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 segments. This method study was carried out to investigate: (1) the amount of myofibrillar proteins extracted from the fiber segment; (2) the effects of temperature on the speed at which actin is propelled by fast and slow myosin; and (3) the effects of myosin isoform expression on motility speed. Approximately 80% of the myosin and myosin-associated proteins were extracted from the fiber segments. while no significant extraction was shown of the thin filament proteins. Fluorescently labeled actin filaments moved with constant speed in a bi-directional motion over the high-density myosin region in the experimental chamber, and motility speed was highly dependent on the myosin 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 microm/s, n = 11) and IIxb (5.81 +/- 0.35 microm/s, n = 6), or llb (6.07 +/- 0.33 microm/s, n = 8) MyHC isoforms. The motility speed and maximum velocity of unloaded shortening (V0) 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 speed were analyzed from 10 to 35 degrees C. The Q10 values, calculated in the 10-25 degrees C temperature range, differed between slow (4.20) and fast (2.38) myosin. In conclusion, this in vitro motility assay offers a unique possibility to compare the regulatory and modulatory influence of myosin isoforms and thin filament proteins on shortening velocity, at the cellular and molecular level in the same muscle fiber.
Hook
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Department of Clinical Neuroscience, Karolinska Hospital, Stockholm, Sweden.
Larsson
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D000199:Actins / Q000032:analysis / Q000378:metabolism; D000205:Actomyosin / Q000378:metabolism*; D000818:Animals; D001681:Biological Assay; D009068:Movement; D009119:Muscle Contraction; D018995:Myosin Heavy Chains; D009218:Myosins / Q000032:analysis / Q000378:metabolism; D020033:Protein Isoforms / Q000032:analysis / Q000378:metabolism; D051381:Rats; D017208:Rats, Wistar; D013696:Temperature
D000199:Actins; D020033:Protein Isoforms; D000205:Actomyosin; D018995:Myosin Heavy Chains; D009218:Myosins