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Gene transfection is a fundamental technique in the fields of biological research and therapeutic innovation. Due to their biocompatibility and membrane-mimetic properties, lipid vectors serve as essential tools in transfection. The successful delivery of genetic material into the cytoplasm is contingent upon the fusion of the vector and cellular membranes, which enables hydrophilic polynucleic acids to traverse the hydrophobic barriers of two intervening membranes. This review examines the critical role of membrane fusion in lipofection efficiency, with a particular focus on the molecular mechanisms that govern lipoplex–membrane interactions. This analysis will examine the key challenges inherent to the fusion process, from achieving initial membrane proximity to facilitating final content release through membrane remodeling. In contrast to viral vectors, which utilize specialized fusion proteins, lipid vectors necessitate a strategic formulation and environmental optimization to enhance their fusogenicity. This review discusses recent advances in vector design and fusion-promoting strategies, emphasizing their potential to improve gene delivery yield. It highlights the importance of understanding lipoplex–membrane fusion mechanisms for developing next-generation delivery systems and emphasizes the need for continued fundamental research to advance lipid-mediated transfection technology. [ABSTRACT FROM AUTHOR]

Key points Somatostatin, a peptide hormone that activates G‐protein‐coupled receptors, inhibits the secretion of many hormones. This study investigated the mechanisms of this inhibition using amperometry recording of Ca2+‐triggered catecholamine secretion from mouse chromaffin cells. Two distinct stimulation protocols, high‐KCl depolarization and caffeine, were used to trigger exocytosis, and confocal fluorescence imaging was used to monitor the rise in intracellular free Ca2+. Analysis of single‐vesicle fusion events (spikes) resolved the action of somatostatin on fusion pores at different stages. Somatostatin reduced spike frequency, and this reduction was accompanied by prolongation of pre‐spike feet and slowing of spike rise times. This indicates that somatostatin stabilizes initial fusion pores and slows their expansion. This action on the initial fusion pore impacted the release mode to favour kiss‐and‐run over full‐fusion. During a spike the permeability of a fusion pore peaks, declines and then settles into a plateau. Somatostatin had no effect on the plateau, suggesting no influence on late‐stage fusion pores. These actions of somatostatin were indistinguishable between exocytosis triggered by high‐KCl and caffeine, and fluorescence imaging showed that somatostatin had no effect on stimulus‐induced rises in cytosolic Ca2+. Our findings thus demonstrate that the signalling cascades activated by somatostatin target the exocytotic machinery that controls the initial and expanding stages of fusion pores, while having no effect on late‐stage fusion pores. As a result of its stronger inhibition of full‐fusion compared to kiss‐and‐run, somatostatin will preferentially inhibit the secretion of large peptides over the secretion of small catecholamines. Somatostatin inhibits the secretion of various hormones by activating G‐protein‐coupled receptors. In this study, we used amperometry to investigate the mechanism by which somatostatin inhibits catecholamine release from mouse chromaffin cells. Somatostatin increased pre‐spike foot lifetime and slowed fusion pore expansion. Somatostatin inhibited full‐fusion more strongly than kiss‐and‐run. Our results suggest that the initial fusion pore is the target of somatostatin‐mediated regulation of hormone release. The stronger inhibition of full‐fusion by somatostatin will result in preferential inhibition of peptide release. [ABSTRACT FROM AUTHOR]

Fusion pore opening is a transient intermediate state of synaptic vesicle exocytosis, which is highly dynamic and precisely regulated by the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex and synaptotagmin-1 (Syt1). Yet, the regulatory mechanism is not fully understood. In this work, using single-channel membrane fusion electrophysiology, we determined that SNAREpins are important for driving fusion pore opening and dilation but incapable of regulating the dynamics. When Syt1 was added, the closing frequency of fusion pores significantly increased, while the radius of fusion pores mildly decreased. In response to Ca2+, SNARE/Syt1 greatly increased the radius of fusion pores and reduced their closing frequency. Moreover, the residue F349 in the C2B domain of Syt1, which mediates Syt1 oligomerization, was required for clamping fusion pore opening in the absence of Ca2+, probably by extending the distance between the two membranes. Finally, in Ca2+-triggered fusion, the primary interface between SNARE and Syt1 plays a critical role in stabilizing and dilating the fusion pore, while the polybasic region of Syt1 C2B domain has a mild effect on increasing the radius of the fusion pore. In summary, our results suggest that Syt1, SNARE, and the anionic membrane synergically orchestrate the dynamics of fusion pore opening in synaptic vesicle exocytosis. [ABSTRACT FROM AUTHOR]

Membrane fusion is not a spontaneous process. Physiologically, the formation of coiled‐coil protein complexes, the SNAREpins, bridges the membrane of a vesicle and a target membrane, brings them in close contact, and provides the energy necessary for their fusion. In this review, we utilize results from in vitro experiments and simple physics and chemistry models to dissect the kinetics and energetics of the fusion process from the encounter of the two membranes to the full expansion of a fusion pore. We find three main energy barriers that oppose the fusion process: SNAREpin initiation, fusion pore opening, and expansion. SNAREpin initiation is inherent to the proteins and makes in vitro fusion kinetic experiments rather slow. The kinetics are physiologically accelerated by effectors. The energy barriers that precede pore opening and pore expansion can be overcome by several SNAREpins acting in concert.

