The Motile Properties of Vimentin Intermediate Filament in B16 Melanoma Cells

Abstract

Background : Intermediate filaments as well as microtubule and microfilament are major components of cytoskeleton of human cells. Melanocytes have vimentin intermediate filament, which have not been well investigated as other cytoskeletons, especially in their function. Objective : The purpose of this study was to observe the motile characteristics of vimentin intermediate filament in living B16 melanoma cells. Methods : The motile properties of vimentin intermediate filament have been studied in living B16 melanoma cells using green fluorescent protein(GFP). cDNA expressing GFP-vimentin fusion protein was cloned and transfected into living B16 melanoma cells. Living cells were observed under fluorescent microscope and confocal microscope. Time-lapse images were collected and analysed. Results : GFP-vimentin is incorporated into the endogenous vimentin networks. Time-lapse observations of vimentin fibrils, demonstrate that they are constantly changing their configurations. Intersecting points of vimentin fibrils, or foci, frequently move towards or away from each other, indicating that the fibrils can lengthen or shorten. Fluorescence recovery after photobleaching shows that bleach zones across fibrils rapidly recover their fluorescence. During this recovery, bleached zones frequently move, indicating translocation of fibrils. Short filamentous structures('squiggle') are also seen actively translocating. Melanosomes also are changing their position back-and-pro constantly. They are co-localized very well with kinesin molecules in B16 melanoma cells. Conclusion : The vimentin intermediate filament and melanosomes in B16 melanoma cells have very active movement, which seem to have close relation with kinesin motor proteins.