Synaptophysin (syp) is a major integral membrane protein of secretory vesicles. Previous work has demonstrated functions for syp in synaptic vesicle cycling, endocytosis, and synaptic plasticity, but ...the role of syp in the process of membrane fusion during Ca
-triggered exocytosis remains poorly understood. Furthermore, although syp resides on both large dense-core and small synaptic vesicles, its role in dense-core vesicle function has received less attention compared with synaptic vesicle function. To explore the role of syp in membrane fusion and dense-core vesicle function, we used amperometry to measure catecholamine release from single vesicles in male and female mouse chromaffin cells with altered levels of syp and the related tetraspanner protein synaptogyrin (syg). Knocking out syp slightly reduced the frequency of vesicle fusion events below wild-type (WT) levels, but knocking out both syp and syg reduced the frequency 2-fold. Knocking out both proteins stabilized initial fusion pores, promoted fusion pore closure (kiss-and-run), and reduced late-stage fusion pore expansion. Introduction of a syp construct lacking its C-terminal dynamin-binding domain in syp knock-outs (KOs) increased the duration and fraction of kiss-and-run events, increased total catecholamine release per event, and reduced late-stage fusion pore expansion. These results demonstrated that syp and syg regulate dense-core vesicle function at multiple stages to initiate fusion, control the choice of mode between full-fusion and kiss-and-run, and influence the dynamics of both initial and late-stage fusion pores. The transmembrane domain (TMD) influences small initial fusion pores, and the C-terminal domain influences large late-stage fusion pores, possibly through an interaction with dynamin.
The secretory vesicle protein synaptophysin (syp) is known to function in synaptic vesicle cycling, but its roles in dense-core vesicle functions, and in controlling membrane fusion during Ca
-triggered exocytosis remain unclear. The present study used amperometry recording of catecholamine release from endocrine cells to assess the impact of syp and related proteins on membrane fusion. A detailed analysis of amperometric spikes arising from the exocytosis of single vesicles showed that these proteins influence fusion pores at multiple stages and control the choice between kiss-and-run and full-fusion. Experiments with a syp construct lacking its C terminus indicated that the transmembrane domain (TMD) influences the initial fusion pore, while the C-terminal domain influences later stages after fusion pore expansion.
Fusion of transmitter-containing vesicles with plasma membranes at the synaptic and neuromuscular junctions mediates neurotransmission and muscle contractions, respectively, thereby underlying all ...thoughts and actions. The fusion process is driven by the coupled folding and assembly of three synaptic SNARE proteins--syntaxin-1 and SNAP-25 on the target plasma membrane (t-SNAREs) and VAMP2 on the vesicular membrane (v-SNARE) into a four-helix bundle. Their assembly is chaperoned by Munc18-1 and many other proteins to achieve the speed and accuracy required for neurotransmission. However, the physiological pathway of SNARE assembly and its coupling to membrane fusion remains unclear. Here, we review recent progress in understanding SNARE assembly and membrane fusion, with a focus on results obtained by single-molecule manipulation approaches and electric recordings of single fusion pores. We describe two pathways of synaptic SNARE assembly, their associated intermediates, energetics, and kinetics. Assembly of the three SNAREs in vitro begins with the formation of a t-SNARE binary complex, on which VAMP2 folds in a stepwise zipper-like fashion. Munc18-1 significantly alters the SNARE assembly pathway: syntaxin-1 and VAMP2 first bind on the surface of Munc18-1 to form a template complex, with which SNAP-25 associates to conclude SNARE assembly and displace Munc18-1. During membrane fusion, multiple trans-SNARE complexes cooperate to open a dynamic fusion pore in a manner dependent upon their copy number and zippering states. Together, these results demonstrate that stepwise and cooperative SNARE assembly drive stagewise membrane fusion.
In laser powder bed fusion (LPBF), melt pool instability can lead to the development of pores in printed parts, reducing the part's structural strength. While camera-based monitoring systems have ...been introduced to improve melt pool stability, these systems only measure melt pool stability in limited, indirect ways. We propose that melt pool stability can be improved by explicitly encoding stability into LPBF monitoring systems through the use of temporal features and pore density modelling. We introduce the temporal features, in the form of temporal variances of common LPBF monitoring features (e.g., melt pool area, intensity), to explicitly quantify printing stability. Furthermore, we introduce a neural network model trained to link these video features directly to pore densities estimated from the CT scans of previously printed parts. This model aims to reduce the number of online printer interventions to only those that are required to avoid porosity. These contributions are then implemented in a full LPBF monitoring system and tested on prints using 316L stainless steel. Results showed that our explicit stability quantification improved the correlation between our predicted pore densities and true pore densities by up to 42%.
Neurons communicate via an essential process called exocytosis. Cholesterol, an abundant lipid in both secretory vesicles and cell plasma membrane can affect this process. In this study, amperometric ...recordings of vesicular dopamine release from two different artificial cell models created from a giant unilamellar liposome and a bleb cell plasma membrane, show that with higher membrane cholesterol the kinetics for vesicular release are decelerated in a concentration dependent manner. This reduction in exocytotic speed was consistent for two observed modes of exocytosis, full and partial release. Partial release events, which only occurred in the bleb cell model due to the higher tension in the system, exhibited amperometric spikes with three distinct shapes. In addition to the classic transient, some spikes displayed a current ramp or plateau following the maximum peak current. These post spike features represent neurotransmitter release from a dilated pore before constriction and show that enhancing membrane rigidity via cholesterol adds resistance to a dilated pore to re-close. This implies that the cholesterol dependent biophysical properties of the membrane directly affect the exocytosis kinetics and that membrane tension along with membrane rigidity can influence the fusion pore dynamics and stabilization which is central to regulation of neurochemical release.