This historical review focuses on the evolution of the knowledge accumulated during the last two centuries on the biology of the adrenal medulla gland and its chromaffin cells (CCs). The review emerged in the context of a series of meetings that started on the Spanish island of Ibiza in 1982 with the name of the International Symposium on Chromaffin Cell Biology (ISCCB). Hence, the review is divided into two periods namely, before 1982 and from this year to 2022, when the 21st ISCCB meeting was just held in Hamburg, Germany. The first historical period extends back to 1852 when Albert Kölliker first described the fine structure and function of the adrenal medulla. Subsequently, the adrenal staining with chromate salts identified the CCs; this was followed by the establishment of the embryological origin of the adrenal medulla, and the identification of adrenaline-storing vesicles. By the end of the nineteenth century, the basic morphology, histochemistry, and embryology of the adrenal gland were known. The twentieth century began with breakthrough findings namely, the experiment of Elliott suggesting that adrenaline was the sympathetic neurotransmitter, the isolation of pure adrenaline, and the deciphering of its molecular structure and chemical synthesis in the laboratory. In the 1950s, Blaschko isolated the catecholamine-storing vesicles from adrenal medullary extracts. This switched the interest in CCs as models of sympathetic neurons with an explosion of studies concerning their functions, i.e., uptake of catecholamines by chromaffin vesicles through a specific coupled transport system; the identification of several vesicle components in addition to catecholamines including chromogranins, ATP, opioids, and other neuropeptides; the calcium-dependence of the release of catecholamines; the underlying mechanism of exocytosis of this release, as indicated by the co-release of proteins; the cross-talk between the adrenal cortex and the medulla; and the emission of neurite-like processes by CCs in culture, among other numerous findings. The 1980s began with the introduction of new high-resolution techniques such as patch-clamp, calcium probes, marine toxins-targeting ion channels and receptors, confocal microscopy, or amperometry. In this frame of technological advances at the Ibiza ISCCB meeting in 1982, 11 senior researchers in the field predicted a notable increase in our knowledge in the field of CCs and the adrenal medulla; this cumulative knowledge that occurred in the last 40 years of history of the CC is succinctly described in the second part of this historical review. It deals with cell excitability, ion channel currents, the exocytotic fusion pore, the handling of calcium ions by CCs, the kinetics of exocytosis and endocytosis, the exocytotic machinery, and the life cycle of secretory vesicles. These concepts together with studies on the dynamics of membrane fusion with super-resolution imaging techniques at the single-protein level were extensively reviewed by top scientists in the field at the 21st ISCCB meeting in Hamburg in the summer of 2022; this frontier topic is also briefly reviewed here. Many of the concepts arising from those studies contributed to our present understanding of synaptic transmission. This has been studied in physiological or pathophysiological conditions, in CCs from animal disease models. In conclusion, the lessons we have learned from CC biology as a peripheral model for brain and brain disease pertain more than ever to cutting-edge research in neurobiology. In the 22nd ISCCB meeting in Israel in 2024 that Uri Asheri is organizing, we will have the opportunity of seeing the progress of the questions posed in Ibiza, and on other questions that undoubtedly will arise. [ABSTRACT FROM AUTHOR]

The sperm acrosome is a large dense-core granule whose contents are secreted by regulated exocytosis at fertilization through the opening of numerous fusion pores between the acrosomal and plasma membranes. In other cells, the nascent pore generated when the membrane surrounding a secretory vesicle fuses with the plasma membrane may have different fates. In sperm, pore dilation leads to the vesiculation and release of these membranes, together with the granule contents. α-Synuclein is a small cytosolic protein claimed to exhibit different roles in exocytic pathways in neurons and neuroendocrine cells. Here, we scrutinized its function in human sperm. Western blot revealed the presence of α-synuclein and indirect immunofluorescence its localization to the acrosomal domain of human sperm. Despite its small size, the protein was retained following permeabilization of the plasma membrane with streptolysin O. α-Synuclein was required for acrosomal release, as demonstrated by the inability of an inducer to elicit exocytosis when permeabilized human sperm were loaded with inhibitory antibodies to human α-synuclein. The antibodies halted calcium-induced secretion when introduced after the acrosome docked to the cell membrane. Two functional assays, fluorescence and transmission electron microscopies revealed that the stabilization of open fusion pores was responsible for the secretion blockage. Interestingly, synaptobrevin was insensitive to neurotoxin cleavage at this point, an indication of its engagement in cis SNARE complexes. The very existence of such complexes during AE reflects a new paradigm. Recombinant α-synuclein rescued the inhibitory effects of the anti-α-synuclein antibodies and of a chimeric Rab3A-22A protein that also inhibits AE after fusion pore opening. We applied restrained molecular dynamics simulations to compare the energy cost of expanding a nascent fusion pore between two model membranes and found it higher in the absence than in the presence of α-synuclein. Hence, our results suggest that α-synuclein is essential for expanding fusion pores.

During exocytosis, the fusion of secretory vesicle with plasma membrane forms a pore that regulates release of neurotransmitter and peptide. Heterogeneity of fusion pore behavior has been attributed to stochastic variation in a common exocytic mechanism, implying a lack of biological control. Using a fluorescent false neurotransmitter (FFN), we imaged dense core vesicle (DCV) exocytosis in primary mouse adrenal chromaffin cells by total internal reflection fluorescence microscopy at millisecond resolution and observed strikingly divergent modes of release, with fast events lasting <30 ms and slow events persisting for seconds. Dual imaging of slow events shows a delay in the entry of external dye relative to FFN release, suggesting exclusion by an extremely narrow pore <1 nm in diameter. Unbiased comprehensive analysis shows that the observed variation cannot be explained by stochasticity alone, but rather involves distinct mechanisms, revealing the bimodal nature of DCV exocytosis. Further, loss of calcium sensor synaptotagmin 7 increases the proportion of slow events without changing the intrinsic properties of either class, indicating the potential for independent regulation. The identification of two distinct mechanisms for release capable of independent regulation suggests a biological basis for the diversity of fusion pore behavior. [ABSTRACT FROM AUTHOR]

The inflexible concept of membrane curvature as an independent property of lipid structures is today obsolete. Lipid bilayers behave as many-body entities with emergent properties that depend on their interactions with the environment. In particular, proteins exert crucial actions on lipid molecules that ultimately condition the collective properties of the membranes. In this review, the potential of enhanced molecular dynamics to address cell-biology problems is discussed. The cases of membrane deformation, membrane fusion, and the fusion pore are analyzed from the perspective of the dimensionality reduction by collective variables. Coupled lipid-protein interactions as fundamental determinants of large membrane remodeling events are also commented. Finally, novel strategies merging cell biology and physics are considered as future lines of research. [ABSTRACT FROM AUTHOR]

