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 Ca2+ levels ([Ca2+] 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 Ca2+-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 Ca2+ 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 [Ca2+]i can be modelled, illustrating how it serves to define rapid exocytosis.
This work was supported by grants from the Spanish Ministry of Education and Culture (MEC, MICINN, BFU2005-02154/BFI and BFU2008-00731) and the Generalitat Valenciana (ACOMP06/036) to L.M.G.; the Spanish Ministry of Education and Culture (BFU2007-67607) and AN (BFU2008-01492) to I.Q.; and the Fundación BBVA and I-MATH project C3-0136 to A.G. and J.S. C.J.T.-H. and I.L.-F. were recipients of fellowships from the MICINN (Spain). V.G-V. thanks CONACyT for its financial support of her PhD scholarship. We also acknowledge the financial support received from the CONSOLIDER programme (CSD07-00023) and CIBERDEM (Instituto de Salud Carlos III).