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, 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.
Highlights
Understanding the role of the cytoskeleton during exocytosis has been a central aspect for research in the neuroscience field in recent years
Confocal microscopy analysis of cells labelled with 4 μM quinacrine has allowed us to make direct observation of vesicle movement throughout the entire cytoplasm of cultured bovine chromaffin cells (Ñeco et al, 2002; Ñeco et al, 2003; Ñeco et al, 2004)
Vesicles tend to stay for periods of 27 seconds in the confocal plane presenting short cycle oscillations in fluorescence, whereas visible light intensity changed in cycles of time ranging from 10 to 20 seconds (Fig. 2C), indicating the slower dynamics of these structures when viewed by transmitted light scanning microscopy
Summary
Understanding the role of the cytoskeleton during exocytosis has been a central aspect for research in the neuroscience field in recent years. Only a small population of docked granules (1-3% of total vesicles) are rapidly released and constitute the ready releasable vesicle pool (Neher and Zucker, 1993; Horrigan and Bookman, 1994), and the rest of the chromaffin vesicles released during continuous or repetitive stimulation are recruited from a reserve pool located behind the F-actin barrier (Vitale et al, 1995; Gil et al, 2000) These vesicles gain access to the plasma membrane to release the stored catecholamines because a local and transient disruption of the F-actin cortical network occurs (Perrin and Aunis, 1985; Cheek and Burgoyne, 1986; Vitale et al, 1995), a process suggested to be controlled by actin-severing proteins such scinderin (Rodriguez del Castillo et al, 1990) or alternatively by network reorganisation based in the interaction between Factin and molecular motors such myosin (Nakanishi et al, 1989; Gutiérrez et al, 1989; Ñeco et al, 2002; Ñeco et al, 2003; Ñeco et al, 2004). These studies performed in living PC12 (Lang et al, 2000) and chromaffin cells (Steyer and Almers, 1999; Oheim and Stühmer, 2000; Johns et al, 2001), have suggested an active role for an actin-myosin transport system in propelling secretory vesicles through the subplasmalemmal zone
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