Drosophila muscle regulation characterized by electron microscopy and three-dimensional reconstruction of thin filament mutants

Abstract

Wild-type and mutant thin filaments were isolated directly from "myosinless" Drosophila indirect flight muscles to study the structural basis of muscle regulation genetically. Negatively stained filaments showed tropomyosin with periodically arranged troponin complexes in electron micrographs. Three-dimensional helical reconstruction of wild-type filaments indicated that the positions of tropomyosin on actin in the presence and absence of Ca(2+) were indistinguishable from those in vertebrate striated muscle and consistent with a steric mechanism of regulation by troponin-tropomyosin in Drosophila muscles. Thus, the Drosophila model can be used to study steric regulation. Thin filaments from the Drosophila mutant heldup(2), which possesses a single amino acid conversion in troponin I, were similarly analyzed to assess the Drosophila model genetically. The positions of tropomyosin in the mutant filaments, in both the Ca(2+)-free and the Ca(2+)-induced states, were the same, and identical to that of wild-type filaments in the presence of Ca(2+). Thus, cross-bridge cycling would be expected to proceed uninhibited in these fibers, even in relaxing conditions, and this would account for the dramatic hypercontraction characteristic of these mutant muscles. The interaction of mutant troponin I with Drosophila troponin C is discussed, along with functional differences between troponin C from Drosophila and vertebrates.

FIGURE 1

Electron micrographs of negatively stained Drosophila IFM thin filaments. A low magnification field showing single thin filaments, paired filaments, and filament rafts isolated from Mhc⁷ myosinless flies. The globular ends of troponin (indicated by black arrows) are evident as bulges repeating at 38-nm intervals and are most easily seen by viewing filaments at a glancing angle. Filament pairs and rafts apparently are joined together by troponin molecules. (Inset) Examples of filaments used for image processing from the IFM of (A and B) Mhc⁷ or (C and D) hdp²; Mhc¹² mutants isolated in EGTA (A and C) or treated with Ca²⁺ (B and D). Single filaments typically were chosen for image processing, but reconstruction of loosely paired filaments was also possible, and invariably both filament types yielded the same results.

FIGURE 2

Surface views of reconstructed thin filaments showing the position of tropomyosin strands on actin. (A) F-actin control (actin subdomains numbered on one monomer); (B–E) Drosophila filaments from Mhc⁷ (B and C) and hdp²; Mhc¹² (D and E) strains; (F and G) reconstituted thin filaments containing mutant CBMII Tn C and otherwise wild-type vertebrate cardiac troponin-tropomyosin components. Reconstructions are of samples maintained in the absence of Ca²⁺ (B, D, and F) or treated with Ca²⁺ (C, E, and G). Tropomyosin strands indicated by black arrows in B and C. Note that the tropomyosin density in B, F, and G is associated with subdomain-1 of each actin monomer and bridges over subdomain-2 of successive monomers along the long-pitch actin helix (on the inner edge of the outer domain of actin (A_o)). In C, D, and E), tropomyosin is associated with subdomain-3 and bridges over subdomain-4 of successive actin monomers (on the outer edge of the inner domain of actin (A_i)). Thus, Ca²⁺-dependent movement of tropomyosin occurs in filaments from Mhc⁷ IFM, but not from hdp²; Mhc¹² mutant IFM or from filaments containing CBMII Tn C. In the case of hdp²; Mhc¹², tropomyosin is trapped on the inner domain of actin and, in the CMBII mutant, on the outer actin domain. The average phase residuals (±SD), a measurement of the agreement among filaments generating reconstructions, for the 15 filaments in each Drosophila data set were 58.0 ± 5.6°, 56.2 ± 7.0°, 55.8 ± 6.0°, and 54.8 ± 5.8°, respectively. The average up-down phase residuals, a measure of filament polarity, were 23.9 ± 6.6°, 24.4 ± 8.2°, 19.1 ± 5.5°, and 28.5 ± 7.9°, respectively, values comparable to those previously found. The average angular rotations between adjacent actin monomers along the genetic helix of the Drosophila filaments were 167.1 ± 0.7°, 167.0 ± 0.9°, 167.3 ± 1.4°, and 167.1 ± 1.3°, respectively, consistent with a 28/13 helix.

FIGURE 3

Helical projections (formed by projecting component densities in reconstructions down the long-pitch actin helices onto a plane perpendicular to the thin-filament axis) showing axially averaged positions of F-actin and tropomyosin (TM) that appear symmetrical on either side of the filament. Densities in all maps shown were significant over background noise at greater than the 99.95% confidence levels. Drosophila filaments from Mhc⁷ (A and B) and hdp²; Mhc¹² mutants (D and E); vertebrate filaments containing wild-type troponin (G and H); or mutant CBMII troponin (I and J). Reconstructions are of samples maintained in the absence of Ca²⁺ (A, D, G, and I) or treated with Ca²⁺ (B, E, H, and J). Again, note that Ca²⁺-dependent movement of tropomyosin occurs in filaments from Mhc⁷ IFM, but not in those from hdp²; Mhc¹² mutants or from reconstituted filaments containing CBMII Tn C instead of control vertebrate troponin. The statistical significance of the differences observed between maps of Mhc⁷ IFM filaments in EGTA or Ca²⁺ conditions was computed. The regions of the two maps that were significantly different from each other at greater than the 99.95% confidence level are displayed as single contour envelopes superimposed on the control map of F-actin (C); densities specific to EGTA (solid black) or Ca²⁺ (open) highlight the tropomyosin movement and the tropomyosin binding positions on actin. The myosin-binding sites on actin are indicated by gray bands in C. No significant differences were detected between contributing densities forming the EGTA and Ca²⁺ maps of the hdp²; Mhc¹² mutants (F), confirming that tropomyosin movement did not occur; an asterisk marks the tropomyosin-binding site on actin in hdp² filaments.