Paramyosin phosphorylation site disruption affects indirect flight muscle stiffness and power generation in Drosophila melanogaster

Abstract

The phosphoprotein paramyosin is a major structural component of invertebrate muscle thick filaments. To investigate the importance of paramyosin phosphorylation, we produced transgenic Drosophila melanogaster in which one, three, or four phosphorylatable serine residues in the N-terminal nonhelical domain were replaced by alanines. Depending on the residues mutated, transgenic lines were either unaffected or severely flight impaired. Flight-impaired strains had decreases in the most acidic paramyosin isoforms, with a corresponding increase in more basic isoforms. Surprisingly, ultrastructure of indirect flight muscle myofibrils was normal, indicating N-terminal phosphorylation is not important for myofibril assembly. However, mechanical studies of active indirect flight muscle fibers revealed that phosphorylation site mutations reduced elastic and viscous moduli by 21-59% and maximum power output by up to 42%. Significant reductions also occurred under relaxed and rigor conditions, indicating that the phosphorylation-dependent changes are independent of strong crossbridge attachment and likely arise from alterations in thick filament backbone properties. Further, normal crossbridge kinetics were observed, demonstrating that myosin motor function is unaffected in the mutants. We conclude that N-terminal phosphorylation of Drosophila paramyosin is essential for optimal force and oscillatory power transduction within the muscle fiber and is key to the high passive stiffness of asynchronous insect flight muscles. Phosphorylation may reinforce interactions between myosin rod domains, enhance thick filament connections to the central M-line of the sarcomere and/or stabilize thick filament interactions with proteins that contribute to fiber stiffness.

Fig. 1.

Structures of paramyosin transgenes used for germ-line transformation. Exons of the paramyosin gene are drawn as open boxes. The first 32 aa of the N-terminal nonhelical domain are emphasized, with four Ser residues that have high potential of phosphorylation marked by ^*. Locations of Ser residues replaced by Alas in mutant transgenes are indicated by “A.” All constructs are cloned in a CaSpeR vector, which has an eye color gene white⁺ used as a marker for selecting transgenic flies.

Fig. 2.

Paramyosin isoform profiles of control and mutant flies on 2D gels. (A) On a Western blot using paramyosin antibody, six paramyosin isoforms are detected in homozygous prm¹ rescued with the normal paramyosin transgene pm, as is the case for silver-stained gels and in yw control flies (data not shown). (B and C) In silver-stained gels, substitution of Ala for Ser-18 in pm^S18A (B) or four Ser residues (Ser-9, -10, -13, and -18) in pm^S-A4 (C) with Alas causes reduction in acidic isoforms and a corresponding increase in basic isoforms. ^* marks isoforms decreased; arrows mark isoforms increased.

Fig. 3.

EM of IFM. Substitution of four Ser residues in the N-terminal nonhelical domain of paramyosin does not affect the ultrastructure of IFMs. Compared with those of wild-type flies (A and B), IFMs of pm^S-A4 flies (C and D) are normal. In longitudinal section (C), pm^S-A4 myofibrils have sarcomeres of constant length and width. In cross section (D Inset), pm^S-A4 thick and thin filaments interdigitate into regular hexagonal arrays, typical of IFMs (B Inset). (Bars: 1 μm.)

Fig. 4.

Elastic modulus (A), viscous modulus (B), and power production (C) values for active IFM across muscle oscillation frequencies for paramyosin mutants (pm^S18A and pm^S-A4) and pm control. Values are means ± SEM. Dashed lines in C represent f_Pmax (frequency of maximum power) from Table 3. ^* indicates a span of frequencies over which there is a significant difference (P < 0.05) between pm and the pm^S18A and pm^S-A4 lines. Temperature = 15°C. Note that the elastic and viscous moduli frequency values were plotted on a log scale over the entire sinusoidal analysis frequency range, whereas the power data were plotted on a linear scale over the power-producing range (where the viscous modulus is negative).

Fig. 5.

Elastic and viscous modulus values for relaxed (A and B) and rigor (C and D) IFM across muscle oscillation frequencies for paramyosin mutants (pm^S18A and pm^S-A4) and pm control. Values are means ± SEM. ^* indicates a span of frequencies over which there is a significant difference (P < 0.05) between pm and the pm^S18A and pm^S-A4 lines. Temperature = 15°C.