Extracellular Matrix for Live Islet Cell Microscopy.

Pancreatic islet ß cells preferentially secrete insulin toward the plasma membrane, making contact with the capillary extracellular matrix (ECM). Isolated islets separated from the exocrine acinar cells are the best system for cell biology studies of primary ß cells, whereas isolated islets lose their capillary network during ex vivo culture. Providing the appropriate extracellular signaling by attaching islets to vascular ECM-coated surfaces can restore the polarized insulin secretion toward the ECM. The guided secretion toward ECM-coated glass coverslips provides a good model for recording insulin secretion in real time to study its regulation. Additionally, ß cells attached to the ECM-coated coverslips are suitable for confocal live imaging of subcellular components including adhesion molecules, cytoskeleton, and ion channels. This procedure is also compatible for total internal reflection fluorescence (TIRF) microscopy, which provides optimal signal-to-noise ratio and high spatial precision of structures close to the plasma membrane. In this article, we describe the optimized protocol for vascular ECM-coating of glass coverslips and the process of attachment of isolated mouse islets on the coverslip. This preparation is compatible with any high-resolution microscopy of live primary ß cells. Key features • Optimized coating procedure to attach isolated islets, compatible for both confocal and TIRF microscopy. • The ECM-coated glass coverslip functions as the artificial capillary surface to guide secretion toward the coated surface for optimal imaging of secretion events. • Shows the process of islets attachment to the ECM-coated surface in a 6-day ex vivo culture.

CAMSAP2 localizes to the Golgi in islet ß-cells and facilitates Golgi-ER trafficking.

Glucose stimulation induces the remodeling of microtubules, which potentiates insulin secretion in pancreatic ß-cells. CAMSAP2 binds to microtubule minus ends to stabilize microtubules in several cultured clonal cells. Here, we report that the knockdown of CAMSAP2 in primary ß-cells reduces total insulin content and attenuates GSIS without affecting the releasability of insulin vesicles. Surprisingly, CAMSAP2 knockdown does not change microtubule stability. Unlike in cultured insulinoma cells, CAMSAP2 in primary ß-cells predominantly localizes to the Golgi apparatus instead of microtubule minus ends. This novel localization is specific to primary ß- but not α-cells and is independent of microtubule binding. Consistent with its specific localization at the Golgi, CAMSAP2 promotes efficient Golgi-ER trafficking in primary ß-cells. Moreover, primary ß-cells and insulinoma cells likely express different CAMSAP2 isoforms. We propose that a novel CAMSAP2 isoform in primary ß-cells has a non-canonical function, which promotes Golgi-ER trafficking to support efficient production of insulin and secretion.

Microtubules regulate pancreatic ß-cell heterogeneity via spatiotemporal control of insulin secretion hot spots.

Heterogeneity of glucose-stimulated insulin secretion (GSIS) in pancreatic islets is physiologically important but poorly understood. Here, we utilize mouse islets to determine how microtubules (MTs) affect secretion toward the vascular extracellular matrix at single cell and subcellular levels. Our data indicate that MT stability in the ß-cell population is heterogenous, and that GSIS is suppressed in cells with highly stable MTs. Consistently, MT hyper-stabilization prevents, and MT depolymerization promotes the capacity of single ß-cell for GSIS. Analysis of spatiotemporal patterns of secretion events shows that MT depolymerization activates otherwise dormant ß-cells via initiation of secretion clusters (hot spots). MT depolymerization also enhances secretion from individual cells, introducing both additional clusters and scattered events. Interestingly, without MTs, the timing of clustered secretion is dysregulated, extending the first phase of GSIS and causing oversecretion. In contrast, glucose-induced Ca2+ influx was not affected by MT depolymerization yet required for secretion under these conditions, indicating that MT-dependent regulation of secretion hot spots acts in parallel with Ca2+ signaling. Our findings uncover a novel MT function in tuning insulin secretion hot spots, which leads to accurately measured and timed response to glucose stimuli and promotes functional ß-cell heterogeneity.

