Cindy Le
- University of Florida
- College of Medicine
Insect flight muscle (IFM) is one of the most highly ordered
muscles in nature. This high degree of order makes it an ideal specimen for
studying different crossbridge states in muscle contraction and for investigating
steric constraints that affect the binding of crossbridges to actin. IFM
is
unique among striated muscles in that not all myosin heads can attach to
actin in the rigor state. Thus, the rigor state of IFM contains both 1-headed
and
2-headed crossbridges. A explanation for this phenomenon has not been found.
The single-headed crossbridges bind actin near the location of the troponin
complex. Thus, one possibility is that the troponin complex in IFM blocks
binding of the second myosin head. If so, troponin may block the binding
of
exogenous S1. Previous results suggested that as many as one actin out of
seven could be free of S1 (Goody et al., Biophys. J. 47, 151-169 (1985)).
To test this possibility we are using electron tomography combined with 3-D
correspondence analysis. Troponin is readily identified in EMs and 3-D reconstructions
of IFM. Specimens consist of rigor IFM which had been soaked in chymotryptic
S1 and were the same specimens in the earlier study. Tomograms were computed
using cross correlation methods to align the images and Whittaker-Shannon
interpolation of the 3-D transform data. Each 38.7 nm thin filament repeat
was extracted from the 3-D reconstruction and subjected to 3-D alignment
and
correspondence analysis. Hierarchical ascendant classification combined with
multireference alignment was used to produce 10 class averages. Each class
average shows significant density added along the entire thin filament but
particularly shows density at the troponin location. Since the resolution
is sufficient to resolve myosin heads, we conclude that troponin does not
block S1 binding. It seems therefore likely that lattice constraints prevent
the second myosin head of 1-headed crossbridges from binding to actin.