Flash and smash: rapid freezing of muscle fibers activated by photolysis of caged ATP.
A new approach was used to study transient structural states of cross-bridges during activation of muscle fibers. Rabbit skinned muscle fibers were rapidly and synchronously activated from the rigor state by photolysis of caged ATP in the presence of Ca2+. At several different times during the switch from rigor to fully active tension development, the fibers were rapidly frozen on a liquid helium-cooled metal block, freeze-substituted, and examined in an electron microscope. The limits of structural preservation and resolution with this technique were analyzed. We demonstrate that the resolution of our images is sufficient to draw the following conclusions about cross-bridge structure. Rigor cross-bridges point away from the Z-line and most of them are wider near the thin filaments than near the backbone of the thick filaments. In contrast, cross-bridges in actively contracting fibers stretch between the thick and thin filaments at a variable angle, and are uniformly thin. Diffraction patterns computed from contracting muscle show layer lines both at 38 and 43 nm indicating that active cross-bridges contribute mass to both the actin- and myosin-based helical periodicities. The images obtained from fibers frozen 20 ms after release of ATP show a mixture of rigor and active type cross-bridge configurations. There is little evidence of cross-bridges with the rigor shape by 50 ms, and the difference in configurations between 50 and 300 ms after photolysis is surprisingly subtle.
Hirose
K
K
Department of Cell and Developmental Biology, University of Pennsylvania, Philadelphia 19104.
Lenart
T D
TD
Murray
J M
JM
Franzini-Armstrong
C
C
Goldman
Y E
YE
D000255:Adenosine Triphosphate / Q000031:analogs & derivatives* / Q000378:metabolism / Q000528:radiation effects; D000818:Animals; D055592:Biophysical Phenomena; D001703:Biophysics; D005615:Freezing; D006652:Histological Techniques / Q000295:instrumentation; D066298:In Vitro Techniques; D007700:Kinetics; D008854:Microscopy, Electron; D009119:Muscle Contraction / Q000502:physiology; D009132:Muscles / Q000378:metabolism / Q000528:radiation effects* / Q000648:ultrastructure*; D010782:Photolysis; D011817:Rabbits
C029273:P(3)-1-(2-nitro)phenylethyladenosine 5'-triphosphate; D000255:Adenosine Triphosphate