Evidence that intermediate filament reorganization is induced by ATP-dependent contraction of the actomyosin cortex in permeabilized fibroblasts

Intermediate filaments (IFs) undergo specific rearrangements in cells, some aspects of which can be induced experimentally. Centripetal aggregation of the IF network, for example, can be produced by a variety of perturbations. However, the source of motive force is clear for neither in vivo nor experimentally generated IF movements, since, unlike microtubules and actin filaments, IFs have no known force-generating system directly associated with them. We recently obtained evidence that the drug-induced aggregation of vimentin IFs in fibroblasts is an active event, which requires ATP and involves the actin cytoskeleton. In the present study, we sought to test the hypothesis that IF aggregation is driven by a centripetally directed contraction of the actomyosin cortex. To that end, we have permeabilized fibroblasts with Triton X-100 in a stabilizing buffer and reactivated cytoskeletal movements in vitro, under defined solution conditions. Upon nucleotide treatment, these permeabilized cells undergo a nucleotide-dependent centripetal aggregation of vimentin IFs similar in appearance and time course to that induced in intact cells by drug treatment. During in vitro IF aggregation, the permeabilized cells remain fully spread and adherent to the substratum, and the distal ends of the microtubules and actin microfilaments retain their positions in the cell periphery, IF aggregation is accompanied by a contraction of F-actin and myosin into focal aggregates in the same perinuclear region in which the IFs accumulate. If permeabilized cells are treated with the actin-severing protein gelsolin prior to the reactivation of IF movement, the actin cytoskeleton is eliminated and IF aggregation fails to occur when ATP is added. These results strongly support a model in which the motive force for IF movement is supplied indirectly by association with a contracting actomyosin network.