Expression of constructs of the neuronal isoform of myosin‐Va interferes with the distribution of melanosomes and other vesicles in melanoma cells

Abstract

Myosin-Va has been implicated in melanosome translocation, but the exact molecular mechanisms underlying this function are not known. In the dilute, S91 melanoma cells, melanosomes move to the cell periphery but do not accumulate in the tips of dendrites as occurs in wild-type B16 melanocytes; rather, they return and accumulate primarily at the pericentrosomal region in a microtubule-dependent manner. Expression of the full-length neuronal isoform of myosin-Va in S91 cells causes melanosomes to disperse, occupying a cellular area approximately twice that observed in non-transfected cells, suggesting a partial rescue of the dilute phenotype. Overexpression of the full tail domain in S91 cells is not sufficient to induce melanosome dispersion, rather it causes melanosomal clumping. Overexpression of the head and head-neck domains of myosin-Va in B16 cells does not alter the melanosome distribution. However, overexpression of the full tail domain in these cells induces melanosome aggregation and the appearance of tail-associated, aggregated particles or vesicular structures that exhibit variable degrees of staining for melanosomal and Golgi beta-COP markers, as well as colocalization with the endogenous myosin-Va. Altogether, the present data suggest that myosin-Va plays a role in regulating the direction of microtubule-dependent melanosome translocation, in addition to promoting the capture of melanosomes at the cell periphery as suggested by previous studies. These studies also reinforce the notion that myosin-V has a broader function in melanocytes by acting on vesicular targeting or intracellular protein trafficking.

Missense mutations in the globular tail of myosin-Va indilutemice partially impair binding of Slac2-a/melanophilin

“…The physiological significance of the latter interaction is evident on the basis of the following observations: (1) deletion of exon F by mutations in the alternative splicing sites causes the dilute phenotype (i.e. perinuclear aggregation of melanosomes) (Seperack et al, 1995;Huang et al, 1998); (2) expression of brain myosin-Va lacking an exon F cannot rescue the phenotype of dilute-derived melanocytes (Wu et al, 2002b), and expression of MC myosin-Va-tail, but not of brain myosin-Va-tail, induces perinuclear aggregation of melanosomes (Wu et al, 2002b;da Silva Bizario et al, 2002;Westbroek et al, 2003); and (3) Slac2-a mutants lacking the exon-F-binding site cannot support normal melanosome distribution in melan-a cells (Kuroda et al, 2003). We therefore concluded that both interaction sites in Slac2-a identified in this study, the myosin-Va-GT-binding site and the myosin-Vaexon-F-binding site, are essential for melanosome transport in melanocytes.…”

Myosin Motors and Not Actin Comets Are Mediators of the Actin-based Golgi-to-Endoplasmic Reticulum Protein Transport

“…that the delay in the Golgi disassembly induced by BFA is much more severe in BDM-than in ML7-treated cells, suggest the participation of a second (processive) motor that subsequently helps move the newly generated transport carriers to their final destination (in this case the ER). In this respect, myosins V and VI are processive motors (which means that it takes successive steps along the actin filament) that are reported to be located in the Golgi complex and involved in vesicular trafficking in some cell types (Nascimento et al, 1997;Buss et al, 1998;DePina and Langford, 1999;Miller and Sheetz, 2000;Schott et al, 2002;da Silva Bizario et al, 2002) and, thus, they could be acting in tandem with myosin II at the Golgi complex. However, we cannot completely rule out the sole implication of myosin II, because despite that this motor does not take successive steps along the actin filament, multiple myosin II moieties could show successive steps along a filament (a processive movement).…”

Myosin V attachment to cargo requires the tight association of two functional subdomains

“…For example, vertebrate myosin Va moves melanosomes in melanocytes ( Provance et al, 1996 ; Nascimento et al, 1997 ; Wu et al, 1997 ; Rogers and Gelfand, 1998 ), the smooth ER in brain Purkinje cells ( Takagishi et al, 1996 ), membranous vesicles in nerve cells ( Evans et al, 1998 ), and chromaffin vesicles ( Rose et al, 2003 ). Moreover, within a single cell type, myosin Va likely moves more than one type of cargo ( da Silva Bizario et al, 2002 ). In S. cerevisiae , the myosin V, Myo2p, is essential and transports secretory vesicles, the vacuole, late Golgi, and peroxisomes ( Govindan et al, 1995 ; Hill et al, 1996 ; Catlett and Weisman, 1998 ; Schott et al, 1999 ; Karpova et al, 2000 ; Hoepfner et al, 2001 ; Rossanese et al, 2001 ).…”