Two Forms of Thick Filament in the Flight Muscle of Drosophila melanogaster

Abstract

Invertebrate striated muscle myosin filaments are highly variable in structure. The best characterized myosin filaments are those found in insect indirect flight muscle (IFM) in which the flight-powering muscles are not attached directly to the wings. Four insect orders, Hemiptera, Diptera, Hymenoptera, and Coleoptera, have evolved IFM. IFM thick filaments from the first three orders have highly similar myosin arrangements but differ significantly among their non-myosin proteins. The cryo-electron microscopy of isolated IFM myosin filaments from the Dipteran Drosophila melanogaster described here revealed the coexistence of two distinct filament types, one presenting a tubular backbone like in previous work and the other a solid backbone. Inside an annulus of myosin tails, tubular filaments show no noticeable densities; solid filaments show four paired paramyosin densities. Both myosin heads of the tubular filaments are disordered; solid filaments have one completely and one partially immobilized head. Tubular filaments have the protein stretchin-klp on their surface; solid filaments do not. Two proteins, flightin and myofilin, are identifiable in all the IFM filaments previously determined. In Drosophila, flightin assumes two conformations, being compact in solid filaments and extended in tubular filaments. Nearly identical solid filaments occur in the large water bug Lethocerus indicus, which flies infrequently. The Drosophila tubular filaments occur in younger flies, and the solid filaments appear in older flies, which fly less frequently if at all, suggesting that the solid filament form is correlated with infrequent muscle use. We suggest that the solid form is designed to conserve ATP when the muscle is not in active use.

Keywords: aging; flightin; myofilin; paramyosin; stretchin-klp.

Figure 1

Original micrographs and class averages illustrating the two filament structures. (A) Solid filaments showing ordered myosin heads. (B) Filaments showing heterogeneous head arrangements including solid filaments with ordered and disordered heads and tubular filaments with disordered heads. (C) Tubular filaments with disordered heads from 30-day-old flies. (D) Class averages from solid filaments showing enhanced details of heads and solid backbones. White arrow points to a visible segment of S2, the initial segment of the myosin tail. (E) Additional class averages from the solid filament dataset showing the enhanced features of ordered heads with solid backbones (top) and disordered heads with hollow backbones. The bottom pair of class averages show almost no myosin head density and a hollow core. (F) Class averages from the tubular dataset of 30-day-old flies showing weak head density and a hollow filament core.

Figure 2

(A–C) Detailed longitudinal and cross-sectional views of segmented reconstructions showing non-myosin protein distribution in disordered- (A) and ordered-head (B) filaments, with a notable presence or absence of stretchin-klp on the surface and paramyosin in the core. (C) The 7.88 Å tubular filament segmented map generated from a class of filaments from the solid filament dataset showing stretchin-klp on the backbone surface and the absence of paramyosin in the core. (D,E) Exploration of the distribution of non-myosin proteins among myosin tails indicating structural variations between disordered- (D) and ordered-head (E) filaments. Density attributable to myofilin is compact when the heads are disordered (D) but larger and more extended when the myosin heads are ordered (E). Note also the greater size and difference in shape of the flightin C-terminal domain density (blue) in the solid filaments (E) and the more compact shape in the tubular filaments (D).

Figure 3

Flightin structural variations. (A,B) Flightin density comparisons. (A) shows the tubular filament flightin (solid) superimposed onto the flightin from the 4.7 Å map (transparent), where the blue and red densities are connected. A trace of a possible connection is present in the tubular filament flightin reconstruction from the present work, but at no contour threshold can a connection be seen. (B) The solid filament flightin superimposed on the tubular filament flightin (transparent). The tubular filament’s possible connection (black arrowheads) is missing in the solid filament version, but a new potential link (yellow arrowhead) is suggested. (C,D) Illustration of flightin’s shape differences. (C) In the tubular filaments, the red density containing the WYR domain and blue density containing the C-terminus are distant from each other, giving their connection, defined by the black arrowheads, an elongated shape. (D) In the solid filaments, the red and blue densities are juxtaposed, with the connection (black arrowhead) close to the WYR domain creating a compact flightin shape. (E) A low-pass-filtered density of the compact flightin structure, in which the WYR and C-terminal domains, both colored red, literally wrap around a myosin tail. In the extended flightin structure, these domains are in the same location but come from separate flightin molecules.

Figure 4

Stretchin-klp, myosin heads, and S2 densities in solid and tubular filaments. (A) Longitudinal view of the solid filament reconstruction (gray) with superposition of myosin head and proximal S2 densities from the tubular filaments (blue). (B,C) Myosin tail layer with proximal S2 average head density from solid filaments (gray) and tubular filaments (blue). (D) Views B and C are superimposed showing the average head and S2 density of tubular filaments (blue) and solid filaments (gray, transparent). (E–G) Stretchin-klp from tubular filaments. (E,F) Stretchin-klp (purple) on the outside of the Drosophila tubular filament following a left-handed helical track across the right-handed myosin helical symmetry. (G) Demonstration of stretchin-klp (purple) helical tracks passing under the location where the IHM “free” head binds the filament backbone, indicating how stretchin-klp potentially blocks free-head binding in tubular filaments.

Figure 5

Paramyosin. (A) Paramyosin in the solid filament’s core, featuring thicker coiled coils near the crown level and a thinner region between the crowns. Color scheme: paramyosin, pink; flightin, red (WYR and adjacent domains) and blue (C-terminus); myofilin, yellow. (B) Segmented, single paramyosin coiled coil. (C,D) Side view and top view, respectively, of paramyosin interactions with non-myosin densities in the filament’s core. (E) Flightin densities go into the paramyosin core, seemingly contacting its outer part, yet higher magnification shows no actual connection to the paramyosin. (F,G) Top and side views show that myofilin density does not contact paramyosin at this resolution.