Myosin I interactions with actin filaments and trans-golgi-derived vesicles in MDCK cell monolayers

RESUMO

In MDCK cell cultured monolayers, as well as in natural and other cultured epithelia, the proper organization of the actin filament ring, tethered to the plasma memebrane at the zonula adhaerens, is apparently necessary for their functioning as a transporting epithelium. It has been proposed that actin filaments, in conjunction with motor proteins, could provide the structural basis that regulates the tight junction (TJ) sealing capacity as well as the transport of memebrane-tagged proteins required for cell polarization. To test this hypothesis, the authors analyzed the localization and possible association ot the actin binding motor protein myosin I with actin filaments during changes in the actin ring position and organization, and also with tran-Golgi-derived vesicle. Modifications of the ring were induced subjecting the cells to external Ca²+ switch), or by treatment with drugs known to depolymerize actin filament (cytochalasin D, CD). The distribution of myosin I and actin, both in intact cells and in cellular fractions, was monitored using heterlogous cross-reacting antibodies and phalloidin. The authors identified an isoform of myosin I of approximately 110-125 KDa, homologus to myosin IB of Acanthamoeba, a fraction of wich colocalized with the peripheral actin ring. The association seems transient as, once the ring retracted as result of Ca²+ depletion, or became disroganized by CD, myosin not longer colocalized with the actin fibers but appeared dispersed in the cytoplasm. Furthermore, a signficant fraction of the total myosin I in the cell was associated to Golgi-derived vesicles which could also associate in vitro with actin filaments. The authors' data support, then, the participation of myosin I, in association with actin filaments, in vesicle translocation to and from the cell membrane as proposed for natural epithelia, and provide a further insigh into the structural organization that maintains epithelial cell polatiry in cultured monolayers