Microtubules and Gαo-signaling modulate the preferential secretion of young insulin secretory granules in islet ß cells via independent pathways.

For sustainable function, each pancreatic islet ß cell maintains thousands of insulin secretory granules (SGs) at all times. Glucose stimulation induces the secretion of a small portion of these SGs and simultaneously boosts SG biosynthesis to sustain this stock. The failure of these processes, often induced by sustained high-insulin output, results in type 2 diabetes. Intriguingly, young insulin SGs are more likely secreted during glucose-stimulated insulin secretion (GSIS) for unknown reasons, while older SGs tend to lose releasability and be degraded. Here, we examine the roles of microtubule (MT) and Gαo-signaling in regulating the preferential secretion of young versus old SGs. We show that both MT-destabilization and Gαo inactivation results in more SGs localization near plasma membrane (PM) despite higher levels of GSIS and reduced SG biosynthesis. Intriguingly, MT-destabilization or Gαo-inactivation results in higher secretion probabilities of older SGs, while combining both having additive effects on boosting GSIS. Lastly, Gαo inactivation does not detectably destabilize the ß-cell MT network. These findings suggest that Gαo and MT can modulate the preferential release of younger insulin SGs via largely parallel pathways.

Glucose Regulates Microtubule Disassembly and the Dose of Insulin Secretion via Tau Phosphorylation.

The microtubule cytoskeleton of pancreatic islet ß-cells regulates glucose-stimulated insulin secretion (GSIS). We have reported that the microtubule-mediated movement of insulin vesicles away from the plasma membrane limits insulin secretion. High glucose-induced remodeling of microtubule network facilitates robust GSIS. This remodeling involves disassembly of old microtubules and nucleation of new microtubules. Here, we examine the mechanisms whereby glucose stimulation decreases microtubule lifetimes in ß-cells. Using real-time imaging of photoconverted microtubules, we demonstrate that high levels of glucose induce rapid microtubule disassembly preferentially in the periphery of individual ß-cells, and this process is mediated by the phosphorylation of microtubule-associated protein tau. Specifically, high glucose induces tau hyper-phosphorylation via glucose-responsive kinases GSK3, PKA, PKC, and CDK5. This causes dissociation of tau from and subsequent destabilization of microtubules. Consequently, tau knockdown in mouse islet ß-cells facilitates microtubule turnover, causing increased basal insulin secretion, depleting insulin vesicles from the cytoplasm, and impairing GSIS. More importantly, tau knockdown uncouples microtubule destabilization from glucose stimulation. These findings suggest that tau suppresses peripheral microtubules turning over to restrict insulin oversecretion in basal conditions and preserve the insulin pool that can be released following stimulation; high glucose promotes tau phosphorylation to enhance microtubule disassembly to acutely enhance GSIS.

Coregulator Sin3a Promotes Postnatal Murine ß-Cell Fitness by Regulating Genes in Ca2+ Homeostasis, Cell Survival, Vesicle Biosynthesis, Glucose Metabolism, and Stress Response.

Swi-independent 3a and 3b (Sin3a and Sin3b) are paralogous transcriptional coregulators that direct cellular differentiation, survival, and function. Here, we report that mouse Sin3a and Sin3b are coproduced in most pancreatic cells during embryogenesis but become much more enriched in endocrine cells in adults, implying continued essential roles in mature endocrine cell function. Mice with loss of Sin3a in endocrine progenitors were normal during early postnatal stages but gradually developed diabetes before weaning. These physiological defects were preceded by the compromised survival, insulin-vesicle packaging, insulin secretion, and nutrient-induced Ca2+ influx of Sin3a-deficient ß-cells. RNA sequencing coupled with candidate chromatin immunoprecipitation assays revealed several genes that could be directly regulated by Sin3a in ß-cells, which modulate Ca2+/ion transport, cell survival, vesicle/membrane trafficking, glucose metabolism, and stress responses. Finally, mice with loss of both Sin3a and Sin3b in multipotent embryonic pancreatic progenitors had significantly reduced islet cell mass at birth, caused by decreased endocrine progenitor production and increased ß-cell death. These findings highlight the stage-specific requirements for the presumed "general" coregulators Sin3a and Sin3b in islet ß-cells, with Sin3a being dispensable for differentiation but required for postnatal function and survival.

