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

Abstract

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, β-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-β-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.

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

“…Our results suggest that recovered cortical actin may act as a barrier to limit granule secretion during a discrete CTL-target encounter and, furthermore, that recovery itself is triggered upon granule fusion, providing a mechanism that facilitates serial killing during an immune response. We also show that both clearance and recovery of cortical actin occurs concomitantly with complementary changes in PIP 2 , suggesting that The role of actin in secretion has been studied in many cell types specializing in exocytosis, including natural killer (NK) cells (35)(36)(37), mast cells (7), pancreatic β cells (38), and adrenal chromaffin cells (39,40). 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).…”

Myosin-Va Regulates Exocytosis through the Submicromolar Ca2+-dependent Binding of Syntaxin-1A

“…Myosin-Va, which mediates F-actindependent conveyance to the plasma membrane (cf. Giner et al, 2005), is on the surface of the vesicle and binds to syntaxin-1A when the intracellular [Ca 2ϩ ] increases to 0.3-0.4 M. After the complex between myosin-Va and syntaxin-1A tethers the vesicle to the membrane, it recruits other SNARE proteins to form the SNARE complex. A second larger elevation in [Ca 2ϩ ] induces vesicular fusion (Rettig and Neher, 2002) and stimulates the exchange of myosin-Va for NSF/␣-SNAP (Our unpublished data; see Figure 5) in the SNARE complex.…”

Neurite extensions in chromaffin cells: study of the influence of the cytoskeletal structure on calcium dynamics and secretion

“…This system permits z-axis reconstruction (0.5-0.55 μm theoretical z slice) and dynamic time-lapse studies, with a time resolution ranging from 0.1 s and with 200 × 150 pixel image acquisition (adequate for regional studies) to about 0.6 s for images of 400 × 300 pixels (for visualization of the entire cell). Simultaneous acquisition of transmitted light images provided information of the distribution of the Factin cytoskeleton and were obtained using the channel implemented in the confocal microscope as well as using bright field optics (the theoretical depth of field is 0.55-0.60 μm, see Giner et al (2005)). The intensity of this channel was adjusted to avoid saturation of the subcortical region and to visualize the lighter cytoplasmic structures.…”

“…The use of scanning light microscopy in confocal microscopes allowed us to study the cytoskeletal organization and its changes during the secretion of "typical" round chromaffin cells (Giner et al 2005) and therefore we were prompted to study these changes during the secretion of neurite-emitting cells. An example of such cells could be observed in Figure 1(a); the long neurite emission ended in a engrossed tip that has been shown to accumulate secretory vesicles (Gutierrez et al 1998).…”