A single-fiber in vitro motility assay. In vitro sliding velocity of F-actin vs. unloaded shortening velocity in skinned muscle fibers

We describe an approach that allows us to form a micro in vitro motility as say with as little myosin as can be retrieved from a short (similar to 10 m m) segment of a single skinned skeletal muscle fiber (diameter some 100 mu m). Myosin is directly extracted from the single fiber segment by a high io nic strength solution in the presence of MgATP, and the extracted myosin is immediately applied to a miniaturized flow cell that has been pretreated w ith BSA. The observed sliding velocities of fluorescently labeled F-actin a re essentially identical with those reported in the literature. Since at th e single fiber level most muscle fibers contain only a single myosin heavy chain isoform this approach allows us to determine without additional purif ication steps, the sliding velocity driven by myosins with different heavy chain isoforms. In addition, this approach can be used to directly correlat e under identical experimental conditions unloaded shortening velocity meas ured in segments of skinned muscle fibers with the in vitro sliding velocit y of fluorescently labeled F-actin by extraction of myosin from the same sk inned fibers. Such direct correlation was performed with different myosin h eavy chain isoforms as well as at different temperatures and ionic strength s. Under all conditions studied, unloaded shortening velocity was 4- to 8-f old faster than sliding velocity in the motility assay even at high tempera ture (22 degrees C) and ionic strengths > 50 mM. This suggests that sliding velocity in the motility assay is limited by additional factors beyond tho se thought to limit velocity of unloaded shortening in muscle fibers. One s uch factor might be unspecific ionic interactions between F-actin and the s ubstrate in the motility assay resulting in somewhat higher sensitivity for ionic strength of sliding velocity in the motility assay. This might becom e of special relevance when using in vitro sliding velocity in assessing fu nctional consequences of mutations involving charged residues of actin or m yosin.