The packing density of a supramolecular membrane protein cluster is controlled by cytoplasmic interactions

Merklinger, Elisa; Schloetel, Jan-Gero; Weber, Pascal; Batoulis, Helena; Holz, Sarah; Karnowski, Nora; Finke, Jérôme; Lang, Thorsten

doi:10.7554/elife.20705

Cited by 24 publications

(46 citation statements)

References 38 publications

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“…This indicates that (in intact cells) the clustering of syntaxin‐1 at granules does not depend on the presence of PtdIns(4,5)P 2 , which is in stark contrast to findings in membrane sheets and artificial membranes . One reason for this discrepancy may be the relative importance of protein‐protein interactions (involving syntaxins N‐terminal Habc domain) over biophysical clustering modes such as hydrophobic mismatch and charge effects . Importantly, our findings contradict models in which clusters of syntaxin‐1 and PtdIns(4,5)P 2 or PtdIns(3,4,5)P 3 act as prearranged acceptor complex or “molecular beacon” during granule docking .…”

Section: Discussioncontrasting

confidence: 74%

PtdIns(4,5)P₂ is not required for secretory granule docking

Omar‐Hmeadi

Gandasi

Barg

2018

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Phosphoinositides (PtdIns) play important roles in exocytosis and are thought to regulate secretory granule docking by co-clustering with the SNARE protein syntaxin to form a docking receptor in the plasma membrane. Here we tested this idea by high-resolution total internal reflection imaging of EGFP-labeled PtdIns markers or syntaxin-1 at secretory granule release sites in live insulin-secreting cells. In intact cells, PtdIns markers distributed evenly across the plasma membrane with no preference for granule docking sites. In contrast, syntaxin-1 was found clustered in the plasma membrane, mostly beneath docked granules. We also observed rapid accumulation of syntaxin-1 at sites where granules arrived to dock. Acute depletion of plasma membrane phosphatidylinositol (4,5) bisphosphate (PtdIns(4,5)P ) by recruitment of a 5'-phosphatase strongly inhibited Ca -dependent exocytosis, but had no effect on docked granules or the distribution and clustering of syntaxin-1. Cell permeabilization by α-toxin or formaldehyde-fixation caused PtdIns marker to slowly cluster, in part near docked granules. In summary, our data indicate that PtdIns(4,5)P accelerates granule priming, but challenge a role of PtdIns in secretory granule docking or clustering of syntaxin-1 at the release site.

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Section: Discussioncontrasting

confidence: 74%

PtdIns(4,5)P₂ is not required for secretory granule docking

Omar‐Hmeadi

Gandasi

Barg

2018

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“…Although the transmembrane domain region of syntaxin-1 alone can form clusters on the plasma membrane (Sieber et al, 2006), the homophilic protein-protein interactions involving the SNARE motif are required to trap syntaxin-1 molecules into nanoclusters (Sieber et al, 2007). Merklinger et al (2017) recently proposed that the transmembrane domain promotes loose clustering of syntaxin-1, whereas the SNARE motif of the cytoplasmic domain facilitates the tight packing of molecules within the clusters.…”

Section: Factors That Regulate Syntaxin-1 Clustering On the Plasma Mementioning

confidence: 99%

“…Fluorescence recovery after photobleaching (FRAP) works by selective bleaching of a fluorescently tagged protein of interest in a small region of the plasma membrane. The recovery of the fluorescence signal in the bleached region is then recorded to assess the mobility of the protein (Halemani et al, 2010;Merklinger et al, 2017;Ribrault et al, 2011;Sieber et al, 2007;Zilly et al, 2011). Stimulated emission depletion (STED) microscopy works by overlapping a doughnut-shaped beam with the excitation beam to selectively switch off fluorescent proteins with the exception of those at the centre of the doughnut.…”

Section: Box 1: a Brief Overview Of Imaging Techniquesmentioning

confidence: 99%

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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“…On the other hand, the smallest maxima may arise from single proteins and therefore are not protein clusters anyway. In any case, the data suggests that most maxima do not arise from single molecules but from larger aggregates as protein clusters, that typically are in the same size range as protein clusters formed by other proteins [29][30][31] .…”

