Local PI(4,5)P2 signaling inhibits fusion pore expansion during exocytosis.

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-kinase-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 inhibits fusion pore expansion and peptide release through an unconventional endocytic mechanism.

Vesicle cholesterol controls exocytotic fusion pore.

In some lysosomal storage diseases (LSD) cholesterol accumulates in vesicles. Whether increased vesicle cholesterol affects vesicle fusion with the plasmalemma, where the fusion pore, a channel between the vesicle lumen and the extracellular space, is formed, is unknown. Super-resolution microscopy revealed that after stimulation of exocytosis, pituitary lactotroph vesicles discharge cholesterol which transfers to the plasmalemma. Cholesterol depletion in lactotrophs and astrocytes, both exhibiting Ca2+-dependent exocytosis regulated by distinct Ca2+sources, evokes vesicle secretion. Although this treatment enhanced cytosolic levels of Ca2+ in lactotrophs but decreased it in astrocytes, this indicates that cholesterol may well directly define the fusion pore. In an attempt to explain this mechanism, a new model of cholesterol-dependent fusion pore regulation is proposed. High-resolution membrane capacitance measurements, used to monitor fusion pore conductance, a parameter related to fusion pore diameter, confirm that at resting conditions reducing cholesterol increases, while enrichment with cholesterol decreases the conductance of the fusion pore. In resting fibroblasts, lacking the Npc1 protein, a cellular model of LSD in which cholesterol accumulates in vesicles, the fusion pore conductance is smaller than in controls, showing that vesicle cholesterol controls fusion pore and is relevant for pathophysiology of LSD.

Fusion pore regulation by cAMP/Epac2 controls cargo release during insulin exocytosis.

Regulated exocytosis establishes a narrow fusion pore as initial aqueous connection to the extracellular space, through which small transmitter molecules such as ATP can exit. Co-release of polypeptides and hormones like insulin requires further expansion of the pore. There is evidence that pore expansion is regulated and can fail in diabetes and neurodegenerative disease. Here, we report that the cAMP-sensor Epac2 (Rap-GEF4) controls fusion pore behavior by acutely recruiting two pore-restricting proteins, amisyn and dynamin-1, to the exocytosis site in insulin-secreting beta-cells. cAMP elevation restricts and slows fusion pore expansion and peptide release, but not when Epac2 is inactivated pharmacologically or in Epac2-/- (Rapgef4-/-) mice. Consistently, overexpression of Epac2 impedes pore expansion. Widely used antidiabetic drugs (GLP-1 receptor agonists and sulfonylureas) activate this pathway and thereby paradoxically restrict hormone release. We conclude that Epac2/cAMP controls fusion pore expansion and thus the balance of hormone and transmitter release during insulin granule exocytosis.

Astrocytic Vesicle-based Exocytosis in Cultures and Acutely Isolated Hippocampal Rodent Slices.

Astrocytes are excitable neural cells that contribute to brain information processing via bidirectional communication with neurons. This involves the release of gliosignaling molecules that affect synapses patterning and activity. Mechanisms mediating the release of these molecules likely consist of non-vesicular and vesicular-based mechanisms. It is the vesicle-based regulated exocytosis that is an evolutionary more complex process. It is well established that the release of gliosignaling molecules has profound effects on information processing in different brain regions (e.g., hippocampal astrocytes contribute to long-term potentiation [LTP]), which has traditionally been considered as one of the cellular mechanisms underlying learning and memory. However, the paradigm of vesicle-based regulated release of gliosignaling molecules from astrocytes is still far from being unanimously accepted. One of the most important questions is to what extent can the conclusions obtained from cultured astrocytes be translated to in vivo conditions. Here, we overview the properties of vesicle mobility and their fusion with the plasma membrane in cultured astrocytes and compare these parameters to those recorded in astrocytes from acute brain hippocampal slices. The results from both experimental models are similar, which validates experiments on isolated astrocytes and further supports arguments in favor of in vivo vesicle-based exocytotic release of gliosignaling molecules. © 2017 Wiley Periodicals, Inc.

Plasma Membrane Phosphatidylinositol 4,5-Bisphosphate Regulates Ca(2+)-Influx and Insulin Secretion from Pancreatic ß Cells.

