Preparation of bead-tailed actin filaments: estimation of the torque produced by the sliding force in an in vitro motility assay.

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RESUMO

By coating covalently the surface of a polystyrene bead (diameter = 1 micron) with gelsolin, we have succeeded in attaching the bead selectively at the barbed end of an actin filament and forming a 1:1 bead-actin filament complex. On a layer of heavy meromyosin on a nitrocellulose-coated coverglass, this bead-actin filament complex slid smoothly, trailing the bead at its end. Therefore we called this preparation "bead-tailed" actin filaments. The sliding velocity was indistinguishable from that of nonbeaded filaments. With use of this system, we tried to detect the axial rotation (rotation around the filament axis) in a sliding actin filament. Although a single bead at the tail end did not serve as the marker for the axial rotation, we occasionally found another bead bound to the tail bead. In this case, the orientation of the bead-aggregate could be followed continuously with a video monitor while the filament was sliding over heavy meromyosin. We observed that actin filaments slid over distances of many tens of micrometers without showing a complete turn of the bead-aggregates. On the basis of the calculation of rotational friction drag on the bead-aggregate, we estimate that the rotational component of the sliding force and the torque produced on a sliding actin filament (length < or = 10 microns) did not accumulate > 1 pN and 5 pN.nm, respectively. In the present system of randomly oriented heavy meromyosin lying on a nitrocellulose film without an external load.

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