Aging Differentially Affects Multiple Aspects of Vesicle Fusion Kinetics

How fusion pore formation during exocytosis affects the subsequent release of vesicle contents remains incompletely understood. It is unclear if the amount released per vesicle is dependent upon the nature of the developing fusion pore and whether full fusion and transient kiss and run exocytosis are regulated by similar mechanisms. We hypothesise that if consistent relationships exist between these aspects of exocytosis then they will remain constant across any age. Using amperometry in mouse chromaffin cells we measured catecholamine efflux during single exocytotic events at P0, 1 month and 6 months. At all ages we observed full fusion (amperometric spike only), full fusion preceded by fusion pore flickering (pre-spike foot (PSF) signal followed by a spike) and pure “kiss and run” exocytosis (represented by stand alone foot (SAF) signals). We observe age-associated increases in the size of all 3 modes of fusion but these increases occur at different ages. The release probability of PSF signals or full spikes alone doesn't alter across any age in comparison with an age-dependent increase in the incidence of “kiss and run” type events. However, the most striking changes we observe are age-associated changes in the relationship between vesicle size and the membrane bending energy required for exocytosis. Our data illustrates that vesicle size does not regulate release probability, as has been suggested, that membrane elasticity or flexural rigidity change with age and that the mechanisms controlling full fusion may differ from those controlling “kiss and run” fusion

The F-Actin Cortex in Chromaffin Granule Dynamics and Fusion: a Minireview

Chromaffin granules are restrained in a dense cortical cytoskeleton before releasing their complex mix of active substances in response to cell stimulation. In recent years, the complex organization and dynamics of the chromaffin cell cortex has been unveiled through its analysis with a range of techniques to visualize this structure, including confocal fluorescence, transmitted light, and evanescent field microscopy. Accordingly, it has become apparent that the cortex is a dense F-actin mesh that contains open polygonal spaces through which vesicles can access the submembrane space. In addition to its retentive role, this structure also influences vesicle motion in both the resting state and during cell stimulation with secretagogues. During secretion, the chromaffin cell cortex undergoes a complex reorganization, helping to replenish the empty fast releasable pool of vesicles. Such changes in the cortical cytoskeleton and in the vesicle motion are governed by the activity of molecular motors, such as myosins II and Va. Interestingly, the F-actin/myosin II network also affects the final stages of exocytosis, which involve the opening and expansion of the fusion pore, and the extrusion of the vesicles contents.This work was supported by grants from the Spanish Ministerio de Investigacion Cientifica e Innovación (MICINN, BFU2008-00731 and BFU2011-25095), and the Generalitat Valenciana (ACOMP2011/090). CT-H was recipient of a fellowship from the MICINN.Peer reviewe

Glucose principally regulates insulin secretion in mouse islets by controlling the numbers of granule fusion events per cell

In dispersed single beta cells the response of each cell to glucose is heterogeneous. In contrast, within an islet, cell-to-cell communication leads to glucose inducing a more homogeneous response. For example, increases in NAD(P)H and calcium are relatively uniform across the cells of the islet. These data suggest that secretion of insulin from single beta cells within an islet should also be relatively homogeneous. The aim of this study was to test this hypothesis by determining the glucose dependence of single-cell insulin responses within an islet