Membrane fusion is not a spontaneous process. Physiologically, the formation of coiled‐coil protein complexes, the SNAREpins, bridges the membrane of a vesicle and a target membrane, brings them in close contact, and provides the energy necessary for their fusion. In this review, we utilize results from in vitro experiments and simple physics and chemistry models to dissect the kinetics and energetics of the fusion process from the encounter of the two membranes to the full expansion of a fusion pore. We find three main energy barriers that oppose the fusion process: SNAREpin initiation, fusion pore opening, and expansion. SNAREpin initiation is inherent to the proteins and makes in vitro fusion kinetic experiments rather slow. The kinetics are physiologically accelerated by effectors. The energy barriers that precede pore opening and pore expansion can be overcome by several SNAREpins acting in concert. [ABSTRACT FROM AUTHOR]

Exocytosis is a universal process of eukaryotic cells, consisting of fusion between the vesicle and the plasma membranes, leading to the formation of a fusion pore, a channel through which vesicle cargo exits into the extracellular space. In 1986, Rand and Parsegian proposed several stages to explain the nature of membrane fusion. Following stimulation, it starts with focused stress destabilization of membranes in contact, followed by the coalescence of two membrane surfaces. In the next fraction of a millisecond, restabilization of fused membranes is considered to occur to maintain the cell’s integrity. This view predicted that once a fusion pore is formed, it must widen abruptly, irreversibly and fully, whereby the vesicle membrane completely integrates with and collapses into the plasma membrane (full fusion exocytosis). However, recent experimental evidence has revealed that once the fusion pore opens, it may also reversibly close (transient or kiss-and-run exocytosis). Here, we present a historical perspective on understanding the mechanisms that initiate the membrane merger and fusion pore formation. Next, post-fusion mechanisms that regulate fusion pore stability are considered, reflecting the state in which the forces of widening and constriction of fusion pores are balanced. Although the mechanisms generating these forces are unclear, they may involve lipids and proteins, including SNAREs, which play a role not only in the pre-fusion but also post-fusion stages of exocytosis. How molecules stabilize the fusion pore in the open state is key for a better understanding of fusion pore physiology in health and disease.

In presynaptic terminals, membrane-delimited Gi/o-mediated presynaptic inhibition is ubiquitous and acts via Gβc to inhibit Ca2+ entry, or directly at SNARE complexes to inhibit Ca21-dependent synaptotagmin-SNARE complex interactions. At CA1-subicular presynaptic terminals, 5-HT1B and GABAB receptors colocalize. GABAB receptors inhibit Ca21 entry, whereas 5-HT1B receptors target SNARE complexes. We demonstrate in male and female rats that GABAB receptors alter Pr, whereas 5-HT1B receptors reduce evoked cleft glutamate concentrations, allowing differential inhibition of AMPAR and NMDAR EPSCs. This reduction in cleft glutamate concentration was confirmed by imaging glutamate release using a genetic sensor (iGluSnFR). Simulations of glutamate release and postsynaptic glutamate receptor currents were made. We tested effects of changes in vesicle numbers undergoing fusion at single synapses, relative placement of fusing vesicles and postsynaptic receptors, and the rate of release of glutamate from a fusion pore. Experimental effects of Pr changes, consistent with GABAB receptor effects, were straightforwardly represented by changes in numbers of synapses. The effects of 5-HT1B receptor-mediated inhibition are well fit by simulated modulation of the release rate of glutamate into the cleft. Colocalization of different actions of GPCRs provides synaptic integration within presynaptic terminals. Train-dependent presynaptic Ca2+ accumulation forces frequency-dependent recovery of neurotransmission during 5-HT1B receptor activation. This is consistent with competition between Ca2+-synaptotagmin and Gβc at SNARE complexes. Thus, stimulus trains in 5-HT1B receptor agonist unveil dynamic synaptic modulation and a sophisticated hippocampal output filter that itself is modulated by colocalized GABAB receptors, which alter presynaptic Ca2+. In combination, these pathways allow complex presynaptic integration. [ABSTRACT FROM AUTHOR]

In the neuron, neurotransmitter release is an essential function that must be both consistent and tightly regulated. The continuity of neurotransmitter release is dependent in large part on vesicle recycling. However, the protein factors that dictate the vesicle recycling pathway are elusive. Here, we use a single vesicle-to-supported bilayer fusion assay to investigate complexin-1 (cpx1)’s influence on SNARE-dependent fusion pore expansion. With total internal reflection (TIR) microscopy using a 10 kDa polymer fluorescence probe, we are able to detect the presence of large fusion pores. With cpx1, however, we observe a significant increase of the probability of the formation of large fusion pores. The domain deletion analysis reveals that the SNARE-binding core domain of cpx1 is mainly responsible for its ability to promote the fusion pore expansion. In addition, the results show that cpx1 helps the pore to expand larger, which results in faster release of the polymer probe. Thus, the results demonstrate a reciprocal relationship between event duration and the size of the fusion pore. Based on the data, a hypothetical mechanistic model can be deduced. In this mechanistic model, the cpx1 binding stabilizes the four-helix bundle structure of the SNARE core throughout the fusion pore expansion, whereby the highly curved bilayer within the fusion pore is stabilized by the SNARE pins.

