Role of cytoplasmic dynein in perinuclear aggregation of phagocytosed melanosomes and supranuclear melanin cap formation in human keratinocytes.

Cytoplasmic dynein is a microtubule-associated motor molecule involved in the retrograde transport of membrane-bound organelles. To determine whether the supranuclear melanin cap of transferred, phagocytosed melanosomes in keratinocytes is associated with cytoplasmic dynein, we performed immunofluorescent confocal microscopy on human keratinocytes in situ. We identified the intermediate chain of cytoplasmic dynein by immunoblotting and examined its distribution by confocal microscopy in relation to microtubules and melano-phagolysosomes in vitro. We also used antisense and sense oligonucleotides of the cytoplasmic dynein heavy chain 1 (Dyh1) and time-lapse and microscopy. The intermediate chain of cytoplasmic dynein was identified in extracts of human foreskin epidermis and in isolated human keratinocytes. The intermediate chain localized with the perinuclear melano-phagolysosomal aggregates in vitro and the supranuclear melanin cap in situ. Antisense oligonucleotides directed towards Dyh1 resulted in dispersal of the keratinocyte perinuclear melano-phagolysosomal aggregates after 24 to 48 h, whereas cells treated with diluent or sense oligonucleotides maintained tight perinuclear aggregates. Taken together, these findings indicate that in human keratinocytes, the retrograde microtubule motor cytoplasmic dynein mediates the perinuclear aggregation of phagocytosed melanosomes, participates in the formation of the supranuclear melanin cap or "microparasol" and serves as a mechanism to help protect the nucleus from ultraviolet-induced DNA damage.

Role of zyxin in differential cell spreading and proliferation of melanoma cells and melanocytes.

Cell spreading, proliferation, and survival are modulated by focal adhesions linking extracellular matrix proteins, integrins, and the cytoskeleton. Zyxin is a focal-adhesion-associated phosphoprotein with one domain involved in the control of actin assembly and three protein-protein adapter domains implicated in the regulation of cell growth and differentiation. We characterized zyxin expression in normal human melanocytes and six melanoma cell lines in relation to cell spreading, growth, and differentiation using Western immunoblotting techniques, image analysis, flow cytometry, and confocal microscopy. We found that zyxin, focal adhesion kinase, and paxillin were significantly upregulated in melanoma cells compared to melanocytes. Zyxin expression directly related to cell spreading and proliferation and inversely related to differentiation, whereas focal adhesion kinase correlated only to cell spreading and paxillin did not significantly correlate with any of the parameters. Treatment of melanoma cells with 12-O-tetradecanoylphorbol-13-acetate downregulated zyxin expression, inhibited cell spreading and proliferation, and promoted differentiation. In contrast, 12-O-tetradecanoylphorbol-13-acetate, a mitogen for melanocytes, induced upregulation of zyxin expression in melanocytes. These findings are consistent with a role of zyxin in modulation of cell spreading, proliferation, and differentiation. Therapies directed at the downregulation of this focal adhesion phosphoprotein in melanoma cells implicate a new approach for controlling melanoma cell growth.