Actomyosin interactions in a novel single muscle fiber in vitro motility assay

A novel in vitro motility assay has been developed to study the actomyosin interaction, in which the molecular motor protein myosin has been extracted and immobilized directly from 2-4 mm single rat skeletal muscle fiber segm ents. This method study was carried out to investigate: (1) the amount of m yofibrillar proteins extracted from the fiber segment; (2) the effects of t emperature on the speed at which actin is propelled by fast and slow myosin ; and (3) the effects of myosin isoform expression on motility speed. Appro ximately 80% of the myosin and myosin-associated proteins were extracted fr om the fiber segments, while no significant extraction was shown of the thi n filament proteins. Fluorescently labeled actin filaments moved with const ant speed in a bi-directional motion over the high-density myosin region in the experimental chamber, and motility speed was highly dependent on the m yosin heavy chain (MyHC) isoform extracted. At 25 degrees C, significant (P < 0.001) differences in motility speed were obtained between type I (1.31 +/- 0.23 mu m/s, n = 11) and IIxb (5.81 +/- 0.35 mu m/s, n = 6), or IIb (6. 07 +/- 0.33 mu m/s, n = 8) MyHC isoforms. The motility speed and maximum ve locity of unloaded shortening (V-0) in single fibers were well correlated, indicating that filament speed is a good molecular analogue to contractile speed at the fiber level. The effects of temperature on filament motility s peed were analyzed from 10 to 35 degrees C. The Q(10) values, calculated in the 10-25 degrees C temperature range, differed between slow (4.20) and fa st (2.38) myosin. In conclusion, this in vitro motility assay offers a uniq ue possibility to compare the regulatory and modulatory influence of myosin isoforms and thin filament proteins on shortening velocity, at the cellula r and molecular level in the same muscle fiber.