The fusion pore is the initial narrow connection that forms between fusing membranes. During vesicular release of hormones or neurotransmitters, the nanometer-sized fusion pore may open-close repeatedly (flicker) before resealing or dilating irreversibly, leading to kiss-and-run or full-fusion events, respectively. Pore dynamics govern vesicle cargo release and the mode of vesicle recycling, but the mechanisms are poorly understood. This is partly due to a lack of reconstituted assays that combine single-pore sensitivity and high time resolution. Total internal reflection fluorescence (TIRF) microscopy offers unique advantages for characterizing single membrane fusion events, but signals depend on effects that are difficult to disentangle, including the polarization of the excitation electric field, vesicle size, photobleaching, orientation of the excitation dipoles of the fluorophores with respect to the membrane, and the evanescent field depth. Commercial TIRF microscopes do not allow control of excitation polarization, further complicating analysis. To overcome these challenges, we built a polarization-controlled total internal reflection fluorescence (pTIRF) microscope and monitored fusion of proteoliposomes with planar lipid bilayers with single molecule sensitivity and ∼15 ms temporal resolution. Using pTIRF microscopy, we detected docking and fusion of fluorescently labeled small unilamellar vesicles, reconstituted with exocytotic/neuronal v-SNARE proteins (vSUVs), with a supported bilayer containing the cognate t-SNAREs (tSBL). By varying the excitation polarization angle, we were able to identify a dye-dependent optimal polarization at which the fluorescence increase upon fusion was maximal, facilitating event detection and analysis of lipid transfer kinetics. An improved algorithm allowed us to estimate the size of the fusing vSUV and the fusion pore openness (the fraction of time the pore is open) for every event. For most events, lipid transfer was much slower than expected for diffusion through an open pore, suggesting that fusion pore flickering limits lipid release. We find a weak correlation between fusion pore openness and vesicle area. The approach can be used to study mechanisms governing fusion pore dynamics in a wide range of membrane fusion processes.

Summary: Many neurotransmitters are organic ions that carry a net charge, and their release from secretory vesicles is therefore an electrodiffusion process. The selectivity of early exocytotic fusion pores is investigated by combining electrodiffusion theory, measurements of amperometric foot signals from chromaffin cells with anion substitution, and molecular dynamics simulation. The results reveal that very narrow fusion pores are cation selective, but more dilated fusion pores become anion permeable. The transition occurs around a fusion pore conductance of ∼300 pS. The cation selectivity of a narrow fusion pore accelerates the release of positively charged transmitters such as dopamine, noradrenaline, adrenaline, serotonin, and acetylcholine, while glutamate release may require a more dilated fusion pore.

SNARE-dependent membrane fusion is essential for neurotransmitter release at the synapse. Recently, α-synuclein has emerged as an important regulator for membrane fusion. Misfolded α-synuclein oligomers are potent fusion inhibitors. However, the function of normal α-synuclein has been elusive. Here, we use the single vesicle-to-supported bilayer fusion assay to dissect the role of α-synuclein in membrane fusion. The assay employs 10 kD Rhodamine B-dextran as the content probe that can detect fusion pores larger than ∼6 nm. We find that the SNARE complex alone is inefficient at dilating fusion pores. However, α-synuclein dramatically increases the probability as well as the duration of large pores. When the SNARE-interacting C-terminal region of α-synuclein was truncated, the mutant behaves the same as the wild-type. However, the double proline mutants compromising membrane-binding show significantly reduced effects on fusion pore expansion. Thus, our results suggest that α-synuclein stimulates fusion pore expansion specifically through its membrane binding.

α-Synuclein (α-synFL) is central to the pathogenesis of Parkinson's disease (PD), in which its nonfunctional oligomers accumulate and result in abnormal neurotransmission. The normal physiological function of this intrinsically disordered protein is still unclear. Although several previous studies demonstrated α-synFL's role in various membrane fusion steps, they produced conFLicting outcomes regarding vesicular secretion. Here, we assess α-synFL's role in directly regulating individual exocytotic release events. We studied the micromillisecond dynamics of single recombinant fusion pores, the crucial kinetic intermediate of membrane fusion that tightly regulates the vesicular secretion in different cell types. α-SynFL accessed v-SNARE within the trans-SNARE complex to form an inhibitory complex. This activity was dependent on negatively charged phospholipids and resulted in decreased open probability of individual pores. The number of trans-SNARE complexes inFLuenced α-synFL's inhibitory action. Regulatory factors that arrest SNARE complexes in different assembly states differentially modulate α-synFL's ability to alter fusion pore dynamics. α-SynFL regulates pore properties in the presence of Munc13-1 and Munc18, which stimulate α-SNAP/NSF-resistant SNARE complex formation. In the presence of synaptotagmin1(syt1), α-synFL contributes with apo-syt1 to act as a membrane fusion clamp, whereas Ca2+•syt1 triggered α-synFL-resistant SNARE complex formation that rendered α-synFL inactive in modulating pore properties. This study reveals a key role of α-synFL in controlling vesicular secretion. [ABSTRACT FROM AUTHOR]

All membrane fusion reactions proceed through an initial fusion pore, including calcium-triggered release of neurotransmitters and hormones. Expansion of this small pore to release cargo is energetically costly and regulated by cells, but the mechanisms are poorly understood. Here, we show that the neuronal/exocytic calcium sensor Synaptotagmin-1 (Syt1) promotes expansion of fusion pores induced by SNARE proteins. Pore dilation relied on calcium-induced insertion of the tandem C2 domain hydrophobic loops of Syt1 into the membrane, previously shown to reorient the C2 domain. Mathematical modelling suggests that C2B reorientation rotates a bound SNARE complex so that it exerts force on the membranes in a mechanical lever action that increases the height of the fusion pore, provoking pore dilation to offset the bending energy penalty. We conclude that Syt1 exerts novel non-local calcium-dependent mechanical forces on fusion pores that dilate pores and assist neurotransmitter and hormone release.

