The Munc18-1 domain 3a hinge-loop controls syntaxin-1A nanodomain assembly and engagement with the SNARE complex during secretory vesicle priming

Kasula, Ravikiran; Chai, Ye Jin; Bademosi, Adekunle T.; Harper, Callista B.; Gormal, Rachel S.; Morrow, Isabel C.; Hosy, Eric; Collins, Brett M.; Choquet, Daniel; Papadopulos, Andreas; Meunier, Frédéric A.

doi:10.1083/jcb.201508118

Cited by 52 publications

(81 citation statements)

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“…As discussed in this review, superresolution imaging techniques have provided critical insights into the nanoscale organization and dynamics of syntaxin-1 in the plasma membrane of neurosecretory cells and neurons. It is becoming clear that activity alters the dynamics and molecular composition of syntaxin-1 nanoclusters on the plasma membrane (Bademosi et al, 2017;Kasula et al, 2016). Future work will focus on elucidating the functional link between the dynamics and molecular composition of individual syntaxin-1 nanoclusters and vesicle docking, priming and fusion events.…”

Section: Discussionmentioning

confidence: 99%

“…The model predicted that syntaxin-1 clusters contain an average of 75 molecules and about 16% of total syntaxin-1 molecules is freely diffusing on the plasma membrane. However, recent SPT experiments have reported a much larger percentage of free syntaxin-1 molecules at neuronal synapses (Ribrault et al, 2011;Schneider et al, 2015), at the NMJs (Bademosi et al, 2017) and in PC12 cells (Kasula et al, 2016). The observed differences in the percentage of free syntaxin-1 molecules could stem from the different imaging techniques and experimental conditions used across studies.…”

Section: Computational Models Of Syntaxin-1 Dynamics On the Plasma Mementioning

confidence: 98%

“…Syntaxin-1 steady-state distribution is predominantly on the plasma membrane, and several studies have shown that it forms nanoclusters in this region (Bademosi et al, 2017;Kasula et al, 2016;Milovanovic and Jahn, 2015). Syntaxin-1 constantly switches between freely diffusing (mobile) and clustered (immobile) states at the plasma membrane which facilitate rapid assembly or disassembly of syntaxin-1 nanoclusters in response to stimulation.…”

Section: Overviewmentioning

confidence: 99%

“…However, whether activity-dependent secretory vesicle release affects the nanocluster organization of syntaxin-1 is not well understood. To address this, Kasula et al (2016) imaged syntaxin-1-GFP using universal point accumulation in nanoscale topography (uPAINT) and Munc18-1-mEos2 using sptPALM in PC12 cells engineered to knock down Munc18-1/2 (DKD-PC12 cells). First, using dual colour super-resolution imaging, the authors demonstrated a partial overlap between syntaxin-1-GFP and Munc18-1-mEos2 nanodomains in fixed DKD-PC12 cells.…”

Section: Syntaxin-1 Dynamics In Neurosecretory Cellsmentioning

confidence: 99%

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Need for speed: Super-resolving the dynamic nanoclustering of syntaxin-1 at exocytic fusion sites

et al. 2020

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Communication between cells relies on regulated exocytosis, a multi-step process that involves the docking, priming and fusion of vesicles with the plasma membrane, culminating in the release of neurotransmitters and hormones. Key proteins and lipids involved in exocytosis are subjected to Brownian movement and constantly switch between distinct motion states which are governed by short-lived molecular interactions. Critical biochemical reactions between exocytic proteins that occur in the confinement of nanodomains underpin the precise sequence of priming steps which leads to the fusion of vesicles. The advent of super-resolution microscopy techniques has provided the means to visualize individual molecules on the plasma membrane with high spatiotemporal resolution in live cells. These techniques are revealing a highly dynamic nature of the nanoscale organization of the exocytic machinery. In this review, we focus on soluble N-ethylmaleimide-sensitive factor attachment receptor (SNARE) syntaxin-1, which mediates vesicular fusion. Syntaxin-1 is highly mobile at the plasma membrane, and its inherent speed allows fast assembly and disassembly of syntaxin-1 nanoclusters which are associated with exocytosis. We reflect on recent studies which have revealed the mechanisms regulating syntaxin-1 nanoclustering on the plasma membrane and draw inferences on the effect of synaptic activity, phosphoinositides, N-ethylmaleimide-sensitive factor (NSF), -soluble NSF attachment protein (-SNAP) and SNARE complex assembly on the dynamic nanoscale organization of syntaxin-1.Abbreviations: SNARE, soluble N-ethylmaleimide-sensitive factor attachment receptor; NSF, N-ethylmaleimide-sensitive factor; -SNAP, -soluble NSF attachment protein; PC12, pheochromocytoma; NMJ, neuromuscular junction; TIRF, total internal reflection fluorescence; STED, stimulated emission depletion; dSTORM, direct stochastic optical resolution microscopy; PALM, photoactivated localization microscopy; SPT, single particle tracking; sptPALM, single particle tracking photoactivated localization microscopy; uPAINT, universal point accumulation in nanoscale topography; PtdIns(4,5)P 2 , phosphatidylinositol (4,5)-biphosphate; PtdIns(3,4,5)P 3 , phosphatidylinositol (3,4,5)-biphosphate; FRAP, fluorescence recovery after bleaching; GFP, green fluorescent protein. Highlights-Syntaxin-1 forms nanoclusters on the plasma membrane.-Multiple molecular mechanisms regulate syntaxin-1 nanoclustering on the plasma membrane.-Synaptic activity, phosphoinositides and SNARE complex assembly differentially control the nanoscale organization of syntaxin-1 on the plasma membrane.-Super-resolution microscopy has the potential to uncover the design principles governing regulated exocytosis.

