Expression of various scinderin domains in chromaffin cells indicates that this protein acts as a molecular switch in the control of actin filament dynamics and exocytosis.

Stimulation-induced chromaffin cell cortical F-actin disassembly allows the movement of vesicles towards exocytotic sites. Scinderin (Sc), a Ca2+-dependent protein, controls actin dynamics. Sc six domains have three actin, two PIP2 and two Ca2+-binding sites. F-actin severing activity of Sc is Ca2+-dependent, whereas Sc-evoked actin nucleation is Ca2+-independent. Sc domain role in secretion was studied by co-transfection of human growth hormone (hGH) reporter gene and green fluorescent protein (GFP)-fusion Sc constructs. Cells over-expressing actin severing Sc1-6 or Sc1-2 (first and second actin binding sites) constructs, increased F-actin disassembly and hGH release upon depolarization. Over-expression of nucleating Sc5-6, Sc5 or ScABP3 (third actin site) constructs decreased F-actin disassembly and hGH release upon stimulation. Over-expression of ScL5-6 or ScL5 (lack of third actin site) produced no changes. During secretion, actin sites 1 and 2 are involved in F-actin severing, whereas site 3 is responsible for nucleation (polymerization). Sc functions as a molecular switch in the control of actin (disassembly left arrow over right arrow assembly) and release (facilitation left arrow over right arrow inhibition). The position of the switch (severing left arrow over right arrow nucleation) may be controlled by [Ca2+]i. Thus, increase in [Ca2+]i produced by stimulation-induced Ca2+ entry would increase Sc-evoked cortical F-actin disassembly. Decrease in [Ca2+]i by either organelle sequestration or cell extrusion would favor Sc-evoked actin nucleation.

Myosins II and V in chromaffin cells: myosin V is a chromaffin vesicle molecular motor involved in secretion.

The presence of myosin II and V in chromaffin cells and their subcellular distribution is described. Myosin II and V distribution in sucrose density gradients showed only a strong correlation between the distribution of myosin V and secretory vesicle markers. Confocal microscopy images demonstrated colocalization of myosin V with dopamine beta-hydroxylase, a chromaffin vesicle marker, whereas myosin II was present mainly in the cell cortex. Cell depolarization induced, in a Ca2+ and time-dependent manner, the dissociation of myosin V from chromaffin vesicles suggesting that this association was not permanent but determined by secretory cycle requirements. Myosin II was also found in the crude granule fraction, however, its distribution was not affected by cell depolarization. Myosin V head antibodies were able to inhibit secretion whereas myosin II antibodies had no inhibitory effect. The pattern of inhibition indicated that these treatments interfered with the transport of vesicles from the reserve to the release-ready compartment, suggesting the involvement of myosin V and not myosin II in this transport process. The results described here suggest that myosin V is a molecular motor involved in chromaffin vesicle secretion. However, these results do not discard an indirect role for myosin II in secretion through its interaction with F-actin networks.

Molecular motors involved in chromaffin cell secretion.

Neurosecretory cells, including chromaffin cells, possess a mesh of filamentous actin underneath the plasma membrane. It has been proposed that filamentous actin network separates the secretory vesicles into two compartments: the reserve pool and the release-ready vesicle pool. Disassembly of chromaffin cell cortical filamentous actin in response to stimulation allows the movement of vesicles from the reserve pool into the release-ready vesicle pool. Electron microscopy of cytoskeletons revealed the presence of polygonal areas almost devoid of actin filaments in stimulated cells. The percentage of stimulated cells showing disrupted cytoskeleton correlates well with the increase in secretion in these cells. Fine filaments also remain in these areas of disassembly, and these reacted with actin antibodies, as demonstrated by immunogold staining. In addition, the movement of vesicles between pools requires Ca(2+) and ATP, a condition for activation of a molecular motor. Confocal microscopy images demonstrated colocalization of myosin Va with dopamine-beta-hydroxylase. Cell depolarization induced the dissociation of myosin Va from chromaffin vesicles. 2,3-Butadione-2-monoxime (BDM), an inhibitor of myosin ATPase, inhibited secretion, suggesting a blockage for chromaffin vesicle transport between the reserve pool and the release-ready vesicle pool. On the other hand, myosin II subcellular distribution was not affected by cell depolarization. Confocal microscopy images show myosin II to be localized in the cell cortex and in some perinuclear structures. Chromaffin vesicles were not stained by myosin II antibody.