The R369 Myosin Residue within Loop 4 Is Critical for Actin Binding and Muscle Function in Drosophila

Abstract

The myosin molecular motor interacts with actin filaments in an ATP-dependent manner to yield muscle contraction. Myosin heavy chain residue R369 is located within loop 4 at the actin-tropomyosin interface of myosin's upper 50 kDa subdomain. To probe the importance of R369, we introduced a histidine mutation of that residue into Drosophila myosin and implemented an integrative approach to determine effects at the biochemical, cellular, and whole organism levels. Substituting the similarly charged but bulkier histidine residue reduces maximal actin binding in vitro without affecting myosin ATPase activity. R369H mutants exhibit impaired flight ability that is dominant in heterozygotes and progressive with age in homozygotes. Indirect flight muscle ultrastructure is normal in mutant homozygotes, suggesting that assembly defects or structural deterioration of myofibrils are not causative of reduced flight. Jump ability is also reduced in homozygotes. In contrast to these skeletal muscle defects, R369H mutants show normal heart ultrastructure and function, suggesting that this residue is differentially sensitive to perturbation in different myosin isoforms or muscle types. Overall, our findings indicate that R369 is an actin binding residue that is critical for myosin function in skeletal muscles, and suggest that more severe perturbations at this residue may cause human myopathies through a similar mechanism.

Keywords: Drosophila melanogaster; cardiomyopathy; muscle; myopathy; myosin.

Figure 1

The R369 residue of myosin loop 4 resides at an interface of F-actin, myosin and tropomyosin (PDB: 6X5Z). The position of R369 (green) is shown within the structure of masseter β-myosin S1 (yellow) bound to α1-actin (cyan) and cardiac αα-tropomyosin (violet) solved by cryo-EM in conjunction with molecular docking of the following high-resolution structures [3]: the crystal structure of squid muscle myosin S1 in rigor (PDB: 3I5G), and cryo-EM structures of human cardiac F-actin (PDB: 6KN8), human cytoplasmic actomyosin (PDB: 5JLH), and rabbit skeletal muscle F-actin with mouse α-tropomyosin (PDB: 5JLF). The loop 4 densities for residues 354–380 were replaced with those of the crystal structure of β-cardiac myosin in the post-rigor state (PDB: 6FSA).

Figure 2

R369H myosin reduces maximal actin binding without affecting ATPase activity. (A–E) Full-length myosin isolated from indirect flight muscle (IFM) of R369H mutants (N = 3) and PwMhc2 wild-type controls (N = 4) was assessed for the following ATPase parameters: (A) Mg²⁺ basal ATPase activity, (B) Ca²⁺ ATPase activity, (C) V_max of actin-stimulated Mg²⁺ ATPase, and (D) actin affinity relative to ATPase (K_m). (E) To determine actin-stimulated activity, Mg²⁺ basal ATPase activities were subtracted from measured ATPase values without actin and over increasing concentrations of F-actin. Data were fit with the Michaelis–Menten equation to determine V_max and K_m. Basal and actin-activated ATPase parameters are unchanged in R369H myosin compared to control myosin. (F) Actin co-sedimentation experiments revealed that the R369H mutation reduces the maximal binding (B_max) of F-actin (N = 3). Bound myosin S1 fractions in actomyosin samples were determined via densitometry and plotted versus F-actin concentration. Bound fractions were calculated as the density of S1 in the insoluble pellet fraction relative to total protein content minus the fraction of pelleted S1 in a control containing S1-alone. The actin-binding dissociation constant for S1 (K_d) was defined as the concentration of F-actin required to reach half B_max. For all assays, data are reported as mean ± SD (see text for specific values). Statistical significance was determined using Student’s t-tests (ns = not significant; * p ≤ 0.05). (G) A representative SDS-polyacrylamide gel containing insoluble pellet (P) fractions of R369H S1 and F-actin-containing samples over increasing concentrations of F-actin (Lanes 5–15: 0.4, 0.6, 0.8, 1, 1.2, 1.4, 1.6, 1.8, 2, 3, and 4 μM). Supernatant (S) and pellet (P) fractions from S1-alone or F-actin-alone controls are shown (Lanes 1–4).

