Synaptotagmin‐7 (Syt‐7) is one of two major calcium sensors for exocytosis in adrenal chromaffin cells, the other being synaptotagmin‐1 (Syt‐1). Despite a broad appreciation for the importance of Syt‐7, questions remain as to its localization, function in mediating discharge of dense core granule cargos, and role in triggering release in response to physiological stimulation. These questions were addressed using two distinct experimental preparations—mouse chromaffin cells lacking endogenous Syt‐7 (KO cells) and a reconstituted system employing cell‐derived granules expressing either Syt‐7 or Syt‐1. First, using immunofluorescence imaging and subcellular fractionation, it is shown that Syt‐7 is widely distributed in organelles, including dense core granules. Total internal reflection fluorescence (TIRF) imaging demonstrates that the kinetics and probability of granule fusion in Syt‐7 KO cells stimulated by a native secretagogue, acetylcholine, are markedly lower than in WT cells. When fusion is observed, fluorescent cargo proteins are discharged more rapidly when only Syt‐1 is available to facilitate release. To determine the extent to which the aforementioned results are attributable purely to Syt‐7, granules expressing only Syt‐7 or Syt‐1 were triggered to fuse on planar supported bilayers bearing plasma membrane SNARE proteins. Here, as in cells, Syt‐7 confers substantially greater calcium sensitivity to granule fusion than Syt‐1 and slows the rate at which cargos are released. Overall, this study demonstrates that by virtue of its high affinity for calcium and effects on fusion pore expansion, Syt‐7 plays a central role in regulating secretory output from adrenal chromaffin cells.

Insulin secretion from β-cells is reduced at the onset of type-1 and during type-2 diabetes. Although inflammation and metabolic dysfunction of β-cells elicit secretory defects associated with type-1 or type-2 diabetes, accompanying changes to insulin granules have not been established. To address this, we performed detailed functional analyses of insulin granules purified from cells subjected to model treatments that mimic type-1 and type-2 diabetic conditions and discovered striking shifts in calcium affinities and fusion characteristics. We show that this behavior is correlated with two subpopulations of insulin granules whose relative abundance is differentially shifted depending on diabetic model condition. The two types of granules have different release characteristics, distinct lipid and protein compositions, and package different secretory contents alongside insulin. This complexity of β-cell secretory physiology establishes a direct link between granule subpopulation and type of diabetes and leads to a revised model of secretory changes in the diabetogenic process.

Fusion and fission of cellular membranes involve dramatic, protein-mediated changes in membrane curvature. Many of the experimental methods useful for investigating curvature sensing or generation require specialized equipment. We have developed a system based on supported lipid bilayers (SLBs) in which lipid tubules are simple to produce and several types of membrane remodeling events can be readily imaged using widely available instrumentation (e.g., tubule fission and/or membrane budding). Briefly, high ionic strength during lipid bilayer deposition results in incorporation of excess lipids in the SLB. After sequentially washing with water and physiological ionic strength buffer solutions, lipid tubules form spontaneously. We find that tubule formation results from solution-dependent spreading of the SLB; washing from water into physiological ionic strength buffer solution leads to expansion of the bilayer and formation of tubules. Conversely, washing from physiological buffer into water results in contraction of the membrane and loss of tubules. We demonstrate the utility of these supported tubulated bilayers, termed "STuBs," with an investigation of Sar1B, a small Ras family G-protein known to influence membrane curvature. The addition of Sar1B to STuBs results in dramatic changes in tubule topology and eventual tubule fission. Overall, STuBs are a simple experimental system, useful for monitoring protein-mediated effects on membrane topology in real time, under physiologically relevant conditions.

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