scholarly journals Visualizing sequential compound fusion and kiss-and-run in live excitable cells

2021 ◽  
Author(s):  
Lihao Ge ◽  
Wonchul Shin ◽  
Ling-Gang Wu
Keyword(s):  
Vesicle Fusion ◽  
Neuroendocrine Cells ◽  
Fusion Pore ◽  
Excitable Cells ◽  
Vesicle Docking ◽  
Vesicular Release ◽  
Flat Membrane ◽  
Pore Closure ◽  
Asynchronous Release

Vesicle fusion is assumed to occur at flat membrane of excitable cells. In live neuroendocrine cells, we visualized vesicle fusion at Ω-shape membrane generated by preceding fusion, termed sequential compound fusion, which may be followed by fusion pore closure, termed compound kiss-and-run. These novel fusion modes contribute to vesicle docking, multi-vesicular release, asynchronous release, and endocytosis. We suggest modifying current models of exo-endocytosis to include these new fusion modes.

Synaptotagmin-1: A Multi-Functional Protein that Mediates Vesicle Docking, Priming, and Fusion

2021 ◽  
Vol 22 ◽  
Author(s):  
Najeeb Ullah ◽  
Ezzouhra El Maaiden ◽  
Md. Sahab Uddin ◽  
Ghulam Md Ashraf
Keyword(s):  
Plasma Membrane ◽  
Secretory Granules ◽  
Secretory Vesicle ◽  
Neuroendocrine Cells ◽  
Snare Complex ◽  
Secretory Vesicles ◽  
Functional Protein ◽  
Vesicle Docking ◽  
Synaptotagmin 1

: The fusion of secretory vesicles with the plasma membrane depends on the assembly of v-SNAREs (VAMP2/synaptobrevin2) and t-SNAREs (SNAP25/syntaxin1) into the SNARE complex. Vesicles go through several upstream steps, referred to as docking and priming, to gain fusion competence. The vesicular protein synaptotagmin-1 (Syt-1) is the principal Ca2+ sensor for fusion in several central nervous system neurons and neuroendocrine cells and part of the docking complex for secretory granules. Syt-1 binds to the acceptor complex such as synaxin1, SNAP-25 on the plasma membrane to facilitate secretory vesicle docking, and upon Ca2+-influx promotes vesicle fusion. This review assesses the role of the Syt-1 protein involved in the secretory vesicle docking, priming, and fusion.

scholarly journals Differential Co-release of Two Neurotransmitters from a Vesicle Fusion Pore in Mammalian Adrenal Chromaffin Cells

Neuron ◽  
2019 ◽  
Vol 102 (1) ◽  
pp. 173-183.e4 ◽  
Author(s):  
Quanfeng Zhang ◽  
Bin Liu ◽  
Qihui Wu ◽  
Bing Liu ◽  
Yinglin Li ◽  
...  
Keyword(s):  
Chromaffin Cells ◽  
Vesicle Fusion ◽  
Fusion Pore ◽  
Adrenal Chromaffin Cells ◽  
Adrenal Chromaffin

scholarly journals Live-cell imaging of exocyst links its spatiotemporal dynamics to various stages of vesicle fusion

2013 ◽  
Vol 201 (5) ◽  
pp. 673-680 ◽  
Author(s):  
Felix Rivera-Molina ◽  
Derek Toomre
Keyword(s):  
Membrane Trafficking ◽  
Live Cell Imaging ◽  
Cell Imaging ◽  
Vesicle Fusion ◽  
Cell Polarization ◽  
Spatiotemporal Dynamics ◽  
Live Cell ◽  
Fusion Pore ◽  
Cell Cortex ◽  
Morphological Criteria

Tethers play ubiquitous roles in membrane trafficking and influence the specificity of vesicle attachment. Unlike soluble N-ethyl-maleimide–sensitive fusion attachment protein receptors (SNAREs), the spatiotemporal dynamics of tethers relative to vesicle fusion are poorly characterized. The most extensively studied tethering complex is the exocyst, which spatially targets vesicles to sites on the plasma membrane. By using a mammalian genetic replacement strategy, we were able to assemble fluorescently tagged Sec8 into the exocyst complex, which was shown to be functional by biochemical, trafficking, and morphological criteria. Ultrasensitive live-cell imaging revealed that Sec8-TagRFP moved to the cell cortex on vesicles, which preferentially originated from the endocytic recycling compartment. Surprisingly, Sec8 remained with vesicles until full dilation of the fusion pore, supporting potential coupling with SNARE fusion machinery. Fluorescence recovery after photobleaching analysis of Sec8 at cell protrusions revealed that a significant fraction was immobile. Additionally, Sec8 dynamically repositioned to the site of membrane expansion, suggesting that it may respond to local cues during early cell polarization.

