Paramyosin phosphorylation site disruption affects indirect flight muscle stiffness and power generation in Drosophila melanogaster.
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.
Liu
Hongjun
H
Department of Biology and Molecular Biology Institute, San Diego State University, 5500 Campanile Drive, San Diego, CA 92182-4614, USA.
Miller
Mark S
MS
Swank
Douglas M
DM
Kronert
William A
WA
Maughan
David W
DW
Bernstein
Sanford I
SI
D000704:Analysis of Variance; D000818:Animals; D030801:Animals, Genetically Modified; D001696:Biomechanical Phenomena; D015153:Blotting, Western; D017931:DNA Primers; D004331:Drosophila melanogaster / Q000378:metabolism / Q000502:physiology*; D015180:Electrophoresis, Gel, Two-Dimensional; D005426:Flight, Animal / Q000502:physiology*; D008854:Microscopy, Electron; D009119:Muscle Contraction / Q000502:physiology*; D016297:Mutagenesis, Site-Directed; D009210:Myofibrils / Q000378:metabolism / Q000502:physiology* / Q000648:ultrastructure; D010766:Phosphorylation; D014335:Tropomyosin / Q000378:metabolism*
D017931:DNA Primers; D014335:Tropomyosin