The analysis of intermediate filament dynamics using transfections and cell fusions.

Abstract

The intermediate filament (IF) proteins have been recently found as dynamic structures that influence several aspects of cell homeostasis. Here, two alternative approaches to study the dynamics of IF proteins are described: the formation of cell hybrids by the fusion of different parental cells, and the transfection of keratin genes in cultured cells. In the first case, the selection of parental cell lines and the use of specific antibodies allow us to study how IF proteins recombine and copolymerize to form the heterokaryon cytoskeleton by immunofluorescence. In the second approach, some modifications of conventional transfection protocols allow the synchronized expression conditions, making it suitable for the analysis of the incorporation of a newly synthesized IF protein into the preexisting IF cytoskeleton of transfected cells.