The position of mitochondria and ER in relation to that of the secretory sites in chromaffin cells.

Knowledge of the distribution of mitochondria and endoplasmic reticulum (ER) in relation to the position of exocytotic sites is relevant to understanding the influence of these organelles in tuning Ca(2+) signals and secretion. Confocal images of probes tagged to mitochondria and the F-actin cytoskeleton revealed the existence of two populations of mitochondria, one that was cortical and one that was perinuclear. This mitochondrial distribution was also confirmed by using electron microscopy. In contrast, ER was sparse in the cortex and more abundant in deep cytoplasmic regions. The mitochondrial distribution might be due to organellar transport, which experiences increasing restrictions in the cell cortex. Further study of organelle distribution in relation to the position of SNARE microdomains and the granule fusion sites revealed that a third of the cortical mitochondria colocalized with exocytotic sites and another third located at a distance closer than two vesicle diameters. ER structures were also present in the vicinity of secretory sites but at a lower density. Therefore, mitochondria and ER have a spatial distribution that suggests a specialized role in modulation of exocytosis that fits with the role of cytosolic Ca(2+) microdomains described previously.

The F-actin cortical network is a major factor influencing the organization of the secretory machinery in chromaffin cells.

We have studied how the F-actin cytoskeleton is involved in establishing the heterogeneous intracellular Ca(2+) levels ([Ca(2+)](i)) and in the organization of the exocytotic machinery in cultured bovine chromaffin cells. Simultaneous confocal visualization of [Ca(2+)](i) and transmitted light studies of the cytoskeleton showed that, following cell stimulation, the maximal signal from the Ca(2+)-sensitive fluorescent dye Fluo-3 was in the empty cytosolic spaces left by cytoskeletal cages. This was mostly due to the accumulation of the dye in spaces devoid of cytoskeletal components, as shown by the use of alternative Ca(2+)-insensitive fluorescent cytosolic markers. In addition to affecting the distribution of such compounds in the cytosol, the cytoskeleton influenced the location of L- and P-Q-type Ca(2+) channel clusters, which were associated with the borders of cytoskeletal cages in resting and stimulated cells. Indeed, syntaxin-1 and synaptotagmin-1, which are components of the secretory machinery, were present in the same location. Furthermore, granule exocytosis took place at these sites, indicating that the organization of the F-actin cytoskeletal cortex shapes the preferential sites for secretion by associating the secretory machinery with preferential sites for Ca(2+) entry. The influence of this cortical organization on the propagation of [Ca(2+)](i) can be modelled, illustrating how it serves to define rapid exocytosis.

Cortical F-actin affects the localization and dynamics of SNAP-25 membrane clusters in chromaffin cells.

It has been proposed recently that the F-actin cytoskeleton organizes the relative disposition of the SNARE proteins and calcium channels that form part of the secretory machinery in chromaffin cells, a neurosecretory model. To test this idea, we used confocal microscopy do determine if DsRed-SNAP-25 microdomains, which define the final sites of exocytosis along with syntaxin-1, preferentially remain in contact with F-actin cortical structures labelled by lifeact-EGFP. A quantitative analysis showed that in cells over-expressing these constructs there is a preferential colocalization, rather than a random distribution of SNAP-25 patches. To analyze the possible interactions between these proteins, we designed FRET experiments and tested whether treatment with agents that affect F-actin mobility would modify SNAP-25 movement. The significant FRET efficiencies detected suggest that direct molecular interactions occur, whereas dynamic experiments using TIRFM revealed that attenuation of cortical F-actin movement clearly diminishes the mobility of SNAP-25 clusters. Taken together, these data can be explained by a model that associates components of the secretory machinery to the F-actin cortex through flexible links.