Eva Lasič1, Matjaž Stenovec2, Marko Kreft3, Phillip J Robinson4, Robert Zorec5. 1. Laboratory of Neuroendocrinology-Molecular Cell Physiology, Institute of Pathophysiology, Faculty of Medicine, University of Ljubljana, Zaloška 4, 1000 Ljubljana, Slovenia. 2. Laboratory of Neuroendocrinology-Molecular Cell Physiology, Institute of Pathophysiology, Faculty of Medicine, University of Ljubljana, Zaloška 4, 1000 Ljubljana, Slovenia; Celica Biomedical, Tehnološki Park 24, 1000 Ljubljana, Slovenia. Electronic address: matjaz.stenovec@mf.uni-lj.si. 3. Laboratory of Neuroendocrinology-Molecular Cell Physiology, Institute of Pathophysiology, Faculty of Medicine, University of Ljubljana, Zaloška 4, 1000 Ljubljana, Slovenia; Celica Biomedical, Tehnološki Park 24, 1000 Ljubljana, Slovenia; University of Ljubljana, Biotechnical Faculty, Department of Biology, CPAE, Večna pot 111, 1000 Ljubljana, Slovenia. 4. Children's Medical Research Institute, The University of Sydney, Australia. 5. Laboratory of Neuroendocrinology-Molecular Cell Physiology, Institute of Pathophysiology, Faculty of Medicine, University of Ljubljana, Zaloška 4, 1000 Ljubljana, Slovenia; Celica Biomedical, Tehnološki Park 24, 1000 Ljubljana, Slovenia. Electronic address: robert.zorec@mf.uni-lj.si.
Abstract
BACKGROUND: Dynamin is a multidomain GTPase exhibiting mechanochemical and catalytic properties involved in vesicle scission from the plasmalemma during endocytosis. New evidence indicates that dynamin is also involved in exocytotic release of catecholamines, suggesting the existence of a dynamin-regulated structure that couples endo- to exocytosis. METHODS: Thus we here employed high-resolution cell-attached capacitance measurements and super-resolution structured illumination microscopy to directly examine single vesicle interactions with the plasmalemma in cultured rat astrocytes treated with distinct pharmacological modulators of dynamin activity. Fluorescent dextrans and the lipophilic plasmalemmal marker DiD were utilized to monitor uptake and distribution of vesicles in the peri-plasmalemmal space and in the cell cytosol. RESULTS: Dynamin inhibition with Dynole™-34-2 and Dyngo™-4a prevented vesicle internalization into the cytosol and decreased fusion pore conductance of vesicles that remained attached to the plasmalemma via a narrow fusion pore that lapsed into a state of repetitive opening and closing - flickering. In contrast, the dynamin activator Ryngo™-1-23 promoted vesicle internalization and favored fusion pore closure by prolonging closed and shortening open fusion pore dwell times. Immunocytochemical staining revealed dextran uptake into dynamin-positive vesicles and increased dextran uptake into Syt4- and VAMP2-positive vesicles after dynamin inhibition, indicating prolonged retention of these vesicles at the plasmalemma. CONCLUSIONS: Our results have provided direct evidence for a role of dynamin in regulation of fusion pore geometry and kinetics of endo- and exocytotic vesicles, indicating that both share a common dynamin-regulated structural intermediate, the fusion pore.
BACKGROUND: Dynamin is a multidomain GTPase exhibiting mechanochemical and catalytic properties involved in vesicle scission from the plasmalemma during endocytosis. New evidence indicates that dynamin is also involved in exocytotic release of catecholamines, suggesting the existence of a dynamin-regulated structure that couples endo- to exocytosis. METHODS: Thus we here employed high-resolution cell-attached capacitance measurements and super-resolution structured illumination microscopy to directly examine single vesicle interactions with the plasmalemma in cultured rat astrocytes treated with distinct pharmacological modulators of dynamin activity. Fluorescent dextrans and the lipophilic plasmalemmal marker DiD were utilized to monitor uptake and distribution of vesicles in the peri-plasmalemmal space and in the cell cytosol. RESULTS: Dynamin inhibition with Dynole™-34-2 and Dyngo™-4a prevented vesicle internalization into the cytosol and decreased fusion pore conductance of vesicles that remained attached to the plasmalemma via a narrow fusion pore that lapsed into a state of repetitive opening and closing - flickering. In contrast, the dynamin activator Ryngo™-1-23 promoted vesicle internalization and favored fusion pore closure by prolonging closed and shortening open fusion pore dwell times. Immunocytochemical staining revealed dextran uptake into dynamin-positive vesicles and increased dextran uptake into Syt4- and VAMP2-positive vesicles after dynamin inhibition, indicating prolonged retention of these vesicles at the plasmalemma. CONCLUSIONS: Our results have provided direct evidence for a role of dynamin in regulation of fusion pore geometry and kinetics of endo- and exocytotic vesicles, indicating that both share a common dynamin-regulated structural intermediate, the fusion pore.
Authors: Lucas Bayonés; María José Guerra-Fernández; Fernando Hinostroza; Ximena Báez-Matus; Jacqueline Vásquez-Navarrete; Luciana I Gallo; Sergio Parra; Agustín D Martínez; Arlek González-Jamett; Fernando D Marengo; Ana M Cárdenas Journal: Int J Mol Sci Date: 2022-09-08 Impact factor: 6.208