Changes in orientation of actin during contraction of muscle.
It is well documented that muscle contraction results from cyclic rotations of actin-bound myosin cross-bridges. The role of actin is hypothesized to be limited to accelerating phosphate release from myosin and to serving as a rigid substrate for cross-bridge rotations. To test this hypothesis, we have measured actin rotations during contraction of a skeletal muscle. Actin filaments of rabbit psoas fiber were labeled with rhodamine-phalloidin. Muscle contraction was induced by a pulse of ATP photogenerated from caged precursor. ATP induced a single turnover of cross-bridges. The rotations were measured by anisotropy of fluorescence originating from a small volume defined by a narrow aperture of a confocal microscope. The anisotropy of phalloidin-actin changed rapidly at first and was followed by a slow relaxation to a steady-state value. The kinetics of orientation changes of actin and myosin were the same. Extracting myosin abolished anisotropy changes. To test whether the rotation of actin was imposed by cross-bridges or whether it reflected hydrolytic activity of actin itself, we labeled actin with fluorescent ADP. The time-course of anisotropy change of fluorescent nucleotide was similar to that of phalloidin-actin. These results suggest that orientation changes of actin are caused by dissociation and rebinding of myosin cross-bridges, and that during contraction, nucleotide does not dissociate from actin.
Borejdo
J
J
Department of Molecular Biology and Immunology, University of North Texas, Fort Worth, Texas, USA. jborejdo@hsc.unt.edu
Shepard
A
A
Dumka
D
D
Akopova
I
I
Talent
J
J
Malka
A
A
Burghardt
T P
TP
D000199:Actins / Q000502:physiology*; D000255:Adenosine Triphosphate / Q000502:physiology*; D000818:Animals; D005456:Fluorescent Dyes / Q000737:chemistry; D018613:Microscopy, Confocal; D009119:Muscle Contraction / Q000502:physiology*; D018482:Muscle, Skeletal / Q000502:physiology*; D009218:Myosins / Q000502:physiology*; D010590:Phalloidine / Q000737:chemistry; D011817:Rabbits
D000199:Actins; D005456:Fluorescent Dyes; D010590:Phalloidine; D000255:Adenosine Triphosphate; D009218:Myosins