• 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.

Figure shows an electron micrograph and computed transform of ifm soaked in myosin s1 which reveal high intensity along 5.9 nm 5.1 layer lines from f-actin. This observation indicates good preservation of the actin structure. Magnification insert on left arrowhead structure expected thin filaments.

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