Impaired insulin release is a hallmark of type 2 diabetes and is closely related to chronically elevated glucose concentrations, known as “glucotoxicity.” However, the molecular mechanisms by which glucotoxicity impairs insulin secretion remain poorly understood. In addition to known kiss-and-run and kiss-and-stay fusion events in INS-1 cells, ultrafast Hessian structured illumination microscopy (Hessian SIM) enables full fusion to be categorized according to the newly identified structures, such as ring fusion (those with enlarged pores) or dot fusion (those without apparent pores). In addition, we identified four fusion intermediates during insulin exocytosis: initial pore opening, vesicle collapse, enlarged pore formation, and final pore dilation. Long-term incubation in supraphysiological doses of glucose reduced exocytosis in general and increased the occurrence of kiss-and-run events at the expense of reduced full fusion. In addition, hyperglycemia delayed pore opening, vesicle collapse, and enlarged pore formation in full fusion events. It also reduced the size of apparently enlarged pores, all of which contributed to the compromised insulin secretion. These phenotypes were mostly due to the hyperglycemia-induced reduction in syntaxin-1A (Stx-1A) and SNAP-25 protein, since they could be recapitulated by the knockdown of endogenous Stx-1A and SNAP-25. These findings suggest essential roles for the vesicle fusion type and intermediates in regulating insulin secretion from pancreatic beta cells in normal and disease conditions.

Summary: Exocytosis of peptides and steroids stored in a dense core vesicular (DCV) form is the final step of every secretory pathway, indispensable for the function of nervous, endocrine and immune systems. The lack of live imaging techniques capable of direct, label‐free visualisation of DCV release makes many aspects of the exocytotic process inaccessible to investigation. We describe the application of correlative scanning ion conductance and fluorescence confocal microscopy (SICM‐FCM) to study the exocytosis of individual granules of insulin from the top, nonadherent, surface of pancreatic β‐cells. Using SICM‐FCM, we were first to directly follow the topographical changes associated with physiologically induced release of insulin DCVs. This allowed us to report the kinetics of the full fusion of the insulin vesicle as well as the subsequent solubilisation of the released insulin crystal. [ABSTRACT FROM AUTHOR]

The formation of the nervous system involves establishing complex networks of synaptic connections between proper partners. This developmental undertaking requires the rapid expansion of the plasma membrane surface area as neurons grow and polarize, extending axons through the extracellular environment. Critical to the expansion of the plasma membrane and addition of plasma membrane material is exocytic vesicle fusion, a regulated mechanism driven by soluble N-ethylmaleimide-sensitive factor attachment proteins receptors (SNAREs). Since their discovery, SNAREs have been implicated in several critical neuronal functions involving exocytic fusion in addition to synaptic transmission, including neurite initiation and outgrowth, axon specification, axon extension, and synaptogenesis. Decades of research have uncovered a rich variety of SNARE expression and function. The basis of SNARE-mediated fusion, the opening of a fusion pore, remains an enigmatic event, despite an incredible amount of research, as fusion is not only heterogeneous but also spatially small and temporally fast. Multiple modes of exocytosis have been proposed, with full-vesicle fusion (FFV) and kiss-and-run (KNR) being the best described. Whereas most in vitro work has reconstituted fusion using VAMP-2, SNAP-25, and syntaxin-1; there is much to learn regarding the behaviors of distinct SNARE complexes. In the past few years, robust heterogeneity in the kinetics and fate of the fusion pore that varies by cell type have been uncovered, suggesting a paradigm shift in how the modes of exocytosis are viewed is warranted. Here, we explore both classic and recent work uncovering the variety of SNAREs and their importance in the development of neurons, as well as historical and newly proposed modes of exocytosis, their regulation, and proteins involved in the regulation of fusion kinetics.

Abstract Background The native pre-fusion structure of gp120/gp41 complex of human immunodeficiency virus type 1 was recently revealed. In the model, the helices of gp41 (α6, α7, α8, and α9) form a four-helix collar underneath trimeric gp120. Gp41 is a class I fusion protein and mediates membrane fusion by forming a post-fusion structure called the six-helix bundle (6HB). The comparison of the pre- and post-fusion structures revealed the large conformational changes in gp41 during the antiparallel packing of the N- and C-terminal heptad repeats (NHRs and CHRs) in membrane fusion. Several mutagenesis studies of gp41 performed in the past were interpreted based on 6HB, the only available structure at that time. To obtain an insight about the current pre-fusion structural model and conformational changes during membrane fusion, alanine insertion mutagenesis of the NHR, CHR and connecting loop regions of HXB2 gp41 was performed. The effects of mutations on biosynthesis and membrane fusion were analyzed by immunoblotting and fusion assays, respectively. The extent of membrane fusion was evaluated by split luciferase-based pore formation and syncytia formation assays, respectively. Results Consistent with the current structural model, drastic negative effects of mutations on biosynthesis and membrane fusion were observed for NHR, loop, and proximal regions of CHR (up to amino acid position 643). The insertions in α9 after it leaves the four-helix collar were tolerable for biosynthesis. These CHR mutants showed varying effects on membrane fusion. Insertion at position 644 or 645 resulted in poor pore and syncytia formation. Efficient pore and syncytia formation almost similar to that of the wild type was observed for insertion at position 647, 648 or 649. However, recovery of virus infectivity was only observed for the insertions beyond position 648. Conclusions The mutagenesis data for HXB2 gp41 is in agreement with the recent pre-fusion structure model. The virus infection data suggested that fusion pores sufficiently large enough for the release of the virus genome complex are formed after the completion of 6HB beyond position 648.

Astrocytes are the key homeostatic cells in the central nervous system; initiation of reactive astrogliosis contributes to neuroinflammation. Pro-inflammatory cytokine interferon γ (IFNγ) induces the expression of the major histocompatibility complex class II (MHCII) molecules, involved in antigen presentation in reactive astrocytes. The pathway for MHCII delivery to the astrocyte plasma membrane, where MHCII present antigens, is unknown. Rat astrocytes in culture and in organotypic slices were exposed to IFNγ to induce reactive astrogliosis. Astrocytes were probed with optophysiologic tools to investigate subcellular localization of immunolabeled MHCII, and with electrophysiology to characterize interactions of single vesicles with the plasmalemma. In culture and in organotypic slices, IFNγ augmented the astrocytic expression of MHCII, which prominently co-localized with lysosomal marker LAMP1-EGFP, modestly co-localized with Rab7, and did not co-localize with endosomal markers Rab4A, EEA1, and TPC1. MHCII lysosomal localization was corroborated by treatment with the lysosomolytic agent glycyl-l-phenylalanine-β-naphthylamide, which reduced the number of MHCII-positive vesicles. The surface presence of MHCII was revealed by immunolabeling of live non-permeabilized cells. In IFNγ-treated astrocytes, an increased fraction of large-diameter exocytotic vesicles (lysosome-like vesicles) with prolonged fusion pore dwell time and larger pore conductance was recorded, whereas the rate of endocytosis was decreased. Stimulation with ATP, which triggers cytosolic calcium signaling, increased the frequency of exocytotic events, whereas the frequency of full endocytosis was further reduced. In IFNγ-treated astrocytes, MHCII-linked antigen surface presentation is mediated by increased lysosomal exocytosis, whereas surface retention of antigens is prolonged by concomitant inhibition of endocytosis. [ABSTRACT FROM AUTHOR]

