Requirement of dynactin p150(Glued) subunit for the functional integrity of the keratinocyte microparasol.

ABSTRACT

The keratinocyte microparasol, composed of a perinuclear microtubular/melano-phagolysosomal complex, protects the nucleus from UV-induced DNA damage. We have previously demonstrated that cytoplasmic dynein is the motor involved in the perinuclear-directed aggregation of phagocytosed melanosomes. Dynactin, of which p150(Glued) is the major subunit, can link directly to microtubules and links organelles to dynein at different domains. To further define the mechanism of the microparasol, we transfected siRNA targeted against p150(Glued) into human keratinocytes cultured with 0.5 mm fluorescent microspheres and performed time-lapse analysis, confocal immunolocalization, and Western immunoblotting after 24 and 48 hours. Western blots revealed a significant knockdown of the p150(Glued) subunit. The knockdown decreased p150(Glued) colocalization with microtubules and decreased perinuclear positioning of the convergent microtubular framework. It also inhibited perinuclear aggregation of phagocytosed fluorescent microspheres and reduced mean centripetal microsphere displacement. The findings provide evidence that dynactin p150(Glued) plays an important role in the functional integrity of the keratinocyte microparasol.