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

Abstract

SummaryWe 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.

The skeleton in the closet: actin cytoskeletal remodeling in β-cell function

“…Additional compelling evidence for direct involvement of actin remodeling in regulated (neuroendocrine cell) secretion comes from chromaffin cells, with colocalization through cortical F-actin of L-type Ca 2ϩ channels and key components of the secretory machinery including granules themselves (120). Another link is provided by a study showing that neuritogenesis depends on the exocytotic machinery, and here again one mechanism was shown to involve the FAK/Src-Arp2/3-VAMP axis (41).…”

Cortical actin recovery at the immunological synapse leads to termination of lytic granule secretion in cytotoxic T lymphocytes

“…One model, known as the "barrier model," suggests that the actin cortex acts physically to prevent vesicles from coming in close enough proximity to the plasma membrane for fusion to occur (38). This model is supported by evidence that actin prevents the access of granules and fusion machinery to the plasma membrane in chromaffin cells (41). Although we have not been able to stabilize actin before lytic granule secretion, because of the effects of actin-stabilizing agents such as Jasplakinolide on TCR signaling (42,43) and T-cell adhesion (44), our observations suggest that the recovery of actin in CTLs after granule fusion may limit the access of lytic granules to the plasma membrane, thus preventing further secretion.…”

The distribution of mitochondria and endoplasmic reticulum in relation with secretory sites in chromaffin cells

“…In summary, our study supports the hypothesis that there are specialized mitochondrial populations serving different roles in the cortical and perinuclear areas of neuroendocrine cells, and gives structural support for idea of fine tuning of Ca 2+ signals by the plasma-membrane-mitochondria-ER triads (Montero et al, 2000) in the proximity of secretory sites. Future studies modeling the influence of the organelle distributions proposed here and the insertion of organelles in the cytoskeletal cages surrounding secretory sites (Torregrosa-Hetland et al, 2011) will be needed to understand the impact of such distributions in the fine tuning of Ca 2+ signals and secretion.…”