No abstract

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.

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Insulin secretion by pancreatic islet β-cells is regulated by glucose levels and is accompanied by proton generation. The voltage-gated proton channel Hv1 is present in pancreatic β-cells and extremely selective for protons. However, whether Hv1 is involved in insulin secretion is unclear. Here we demonstrate that Hv1 promotes insulin secretion of pancreatic β-cells and glucose homeostasis. Hv1-deficient mice displayed hyperglycemia and glucose intolerance because of reduced insulin secretion but retained normal peripheral insulin sensitivity. Moreover, Hv1 loss contributed much more to severe glucose intolerance as the mice got older. Islets of Hv1-deficient and heterozygous mice were markedly deficient in glucose- and K+-induced insulin secretion. In perifusion assays, Hv1 deletion dramatically reduced the first and second phase of glucose-stimulated insulin secretion. Islet insulin and proinsulin content was reduced, and histological analysis of pancreas slices revealed an accompanying modest reduction of β-cell mass in Hv1 knockout mice. EM observations also indicated a reduction in insulin granule size, but not granule number or granule docking, in Hv1-deficient mice. Mechanistically, Hv1 loss limited the capacity for glucose-induced membrane depolarization, accompanied by a reduced ability of glucose to raise Ca2+ levels in islets, as evidenced by decreased durations of individual calcium oscillations. Moreover, Hv1 expression was significantly reduced in pancreatic β-cells from streptozotocin-induced diabetic mice, indicating that Hv1 deficiency is associated with β-cell dysfunction and diabetes. We conclude that Hv1 regulates insulin secretion and glucose homeostasis through a mechanism that depends on intracellular Ca2+ levels and membrane depolarization.

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In fluorescence microscopy, computational algorithms have been developed to suppress noise, enhance contrast, and even enable super-resolution (SR). However, the local quality of the images may vary on multiple scales, and these differences can lead to misconceptions, which is especially intractable in emerging deep-learning ones. Current mapping methods fail to finely estimate the local quality, challenging to associate the SR scale content. Here, we develop a rolling Fourier ring correlation (rFRC) framework to evaluate the reconstruction uncertainties down to SR scale. To visually pinpoint regions with low reliability, a filtered rFRC is combined with a modified resolution scaled error map (RSM), offering a comprehensive and concise map for further examination. We demonstrate their performances on various SR imaging modalities, and the resulting quantitative maps enable better SR images integrated from different reconstructions. Beyond that, we provide a strategy for learning-based restorations, allowing a direct detection of both data and model uncertainties, and expect the representative cases can inspire further advances in this rapidly developing field.

No abstract

Despite the wide application of super-resolution (SR) microscopy in biological studies of cells, the technology is rarely used to monitor functional changes in live cells. By combining fast spinning disc-confocal structured illumination microscopy (SD-SIM) with loading of cytosolic fluorescent Ca 2+ indicators, we have developed an SR method for visualization of regional Ca 2+ dynamics and related cellular organelle morphology and dynamics, termed SR calcium lantern imaging. In COS-7 cells stimulated with ATP, we have identified various calcium macrodomains characterized by different types of Ca 2+ release from endoplasmic reticulum (ER) stores. Finally, we demonstrated various roles of mitochondria in mediating calcium signals from different sources; while mitochondria can globally potentiate the Ca 2+ entry associated with store release, mitochondria also locally control Ca 2+ release from the neighboring ER stores and assist in their refilling processes.