POLARITY SORTING OF ACTIN-FILAMENTS IN CYTOCHALASIN-TREATED FIBROBLASTS

The polarity of actin filaments is fundamental for the subcellular mec hanics of actin-myosin interaction; however, little is known about how actin filaments are oriented with respect to myosin in non-muscle cel ls and how actin polarity organization is established and maintained, Here we approach these questions by investigating changes in the organ ization and polarity of actin relative to myosin II during actin filam ent translocation, Actin and myosin II reorganization was followed bot h kinetically, using microinjected fluorescent analogs of actin and my osin, and ultrastructurally, using myosin S1 decoration and immunogold labelling, in cultured fibroblasts that were induced to contract by t reatment with cytochalasin D. We observed rapid (within 15 minutes) fo rmation of ordered actin filament arrays: short tapered bundles and as ter-like assemblies, in which filaments had uniform polarity with thei r barbed ends oriented toward the aggregate of myosin II at the base o f a bundle or in the center of an aster, The resulting asters further interacted with each other and aggregated into bigger asters, The arra ngement of actin in asters was in sharp contrast to the mixed polarity of actin filaments relative to myosin in non-treated cells. At the ed ge 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 nontreated cells, This rearrangement is i ndicative of relative translocation of actin and myosin II and of the ability of myosin II to sort actin filaments with respect to their pol arity during translocation, The results suggest that the myosin II-act in system of non-muscle cells is organized as a dynamic network where actin filament arrangement is defined in the course of its interaction with myosin II.