Microtubules Regulate Localization and Availability of Insulin Granules in Pancreatic Beta Cells.

Two key prerequisites for glucose-stimulated insulin secretion (GSIS) in ß cells are the proximity of insulin granules to the plasma membrane and their anchoring or docking to the plasma membrane (PM). Although recent evidence has indicated that both of these factors are altered in the context of diabetes, it is unclear what regulates localization of insulin granules and their interactions with the PM within single cells. Here, we demonstrate that microtubule (MT)-motor-mediated transport dynamics have a critical role in regulating both factors. Super-resolution imaging shows that whereas the MT cytoskeleton resembles a random meshwork in the cells' interior, MTs near the cell surface are preferentially aligned with the PM. Computational modeling suggests two consequences of this alignment. First, this structured MT network preferentially withdraws granules from the PM. Second, the binding and transport of insulin granules by MT motors prevents their stable anchoring to the PM. These findings suggest the MT cytoskeleton may negatively regulate GSIS by both limiting the amount of insulin proximal to the PM and preventing or breaking interactions between the PM and the remaining nearby insulin granules. These results predict that altering MT network structure in ß cells can be used to tune GSIS. Thus, our study points to the potential of an alternative therapeutic strategy for diabetes by targeting specific MT regulators.

Localization and function of budding yeast CENP-A depends upon kinetochore protein interactions and is independent of canonical centromere sequence.

In many eukaryotes, the centromere is epigenetically specified and not strictly defined by sequence. In contrast, budding yeast has a specific 125 bp sequence required for kinetochore function. Despite the difference in centromere specification, budding yeast and multicellular eukaryotic centromeres contain a highly conserved histone H3 variant, CENP-A. The localization of budding yeast CENP-A, Cse4, requires the centromere DNA binding components, which are not conserved in multicellular eukaryotes. Here, we report that Cse4 localizes and functions at a synthetic kinetochore assembly site that lacks centromere sequence. The outer kinetochore Dam1-DASH and inner kinetochore CBF3 complexes are required for Cse4 localization to that site. Furthermore, the natural kinetochore also requires the outer kinetochore proteins for full Cse4 localization. Our results suggest that Cse4 localization at a functional kinetochore does not require the recognition of a specific DNA sequence by the CBF3 complex; rather, its localization depends on stable interactions among kinetochore proteins.

Mutation at the cargo-receptor binding site of Atg8 also affects its general autophagy regulation function.

Autophagy is a highly conserved degradation pathway for intracellular macromolecules and organelles. Among those characterized autophagy regulators, the ubiquitin-like protein Atg8 is found to be a membrane modifier that both regulates biogenesis of transport vesicles and interacts with the cargo receptor Atg19 for selective autophagic transport of the vacuolar enzyme prApe1 in budding yeast. The role of Atg8 in the enlargement of vesicle membrane during autophagosome biogenesis has been well documented,but how Atg8 coordinates vesicle formation and sorting of selective cargo is largely unknown. Identification of the cargo-receptor binding site of Atg8 would provide information to solve this issue.Here we characterized Atg8 mutants that were defective in interaction with the prApe1 receptor Atg19 and found that the vesicle formation function of these Atg8 mutants was also compromised to different extents. Atg8 mutants with single-residue substitution at the Atg19-binding site were defective in lipid conjugation and/or subcellular localization. Additional Atg8 mutants were found defective in autophagosome formation without affecting their interaction with Atg19, suggesting partially overlapping of the cargo-sorting site and its domains critical for autophagy control. Our observation paves the road for a more comprehensive understanding on how Atg8 coordinates cargo sorting and vesicle formation in selective autophagic pathways.