Regulation of melanosome movement in the cell cycle by reversible association with myosin V

Abstract

Previously, we have shown that melanosomes of Xenopus laevis melanophores are transported along both microtubules and actin filaments in a coordinated manner, and that myosin V is bound to purified melanosomes (Rogers, S., and V.I. Gelfand. 1998. Curr. Biol. 8:161-164). In the present study, we have demonstrated that myosin V is the actin-based motor responsible for melanosome transport. To examine whether myosin V was regulated in a cell cycle-dependent manner, purified melanosomes were treated with interphase- or metaphase-arrested Xenopus egg extracts and assayed for in vitro motility along Nitella actin filaments. Motility of organelles treated with mitotic extract was found to decrease dramatically, as compared with untreated or interphase extract-treated melanosomes. This mitotic inhibition of motility correlated with the dissociation of myosin V from melanosomes, but the activity of soluble motor remained unaffected. Furthermore, we find that myosin V heavy chain is highly phosphorylated in metaphase extracts versus interphase extracts. We conclude that organelle transport by myosin V is controlled by a cell cycle-regulated association of this motor to organelles, and that this binding is likely regulated by phosphorylation of myosin V during mitosis.

Figure 1

Expression of the dominant-negative MST construct induces uninduced pigment aggregation in melanophores. A, Transfected cell stained with an mAb against the myc epitope-tag to verify expression of MST. Expressed MST is present throughout the cytoplasm, but staining in the center of the cell is obscured by the pigment mass. B, This is a bright-field image of the cell in A, demonstrating that its pigment is aggregated to the center of the cell. C, Control cells transfected with GFP grow in culture with dispersed pigment (D). Bar, 20 μm.

Figure 2

Phase-contrast microscopy of an amelanotic melanophore possessing numerous vesicles that aggregate to the center of the cell upon treatment with melatonin (A) and disperse following treatment with MSH (B). In these cells, myosin V immunolocalizes to unmelanized melanosomes and comigrates with these organelles during aggregation (C) and dispersion (D). Bar, 20 μm.

Figure 3

Treatment of melanosomes with metaphase-, but not interphase-arrested frog egg extracts inhibits actin-based motility along Nitella actin filaments in vitro. The histogram shows the numbers of motile (black bar) and immotile (white bar) melanosomes treated with interphase extracts (I), metaphase extracts (M), or untreated control organelles (MS). Treatment with mitotic extracts inhibited motility ∼10-fold, compared with the other two treatments. The results of two independent experiments are shown.

Figure 4

A, Immunoblot for myosin V of melanosome fractions treated with mitotic extracts (M), interphase extracts (I), or untreated organelles (MS). Myosin V is absent from metaphase treated organelles. The samples for each lane were normalized to load equivalent numbers of melanosomes by measuring the absorbance of melanin at 550 nm. Myosin V is designated by MV. B, Coomassie blue stained gel of the samples shown for A, demonstrating that the protein loaded in each lane is approximately equal. C, Immunoblot for myosin V in interphase extracts (I_ex), metaphase extracts (M_ex), interphase organelles (I_o), and metaphase organelles (M_o) from frog extracts. Equal amounts of protein were loaded for each sample. D, Immunoblot for myosin V on melanosomes treated with interphase (I) and metaphase (M) high-speed supernatants prepared from egg extracts.

Figure 5

Immunoblots of microtubule motors present on metaphase-treated (M), interphase-treated (I), or untreated (MS) melanosomes. A, Detection of cytoplasmic dynein by immunoblotting for dynein intermediate chain (DIC) with the m74-1 mAb. Dynein is dissociated from melanosomes in mitotic frog egg extract. B, Kinesin-II (KII) was detected in similarly treated melanosomes with an antibody against the motor's 85-kD motor subunit, the monoclonal K2.4. The amounts of this motor on melanosomes are unaffected by treatment with extracts. The load for each lane was equalized for the number of melanosomes by absorbance.

Figure 6

Immunoadsorbed myosin V from interphase (A) or metaphase (B) extracts is able to transport Sepharose beads in the Nitella motility assay. The beads appear as large, refractile spheres, whereas the smaller oval objects are the underlying Nitella chloroplasts. The centers of three beads in each panel were tracked over 30 min; each white dot overlaid on the video frames represents the position of the beads in 1 min increments. Bar, 20 μm.

Figure 7

Phosphorylation of myosin V in cell cycle-arrested frog egg extracts. A, Extracts were ³²P-labeled, and myosin V was immunoprecipitated from interphase- and metaphase-arrested extracts using affinity-purified DIL2 antibody (I and M, respectively), separated by SDS-PAGE, and analyzed by autoradiography. Myosin V (MV) is more highly phosphorylated in metaphase extracts. B, Coomassie blue stained gel as in A, to demonstrate approximately equal protein load for both treatments.