Multiple Mechanisms Driving F-actin-Dependent Transport of Organelles to and From Secretory Sites in Bovine Chromaffin Cells.

Neuroendocrine chromaffin cells represent an excellent model to study the molecular mechanisms associated with the exo-endocytotic cycle of neurotransmitter release. In this study, EGFP-Lifeact and confocal microscopy has been used to analyze the re-organization of the cortical F-actin cytoskeleton associated to organelle transport during secretion with unprecedented detail. In these cells secretory events accumulate in temperature-sensitive and myosin II-dependent F-actin expansions and retractions affecting specific regions of the sub-membrane space. Interestingly, not only vesicles but also mitochondria are transported toward the plasmalemma during these expansions. Simultaneously, we found F-actin cytoskeletal retraction withdraws vesicles from the sub-plasmalemmal space, forming novel empty internal spaces into which organelles can be transported. In addition to these well-coordinated, F-actin-myosin II dependent processes that drive the transport of the majority of vesicles, fast transport of chromaffin vesicles was observed, albeit less frequently, which used F-actin comet tails nucleated from the granular membrane. Thus, upon cell stimulation F-actin structures use diverse mechanisms to transport organelles to and from the membrane during the exo-endocytotic cycle taking place in specific areas of cell periphery.

The Differential Organization of F-Actin Alters the Distribution of Organelles in Cultured When Compared to Native Chromaffin Cells.

Cultured bovine chromaffin cells have been used extensively as a neuroendocrine model to study regulated secretion. In order to extend such experimental findings to the physiological situation, it is necessary to study mayor cellular structures affecting secretion in cultured cells with their counterparts present in the adrenomedullary tissue. F-actin concentrates in a peripheral ring in cultured cells, as witnessed by phalloidin-rodhamine labeling, while extends throughout the cytoplasm in native cells. This result is also confirmed when studying the localization of α-fodrin, a F-actin-associated protein. Furthermore, as a consequence of this redistribution of F-actin, we observed that chromaffin granules and mitochondria located into two different cortical and internal populations in cultured cells, whereas they are homogeneously distributed throughout the cytoplasm in the adrenomedullary tissue. Nevertheless, secretion from isolated cells and adrenal gland pieces is remarkably similar when measured by amperometry. Finally, we generate mathematical models to consider how the distribution of organelles affects the secretory kinetics of intact and cultured cells. Our results imply that we have to consider F-actin structural changes to interpret functional data obtained in cultured neuroendocrine cells.

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.

Real-time dynamics of the F-actin cytoskeleton during secretion from chromaffin cells.

Transmitted light images showed an intricate and dynamic cytoplasmic structural network in cultured bovine chromaffin cells observed under high magnification. These structures were sensitive to chemicals altering F-actin-myosin and colocalised with peripheral F-actin, beta-actin and myosin II. Interestingly, secretagogues induced a Ca2+-dependent, rapid (>10 second) and transitory (60-second cycle) disassembling of these cortical structures. The simultaneous formation of channel-like structures perpendicular to the plasmalemma conducting vesicles to the cell limits and open spaces devoid of F-actin in the cytoplasm were also observed. Vesicles moved using F-actin pathways and avoided diffusion in open, empty zones. These reorganisations representing F-actin transfer from the cortical barrier to the adjacent cytoplasmic area have been also confirmed by studying fluorescence changes in cells expressing GFP-beta-actin. Thus, these data support the function of F-actin-myosin II network acting simultaneously as a barrier and carrier system during secretion, and that transmitted light images could be used as an alternative to fluorescence in the study of cytoskeleton dynamics in neuroendocrine cells.