The masticatory system under varying functional load. Part 1: Structural adaptation of rabbit jaw muscles to reduced masticatory load.

Skeletal muscle fibres can change their myosin heavy-chain (MyHC) isoform and cross-sectional area, which determine their contraction velocity and maximum force generation, respectively, to adapt to varying functional loads. In general, reduced muscle activity induces transition towards faster fibres and a decrease in fibre cross-sectional area. In order to investigate the effect of a reduction in masticatory load on three functionally different jaw muscles, the MyHC composition and the corresponding cross-sectional area of fibres were determined in the superficial masseter, superficial temporalis, and digastric muscles of male juvenile New Zealand White rabbits that had been raised on a soft diet (n=8) from 8 to 20 weeks of age and in those of normal diet controls (n=8). Differences between groups were tested for statistical significance using a Mann-Whitney rank sum test. The proportion and cross-sectional area of fibres co-expressing MyHC-I and MyHC-cardiac alpha were significantly smaller in the masseter muscles of the animals that had been fed soft food than in those of the controls. In contrast, the proportions and cross-sectional areas of the various fibre types in the temporalis and digastric muscles did not differ significantly between the groups. The results suggest that reducing the masticatory load during development affects the contraction velocity and maximum force generation of the jaw-closing muscles that are primarily responsible for force generation during chewing. These muscles adapt structurally to the reduced functional load with changes in the MyHC composition and cross-sectional area mainly within their slow fibre compartment.

X-ray Crystallographic and Molecular Dynamic Analyses of Drosophila melanogaster Embryonic Muscle Myosin Define Domains Responsible for Isoform-Specific Properties.

Drosophila melanogaster is a powerful system for characterizing alternative myosin isoforms and modeling muscle diseases, but high-resolution structures of fruit fly contractile proteins have not been determined. Here we report the first x-ray crystal structure of an insect myosin: the D melanogaster skeletal muscle myosin II embryonic isoform (EMB). Using our system for recombinant expression of myosin heavy chain (MHC) proteins in whole transgenic flies, we prepared and crystallized stable proteolytic S1-like fragments containing the entire EMB motor domain bound to an essential light chain. We solved the x-ray crystal structure by molecular replacement and refined the resulting model against diffraction data to 2.2 Å resolution. The protein is captured in two slightly different renditions of the rigor-like conformation with a citrate of crystallization at the nucleotide binding site and exhibits structural features common to myosins of diverse classes from all kingdoms of life. All atom molecular dynamics simulations on EMB in its nucleotide-free state and a derivative homology model containing 61 amino acid substitutions unique to the indirect flight muscle isoform (IFI) suggest that differences in the identity of residues within the relay and the converter that are encoded for by MHC alternative exons 9 and 11, respectively, directly contribute to increased mobility of these regions in IFI relative to EMB. This suggests the possibility that alternative folding or conformational stability within these regions contribute to the observed functional differences in Drosophila EMB and IFI myosins.

alphaB-crystallin maintains skeletal muscle myosin enzymatic activity and prevents its aggregation under heat-shock stress.

Here, we provide functional and direct structural evidence that alphaB-crystallin, a member of the small heat-shock protein family, suppresses thermal unfolding and aggregation of the myosin II molecular motor. Chicken skeletal muscle myosin was thermally unfolded at heat-shock temperature (43 degrees C) in the absence and in the presence of alphaB-crystallin. The ATPase activity of myosin at 25 degrees C was used as a parameter to monitor its unfolding. Myosin retained only 65% and 8% of its ATPase activity when incubated at heat-shock temperature for 15 min and 30 min, respectively. However, 84% and 58% of the myosin ATPase activity was maintained when it was incubated with alphaB-crystallin under the same conditions. Furthermore, actin-stimulated ATPase activity of myosin was reduced by approximately 90%, when myosin was thermally unfolded at 43 degrees C for 30 min, but was reduced by only approximately 42% when it was incubated with alphaB-crystallin under the same conditions. Light-scattering assays and bound thioflavin T fluorescence indicated that myosin aggregates when incubated at 43 degrees C for 30 min, while alphaB-crystallin suppressed this thermal aggregation. Photo-labeled bis-ANS alphaB-crystallin fluorescence studies confirmed the transient interaction of alphaB-crystallin with myosin. These findings were further supported by electron microscopy of rotary shadowed molecules. This revealed that approximately 94% of myosin molecules formed inter and intra-molecular aggregates when incubated at 43 degrees C for 30 min. alphaB-Crystallin, however, protected approximately 48% of the myosin molecules from thermal aggregation, with protected myosin appearing identical to unheated molecules. These results are the first to show that alphaB-crystallin maintains myosin enzymatic activity and prevents the aggregation of the motor under heat-shock conditions. Thus, alphaB-crystallin may be critical for nascent myosin folding, promoting myofibrillogenesis, maintaining cytoskeletal integrity and sustaining muscle performance, since heat-shock temperatures can be produced during multiple stress conditions or vigorous exercise.

Highly selective directed assembly of functional actomyosin on Au surfaces.

The capability of assembling biomotors onto specific locations of solid substrates is a key for development of biomotor-based nanomechanical systems. We developed a method to direct the assembly of the heavy meromyosin fragment from rabbit skeletal muscle myosin onto specific locations of Au substrates utilizing surface molecular patterns. In this strategy, chemically directed patterns of streptavidin were achieved to direct highly specific assembly of biotinylated heavy meromyosin on the substrates--a strategy applicable for patterning a variety of biotinylated molecules--while BSA was utilized to avoid nonspecific adsorption. In vitro motility assays of filament sliding were used to confirm functionality of assembled actomyosin.