scholarly journals Syndapin 3 modulates fusion pore expansion in mouse neuroendocrine chromaffin cells

2014 ◽  
Vol 306 (9) ◽  
pp. C831-C843 ◽  
Author(s):  
Prattana Samasilp ◽  
Kyle Lopin ◽  
Shyue-An Chan ◽  
Rajesh Ramachandran ◽  
Corey Smith
Keyword(s):  
Chromaffin Cells ◽  
Control Point ◽  
Peripheral Circulation ◽  
Neuroendocrine Cells ◽  
Fusion Pore ◽  
Cell Periphery ◽  
Hormone Release ◽  
Excitatory Synaptic Input ◽  
Activity Dependent ◽  
Pore Expansion

Adrenal neuroendocrine chromaffin cells receive excitatory synaptic input from the sympathetic nervous system and secrete hormones into the peripheral circulation. Under basal sympathetic tone, modest amounts of freely soluble catecholamine are selectively released through a restricted fusion pore formed between the secretory granule and the plasma membrane. Upon activation of the sympathoadrenal stress reflex, elevated stimulation drives fusion pore expansion, resulting in increased catecholamine secretion and facilitating release of copackaged peptide hormones. Thus regulated expansion of the secretory fusion pore is a control point for differential hormone release of the sympathoadrenal stress response. Previous work has shown that syndapin 1 deletion alters transmitter release and that the dynamin 1-syndapin 1 interaction is necessary for coupled endocytosis in neurons. Dynamin has also been shown to be involved in regulation of fusion pore expansion in neuroendocrine chromaffin cells through an activity-dependent association with syndapin. However, it is not known which syndapin isoform(s) contributes to pore dynamics in neuroendocrine cells. Nor is it known at what stage of the secretion process dynamin and syndapin associate to modulate pore expansion. Here we investigate the expression and localization of syndapin isoforms and determine which are involved in mediating fusion pore expansion. We show that all syndapin isoforms are expressed in the adrenal medulla. Mutation of the SH3 dynamin-binding domain of all syndapin isoforms shows that fusion pore expansion and catecholamine release are limited specifically by mutation of syndapin 3. The mutation also disrupts targeting of syndapin 3 to the cell periphery. Syndapin 3 exists in a persistent colocalized state with dynamin 1.

scholarly journals A triggered mechanism retrieves membrane in seconds after Ca(2+)-stimulated exocytosis in single pituitary cells

1994 ◽  
Vol 124 (5) ◽  
pp. 667-675 ◽  
Author(s):  
P Thomas ◽  
AK Lee ◽  
JG Wong ◽  
W Almers
Keyword(s):  
Plasma Membrane ◽  
Cell Surface ◽  
Time Constant ◽  
Flash Photolysis ◽  
Membrane Capacitance ◽  
Coated Vesicles ◽  
Neuroendocrine Cells ◽  
Pituitary Cells ◽  
Excitable Cells ◽  
Late Step

In neuroendocrine cells, cytosolic Ca2+ triggers exocytosis in tens of milliseconds, yet known pathways of endocytic membrane retrieval take minutes. To test for faster retrieval mechanisms, we have triggered short bursts of exocytosis by flash photolysis of caged Ca2+, and have tracked subsequent retrieval by measuring the plasma membrane capacitance. We find that a limited amount of membrane can be retrieved with a time constant of 4 s at 21-26 degrees C, and that this occurs partially via structures larger than coated vesicles. This novel mechanism may be arrested at a late step. Incomplete retrieval structures then remain on the cell surface for minutes until the consequences of a renewed increase in cytosolic [Ca2+] disconnect them from the cell surface in < 1 s. Our results provide evidence for a rapid, triggered membrane retrieval pathway in excitable cells.

scholarly journals Proteolytic maturation of α2δ controls the probability of synaptic vesicular release

eLife ◽  
2018 ◽  
Vol 7 ◽  
Author(s):  
Laurent Ferron ◽  
Ivan Kadurin ◽  
Annette C Dolphin
Keyword(s):  
Calcium Channels ◽  
Inhibitory Effect ◽  
Wild Type ◽  
Voltage Gated ◽  
Synaptic Release ◽  
Voltage Gated Calcium Channels ◽  
Vesicular Release ◽  
Active Zones ◽  
Asynchronous Release