The formation of the nervous system involves establishing complex networks of synaptic connections between proper partners. This developmental undertaking requires the rapid expansion of the plasma membrane surface area as neurons grow and polarize, extending axons through the extracellular environment. Critical to the expansion of the plasma membrane and addition of plasma membrane material is exocytic vesicle fusion, a regulated mechanism driven by soluble N-ethylmaleimide-sensitive factor attachment proteins receptors (SNAREs). Since their discovery, SNAREs have been implicated in several critical neuronal functions involving exocytic fusion in addition to synaptic transmission, including neurite initiation and outgrowth, axon specification, axon extension, and synaptogenesis. Decades of research have uncovered a rich variety of SNARE expression and function. The basis of SNARE-mediated fusion, the opening of a fusion pore, remains an enigmatic event, despite an incredible amount of research, as fusion is not only heterogeneous but also spatially small and temporally fast. Multiple modes of exocytosis have been proposed, with full-vesicle fusion (FFV) and kiss-and-run (KNR) being the best described. Whereas most in vitro work has reconstituted fusion using VAMP-2, SNAP-25, and syntaxin-1; there is much to learn regarding the behaviors of distinct SNARE complexes. In the past few years, robust heterogeneity in the kinetics and fate of the fusion pore that varies by cell type have been uncovered, suggesting a paradigm shift in how the modes of exocytosis are viewed is warranted. Here, we explore both classic and recent work uncovering the variety of SNAREs and their importance in the development of neurons, as well as historical and newly proposed modes of exocytosis, their regulation, and proteins involved in the regulation of fusion kinetics. [ABSTRACT FROM AUTHOR]

There is a general consensus that synaptic vesicular release by a full collapse process is the primary machinery of synaptic transmission. However, competing view suggests that synaptic vesicular release operates via a kiss-and-run mechanism. By monitoring the release dynamics of a synaptic vesicular marker, FM1-43 from individual synapses in hippocampal neurons, we found evidence that the release of synaptic vesicle was delayed by several seconds after the start of field stimulation. This phenomenon was associated with modified opening kinetics of fusion pores. Detailed analysis revealed that some synapses were completely inactive for a few seconds after stimulation, despite immediate calcium influx. This delay in vesicular release was modulated by various stimulation protocols and different frequencies, indicating an activity-dependent regulation mechanism for neurotransmitter exocytosis. Staurosporine, a drug known to induce “kiss-and-run” exocytosis, increased the proportion of delayed synapses as well as the delay duration, while fluoxetine acted contrarily. Besides being a serotonin reuptake inhibitor, it directly enhanced vesicle mobilization and reduced synaptic fatigue. Exocytosis was never delayed, when it was monitored with pH-sensitive probes, synaptopHlourin and αSyt-CypHerE5 antibody, indicating an instantaneous formation of a fusion pore that allowed rapid equilibration of vesicular lumenal pH but prevented FM1-43 release because of its slow dissociation from the inner vesicular membrane. Our observations suggest that synapses operate via a sequential “kiss-and-run” and “full-collapse” exocytosis mechanism. The initially narrow vesicular pore allows the equilibration of intravesicular pH which then progresses toward full fusion, causing FM1-43 release.

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.

Aim: Astrocytes play a homeostatic role in the central nervous system and influence numerous aspects of neurophysiology via intracellular trafficking of vesicles. Intermediate filaments (IFs), also known as nanofilaments, regulate a number of cellular processes including organelle trafficking and adult hippocampal neurogenesis. We have recently demonstrated that the IF protein nestin, a marker of neural stem cells and immature and reactive astrocytes, is also expressed in some astrocytes in the unchallenged hippocampus and regulates neurogenesis through Notch signalling from astrocytes to neural stem cells, possibly via altered trafficking of vesicles containing the Notch ligand Jagged‐1. Methods: We thus investigated whether nestin affects vesicle dynamics in astrocytes by examining single vesicle interactions with the plasmalemma and vesicle trafficking with high‐resolution cell‐attached membrane capacitance measurements and confocal microscopy. We used cell cultures of astrocytes from nestin‐deficient (Nes−/−) and wild‐type (wt) mice, and fluorescent dextran and Fluo‐2 to examine vesicle mobility and intracellular Ca2+ concentration respectively. Results: Nes−/− astrocytes exhibited altered sizes of vesicles undergoing full fission and transient fusion, altered vesicle fusion pore geometry and kinetics, decreased spontaneous vesicle mobility and altered ATP‐evoked mobility. Purinergic stimulation evoked Ca2+ signalling that was slightly attenuated in Nes−/− astrocytes, which exhibited more oscillatory Ca2+ responses than wt astrocytes. Conclusion: These results demonstrate at the single vesicle level that nestin regulates vesicle interactions with the plasmalemma and vesicle trafficking, indicating its potential role in astrocyte vesicle‐based communication. [ABSTRACT FROM AUTHOR]

