COLOCALIZATION OF NESTIN AND VIMENTIN DESMIN IN SKELETAL-MUSCLE CELLSDEMONSTRATED BY 3-DIMENSIONAL FLUORESCENCE DIGITAL IMAGING MICROSCOPY/

During skeletal muscle development three intermediate filament protein s are expressed: nestin, vimentin, and desmin. Vimentin and desmin bel ong to the class III intermediate filaments and are closely related to each other, whereas nestin is a more distantly related, class VI, int ermediate filament. It was previously observed by conventional immunoc ytochemistry that the intracellular patterns of nestin, desmin, and vi mentin appeared indistinguishable, despite nestin's more distant evolu tionary relationship. We here extend this analysis by applying three-d imensional fluorescence digital imaging microscopy to compare the intr acellular distribution of nestin with that of desmin, vimentin, actin, and tubulin in G6 human fetal skeletal muscle cells. We show that in vitro differentiation of G6 cells caa produce an intermediate filament expression pattern similar to that observed during myogenesis in vivo , i.e., downregulation of vimentin but not of nestin and desmin during myotube maturation. The image analysis demonstrated that the degree o f overlap between nestin and desmin/vimentin was very extensive in myo blasts and in multinucleate myotubes in all regions of the cells. In c ontrast, nestin did not colocalize with tubulin or actin in G6 myoblas ts. In particular, nestin immunoreactivity was not detected at the mic rotubule-organizing center, and it was only sparsely observed at the c ell periphery where actin stress fibers were seen. Our data lend furth er support to the notion that nestin interacts very closely with the t wo more distantly related class III intermediate filament proteins des min and vimentin in the entire muscle cell, before and after myotube f ormation. A comparison of conserved amino acid residues in the differe nt Ifs suggests that charged amino acid residues in the alpha-helical rod domain may play a role in the interaction. (C) 1994 Academic Press , Inc.