Rotation of actin monomers during isometric contraction of skeletal muscle.
Cyclic interactions of myosin and actin are responsible for contraction of muscle. It is not self-evident, however, that the mechanical cycle occurs during steady-state isometric contraction where no work is produced. Studying cross-bridge dynamics during isometric steady-state contraction requires an equilibrium time-resolved method (not involving application of a transient). This work introduces such a method, which analyzes fluctuations of anisotropy of a few actin molecules in muscle. Fluorescence anisotropy, indicating orientation of an actin protomer, is collected from a volume of a few attoliters (10(-18) L) by confocal total internal reflection (CTIR) microscopy. In this method, the detection volume is made shallow by TIR illumination, and narrow by confocal aperture inserted in the conjugate image plane. The signal is contributed by approximately 12 labeled actin molecules. Shortening of a myofibril during contraction is prevented by light cross-linking with 1-ethyl-3-[3-dimethylamino)-propyl]-carbodiimide. The root mean-squared anisotropy fluctuations are greater in isometrically contracting than in rigor myofibrils. The results support the view that during isometric contraction, cross-bridges undergo a mechanical cycle.
Borejdo
Julian
J
University of North Texas Health Science Center, Department of Molecular Biology and Immunology, Fort Worth, Texas 76107, USA. jborejdo@hsc.unt.edu
Muthu
Priya
P
Talent
John
J
Akopova
Irina
I
Burghardt
Thomas P
TP
D000199:Actins / Q000502:physiology* / Q000648:ultrastructure*; D007537:Isometric Contraction / Q000502:physiology*; D020409:Molecular Motor Proteins / Q000502:physiology / Q000648:ultrastructure; D018482:Muscle, Skeletal / Q000502:physiology* / Q000648:ultrastructure*; D009210:Myofibrils / Q000502:physiology* / Q000648:ultrastructure*; D011487:Protein Conformation; D012399:Rotation
D000199:Actins; D020409:Molecular Motor Proteins