Auxiliary α2δ subunits are important proteins for trafficking of voltage-gated calcium channels (CaV) at the active zones of synapses. We have previously shown that the post-translational proteolytic cleavage of α2δ is essential for their modulatory effects on the trafficking of N-type (CaV2.2) calcium channels (Kadurin et al., 2016). We extend these results here by showing that the probability of presynaptic vesicular release is reduced when an uncleaved α2δ is expressed in rat neurons and that this inhibitory effect is reversed when cleavage of α2δ is restored. We also show that asynchronous release is influenced by the maturation of α2δ−1, highlighting the role of CaV channels in this component of vesicular release. We present additional evidence that CaV2.2 co-immunoprecipitates preferentially with cleaved wild-type α2δ. Our data indicate that the proteolytic maturation increases the association of α2δ−1 with CaV channel complex and is essential for its function on synaptic release.

scholarly journals Cdc42 controls the dilation of the exocytotic fusion pore by regulating membrane tension

2014 ◽  
Vol 25 (20) ◽  
pp. 3195-3209 ◽  
Author(s):  
Marine Bretou ◽  
Ouardane Jouannot ◽  
Isabelle Fanget ◽  
Paolo Pierobon ◽  
Nathanaël Larochette ◽  
...  
Keyword(s):  
Membrane Fusion ◽  
Small Gtpase ◽  
Dominant Negative ◽  
Neuroendocrine Cells ◽  
Fusion Pore ◽  
Membrane Tension ◽  
Release Process ◽  
Vesicle Exocytosis ◽  
Pore Dilation ◽  
Pore Enlargement

Membrane fusion underlies multiple processes, including exocytosis of hormones and neurotransmitters. Membrane fusion starts with the formation of a narrow fusion pore. Radial expansion of this pore completes the process and allows fast release of secretory compounds, but this step remains poorly understood. Here we show that inhibiting the expression of the small GTPase Cdc42 or preventing its activation with a dominant negative Cdc42 construct in human neuroendocrine cells impaired the release process by compromising fusion pore enlargement. Consequently the mode of vesicle exocytosis was shifted from full-collapse fusion to kiss-and-run. Remarkably, Cdc42-knockdown cells showed reduced membrane tension, and the artificial increase of membrane tension restored fusion pore enlargement. Moreover, inhibiting the motor protein myosin II by blebbistatin decreased membrane tension, as well as fusion pore dilation. We conclude that membrane tension is the driving force for fusion pore dilation and that Cdc42 is a key regulator of this force.

scholarly journals Fusion Pore Formation Observed During SNARE-Mediated Vesicle Fusion with Pore-Spanning Membranes

2020 ◽  
Author(s):  
P. Mühlenbrock ◽  
K. Herwig ◽  
L. Vuong ◽  
I. Mey ◽  
C. Steinem
Keyword(s):  
Fluorescence Microscopy ◽  
Pore Formation ◽  
Three Dimensional ◽  
Rare Event ◽  
Vesicle Fusion ◽  
Fusion Process ◽  
Fusion Pore ◽  
Dual Color ◽  
Fusion Pores

ABSTRACTPlanar pore-spanning membranes (PSMs) have been shown to be a versatile tool to resolve docking and elementary steps of the fusion process with single large unilamellar vesicles (LUVs). However, in previous studies, we monitored only lipid mixing and did not gather information about the formation of fusion pores. To address this important step of the fusion process, we entrapped sulforhodamine B at self-quenching concentrations into LUVs containing the v-SNARE synaptobrevin 2, which were docked and fused with lipid-labeled PSMs containing the t-SNARE acceptor complex ΔN49 prepared on porous silicon substrates. By dual color spinning disc fluorescence microcopy with a time resolution of 20 ms, we could unambiguously distinguish between bursting vesicles and fusion pore formation. Owing to the aqueous compartment underneath the PSMs, vesicle bursting turned out to be an extremely rare event (< 0.01 %). From the time-resolved dual color fluorescence time traces, we were able to identify different fusion pathways including remaining three-dimensional postfusion structures with released content and flickering fusion pores. Our results on fusion pore formation and lipid diffusion from the PSM into the fusing vesicle let us conclude that the content release, i.e., fusion pore formation follows the merger of the two lipid membranes by only about 40 ms.STATEMENT OF SIGNIFICANCEDespite great efforts to develop in vitro fusion assays to understand the process of neuronal fusion, there is still a huge demand to provide single vesicle fusion assays that simultaneously report on all intermediate states including three-dimensional postfusion structures and fusion pore formation including flickering pores without the underlying artifact of vesicle bursting. Here, we show that pore-spanning membranes (PSMs) are ideal candidates to fulfill these demands. Owing to their planarity and the second aqueous compartments, they are readily accessible by fluorescence microscopy and provide sufficient space so that vesicle bursting becomes negligible. Dual color fluorescence microscopy allows distinguishing between different fusion intermediates and fusion pathways such as “kiss and run” fusion as well as flickering fusion pores.

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