NON-SARCOMERIC MODE OF MYOSIN-II ORGANIZATION IN THE FIBROBLAST LAMELLUM

The organization of myosin in the fibroblast lamellum was studied by c orrelative fluorescence and electron microscopy after a novel procedur e to reveal its underlying morphology. An X-rhodamine analog of conven tional smooth muscle myosin (myosin II) that colocalized after microin jection with endogenous myosin was used to trace myosin distribution i n living fibroblasts. Then, the same cells were examined by EM of plat inum replicas. To visualize the structural arrangement of myosin, othe r cytoskeletal fibrillar structures had to be removed: microtubules we re depolymerized by nocodazole treatment of the living cells before in jection of myosin; continued nocodazole treatment also induced the int ermediate filaments to concentrate near the nucleus, thus removing the m from the lamellar region; actin filaments were removed after lysis o f the cells by incubation of the cytoskeletons with recombinant gelsol in. Possible changes in myosin organization caused by this treatment w ere examined by fluorescence microscopy. No significant differences in myosin distribution patterns between nocodazole-treated and control c ells were observed. Cell lysis and depletion of actin also did not ind uce reorganization of myosin as was shown by direct comparison of myos in distribution in the same cells in the living state and after gelsol in treatment. EM of the well-spread, peripheral regions of actin-deple ted cytoskeletons revealed a network of bipolar myosin mini-filaments, contacting each other at their terminal, globular regions. The morpho logy of this network corresponded well to the myosin distribution obse rved by fluorescence microscopy. A novel mechanism of cell contraction by folding of the myosin filament network is proposed.