The polarity of actin filaments is fundamental for the subcellular mechanics of actin-myosin interaction; however, little is known about how actin
filaments are oriented with respect to myosin in nonmuscle cells and how actin polarity organization is established and maintained. Here we
approach these questions by investigating changes in the organization and polarity of actin relative to myosin II during actin filament
translocation. Actin and myosin II reorganization was followed both kinetically, using microinjected fluorescent analogs of actin and myosin, and
ultrastructurally, using myosin S1 decoration and immunogold labelling, in cultured fibroblasts which were induced to contract by treatment with
cytochalasin D. We observed rapid (within 15 min) formation of ordered actin filament arrays: short tapered bundles and aster-like assemblies in
which filaments had uniform polarity with their barbed ends oriented toward the aggregate of myosin II at the base of a bundle or in the center of
an aster. The resulting asters further interacted with each other and aggregated into bigger asters. The arrangement of actin in asters was in sharp
contrast to the mixed polarity of actin filaments relative to myosin in non-treated cells. At the edge of the cell, actin filaments became oriented with their barbed ends toward the cell center; that is, the orientation was opposite to what was observed at the edge of non-treated cells. This
rearrangement is indicative of relative translocation of actin and myosin II and of the ability of myosin II to sort actin filaments with respect to
their polarity during translocation. The results suggest that the myosin II-actin system of nonmuscle cells is organized as a dynamic network
where actin filament arrangement is defined in the course of its interaction with myosin II.
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Sequence 1. (704k) -
Dynamics of microinjected tetramethylrhodamine-myosin II in polarized REF-52 cell treated with cytochalasin D (2 µM). Contraction of stress fibers containing parallel myosin ribbons was followed by formation of tight myosin foci. Myosin foci also appeared de novo during cytochalasin treatment in gaps between contracted stress fibers. Myosin foci exhibited complex irregular motility with the overall trend being the fusion of foci with each other to form larger foci. Frames were taken at 1 minute intervals, cytochalasin addition was at 5 min, field of view approximately 30 µm by 26 µm.
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Sequence 2. (638k) -
Dynamics of microinjected tetramethylrhodamine-myosin II in radially spreading REF-52 cell treated with cytochalasin D. Formation of myosin foci from preexisting myosin ribbons in the lamellum as well as de novo is similar to sequence 1. Frames were taken at 45 second intervals, cytochalasin addition was at 3 min, field of view approximately 30 µm by 25 µm.
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Sequence 3. (1000k) - Dynamics of microinjected actin labeled with 5-(and 6)-carboxytetramethylrhodamine succinimidyl ester in cytochalasin D-treated REF-52 cell. Contraction of stress fibers and aggregation of actin in lamellipodia eventually resulted in the formation of actin asters with bright centers and radiating short bundles of actin filaments. Actin asters frequently approached each other and fused together. Frames were taken at 1 minute intervals, cytochalasin addition was at 3 min, field of view approximately 55 µm by 35 µm.
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Sequence 4.
(1100k) - Similar to sequence 3, but actin asters formed in the lamellum showing diffuse distribution of actin prior to addition of cytochalasin. Frames were taken at 1 minute intervals, cytochalasin addition was at 6 min, field of view approximately 35 µm by 30 µm.
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