Wen Du

Graded activation of CRAC channel by binding of different numbers of STIM1 to Orai1 subunits.

The Ca2+ release-activated Ca2+ (CRAC) channel pore is formed by Orai1 and gated by STIM1 after intracellular Ca2+ store depletion. To resolve how many STIM1 molecules are required to open a CRAC channel, we fused different numbers of Orai1 subunits with functional two-tandem cytoplasmic domains of STIM1 (residues 336-485, designated as S domain). Whole-cell patch clamp recordings of these chimeric molecules revealed that CRAC current reached maximum at a stoichiometry of four Orai1 and eight S domains. Further experiments indicate that two-tandem S domains specifically interact with the C-terminus of one Orai1 subunit, and CRAC current can be gradually increased as more Orai1 subunits can interact with S domains or STIM1 proteins. Our data suggest that maximal opening of one CRAC channel requires eight STIM1 molecules, and support a model that the CRAC channel activation is not in an “all-or-none” fashion but undergoes a graded process via binding of different numbers of STIM1.

Contribution of Different Mechanisms to Pancreatic Beta-cell Hyper-secretion in Non-obese Diabetic (NOD) Mice during Pre-diabetes

Background: Pre-diabetic islet hyper-secretion is crucial to the development of the disease but the mechanisms remain unknown. Results: We reveal dynamic changes in beta-cell mass and function in non-obese diabetic (NOD) mice of different ages. Conclusion: Beta-cell mass increase and individual beta-cell secretory ability enhancement contribute to islet hyperactivity at different stages. Significance: This may provide insights into alteration of beta-cell function during the disease progression. The development of insulin-dependent diabetes mellitus (IDDM) results from the selective destruction of pancreatic beta-cells. Both humans and spontaneous models of IDDM, such as NOD mice, have an extended pre-diabetic stage. Dynamic changes in beta-cell mass and function during pre-diabetes, such as insulin hyper-secretion, remain largely unknown. In this paper, we evaluated pre-diabetic female NOD mice at different ages (6, 10, and 14 weeks old) to illustrate alterations in beta-cell mass and function as disease progressed. We found an increase in beta-cell mass in 6-week-old NOD mice that may account for improved glucose tolerance in these mice. As NOD mice aged, beta-cell mass progressively reduced with increasing insulitis. In parallel, secretory ability of individual beta-cells was enhanced due to an increase in the size of slowly releasable pool (SRP) of vesicles. Moreover, expression of both SERCA2 and SERCA3 genes were progressively down-regulated, which facilitated depolarization-evoked secretion by prolonging Ca2+ elevation upon glucose stimulation. In summary, we propose that different mechanisms contribute to the insulin hyper-secretion at different ages of pre-diabetic NOD mice, which may provide some new ideas concerning the progression and management of type I diabetes.

Diacylglycerol Guides the Hopping of Clathrin-Coated Pits along Microtubules for Exo-Endocytosis Coupling

Many receptor-mediated endocytic processes are mediated by constitutive budding of clathrin-coated pits (CCPs) at spatially randomized sites before slowly pinching off from the plasma membrane (60-100 s). In contrast, clathrin-mediated endocytosis (CME) coupled with regulated exocytosis in excitable cells occurs at peri-exocytic sites shortly after vesicle fusion (∼10 s). The molecular mechanism underlying this spatiotemporal coupling remains elusive. We show that coupled endocytosis makes use of pre-formed CCPs, which hop to nascent fusion sites nearby following vesicle exocytosis. A dynamic cortical microtubular network, anchored at the cell surface by the cytoplasmic linker-associated protein on microtubules and the LL5β/ELKS complex on the plasma membrane, provides the track for CCP hopping. Local diacylglycerol gradients generated upon exocytosis guide the direction of hopping. Overall, the CCP-cytoskeleton-lipid interaction demonstrated here mediates exocytosis-coupled fast recycling of both plasma membrane and vesicular proteins, and it is required for the sustained exocytosis during repetitive stimulations.

HID-1 is a peripheral membrane protein primarily associated with the medial- and trans- Golgi apparatus.

Caenorhabditis elegans hid-1 gene was first identified in a screen for mutants with a high-temperature-induced dauer formation (Hid) phenotype. Despite the fact that the hid-1 gene encodes a novel protein (HID-1) which is highly conserved from Caenorhabditis elegans to mammals, the domain structure, subcellular localization, and exact function of HID-1 remain unknown. Previous studies and various bioinformatic softwares predicted that HID-1 contained many transmembrane domains but no known functional domain. In this study, we revealed that mammalian HID-1 localized to the medial- and trans- Golgi apparatus as well as the cytosol, and the localization was sensitive to brefeldin A treatment. Next, we demonstrated that HID-1 was a peripheral membrane protein and dynamically shuttled between the Golgi apparatus and the cytosol. Finally, we verified that a conserved N-terminal myristoylation site was required for HID-1 binding to the Golgi apparatus. We propose that HID-1 is probably involved in the intracellular trafficking within the Golgi region.