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Section: Discussionmentioning

confidence: 99%

Section: Computational Models Of Syntaxin-1 Dynamics On the Plasma Mementioning

confidence: 98%

Section: Overviewmentioning

confidence: 99%

Section: Syntaxin-1 Dynamics In Neurosecretory Cellsmentioning

confidence: 99%

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Need for speed: Super-resolving the dynamic nanoclustering of syntaxin-1 at exocytic fusion sites

et al. 2020

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“…For stochastic photoconversion of mEos2 molecules, low amount (3-5%) of 405 nm laser and 75-80% of 561 nm laser was used. Data analysis was carried out as previously described 47,48 using PALM-Tracer, a plugin in Metamorph software (Molecular Devices).…”

Section: Single Molecule Tracking and Quantitationmentioning

confidence: 99%

Dissecting the nanoscale lipid profile of caveolae

Zhou

Ariotti

et al. 2020

Preprint

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words)Caveolae are specialized domains of the vertebrate cell surface with a well-defined morphology and crucial roles in cell migration and mechanoprotection. Unique compositions of proteins and lipids determine membrane architectures. The precise caveolar lipid profile and the roles of the major caveolar structural proteins, caveolins and cavins, in selectively sorting lipids have not been defined. Here we used quantitative nanoscale lipid mapping together with molecular dynamic simulations to define the caveolar lipid profile. We show that caveolin1 (CAV1) and cavin1 individually sort distinct plasma membrane lipids. Intact caveolar structures composed of both CAV1 and cavin1 further generate a unique lipid nanoenvironment. The caveolar lipid sorting capability includes selectivities for lipid headgroups and acyl chains. Because lipid headgroup metabolism and acyl chain remodelling are tightly regulated, this selective lipid sorting may allow caveolae to act as transit hubs to direct communications among lipid metabolism, vesicular trafficking and signalling.Caveolae are a striking morphological feature of the plasma membrane of many vertebrate cells. Caveolae have been implicated in mechanoprotection, endocytosis, signal transduction and lipid regulation 1-6 . The characteristic morphology of caveolae, with a bulb connected to the plasma membrane by a highly-curved neck, is generated by integral membrane proteins termed caveolins and by lipid binding peripheral membrane proteins, the cavins 7-14 .Specifically, caveolin-1 (CAV1) and caveolin-3 (CAV3, in striated muscle) and cavin1 (or polymerase I and transcript release factor, PTRF) are essential for caveola formation 7,10 .Another set of key caveolar components comprise the plasma membrane (PM) lipids. While biophysical studies have consistently suggested that the lateral distribution of lipids determines and/or responds to changing membrane morphology, our understanding of the lipid composition of caveolae and how this contributes to caveola formation, function, and disassembly, is still relatively primitive. A detailed molecular understanding of the lipid constituents of caveolae, defined both by their headgroups and acyl chains, is crucial for understanding the formation of caveolae, as well as their disassembly, processes that are crucial for caveola function. Moreover, caveolae can provide a paradigm for understanding how local concentrations of specific lipid species contribute to membrane morphogenesis.

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Exploring the conformational changes of the Munc18‐1/syntaxin 1a complex

Stefani,

Iwaszkiewicz,

Fasshauer

2024

Protein Science

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Neurotransmitters are released from synaptic vesicles, the membrane of which fuses with the plasma membrane upon calcium influx. This membrane fusion reaction is driven by the formation of a tight complex comprising the plasma membrane SNARE proteins syntaxin‐1a and SNAP‐25 with the vesicle SNARE protein synaptobrevin. The neuronal protein Munc18‐1 forms a stable complex with syntaxin‐1a. Biochemically, syntaxin‐1a cannot escape the tight grip of Munc18‐1, so formation of the SNARE complex is inhibited. However, Munc18‐1 is essential for the release of neurotransmitters in vivo. It has therefore been assumed that Munc18‐1 makes the bound syntaxin‐1a available for SNARE complex formation. Exactly how this occurs is still unclear, but it is assumed that structural rearrangements occur. Here, we used a series of mutations to specifically weaken the complex at different positions in order to induce these rearrangements biochemically. Our approach was guided through sequence and structural analysis and supported by molecular dynamics simulations. Subsequently, we created a homology model showing the complex in an altered conformation. This conformation presumably represents a more open arrangement of syntaxin‐1a that permits the formation of a SNARE complex to be initiated while still bound to Munc18‐1. In the future, research should investigate how this central reaction for neuronal communication is controlled by other proteins.This article is protected by copyright. All rights reserved.

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The Munc18-1 domain 3a hinge-loop controls syntaxin-1A nanodomain assembly and engagement with the SNARE complex during secretory vesicle priming

Cited by 52 publications

References 46 publications

Need for speed: Super-resolving the dynamic nanoclustering of syntaxin-1 at exocytic fusion sites

Need for speed: Super-resolving the dynamic nanoclustering of syntaxin-1 at exocytic fusion sites

Dissecting the nanoscale lipid profile of caveolae

Exploring the conformational changes of the Munc18‐1/syntaxin 1a complex

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