Visualization of Melanosome Dynamics within Wild-Type and Dilute Melanocytes Suggests a Paradigm for Myosin V Function In Vivo

Abstract

Unlike wild-type mouse melanocytes, where melanosomes are concentrated in dendrites and dendritic tips, melanosomes in dilute (myosin Va−) melanocytes are concentrated in the cell center. Here we sought to define the role that myosin Va plays in melanosome transport and distribution. Actin filaments that comprise a cortical shell running the length of the dendrite were found to exhibit a random orientation, suggesting that myosin Va could drive the outward spreading of melanosomes by catalyzing random walks. In contrast to this mechanism, time lapse video microscopy revealed that melanosomes undergo rapid (∼1.5 μm/s) microtubule-dependent movements to the periphery and back again. This bidirectional traffic occurs in both wild-type and dilute melanocytes, but it is more obvious in dilute melanocytes because the only melanosomes in their periphery are those undergoing this movement. While providing an efficient means to transport melanosomes to the periphery, this component does not by itself result in their net accumulation there. These observations, together with previous studies showing extensive colocalization of myosin Va and melanosomes in the actin-rich periphery, suggest a mechanism in which a myosin Va–dependent interaction of melanosomes with F-actin in the periphery prevents these organelles from returning on microtubules to the cell center, causing their distal accumulation. This “capture” model is supported by the demonstration that (a) expression of the myosin Va tail domain within wild-type cells creates a dilute-like phenotype via a process involving initial colocalization of tail domains with melanosomes in the periphery, followed by an ∼120-min, microtubule-based redistribution of melanosomes to the cell center; (b) microtubule-dependent melanosome movement appears to be damped by myosin Va; (c) intermittent, microtubule-independent, ∼0.14 μm/s melanosome movements are seen only in wild-type melanocytes; and (d) these movements do not drive obvious spreading of melanosomes over 90 min. We conclude that long-range, bidirectional, microtubule-dependent melanosome movements, coupled with actomyosin Va–dependent capture of melanosomes in the periphery, is the predominant mechanism responsible for the centrifugal transport and peripheral accumulation of melanosomes in mouse melanocytes. This mechanism represents an alternative to straightforward transport models when interpreting other myosin V mutant phenotypes.

The Dilute Locus and Griscelli Syndrome: Gateways Towards a Better Understanding of Melanosome Transport

“…First, by expressing myosin Va tail domain-GFP fusion proteins in wild-type melanocytes, displacing endogenous myosin Va from the melanosomes, melanosomes redistributed on microtubules to the cell center, creating a dominant-negative dilute-like phenotype. By comparing the dynamics of melanosomes in microtubule-depleted wild-type and dilute melanocytes, a class of slow ( 0.14 mm/s) myosin Va-dependent (short range) in vivo melanosome movements was identified (10). Recently direct interactions could be demonstrated between microtubule-associated and actin-associated motor proteins (37), supporting their interactive way of co-operation.…”

“…once the melanosomes reach this subcortical area, is an emerging field of research. Overexpression of green fluorescent protein (GFP)-tagged myosin Va-tail domain fusion proteins shows colocalization with black end-stage melanosomes, indicating that indeed this tail domain is the cargo-binding domain, and moreover, induces a dilute-like dominant-negative image of perinuclear melanosome accumulation through competition with native myosin Va for the melanosome cargo (10,12). These experiments also revealed that, in melanoma cells, myosin Va also colocalizes with, e.g.…”

Mouse coat color mutations: From fancy mice to functional genomics

“…Although melanosomes are transported along both actin-based and microtubule-based motors in wild-type melanocytes, these mutations affect the actin-based movements only. The bidirectional microtubule-based movements between the center of the cell and its periphery still take place in mutant melanocytes, but a failure in melanosome capture at the periphery results in their accumulation at the center (Wu et al, 1998). The d, ash, and ln loci encode for the Myosin Va, Rab27a and Melanophilin proteins, respectively (Mercer et al, 1991;Wilson et al, 2000;Matesic et al, 2001).…”

Head-to-tail regulation is critical for the in vivo function of myosin V

“…In mammalian cells, myosin V motors have been implicated in transporting melanosomes (Wu et al, 1998), recycling endosomes (Lapierre et al, 2001), and the endoplasmic reticulum (Wagner et al, 2011). To perform these functions, all myosin V motors consist of two heavy chains with N-terminal motors dimerizing along a coiledcoil stalk, six associated light chains decorating each forcetransducing lever arm, and two cargo-binding globular tail domains at the C terminus (Hammer and Sellers, 2012).…”