F-actin-myosin II inhibitors affect chromaffin granule plasma membrane distance and fusion kinetics by retraction of the cytoskeletal cortex.

José Villanueva, Vanesa Torres, Cristina J Torregrosa-Hetland, Virginia Garcia-Martinez, Inmaculada López-Font, Salvador Viniegra, Luis M Gutiérrez

Index: J. Mol. Neurosci. 48(2) , 328-38, (2012)

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Abstract

Chromaffin cell catecholamines are released when specialized secretory vesicles undergo exocytotic membrane fusion. Evidence indicates that vesicle supply and fusion are controlled by the activity of the cortical F-actin-myosin II network. To study in detail cell cortex and vesicle interactions, we use fluorescent labeling with GFP-lifeact and acidotropic dyes in confocal and evanescent wave microscopy. These techniques provide structural details and dynamic images of chromaffin granules caged in a complex cortical structure. Both the movement of cortical structures and granule motion appear to be linked, and this motion can be restricted by the myosin II-specific inhibitor, blebbistatin, and the F-actin stabilizer, jasplakinolide. These treatments also affect the position of the vesicles in relation to the plasma membrane, increasing the distance between them and the fusion sites. Consequently, we observed slower single vesicle fusion kinetics in treated cells after neutralization of acridine orange-loaded granules during exocytosis. Increasing the distance between the granules and the fusion sites appears to be linked to the retraction of the F-actin cytoskeleton when treated with jasplakinolide. Thus, F-actin-myosin II inhibitors appear to slow granule fusion kinetics by altering the position of vesicles after relaxation of the cortical network.