高速成像揭示了致密核心囊泡胞吐的双重模态性质。

High-speed imaging reveals the bimodal nature of dense core vesicle exocytosis.

机构信息

Department of Neurology, School of Medicine, University of California San Francisco, San Francisco, CA 94158.

Department of Physiology, School of Medicine, University of California San Francisco, San Francisco, CA 94158.

出版信息

Proc Natl Acad Sci U S A. 2023 Jan 3;120(1):e2214897120. doi: 10.1073/pnas.2214897120. Epub 2022 Dec 27.

Abstract

During exocytosis, the fusion of secretory vesicle with plasma membrane forms a pore that regulates release of neurotransmitter and peptide. Heterogeneity of fusion pore behavior has been attributed to stochastic variation in a common exocytic mechanism, implying a lack of biological control. Using a fluorescent false neurotransmitter (FFN), we imaged dense core vesicle (DCV) exocytosis in primary mouse adrenal chromaffin cells by total internal reflection fluorescence microscopy at millisecond resolution and observed strikingly divergent modes of release, with fast events lasting <30 ms and slow events persisting for seconds. Dual imaging of slow events shows a delay in the entry of external dye relative to FFN release, suggesting exclusion by an extremely narrow pore <1 nm in diameter. Unbiased comprehensive analysis shows that the observed variation cannot be explained by stochasticity alone, but rather involves distinct mechanisms, revealing the bimodal nature of DCV exocytosis. Further, loss of calcium sensor synaptotagmin 7 increases the proportion of slow events without changing the intrinsic properties of either class, indicating the potential for independent regulation. The identification of two distinct mechanisms for release capable of independent regulation suggests a biological basis for the diversity of fusion pore behavior.

摘要

在胞吐作用过程中,分泌囊泡与质膜融合形成一个调节神经递质和肽类释放的孔道。融合孔道行为的异质性归因于常见胞吐机制中的随机变化,这意味着缺乏生物学控制。使用荧光假神经递质(FFN),我们通过全内反射荧光显微镜以毫秒分辨率对原代小鼠肾上腺嗜铬细胞中的致密核心囊泡(DCV)胞吐作用进行成像,并观察到释放的明显不同模式,快速事件持续时间<30ms,而缓慢事件持续数秒。对缓慢事件的双重成像显示,外部染料的进入相对于 FFN 释放存在延迟,这表明存在直径<1nm 的极窄孔道的排斥。无偏综合分析表明,观察到的变异不能仅用随机性来解释,而是涉及不同的机制,揭示了 DCV 胞吐作用的双峰性质。此外,钙传感器突触结合蛋白 7 的缺失增加了缓慢事件的比例,而不改变任一类事件的固有特性,表明独立调节的可能性。两种能够独立调节的释放机制的识别为融合孔道行为的多样性提供了生物学基础。

https://cdn.ncbi.nlm.nih.gov/pmc/blobs/21ef/9910497/efe226c23db0/pnas.2214897120fig01.jpg

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