ANALYSIS OF THE ACTIN-MYOSIN-II SYSTEM IN FISH EPIDERMAL KERATOCYTES - MECHANISM OF CELL BODY TRANSLOCATION

While the protrusive event of cell locomotion is thought to be driven by actin polymerization, the mechanism of forward translocation of the cell body is unclear. To elucidate the mechanism of cell body translo cation, we analyzed the supramolecular organization of the actin-myosi n II system and the dynamics of myosin II in fish epidermal keratocyte s. In lamellipodia, long actin filaments formed dense networks with nu merous free ends in a brushlike manner near the leading edge. Shorter actin filaments often formed T junctions with longer filaments in the brushlike area, suggesting that new filaments could be nucleated at si des of preexisting filaments or linked to them immediately after nucle ation. The polarity of actin filaments was almost uniform, with barbed ends forward throughout most of the lamellipodia but mixed in arc-sha ped filament bundles at the lamellipodial/cell body boundary. Myosin I I formed discrete clusters of bipolar mini-filaments in lamellipodia t hat increased in size and density towards the cell body boundary and c olocalized with actin in boundary bundles. Time-lapse observation demo nstrated that myosin clusters appeared in the lamellipodia and remaine d stationary with respect to the substratum in locomoting cells, but t hey exhibited retrograde flow in cells tethered in epithelioid colonie s, Consequently, both in locomoting and stationary cells, myosin clust ers approached the cell body boundary, where they became compressed an d aligned, resulting in the formation of boundary bundles. In locomoti ng cells, the compression was associated with forward displacement of myosin features. These data are not consistent with either sarcomeric or polarized transport mechanisms of cell body translocation. We propo se that ?he forward translocation of the cell body and retrograde flow in the lamellipodia are both driven by contraction of an actin-myosin network in the lamellipodial/cell body transition zone.