Probing myosin structural conformation in vivo by second-harmonic generation microscopy.
Understanding of complex biological processes requires knowledge of molecular structures and measurement of their dynamics in vivo. The collective chemomechanical action of myosin molecules (the molecular motors) in the muscle sarcomere represents a paradigmatic example in this respect. Here, we describe a label-free imaging method sensitive to protein conformation in vivo. We employed the order-based contrast enhancement by second-harmonic generation (SHG) for the functional imaging of muscle cells. We found that SHG polarization anisotropy (SPA) measurements report on the structural state of the actomyosin motors, with significant sensitivity to the conformation of myosin. In fact, each physiological/biochemical state we probed (relaxed, rigor, isometric contraction) produced a distinct value of polarization anisotropy. Employing a full reconstruction of the contributing elementary SHG emitters in the actomyosin motor array at atomic scale, we provide a molecular interpretation of the SPA measurements in terms of myosin conformations. We applied this method to the discrimination between attached and detached myosin heads in an isometrically contracting intact fiber. Our observations indicate that isometrically contracting muscle sustains its tetanic force by steady-state commitment of 30% of myosin heads. Applying SPA and molecular structure modeling to the imaging of unstained living tissues provides the basis for a generation of imaging and diagnostic tools capable of probing molecular structures and dynamics in vivo.
Nucciotti
V
V
Laboratory of Physiology, Department of Evolutionary Biology, University of Florence, 50125 Florence, Italy.
Stringari
C
C
Sacconi
L
L
Vanzi
F
F
Fusi
L
L
Linari
M
M
Piazzesi
G
G
Lombardi
V
V
Pavone
F S
FS
D000818:Animals; D016880:Anisotropy; D016764:Cell Polarity / Q000502:physiology; D008954:Models, Biological; D057054:Molecular Imaging / Q000379:methods*; D032342:Muscle Cells / Q000737:chemistry*; D009119:Muscle Contraction / Q000502:physiology*; D009218:Myosins / Q000737:chemistry* / Q000648:ultrastructure; D011487:Protein Conformation; D016658:Psoas Muscles / Q000502:physiology; D011817:Rabbits
D009218:Myosins
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