Insulin secretion from pancreatic ß cells is regulated by the blood glucose concentration and occurs through Ca(2+)-triggered exocytosis. The activities of multiple ion channels in the ß cell plasma membrane are required to fine-tune insulin secretion in order to maintain normoglycemia. Phosphoinositide lipids in the plasma membrane often gate ion channels, and variations in the concentration of these lipids affect ion-channel open probability and conductance. Using light-regulated synthesis or depletion of plasma membrane phosphatidylinositol 4,5-bisphosphate (PI[4,5]P2), we found that this lipid positively regulated both depolarization- and glucose-triggered Ca(2+) influx in a dose-dependent manner. Small reductions of PI(4,5)P2 caused by brief illumination resulted in partial suppression of Ca(2+) influx that followed the kinetics of the lipid, whereas depletion resulted in marked inhibition of both Ca(2+) influx and insulin secretion.

Dominant negative SNARE peptides stabilize the fusion pore in a narrow, release-unproductive state.

Key support for vesicle-based release of gliotransmitters comes from studies of transgenic mice with astrocyte-specific expression of a dominant-negative domain of synaptobrevin 2 protein (dnSNARE). To determine how this peptide affects exocytosis, we used super-resolution stimulated emission depletion microscopy and structured illumination microscopy to study the anatomy of single vesicles in astrocytes. Smaller vesicles contained amino acid and peptidergic transmitters and larger vesicles contained ATP. Discrete increases in membrane capacitance, indicating single-vesicle fusion, revealed that astrocyte stimulation increases the frequency of predominantly transient fusion events in smaller vesicles, whereas larger vesicles transitioned to full fusion. To determine whether this reflects a lower density of SNARE proteins in larger vesicles, we treated astrocytes with botulinum neurotoxins D and E, which reduced exocytotic events of both vesicle types. dnSNARE peptide stabilized the fusion-pore diameter to narrow, release-unproductive diameters in both vesicle types, regardless of vesicle diameter.

Local electrostatic interactions determine the diameter of fusion pores.

In regulated exocytosis vesicular and plasma membranes merge to form a fusion pore in response to stimulation. The nonselective cation HCN channels are involved in the regulation of unitary exocytotic events by at least 2 mechanisms. They can affect SNARE-dependent exocytotic activity indirectly, via the modulation of free intracellular calcium; and/or directly, by altering local cation concentration, which affects fusion pore geometry likely via electrostatic interactions. By monitoring membrane capacitance, we investigated how extracellular cation concentration affects fusion pore diameter in pituitary cells and astrocytes. At low extracellular divalent cation levels predominantly transient fusion events with widely open fusion pores were detected. However, fusion events with predominately narrow fusion pores were present at elevated levels of extracellular trivalent cations. These results show that electrostatic interactions likely help determine the stability of discrete fusion pore states by affecting fusion pore membrane composition.

Hyperpolarization-activated cyclic nucleotide-gated channels and cAMP-dependent modulation of exocytosis in cultured rat lactotrophs.

Hormone and neurotransmitter release from vesicles is mediated by regulated exocytosis, where an aqueous channel-like structure, termed a fusion pore, is formed. It was recently shown that second messenger cAMP modulates the fusion pore, but the detailed mechanisms remain elusive. In this study, we asked whether the hyperpolarization-activated cyclic nucleotide-gated (HCN) channels, which are activated by cAMP, are involved in the regulation of unitary exocytic events. By using the Western blot technique, a real-time PCR, immunocytochemistry in combination with confocal microscopy, and voltage-clamp measurements of hyperpolarizing currents, we show that HCN channels are present in the plasma membrane and in the membrane of secretory vesicles of isolated rat lactotrophs. Single vesicle membrane capacitance measurements of lactotrophs, where HCN channels were either augmented by transfection or blocked with an HCN channel blocker (ZD7288), show modulated fusion pore properties. We suggest that the changes in local cation concentration, mediated through HCN channels, which are located on or near secretory vesicles, have an important role in modulating exocytosis.

High-resolution membrane capacitance measurements for the study of exocytosis and endocytosis.