Dysregulation of lipid homeostasis leads to the development of metabolic disorders including obesity, diabetes, cardiovascular disease and cancer. Lipid droplets (LDs) are subcellular organelles vital in the maintenance of lipid homeostasis by coordinating lipid synthesis, lipid storage, lipid secretion and lipolysis. Under fed condition, free fatty acids (FFAs) are remodeled and esterified into neutral lipids by lipogenesis and stored in the LDs. The lipid storage capacity of LDs is controlled by its growth via local lipid synthesis or by LD fusion. During fasting, neutral lipids are hydrolyzed by lipolysis, released as FFAs and secreted to meet energy demand. Cell death‐inducing DNA fragmentation factor alpha (DFFA)‐like effector (CIDE) family proteins composed of Cidea, Cideb and Cidec/Fsp27 are ER‐ and LD‐associated proteins and have emerged as important regulators of lipid homeostasis. Notably, when localized on the LDs, CIDE proteins enrich at the LD‐LD contact sites (LDCSs) and control LD fusion and growth. Here, we summarize these recent advances made on the role of CIDE proteins in the regulation of lipid metabolism with a particular focus on the molecular mechanisms underlying CIDE‐mediated LD fusion and growth. [ABSTRACT FROM AUTHOR]

There is a general consensus that synaptic vesicular release by a full collapse process is the primary machinery of synaptic transmission. However, competing view suggests that synaptic vesicular release operates via a kiss-and-run mechanism. By monitoring the release dynamics of a synaptic vesicular marker, FM1-43 from individual synapses in hippocampal neurons, we found evidence that the release of synaptic vesicle was delayed by several seconds after the start of field stimulation. This phenomenon was associated with modified opening kinetics of fusion pores. Detailed analysis revealed that some synapses were completely inactive for a few seconds after stimulation, despite immediate calcium influx. This delay in vesicular release was modulated by various stimulation protocols and different frequencies, indicating an activity-dependent regulation mechanism for neurotransmitter exocytosis. Staurosporine, a drug known to induce "kiss-and-run" exocytosis, increased the proportion of delayed synapses as well as the delay duration, while fluoxetine acted contrarily. Besides being a serotonin reuptake inhibitor, it directly enhanced vesicle mobilization and reduced synaptic fatigue. Exocytosis was never delayed, when it was monitored with pH-sensitive probes, synaptopHlourin and αSyt-CypHerE5 antibody, indicating an instantaneous formation of a fusion pore that allowed rapid equilibration of vesicular lumenal pH but prevented FM1-43 release because of its slow dissociation from the inner vesicular membrane. Our observations suggest that synapses operate via a sequential "kiss-and-run" and "full-collapse" exocytosis mechanism. The initially narrow vesicular pore allows the equilibration of intravesicular pH which then progresses toward full fusion, causing FM1-43 release. [ABSTRACT FROM AUTHOR]

The protein α‐synuclein has a central role in the pathogenesis of Parkinson's disease (PD). In this review, we discuss recent results concerning its primary function, which appears to be on cell membranes. The pre‐synaptic location of synuclein has suggested a role in neurotransmitter release and it apparently associates with synaptic vesicles because of their high curvature. Indeed, synuclein over‐expression inhibits synaptic vesicle exocytosis. However, loss of synuclein has not yet been shown to have a major effect on synaptic transmission. Consistent with work showing that synuclein can promote as well as sense membrane curvature, recent analysis of synuclein triple knockout mice now shows that synuclein accelerates dilation of the exocytic fusion pore. This form of regulation affects primarily the release of slowly discharged lumenal cargo such as neural peptides, but presumably also contributes to maintenance of the release site. This article is part of the Special Issue "Synuclein". [ABSTRACT FROM AUTHOR]

Synaptic release of neuropeptides packaged in dense-core vesicles (DCVs) regulates synapses, circuits, and behaviors including feeding, sleeping, and pain perception. Here, synaptic DCV fusion pore openings are imaged without interference from cotransmitting small synaptic vesicles (SSVs) with the use of a fluorogen-activating protein (FAP). Activity-evoked kiss and run exocytosis opens synaptic DCV fusion pores away from active zones that readily conduct molecules larger than most native neuropeptides (i.e., molecular weight [MW] up to, at least, 4.5 kDa). Remarkably, these synaptic fusion pores also open spontaneously in the absence of stimulation and extracellular Ca2+. SNARE perturbations demonstrate different mechanisms for activity-evoked and spontaneous fusion pore openings with the latter sharing features of spontaneous small molecule transmitter release by active zone-associated SSVs. Fusion pore opening at resting synapses provides a mechanism for activity-independent peptidergic transmission. [ABSTRACT FROM AUTHOR]

Highlights • Question: does the membrane-curving ability of α-synuclein alter fusion pore expansion and secretory protein discharge? • Endogenous β-synuclein but not α-synuclein is associated with secretory granules in bovine adrenal chromaffin cells. • Overexpression of α-synuclein but not β-synuclein quickened the post- fusion discharge of BDNF-pHluorin by ∼30%. • Neither α- nor β-synuclein significantly altered curvature dynamics associated with fusion pore expansion measured by pTIRFM. • The physiological significance of the small increased rate of post-fusion protein discharge is uncertain. Abstract α-Synuclein is strongly implicated in the pathogenesis of Parkinson's disease as well as in other neurodegenerative diseases. However, its normal function in cells is not understood. The N-termini of α-, β-, and γ-synuclein contains six to seven 11-amino acid repeats that are predicted to form amphipathic helices. Membrane-binding and membrane-curving abilities of synuclein raise the possibility that synuclein could alter cellular processes that involve highly curved structures. In the present study we examined the localization of endogenous synuclein in bovine chromaffin cells by immunocytochemistry and its possible function to control protein discharge upon fusion of the granule with the plasma membrane by regulating the fusion pore. We found with quantitative immunocytochemistry that endogenous β-synuclein associates with secretory granules. Endogenous α-synuclein only rarely co-localizes with secretory granules. Overexpression of α-synuclein but not β-synuclein quickened the post- fusion discharge of BDNF-pHluorin by approxinately 30%. However, neither α- nor β-synuclein significantly altered curvature dynamics associated with fusion pore expansion that were measured by the combination of polarization and total internal reflection fluorescence microscopy (pTIRFM). Whatever the mechanism, the physiological significance of the small increased rate of post-fusion protein discharge caused by α-synuclein remains to be demonstrated, especially since endogenous β-, but not α-synuclein is the predominant synuclein isoform associated with chromaffin granules. [ABSTRACT FROM AUTHOR]

