Rotation of the lever arm of Myosin in contracting skeletal muscle fiber measured by two-photon anisotropy.
The rotation of the lever arm of myosin cross-bridges is believed to be responsible for muscle contraction. To resolve details of this rotation, it is necessary to observe a single cross-bridge. It is still impossible to do so in muscle fiber, but it is possible to investigate a small population of cross-bridges by simultaneously activating myosin in a femtoliter volume by rapid release of caged ATP. In earlier work, in which the number of observed cross-bridges was limited to approximately 600 by confocal microscopy, we were able to measure the rates of cross-bridge detachment and rebinding. However, we were unable to resolve the power stroke. We speculated that the reason for this was that the number of observed cross-bridges was too large. In an attempt to decrease this number, we used two-photon microscopy which permitted observation of approximately 1/2 as many cross-bridges as before with the same signal/noise ratio. With the two-photon excitation, the number of cross-bridges was small enough to resolve the beginning of the power stroke. The results indicated that the power stroke begins approximately 170 ms after the rigor cross-bridge first binds ATP.
Borejdo
J
J
Department of Molecular Biology and Immunology, University of North Texas, Fort Worth, Texas 76107, USA. jborejdo@hsc.unt.edu
Shepard
A
A
Akopova
I
I
Grudzinski
W
W
Malicka
J
J
D000818:Animals; D016880:Anisotropy; D002478:Cells, Cultured; D036681:Fluorescence Recovery After Photobleaching / Q000379:methods; D036641:Microscopy, Fluorescence, Multiphoton / Q000379:methods*; D020409:Molecular Motor Proteins / Q000502:physiology / Q000648:ultrastructure; D009119:Muscle Contraction / Q000502:physiology*; D018485:Muscle Fibers, Skeletal / Q000502:physiology* / Q000648:ultrastructure*; D018482:Muscle, Skeletal / Q000502:physiology* / Q000648:ultrastructure*; D009218:Myosins / Q000502:physiology* / Q000648:ultrastructure*; D011817:Rabbits; D012399:Rotation
D020409:Molecular Motor Proteins; D009218:Myosins