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Actin dynamics provides membrane tension to merge fusing vesicles into the plasma membrane.

ABSTRACT: Vesicle fusion is executed via formation of an ?-shaped structure (?-profile), followed by closure (kiss-and-run) or merging of the ?-profile into the plasma membrane (full fusion). Although ?-profile closure limits release but recycles vesicles economically, ?-profile merging facilitates release but couples to classical endocytosis for recycling. Despite its crucial role in determining exocytosis/endocytosis modes, how ?-profile merging is mediated is poorly understood in endocrine cells and neurons containing small ?30-300?nm vesicles. Here, using confocal and super-resolution STED imaging, force measurements, pharmacology and gene knockout, we show that dynamic assembly of filamentous actin, involving ATP hydrolysis, N-WASP and formin, mediates ?-profile merging by providing sufficient plasma membrane tension to shrink the ?-profile in neuroendocrine chromaffin cells containing ?300?nm vesicles. Actin-directed compounds also induce ?-profile accumulation at lamprey synaptic active zones, suggesting that actin may mediate ?-profile merging at synapses. These results uncover molecular and biophysical mechanisms underlying ?-profile merging.

SUBMITTER: Wen PJ

PROVIDER: S-EPMC5013665 | biostudies-literature | 2016 Aug

REPOSITORIES: biostudies-literature

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Actin dynamics provides membrane tension to merge fusing vesicles into the plasma membrane.

Wen Peter J PJ Grenklo Staffan S Arpino Gianvito G Tan Xinyu X Liao Hsien-Shun HS Heureaux Johanna J Peng Shi-Yong SY Chiang Hsueh-Cheng HC Hamid Edaeni E Zhao Wei-Dong WD Shin Wonchul W Näreoja Tuomas T Evergren Emma E Jin Yinghui Y Karlsson Roger R Ebert Steven N SN Jin Albert A Liu Allen P AP Shupliakov Oleg O Wu Ling-Gang LG

Nature communications 20160831

Vesicle fusion is executed via formation of an Ω-shaped structure (Ω-profile), followed by closure (kiss-and-run) or merging of the Ω-profile into the plasma membrane (full fusion). Although Ω-profile closure limits release but recycles vesicles economically, Ω-profile merging facilitates release but couples to classical endocytosis for recycling. Despite its crucial role in determining exocytosis/endocytosis modes, how Ω-profile merging is mediated is poorly understood in endocrine cells and ne ...[more]

PMID: 27576662

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Project description:The intracellular trafficking of connexin 43 (Cx43) hemichannels presents opportunities to regulate cardiomyocyte gap junction coupling. Although it is known that Cx43 hemichannels are transported along microtubules to the plasma membrane, the role of actin in Cx43 forward trafficking is unknown.We explored whether the actin cytoskeleton is involved in Cx43 forward trafficking.High-resolution imaging reveals that Cx43 vesicles colocalize with nonsarcomeric actin in adult cardiomyocytes. Live-cell fluorescence imaging reveals Cx43 vesicles as stationary or traveling slowly (average speed 0.09 ?m/s) when associated with actin. At any time, the majority (81.7%) of vesicles travel at subkinesin rates, suggesting that actin is important for Cx43 transport. Using Cx43 containing a hemagglutinin tag in the second extracellular loop, we developed an assay to detect transport of de novo Cx43 hemichannels to the plasma membrane after release from Brefeldin A-induced endoplasmic reticulum/Golgi vesicular transport block. Latrunculin A (for specific interference of actin) was used as an intervention after reinitiation of vesicular transport. Disruption of actin inhibits delivery of Cx43 to the cell surface. Moreover, using the assay in primary cardiomyocytes, actin inhibition causes an 82% decrease (P<0.01) in de novo endogenous Cx43 delivery to cell-cell borders. In Langendorff-perfused mouse heart preparations, Cx43/?-actin complexing is disrupted during acute ischemia, and inhibition of actin polymerization is sufficient to reduce levels of Cx43 gap junctions at intercalated discs.Actin is a necessary component of the cytoskeleton-based forward trafficking apparatus for Cx43. In cardiomyocytes, Cx43 vesicles spend a majority of their time pausing at nonsarcomeric actin rest stops when not undergoing microtubule-based transport to the plasma membrane. Deleterious effects on this interaction between Cx43 and the actin cytoskeleton during acute ischemia contribute to losses in Cx43 localization at intercalated discs.

Dataset Information

Actin dynamics provides membrane tension to merge fusing vesicles into the plasma membrane.

Publications

Actin dynamics provides membrane tension to merge fusing vesicles into the plasma membrane.

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