Abstract Vesicular exocytosis is a ubiquitous bio-process involved in plenty of normal and pathologic events in living cells and has received increasing research attention. After decades of progress, amperometry has been demonstrated to be one of the most powerful techniques to monitor the exocytotic release in real time because of its remarkable advantages such as facility to implement, high sensitivity and sub-millisecond temporal resolution. In this review, we provide an overview of recent advances in electrochemical courses related to exocytotic secretions. The principle for amperometric characterization of quantal exocytosis is briefly introduced. Development of mircrofabricated architectures for amperometric detection and their contributions are summarized. An intimate coupling of amperometry and fluorescence imaging for in-situ exocytosis tracking are highlighted. Highlights • Amperometry provides quantitative information of exocytosis at sub-millisecond level. • Lab-on-chips satisfies different requirements for exocytosis monitoring. • Coupling electrochemistry with fluorescence imaging promises high spatiotemporal resolution. • Transparent electrode is the key for the combined method. [ABSTRACT FROM AUTHOR]

Ketamine, a non-competitive N–methyl–d–aspartate receptor (NMDAR) antagonist, exerts a rapid, potent and long-lasting antidepressant effect, although the cellular and molecular mechanisms of this action are yet to be clarified. In addition to targeting neuronal NMDARs fundamental for synaptic transmission, ketamine also affects the function of astrocytes, the key homeostatic cells of the central nervous system that contribute to pathophysiology of major depressive disorder. Here, I review studies revealing that (sub)anesthetic doses of ketamine elevate intracellular cAMP concentration ([cAMP]i) in astrocytes, attenuate stimulus-evoked astrocyte calcium signaling, which regulates exocytotic secretion of gliosignaling molecules, and stabilize the vesicle fusion pore in a narrow configuration, possibly hindering cargo discharge or vesicle recycling. Next, I discuss how ketamine affects astrocyte capacity to control extracellular K+ by reducing vesicular delivery of the inward rectifying potassium channel (Kir4.1) to the plasmalemma that reduces the surface density of Kir4.1. Modified astroglial K+ buffering impacts upon neuronal firing pattern as demonstrated in lateral habenula in a rat model of depression. Finally, I highlight the discovery that ketamine rapidly redistributes cholesterol in the astrocyte plasmalemma, which may alter the flux of cholesterol to neurons. This structural modification may further modulate a host of processes that synergistically contribute to ketamine’s rapid antidepressant action.

The pathogenesis of degeneration in Parkinson's disease (PD) remains poorly understood but multiple lines of evidence have converged on the presynaptic protein α-synuclein (αsyn). αSyn has been shown to regulate several cellular processes, however, its normal function remains poorly understood. In this review, we will specifically focus on its role in exocytosis. [ABSTRACT FROM AUTHOR]

SNAP-25 is an essential component of SNARE complexes driving fast Ca2+-dependent exocytosis. Yet, the functional implications of the tandem-like structure of SNAP-25 are unclear. Here, we have investigated the mechanistic role of the acylated “linker” domain that concatenates the two SNARE motifs within SNAP-25. Refuting older concepts of an inert connector, our detailed structure-function analysis in murine chromaffin cells demonstrates that linker motifs play a crucial role in vesicle priming, triggering, and fusion pore expansion. Mechanistically, we identify two synergistic functions of the SNAP-25 linker: First, linker motifs support t-SNARE interactions and accelerate ternary complex assembly. Second, the acylated N-terminal linker segment engages in local lipid interactions that facilitate fusion triggering and pore evolution, putatively establishing a favorable membrane configuration by shielding phospholipid headgroups and affecting curvature. Hence, the linker is a functional part of the fusion complex that promotes secretion by SNARE interactions as well as concerted lipid interplay.

Dynamin 1 (dynl) is required for clathrin-mediated endocytosis in most secretory (neuronal and neuroendocrine) cells. There are two modes of Ca2+-dependent catecholamine release from single dense-core vesicles: full-quantal (quantal) and subquantal in adrenal chromaffin cells, but their relative occurrences and impacts on total secretion remain unclear. To address this fundamental question in neurotransmission area using both sexes of animals, here we report the following: (1) dynl -KO increased quantal size (QS, but not vesicle size/content) by⩾250% in dynl-KO mice; (2) the KO-increased QS was rescued by dynl (but not its deficient mutant or dyn2); (3) the ratio of quantal versus subquantal events was increased by KO; (4) following a release event, more protein contents were retained in WT versus KO vesicles; and (5) the fusion pore size (dp) was increased from ⩽9 to ⩾9 nm by KO. Therefore, Ca2+ -induced exocytosis is generally a subquantal release in sympathetic adrenal chromaffin cells, implying that neurotransmitter release is generally regulated by dynamin in neuronal cells. [ABSTRACT FROM AUTHOR]

Membrane fusion and fission are fundamental processes in living organisms. Membrane fusion occurs through the formation of a fusion pore, which is the structure that connects two lipid membranes during their fusion. Fusion pores can form spontaneously, but cells endow themselves with a set of proteins that make the process of fusion faster and regulatable. The fusion pore starts with a narrow diameter and dilates relatively slowly; it may fluctuate in size or can even close completely, producing a transient vesicle fusion (kiss‐and‐run), or can finally expand abruptly to release all vesicle contents. A set of proteins control the formation, dilation, and eventual closure of the fusion pore and, therefore, the velocity at which the contents of secretory vesicles are released to the extracellular medium. Thus, the regulation of fusion pore expansion or closure is key to regulate the release of neurotransmitters and hormones. Here, we review the phases of the fusion pore and discuss the implications in the modes of exocytosis. [ABSTRACT FROM AUTHOR]