Structure of the Flight Muscle Thick Filament from the Bumble Bee, Bombus ignitus, at 6 Å Resolution

Abstract

Four insect orders have flight muscles that are both asynchronous and indirect; they are asynchronous in that the wingbeat frequency is decoupled from the frequency of nervous stimulation and indirect in that the muscles attach to the thoracic exoskeleton instead of directly to the wing. Flight muscle thick filaments from two orders, Hemiptera and Diptera, have been imaged at a subnanometer resolution, both of which revealed a myosin tail arrangement referred to as “curved molecular crystalline layers”. Here, we report a thick filament structure from the indirect flight muscles of a third insect order, Hymenoptera, the Asian bumble bee Bombus ignitus. The myosin tails are in general agreement with previous determinations from Lethocerus indicus and Drosophila melanogaster. The Skip 2 region has the same unusual structure as found in Lethocerus indicus thick filaments, an α-helix discontinuity is also seen at Skip 4, but the orientation of the Skip 1 region on the surface of the backbone is less angled with respect to the filament axis than in the other two species. The heads are disordered as in Drosophila, but we observe no non-myosin proteins on the backbone surface that might prohibit the ordering of myosin heads onto the thick filament backbone. There are strong structural similarities among the three species in their non-myosin proteins within the backbone that suggest how one previously unassigned density in Lethocerus might be assigned. Overall, the structure conforms to the previously observed pattern of high similarity in the myosin tail arrangement, but differences in the non-myosin proteins.

Structure of the Drosophila melanogaster Flight Muscle Myosin Filament at 4.7 Å Resolution Reveals New Details of Non-Myosin Proteins

“…In a tangential view revealing the radial disposition, both reconstructions are superimposed (Figure 4e). The proximity of the floating density to the beginning of the proximal S2 suggests that the floating density represents disordered myosin RLCs, as previously suggested for the similar density observed in Bombus [34].…”

“…Myofilin (always shown in yellow) was tentatively identified as the second non-myosin density in Lethocerus [30]. The Drosophila myofilin amino acid sequence is the smallest of the three species so far reconstructed [34]. Myofilin is predominately found on the inside surface of the myosin tail annulus (Figure 6a-d).…”

“…The myosin tails are arranged around a hollow core with a diameter of 180 Å, unlike the thick filaments of Lethocerus and Bombus, which contained eight densities believed to correspond to paramyosin [30,34]. Myosin tails in Drosophila extend from the N-terminus, located outside the tail annulus, to the C-terminus, located inside the tail annulus, giving an outward tilt of 1.8 • relative to the filament axis.…”

“…Flightin (always shown in red) was first visualized in Lethocerus as a small, folded domain, since identified as the WYR domain [34], followed by an extended peptide chain that reaches outside of the myosin tail annulus to contact the proximal S2 [30]. Recently, a flight muscle thick filament reconstruction from Bombus ignitus showed that a previously unidentified density, the so-called "blue protein" [30], was connected to the WYR domain via an extended density [34].…”

“…An RLC mutant that could not be phosphorylated [33] also had disordered heads, thus ruling out phosphorylation as the cause. More recently, a bumble bee thick filament from Bombus ignitus was reported to also have disordered heads [34]. It remains unclear whether Drosophila flight muscle thick filaments form an IHM that is disordered because it cannot attach to the filament backbone or whether a disordered IHM forms at all.…”