Author/Editor		Lasič, Eva; Stenovec, Matjaž; Kreft, Marko; Robinson, Phillip J.; Zorec, Robert
Title		Dynamin regulates the fusion pore of endo- and exocytotic vesicles as revealed by membrane capacitance measurements
Type		članek
Publication year		2017
Volume		str. str.
ISSN		0304-4165 - BBA - General Subjects
Language		eng
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 DynoleTM-34-2 and DyngoTM-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 RyngoTM-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. Abbreviations Cm, membrane capacitance; Cv, vesicle capacitance; Gp, fusion pore conductance; Im, imaginary part of the admittance signal; Re, real part of the admittance signal; SIM, structured illumination microscopy; Syt4, synaptotagmin 4; VAMP2, vesicle-associated membrane protein 2.
Keywords		exocytosis endocytosis membrane capacitance eksocitoza endocitoza membranska kapacitivnost