title: Spatiotemporal detection and analysis of exocytosis reveal fusion "hotspots" organized by the cytoskeleton in endocrine cells.
abstract: Total internal reflection fluorescence microscope has often been used to study the molecular mechanisms underlying vesicle exocytosis. However, the spatial occurrence of the fusion events within a single cell is not frequently explored due to the lack of sensitive and accurate computer-assisted programs to analyze large image data sets. Here, we have developed an image analysis platform for the nonbiased identification of different types of vesicle fusion events with high accuracy in different cell types. By performing spatiotemporal analysis of stimulus-evoked exocytosis in insulin-secreting INS-1 cells, we statistically prove that individual vesicle fusion events are clustered at hotspots. This spatial pattern disappears upon the disruption of either the actin or the microtubule network; this disruption also severely inhibits evoked exocytosis. By demonstrating that newcomer vesicles are delivered from the cell interior to the surface membrane for exocytosis, we highlight a previously unappreciated mechanism in which the cytoskeleton-dependent transportation of secretory vesicles organizes exocytosis hotspots in endocrine cells.
authors.affiliation: The State Key Laboratory of Biomembrane and Membrane Biotechnology, Beijing Key Laboratory of Cardiometabolic Molecular Medicine, Institute of Molecular Medicine, Peking University and National Center for Nanoscience and Technology, Beijing, China.
authors.forename: Tianyi
authors.initials: T
authors.lastname: Yuan
authors.affiliation: National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing, China.
authors.forename: Jingze
authors.initials: J
authors.lastname: Lu
authors.affiliation: National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing, China.
authors.forename: Jinzhong
authors.initials: J
authors.lastname: Zhang
authors.affiliation: National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing, China. Electronic address: zyd0903@gmail.com.
authors.forename: Yongdeng
authors.initials: Y
authors.lastname: Zhang
authors.affiliation: The State Key Laboratory of Biomembrane and Membrane Biotechnology, Beijing Key Laboratory of Cardiometabolic Molecular Medicine, Institute of Molecular Medicine, Peking University and National Center for Nanoscience and Technology, Beijing, China. Electronic address: lychen@pku.edu.cn.
authors.forename: Liangyi
authors.initials: L
authors.lastname: Chen
mesh_terms: D008841:Actin Cytoskeleton / Q000378:metabolism*; D000818:Animals; D002460:Cell Line; D005089:Exocytosis; D050417:Insulin-Secreting Cells / Q000378:metabolism*; D008561:Membrane Fusion; D008856:Microscopy, Fluorescence / Q000379:methods; D051381:Rats; D022142:Secretory Vesicles / Q000378:metabolism