HID-1 is required for homotypic fusion of immature secretory granules during maturation

Secretory granules, also known as dense core vesicles, are generated at the trans-Golgi network and undergo several maturation steps, including homotypic fusion of immature secretory granules (ISGs) and processing of prehormones to yield active peptides. The molecular mechanisms governing secretory granule maturation are largely unknown. Here, we investigate a highly conserved protein named HID-1 in a mouse model. A conditional knockout of HID-1 in pancreatic β cells leads to glucose intolerance and a remarkable increase in the serum proinsulin/insulin ratio caused by defective proinsulin processing. Large volume three-dimensional electron microscopy and immunofluorescence imaging reveal that ISGs are much more abundant in the absence of HID-1. We further demonstrate that HID-1 deficiency prevented secretory granule maturation by blocking homotypic fusion of immature secretory granules. Our data identify a novel player during the early maturation of immature secretory granules. DOI: http://dx.doi.org/10.7554/eLife.18134.001

Ultra-structural study of insulin granules in pancreatic β-cells of db/db mouse by scanning transmission electron microscopy tomography

Insulin granule trafficking is a key step in the secretion of glucose-stimulated insulin from pancreatic β-cells. The main feature of type 2 diabetes (T2D) is the failure of pancreatic β-cells to secrete sufficient amounts of insulin to maintain normal blood glucose levels. In this work, we developed and applied tomography based on scanning transmission electron microscopy (STEM) to image intact insulin granules in the β-cells of mouse pancreatic islets. Using three-dimensional (3D) reconstruction, we found decreases in both the number and the grey level of insulin granules in db/db mouse pancreatic β-cells. Moreover, insulin granules were closer to the plasma membrane in diabetic β-cells than in control cells. Thus, 3D ultra-structural tomography may provide new insights into the pathology of insulin secretion in T2D.

Impaired maturation of large dense-core vesicles in muted-deficient adrenal chromaffin cells.

ABSTRACT The large dense-core vesicle (LDCV), a type of lysosome-related organelle, is involved in the secretion of hormones and neuropeptides in specialized secretory cells. The granin family is a driving force in LDCV biogenesis, but the machinery for granin sorting to this biogenesis pathway is largely unknown. The mu mutant mouse, which carries a spontaneous null mutation on the Muted gene (also known as Bloc1s5), which encodes a subunit of the biogenesis of lysosome-related organelles complex-1 (BLOC-1), is a mouse model of Hermansky–Pudlak syndrome. Here, we found that LDCVs were enlarged in mu adrenal chromaffin cells. Chromogranin A (CgA, also known as CHGA) was increased in mu adrenals and muted-knockdown cells. The increased CgA in mu mice was likely due a failure to export this molecule out of immature LDCVs, which impairs LDCV maturation and docking. In mu chromaffin cells, the size of readily releasable pool and the vesicle release frequency were reduced. Our studies suggest that the muted protein is involved in the selective export of CgA during the biogenesis of LDCVs.

Alterations of the Ca2+ signaling pathway in pancreatic beta-cells isolated from db/db mice

Upon glucose elevation, pancreatic beta-cells secrete insulin in a Ca2+-dependent manner. In diabetic animal models, different aspects of the calcium signaling pathway in beta-cells are altered, but there is no consensus regarding their relative contributions to the development of beta-cell dysfunction. In this study, we compared the increase in cytosolic Ca2+ ([Ca2+]i) via Ca2+ influx, Ca2+ mobilization from endoplasmic reticulum (ER) calcium stores, and the removal of Ca2+ via multiple mechanisms in beta-cells from both diabetic db/db mice and non-diabetic C57BL/6J mice. We refined our previous quantitative model to describe the slow [Ca2+]i recovery after depolarization in beta-cells from db/db mice. According to the model, the activity levels of the two subtypes of the sarco-endoplasmic reticulum Ca2+-ATPase (SERCA) pump, SERCA2 and SERCA3, were severely down-regulated in diabetic cells to 65% and 0% of the levels in normal cells. This down-regulation may lead to a reduction in the Ca2+ concentration in the ER, a compensatory up-regulation of the plasma membrane Na+/Ca2+ exchanger (NCX) and a reduction in depolarization-evoked Ca2+ influx. As a result, the patterns of glucose-stimulated calcium oscillations were significantly different in db/db diabetic beta-cells compared with normal cells. Overall, quantifying the changes in the calcium signaling pathway in db/db diabetic beta-cells will aid in the development of a disease model that could provide insight into the adaptive transformations of beta-cell function during diabetes development.

Proteomic analysis of insulin secretory granules in INS-1 cells by protein correlation profiling

Insulin secretory granules (ISGs), a group of distinguishing organelles in pancreatic β cells, are responsible for the storage and secretion of insulin to maintain blood glucose homeostasis. The molecular mechanisms of ISG biogenesis, maturation, transportation, and exocytosis are still largely unknown because the proteins involved in these distinct steps have not been fully identified. Subcellular fractionation by density gradient centrifugation has been successfully employed to analyze the proteomes of numerous organelles. However, use of this method to elucidate the ISG proteome is limited by co-fractionated contaminants because ISGs are very dynamic and have abundant exchanges or contacts with other organelles, such as the Golgi apparatus, lysosomes, and endosomes. In this study, we developed a new strategy for identifying ISG proteins by protein correlation profiling (PCP)-based proteomics, which included ISG purification by OptiPrep density gradient centrifugation, label-free quantitative proteome, and identification of ISG proteins by correlating fractionation profiles between candidates and known ISG markers. Using this approach, we were able to identify 81 ISG proteins. Among them, TM9SF3, a nine-transmembrane protein, was considered a high confidence ISG candidate protein highlighted in the PCP network. Further biochemical and immunofluorescence assays indicated that TM9SF3 localized in ISGs, suggesting that it is a potential new ISG marker.Graphical abstract