In order to understand exocytosis and endocytosis, it is necessary to study these processes directly. An elegant way to do this is by measuring plasma membrane capacitance (C(m)), a parameter proportional to cell surface area, the fluctuations of which are due to fusion and fission of secretory and other vesicles. Here we describe protocols that enable high-resolution C(m) measurements in macroscopic and microscopic modes. Macroscopic mode, performed in whole-cell configuration, is used for measuring bulk C(m) changes in the entire membrane area, and it enables the introduction of exocytosis stimulators or inhibitors into the cytosol through the patch pipette. Microscopic mode, performed in cell-attached configuration, enables measurements of C(m) with attofarad resolution and allows characterization of fusion pore properties. Although we usually apply these protocols to primary pituitary cells and astrocytes, they can be adapted and used for other cell types. After initial hardware setup and culture preparation, several C(m) measurements can be performed daily.

Comparison of unitary exocytic events in pituitary lactotrophs and in astrocytes: modeling the discrete open fusion-pore states.

In regulated exocytosis the merger between the vesicle and the plasma membranes leads to the formation of an aqueous channel (a fusion-pore), through which vesicular secretions exit into the extracellular space. A fusion pore was thought to be a short-lived intermediate preceding full-fusion of the vesicle and the plasma membranes (full-fusion exocytosis). However, transient exocytic events were also observed, where the fusion-pore opens and closes, repetitively. Here we asked whether there are different discrete states of the open fusion-pore. Unitary exocytic events were recorded by the high-resolution cell-attached patch-clamp method in pituitary lactotrophs and brain astrocytes. We monitored reversible unitary exocytic events, characterized by an on-step, which is followed by an off-step in membrane capacitance (C m ), a parameter linearly related to the membrane area. The results revealed three categories of reversible exocytic events (transient fusion-pore openings), which do not end with the complete integration of the vesicle membrane into the plasma membrane. These were categorized according to the observed differences in the amplitude and sign of the change in the real (Re) parts of the admittance signals: in case I events (Re ≈ 0) fusion pores are relatively wide; in case II (Re > 0) and case III (Re < 0) events fusion pores are relatively narrow. We show that case III events are more likely to occur for small vesicles, whereas, case II events are more likely to occur for larger vesicles. Case III events were considerably more frequent in astrocytes than in lactotrophs.

Cholesterol and regulated exocytosis: a requirement for unitary exocytotic events.

Since the 1970s, much effort was been expended researching mechanisms of regulated exocytosis. Early work focused mainly on the role of proteins. Most notably the discovery of SNARE proteins in the 1980s and the zippering hypothesis brought us much closer to understanding the complex interactions in membrane fusion between vesicle and plasma membranes, a pivotal component of regulated exocytosis. However, most likely due to the predictions of the Singer-Nicholson fluid mosaic membrane model, the lipid components of the exocytotic machinery remained largely overlooked. Lipids were considered passive constituents of cellular membranes, not contributing much, if anything, to the process of exocytosis and membrane fusion. Since the 1990s, this so-called proteocentric view has been gradually giving way to the new perspective best described with the term proteolipidic. Many lipids were found to be of great importance in the regulation of exocytosis. Here we highlight the role of cholesterol. Furthermore, by using high-resolution cell-attached membrane capacitance measurements, we have monitored unitary exocytotic events in cholesterol-depleted membranes. We show that the frequency of these events is attenuated, providing evidence at the single vesicle level that cholesterol directly influences the merger of the vesicle and the plasma membranes.

Exocytosis in astrocytes: transmitter release and membrane signal regulation.

Astrocytes, a type of glial cells in the brain, are eukaryotic cells, and a hallmark of these are subcellular organelles, such as secretory vesicles. In neurons vesicles play a key role in signaling. Upon a stimulus-an increase in cytosolic concentration of free Ca(2+) ([Ca(2+)](i))-the membrane of vesicle fuses with the presynaptic plasma membrane, allowing the exit of neurotransmitters into the extracellular space and their diffusion to the postsynaptic receptors. For decades it was thought that such vesicle-based mechanisms of gliotransmitter release were not present in astrocytes. However, in the last 30 years experimental evidence showed that astrocytes are endowed with mechanisms for vesicle- and non-vesicle-based gliotransmitter release mechanisms. The aim of this review is to focus on exocytosis, which may play a role in gliotransmission and also in other forms of cell-to-cell communication, such as the delivery of transporters, ion channels and antigen presenting molecules to the cell surface.