Section: Discussionmentioning

confidence: 90%

Anatomy of a viral entry platform differentially functionalized by integrins α3 and α6

Finke

Mikuličić

Loster

et al. 2020

Sci Rep

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www.nature.com/scientificreports www.nature.com/scientificreports/ cluster increases at viral particle attachment sites. Our data suggests that viral particles use an entry platform containing integrin α6 for virus attachment and integrin α3 for internalisation. Resultsintegrin α3 is required for HPV16 infection of keratinocytes. To clarify the role of integrin α3 in the infection of keratinocytes, we infected integrin α3 depleted HaCaT cells by incubation with HPV16 pseudovirions (PsVs). For positive control, cells were depleted from integrin α6, which is required for infection of HeLa and KH-SV cells 5,21 . In Western blot analysis, knockdown reduced integrin α6 and integrin α3 protein levels by 87% and 97%, respectively ( Fig. S1). Intracellular processing of the L1 virus capsid protein was monitored by Western blot analysis and by microscopy. In the Western blots, we quantified the ~25 kDa cleavage product of L1, which is generated only after internalization and lysosomal degradation of the virus 22 . As seen in Fig. 1A, knockdown of integrin α3 and integrin α6 reduced the cleavage product by 73% and 44%, respectively. For microscopy, we employed antibody-detection of the L1-7 epitope, which only becomes detectable after intracellular capsid disassembly 4,23 . Knockdown of either one of the integrins reduced the number of L1-7 positive organelles to about 65% ( Fig. 1B,C). Moreover, organelles were slightly dimmer ( Fig. S2), suggesting that apart from less forming endocytic organelles the viral load per organelle diminishes.Next, we employed a luciferase-based infection assay to test whether less uptake and processing of the L1 protein would be associated with a lower infection rate. Infection is inhibited by 88% and 67% after integrin α3 and integrin α6 knockdown, respectively ( Fig. 1D). It should be noted that integrin knockdown has secondary effects ( Fig. S3). Still, after correcting for these secondary effects, knockdown of each integrin more than halves the infection rate ( Fig. S3). These experiments demonstrate that apart from integrin α6, integrin α3 plays a role in HPV infection of HaCaT cells as well. However, in Western blot analysis testing PsVs cell-surface primary binding only the knockdown of integrin α6 strongly inhibits binding by 40% ( Fig. 2), suggesting each of these integrins is differently involved during infection.PsVs associate with cluster crowds. Next, we asked whether PsV particles are closer to their potential binding partner integrin α6, and analysed the distances between PsVs and CD151/integrin maxima. On membrane sheets, PsVs are often close to or overlap with CD151 or integrin α6 maxima (Fig. 4A,B). Most distances are in the range of several 100 nm ( Fig. 4C), with no trend towards shorter distances to integrin α6 maxima. Also, no maxima preference is observed on PsVs/CD151/integrin α3 stainings of cells ( Fig. 4D-F), or PsVs/ CD151-GFP/integrin α3 stainings on membrane sheets (Fig. S11). Moreover, it should be noted that PsVs diminish the level of CD151 and integrins by 10-...

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The packing density of a supramolecular membrane protein cluster is controlled by cytoplasmic interactions

Cited by 24 publications

References 38 publications

PtdIns(4,5)P₂ is not required for secretory granule docking

PtdIns(4,5)P₂ is not required for secretory granule docking

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

Anatomy of a viral entry platform differentially functionalized by integrins α3 and α6

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The packing density of a supramolecular membrane protein cluster is controlled by cytoplasmic interactions

Cited by 24 publications

References 38 publications

PtdIns(4,5)P2 is not required for secretory granule docking

PtdIns(4,5)P2 is not required for secretory granule docking

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

Anatomy of a viral entry platform differentially functionalized by integrins α3 and α6

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PtdIns(4,5)P₂ is not required for secretory granule docking

PtdIns(4,5)P₂ is not required for secretory granule docking