ELECTRON TOMOGRAPHY OF INSECT FLIGHT-MUSCLE IN RIGOR AND AMPPNP AT 23-DEGREES-C

Treatment of rigor fibers of insect flight muscle (IFM) with AMPPNP at 23 degrees C causes a 70% drop in tension with little change in stiff ness. In order to visualize the changes in crossbridge conformation an d distribution that give rise to the mechanical response, we have prod uced three-dimensional reconstructions by tomography of both rigor and AMPPNP-treated muscle that do not average the repeating motifs of cro ssbridges, and thereby retain information on variability of crossbridg e structure and distribution. Tomograms can be averaged when display o f only the regular features is wanted. Tomograms of rigor IFM show dou ble-headed lead and single-headed rear crossbridges. Tomograms of IFM treated with AMPPNP at 23 degrees C reveal many double-headed and some single-headed ''lead'' bridges but few crossbridges corresponding to the rear bridges of rigor. Instead, new non-rigor forms of variably an gled crossbridges are found bound to actin sites not labeled with myos in heads in rigor. This indicates that the rear bridges of rigor have redistributed during the transition from rigor to the AMPPNP state, wh ich could explain the maintenance of rigor stiffness despite the loss of tension. Comparison of in situ crossbridges in tomograms of rigor w ith atomic model of acto-S1, the complex formed by myosin subfragment 1 and actin, reveals that the regulatory domain of S1 would require si gnificant bending and realignment to fit into both types of rigor cros sbridges. The modifications are particularly significant for the rear bridges and suggest that differential strain in the regulatory domain of rear bridges may be the basis for their detachment and redistributi on upon binding AMPPNP. Similar comparison using lead-type crossbridge s in AMPPNP reveals departures from the rigor acto-S1 atomic. model th at include azimuthal straightening and a slight M-ward bending in the regulatory domain. Both the motor and regulatory domains of the new no n-rigor crossbridges differ from those in the atomic model of acto-SZ. A new crossbridge motif identified in AMPPNP-treated muscle consists of paired rigor-like and non-rigor crossbridges and suggests possible transitions in the myosin working stroke. (C) 1996 Academic Press Limi ted