Figure 3

The R369H myosin mutation reduces locomotion in Drosophila. R369H mutant lines (L1, L2, and L3) were crossed into a Mhc¹⁰ background that is null for endogenous myosin in IFMs and jump muscles. (A,B) Flight indices were calculated as 6*U/T +4*H/T +2*D/T +0*N/T, where each fly was scored for flight upward (U), horizontally (H), downward (D) or the inability to fly (N), and T represents the total number of flies tested. N ≥ 100 flies for each line/age. (A) Homozygous mutants exhibit declines in flight ability that are progressive with age. The statistical significance of differences between groups arising from effects of age and genotype was determined using a two-way ANOVA (Age: p < 0.0001, Genotype: p < 0.0001, Interaction: p < 0.0001). Multiple comparisons between mutants and PwMhc2 controls are shown (**** p < 0.0001). (B) Heterozygous mutants exhibit reduced flight ability at 3 weeks of age. A one-way ANOVA determined statistical significance between genotypes (* p ≤ 0.05, *** p < 0.001, **** p < 0.0001). (C) Jumping distances are reduced in 2-day-old homozygous mutants. The average values of the top 3 of 10 jump distances are reported (N = 40 per line). A one-way ANOVA determined statistical significance of differences between genotypes (**** p < 0.0001). Values represent mean ±S.E.M. Full genotypes are defined as: R369H homozygotes (Mhc¹⁰/Mhc¹⁰; P[R369H]/P[R369H]), PwMhc2 homozygotes (P[PwMhc2]/P[PwMhc2]; Mhc¹⁰/Mhc¹⁰), R369H heterozygotes (Mhc¹⁰/+; P[R369H]/-), and PwMhc2 heterozygotes (P[PwMhc2]/-; Mhc¹⁰/+), where “-” indicates the absence of a transgene on the homologous chromosome.

Figure 4

R369H myosin does not affect the stability of IFM myofibrils. Transmission electron micrographs of thin-sectioned IFMs in longitudinal orientation were obtained from 3-week-old homozygous PwMhc2 control or R369H mutant flies in a Mhc¹⁰ background that is null for endogenous myosin in IFMs and jump muscles. (A,B) Low-magnification micrographs of transverse sections show that the morphology of IFM myofibrils is normal in R369H mutant flies compared to controls. Scale bar, 0.5 µm. (C,D) High-magnification micrographs of transverse sections show a normal hexagonal pattern of thick and thin filaments in mutants and controls. Scale bar, 0.1 µm. (E,F) Low magnification images of myofibrils in longitudinal orientation show normal myofibrillar organization in R369H mutant IFMs. Scale bar, 2 μm. (G,H) High magnification images of longitudinal sections show normal sarcomeres bordered by Z-disks (arrowheads) in mutants. Scale bar, 0.5 μm. (I) Mean inter–thick filament spacing values were determined from images of transverse sections using a custom Python script. ≥700 thick filament centers per biological replicate (N = 3). (J) Mean sarcomere lengths were calculated from images of longitudinal sections using Image-J software. Fifty sarcomeres, N = 3 for each line. Data are reported as mean ± SD. Statistical significance was determined using Student’s t-tests, where ns = non-significant difference compared to controls. MF: myofibril, M: mitochondrion. Full genotypes are defined as follows: R369H homozygotes (Mhc¹⁰/Mhc¹⁰; P[R369H]/P[R369H]); PwMhc2 homozygotes (P[PwMhc2]/P[PwMhc2]; Mhc¹⁰/Mhc¹⁰).

Figure 5

The R369H mutation does not affect cardiac physiological parameters in Drosophila. Homozygous or heterozygous mutant R369H lines (L1 or L2) and PwMhc2 controls were crossed into a myosin-null Mhc^1/Mhc¹ or Mhc^1/+ background, respectively. (A,B) Cardiac diameters and (C) fractional shortening values do not differ in 4-day-old homozygous and heterozygous lines compared to controls. R369H-L2 homozygous flies display (D) reduced diastolic intervals and (F) elevated heart rates, while R369H-L1 homozygous flies display no differences in cardiac dynamics (D–F) compared to controls. N ≥ 30 flies for each genotype. Values represent mean ± S.E.M. One-way ANOVAs determined statistical significance compared to controls (* p ≤ 0.05, ** p < 0.01, and ns = non-significant). Full genotypes are defined as follows: R369H homozygotes (Mhc¹/Mhc¹; P[R369H]/P[R369H]); PwMhc2 homozygotes (P[PwMhc2]/P[PwMhc2]; Mhc¹/Mhc¹); R369H heterozygotes (Mhc¹/+; P[R369H]/-); and PwMhc2/+ heterozygotes (P[PwMhc2]/-; Mhc¹/+), where “-” indicates the absence of a transgene on the homologous chromosome.

Figure 6

The R369H mutation does not affect assembly and stability of cardiac myofibrils in Drosophila. Homozygous mutant R369H lines (L1 or L2) and PwMhc2 controls were crossed into a myosin-null Mhc^1/Mhc¹ background. (A,B) Transmission electron micrographs of transverse sections of the heart between the 3rd and 4th sets of ostia show no differences in the organization or stability of myofibrils in 4-day-old R369H homozygous lines compared to controls. The arrows indicate that Z-disks are discontinuous, as standardly observed in Drosophila cardiac myofibrils [11]. MF: myofibril. M: mitochondrion. VL: ventral-longitudinal skeletal muscle fibers. Scale bar, 0.5 µm. (C,D) Cardiac thickness values of dorsal-side (C) and ventral-side (D) areas of the heart do not differ in R369H lines compared to controls (N = 3 for each line). Values represent mean ± SD. Statistical significance was assessed using Student’s t tests, where ns = non-significant. Full genotypes are defined as follows: R369H homozygotes (Mhc¹/Mhc¹; P[R369H]/P[R369H]); PwMhc2 homozygotes (P[PwMhc2]/P[PwMhc2]; Mhc¹/Mhc¹).