NOVEL FEATURES OF INTERMEDIATE FILAMENT DYNAMICS REVEALED BY GREEN FLUORESCENT PROTEIN CHIMERAS

In order to study the dynamic behavior of intermediate filament networ ks in living cells, we have prepared constructs fusing green fluoresce nt protein to intermediate filament proteins. Vimentin fused to green fluorescent protein labeled the endogenous intermediate filament netwo rk. We generated stable SW13 and NIH3T3 cell lines that express an enh anced green fluorescent protein fused to the N-terminus of full-length vimentin, We were able to observe the dynamic behavior of the interme diate filament network in these cells for periods as long as 4 hours ( images acquired every 2 minutes). In both cell lines, the vimentin net work constantly moves in a wavy manner, In the NIH3T3 cells, we observ ed extension of individual vimentin filaments at the edge of the cell. This movement is dependent on microtubules, since the addition of noc odazole stopped the extension of the intermediate filaments. Injection of anti-IFA causes the redistribution or 'collapse' of intermediate f ilaments. We injected anti-IFA antibodies into NIH3T3 cells stably exp ressing green fluorescent protein fused to vimentin and found that ind ividual intermediate filaments move slowly towards the perinuclear are a without obvious disassembly, These results demonstrate that individu al intermediate filaments are translocated during the collapse, rather than undergoing disassembly-induced redistribution. Injections of tub ulin antibodies disrupt the interactions between intermediate filament s and stable microtubules and cause the collapse of the vimentin netwo rk showing that these interactions play an important role in keeping t he intermediate filament network extended. The nocodazole inhibition o f intermediate filament extension and the anti-IFA microinjection expe riments are consistent with a model in which intermediate filaments ex hibit an extended distribution when tethered to microtubules, but are translocated to the perinuclear area when these connections are severe d.