Transient Transfections and Heterokaryons as Tools for the Analysis of Keratin IF Dynamics

All eukaryotic cells contain three distinct cytoskeletal networks: microtubules, microfilaments, and intermediate filaments (IF). In epithelial cells in vivo, the IF cytoskeleton is composed of heteropolymers of type I (acidic) and type II (neutral-basic) keratins, whose expression is carefully controlled in a cell type- and differentiation-specific manner (1 –3 ). In addition, many epithelial cells in culture also induce the synthesis of the IF protein vimentin. In contrast to the other cytoskeletal proteins, which have been shown to be highly dynamic (4 –8 ), IF in general, and keratins in particular, have long been considered the most stable and consequently the least dynamic components of the cell cytoskeleton (9 ,10 ). The majority of the dynamic studies on subcellular structures have been performed by microinjecting the proteins under study after their in vitro labeling. This approach, however, is very difficult with keratins due to the high insolubility shown by these proteins over a wide range of physiological conditions. In addition, this technique could lead to alteration in the dynamic process due to the large amount of protein injected into the cells (11 ) and because the incorporation of new molecules into IF structures also differs between preexisting and newly assembling filaments (12 ). As an alternative to microinjection, transfections can be used. However, in standard transient transfection experiments, expression of the transgene takes place during a broad range of time (12–96 h), and it is impossible to know the precise moment at which the expression started in any particular cell, thus making conventional transfection techniques unfavorable for dynamic studies. However, the standard transfection technique is very useful for investigating the behavior of mutated genes (13 –15 ).