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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 b-hydroxylase, a chromaffin vesicle marker, whereas myosin II was present mainly in the cell cortex. Cell depolarization induced, in a Ca 2+ 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.

MARCKS binds and crosslinks F-actin, the latter is inhibited by protein kinase C-induced MARCKS phosphorylation. MARCKS was found in chromaffin cells by immunoblotting. MARCKS was also detected by immunoprecipitation. In intact or permeabilized cells MARCKS phosphorylation increased upon stimulation with 10؊7 M phorbol 12-myristate 13-acetate. This was accompanied by cortical F-actin disassembly and potentiation of secretion. MARCKS phosphorylation, cortical F-actin disassembly, and potentiation of Ca 2؉ -evoked secretion were inhibited by a peptide (MARCKS phosphorylation site domain sequence (MPSD)) with amino acid sequence corresponding to MARCKS phosphorylation site. MPSD was phosphorylated in the process. A similar peptide (alanine-substituted phosphorylated site domain) with four serine residues of MPSD substituted by alanines was ineffective. These results provide the first evidence for MARCKS involvement in chromaffin cell secretion and suggest that regulation of cortical F-actin crosslinking might be involved in this process.

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Stimulation-induced chromaffin cell cortical F-actin disassembly allows the movement of vesicles towards exocytotic sites. Scinderin (Sc), a Ca 2+ -dependent protein, controls actin dynamics. Sc six domains have three actin, two PIP 2 and two Ca 2+ -binding sites. F-actin severing activity of Sc is Ca 2+ -dependent, whereas Sc-evoked actin nucleation is Ca 2+ -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 ScABP 3 (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 « assembly) and release (facilitation « inhibition). Neurons and neurosecretory cells store neurotransmitters, nucleotides and peptides in a membrane-bound organelle: the secretory vesicle (Trifaró and Poisner 1982). Upon cell stimulation, the soluble content of the vesicles is released to the cell exterior by exocytosis. Experimental evidence has suggested that cortical F-actin microfilament network plays an important role in the regulation of exocytosis in several secretory systems (Trifaró et al. 1984a;Cheek and Burgoyne 1986;Perrin et al. 1992;Li et al. 1994;Price et al. 1995;Morales et al. 2000;Yoneda et al. 2000). Chromaffin cells possess a mesh of cortical actin filaments (F-actin) which excludes vesicles from release sites at the plasma membrane. Chromaffin cell stimulation induces cortical F-actin disassembly and this (Cheek and Burgoyne 1986;Burgoyne et al. 1989; Trifaró et al. 1989;Vitale et al. 1991) precedes exocytosis (Trifaró et al. 1984a;Cheek and Burgoyne 1986;Burgoyne and Cheek 1987;Burgoyne 1991). Stimulation-induced cortical F-actin disassembly is a Ca 2+ -dependent process. It has been shown that scinderin (Sc), a protein that severs F-actin in a Ca 2+ -dependent manner (Rodriguez Del Castillo et al. 1990), is involved in F-actin network regulation (Vitale et al. 1991), playing an important role in the reorganization of cortical actin filaments brought about by cell stimulation. Work from our laboratory has demonstrated that recombinant scinderin potentiates Ca 2+ -evoked exocytosis from permeabilized chromaffin cells and that antisense targeted to the Abbreviations used: BSA, bovine serum albumin; DBH, dopamine b-hydroxylase; GFP, green fluorescent protein; hGH, human growth hormone; PBS, phosphate-buffered saline; RT, room temperature; Sc, scinderin; SDS-PAGE, sodium dodecyl sulfate -polyacrylamide gel electrophoresis.