KERATIN INTERMEDIATE FILAMENT DYNAMICS IN CELL HETEROKARYONS REVEALS DIVERSE BEHAVIOR OF DIFFERENT KERATINS

To study the dynamics of keratin intermediate filaments, we fused two different types of epithelial cells (PtK2 and BMGE+H) and studied how the keratins from the parental cells recombine and copolymerize to for m the heterokaryon cytoskeleton. The behaviour of the keratins during this process was followed by immunofluorescence using specific antibod ies. After fusion, the parental cytoskeletons undergo a depolymerizati on process most apparent in the region adjacent to the fusion area. Th e depolymerized subunits spread throughout the heterokaryon and copoly merize into a new hybrid cytoskeleton. The complete process is very ra pid, occurring in 3-4 hours, thus demonstrating the highly dynamic nat ure of the keratin cytoskeleton. Although newly synthesised subunits c ontribute to the formation of the hybrid cytoskeleton, the process tak es place with similar kinetics in the absence of protein synthesis, sh owing the dynamic nature of the keratins from pre-existing cytoskeleto ns. During this process, specific keratins behave differently. Keratin s K8, K18, K5 and K10 are mobilised from the parental cytoskeletons an d reassemble rapidly into the hybrid cytoskeleton (3-6 hours), whereas K14 requires a substantially longer period (9-24 hours). Thus, differ ent keratins, even when they form part of the same heterodimeric/tetra meric complexes, as is the case for K5 and K14, exhibit different dyna mics. This suggests that individual polypeptides or homopolymeric comp lexes rather than exclusively heterodimeric/tetrameric subunits, as is currently thought, can also take part in keratin intermediate filamen t assembly and dynamics. Biochemical analysis performed in the absence of protein synthesis revealed greater amounts of K5 than of K14 in th e soluble pool of BMGE+H cells. Crosslinking and immunoprecipitation e xperiments indicated an excess of monomeric K5, as well as of K5/K14 h eterodimers and K5 homodimers in the soluble pool. These results are i n agreement with the different dynamic behaviour of these keratins obs erved in immunofluorescence. On the contrary, the phosphorylation leve ls of K5 and K14 are similar in both the soluble pool and the polymeri zed fraction, suggesting that phosphorylation does not play an importa nt role in the different dynamics displayed by these two proteins. In summary our results demonstrate that, following fusion, the keratin in termediate filament network reshapes rather rapidly and that keratins are highly dynamic proteins, although this mobility depends on each pa rticular polypeptide.