Synaptotagmins (syts) are abundant, evolutionarily conserved integral membrane proteins that play essential roles in regulated exocytosis in nervous and endocrine systems. There are at least 17 syt ...isoforms in mammals, all with tandem C-terminal C2 domains with highly variable capacities for Ca(2+) binding. Many syts play roles in neurotransmitter release or hormone secretion or both, and a growing body of work supports a role for some syts as Ca(2+) sensors of exocytosis. Work in many types of endocrine cells has documented the presence of a number of syt isoforms on dense-core vesicles containing various hormones. Syts can influence the kinetics of exocytotic fusion pores and the choice of release mode between kiss-and-run and full-fusion. Vesicles harboring different syt isoforms can preferentially undergo distinct modes of exocytosis with different forms of stimulation. The diverse properties of syt isoforms enable these proteins to shape Ca(2+) sensing in endocrine cells, thus contributing to the regulation of hormone release and the organization of complex endocrine functions.
Electrochemical cytometry is a method developed recently to determine the content of an individual cell vesicle. The mechanism of vesicle rupture at the electrode surface involves the formation of a ...pore at the interface between a vesicle and the electrode through electroporation, which leads to the release and oxidation of the vesicle's chemical cargo. We have manipulated the membrane properties using excited fluorophores conjugated to lipids, which appears to make the membrane more susceptible to electroporation. We propose that by having excited fluorophores in close contact with the membrane, membrane lipids (and perhaps proteins) are oxidized upon production of reactive oxygen species, which then leads to changes in membrane properties and the formation of water defects. This is supported by experiments in which the fluorophores were placed on the lipid tail instead of the headgroup, which leads to a more rapid onset of vesicle opening. Additionally, application of DMSO to the vesicles, which increases the membrane area per lipid, and decreasing the membrane thickness result in the same enhancement in vesicle opening, which confirms the mechanism of vesicle opening with excited fluorophores in the membrane. Light‐induced manipulation of membrane vesicle pore opening might be an attractive means of controlling cell activity and exocytosis. Additionally, our data confirm that in experiments in which cells or vesicle membranes are labeled for fluorescence monitoring, the properties of the excited membrane change substantially.
Vesicle electrochemical cytometry has been developed as a novel technique to measure the content of individual vesicles. It is proposed that bringing an excited fluorophore into close contact with the membrane enhances vesicle opening through the oxidation of membrane lipids (and perhaps proteins), which leads to a more disrupted membrane and both better adsorption of vesicles to the electrode surface and an electroporation‐formed fusion pore.
Lactotrophs are one of the five secretory anterior pituitary cell types specialized to synthesize and release prolactin. In vitro, these cells fire action potentials (APs) spontaneously and the ...accompanied Ca(2+) transients are of sufficient amplitude to keep the exocytotic pathway, the transcription of prolactin gene, and de novo hormone synthesis continuously active. Basal cyclic nucleotide production is also substantial in cultured cells but not critical for the APs secretion/transcription coupling in lactotrophs. However, elevated intracellular cAMP levels enhance the excitability of lactotrophs by stimulating the depolarizing non-selective cationic hyperpolarization-activated cyclic nucleotide-regulated and background channels, whereas cGMP inhibits it by activating Ca(2+)-controlled K(+) channels. Elevated cAMP also modulates prolactin release downstream of Ca(2+) influx by changing the kinetic of secretory pores: stimulate at low and inhibit at high concentrations. Induction of prolactin gene and lactotroph proliferation is also stimulated by elevated cAMP through protein kinase A. Together, these observations suggest that in lactotrophs cAMP exhibits complex regulatory effects on voltage-gated Ca(2+) influx and Ca(2+)-dependent cellular processes.
Dynamin is a GTPase mechanochemical enzyme involved in the late steps of endocytosis, where it separates the endocytotic vesicle from the cell membrane. However, recent reports have emphasized its ...role in exocytosis. In this case, dynamin may contribute to the control of the exocytotic pore, thus suggesting a direct control on the efflux of neurotransmitters. Dynasore, a selective inhibitor of the GTPase activity of dynamin, was used to investigate the role of dynamin in exocytosis. Exocytosis was analyzed by amperometry, thus revealing that dynasore inhibits exocytosis in a dose-dependent manner. Analysis of the exocytotic peaks shows that the inhibition of the GTPase activity of dynamin leads to shorter, smaller events. This observation, together with the rapid effect of dynasore, suggests that the blocking of the GTPase induces the formation of a more narrow and short-lived fusion pore. These results suggest that the GTPase properties of dynamin are involved in the duration and kinetics of exocytotic release. Interestingly, and in strong contrast with its role in endocytosis, the mechanochemical properties of dynamin appear to contribute to the dilation and stability of the pore during exocytosis.
We have amperometrically measured dopamine release from rat pheochromocytoma cells (PC12 cells) in high osmolarity conditions with and without L-3,4-dihydroxyphenylalanine (L-DOPA) treatment. We ...observe an increase in the number of release events displaying a prespike feature or "foot" when the cells are stimulated in high osmolarity saline. We also see an increase in foot area and duration when cells are stimulated in high osmolarity saline, or high osmolarity saline subsequent to incubation with the dopamine precursor L-DOPA in isotonic saline, which serves to increase the vesicle size. The data suggest that membrane biophysics are an important component in defining the rate, duration and amount of neurotransmitter release via the fusion pore.
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