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First published online 2 September 2003
doi: 10.1242/jcs.00709

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Mutations in the motor domain modulate myosin activity and myofibril organization

¹ Department of Pathology and Laboratory Medicine, Robert Wood Johnson Medical School, Piscataway, NJ 08854, USA
² Department of Molecular and Cellular Biochemistry, University of Kentucky, Lexington, KY 40536, USA

View larger version (42K): [in a new window]   Fig. 1. A molecular model showing the location of the GFP fusion to the myosin motor domain. This ribbon representation of the myosin motor domain (blue) and GFP (green) illustrates the linkage (arrowhead) of the C terminus of GFP to the N terminus of myosin. This model illustrates the important functional sites on the myosin motor domain in relation to the GFP attachment point. The three point mutations in the motor domain (R403Q, R453C and G584R) are identified with CPK models (red) of the WT residues. The essential light chain (yellow) and the regulatory light chain (magenta) are bound to the myosin lever arm. Model coordinates are for adult pectoralis muscle myosin subfragment-1 (Rayment et al., 1993) and GFP (Yang et al., 1996).

View larger version (106K): [in a new window]   Fig. 2. Transient expression and assembly of GFP-myosin in embryonic cardiomyocytes. (A) In this live cardiomyocyte imaged 26 hours post-transfection the GFP-myosin is found assembled in striated myofibrils, in non-striated myofilaments, and in scattered thick filaments (white arrowheads) and non-aligned A-bands. (B) The same cell was examined 18 hours later (44 hours post-transfection). The organization and number of myofibrils in the cell have increased with a concomitant decrease in the non-striated fluorescence. (C) A cardiomyocyte expressing GFP-myosin (green) was fixed 72 hours post-transfection and stained with mAb RT11 (red) that labels the titin PEVK domain at the A-I junction (Moncman and Wang, 1996). The boxed region is enlarged (inset) to show the ordered myofibril structure. (D) A DIC image overlaid in green with the fluorescent image of GFP-myosin. Two adjacent cells are expressing GFP-myosin and a third neighboring cell is not. The GFP-myosin-expressing cells have formed a prominent intercalated disk (white arrowheads) and the myofibrils are arranged co-linearly across the disk. The magnification in A-C is the same.

View larger version (54K): [in a new window]   Fig. 3. The contractile activity of GFP-myosin-expressing cells. (A) DIC image of a small area of an R403Q mutant GFP-myosin-expressing cardiomyocyte showing a clearly defined myofibril. Two z-lines spaced 7 sarcomeres apart are marked with small bulls eyes. The fluorescent cell was selected then the DIC image was recorded at 30 frames/second. (B) The positions of the two marked z-lines were tracked over time and the relative length changes plotted. The velocities of the contraction and relaxation phases (dL/dT) were determined and summarized in Table 1. Cells were maintained at 30°C on a heated microscope stage. Movies of this cell and another can be found at http://jcs.biologists.org/supplemental.

View larger version (77K): [in a new window]   Fig. 4. Organization of the contractile cytoskeleton in transfected cardiomyocytes. The transfected cardiomyocytes can be grouped into four classes based on the degree of organization of the contractile cytoskeleton. Non-striated cells contain brightly fluorescent myofilaments but lack the organized repeating units characteristic of myofibrils. Striated-1 cells contain a mixture of non-striated myofilaments interspersed with thin and misaligned striated myofibrils. Striated-2 cells contain predominantly striated myofibrils; however, this class lacks lateral alignment of the myofibrils. Striated-3 cells are packed with laterally aligned striated myofibrils that stretch between cell attachment points. The magnification is the same in all four panels.

View larger version (25K): [in a new window]   Fig. 5. Analysis of the organization of the contractile cytoskeleton in embryonic cardiomyocytes. Cells were transfected with the WT GFP-myosin and the three FHC mutants and fixed 72 hours post-transfection. The fixed cells were counter-stained with antibodies to muscle specific markers (either anti-titin, RT11, or anti-myosin, F18). The GFP-myosin-positive cells were scored in one of the four classifications illustrated in Fig. 4 and described in the text. The counterstaining for other muscle-specific protein was used to confirm the myofilament organization. The non-transfected control cells were scored after staining with anti-titin or anti-myosin. These data summarize the results from four separate experiments and include over 4400 cells. The number of cells in each group varied between 200-400 cells for a total of 1100 cells/experiment. Pairwise comparison of the distribution of cells in classes for the different groups was done using Student's t-test with a confidence level of P<0.05. Error bars correspond to the standard deviation within each class of the four experiments.

View larger version (151K): [in a new window]   Fig. 6. Replication-defective adenovirus induced expression of GFP-myosin in C2C12 myotubes. Post-mitotic C2C12 myocytes were infected 48 hours after transfer to fusion medium and maintained in culture for an additional 4 days. Almost all of the large myotubes are brightly fluorescent from the expressed GFP-myosin. When viewed at higher magnification (inset) it is clear that the WT GFP-myosin has assembled with the endogenous C2C12 myosin into ordered striated myofibrils.

View larger version (51K): [in a new window]   Fig. 7. Isolation and analysis of the GFP-myosin expressed in C2C12 myotubes. (A) SDS-PAGE of myosin isolated from uninfected C2C12 myotubes (lane 1), myotubes infected with WT AdGFPMHC and expressing GFP-myosin (lane 2), and purified GFP-myosin sample after ion-exchange chromatography (lane 3). The myosin heavy and light chains are resolved in this 12.5% acrylamide gel. The GFP-myosin heavy chain migrates just above the C2C12 myosin heavy chain in lane 2. (B) A 6% acrylamide gel clearly resolves the GFP-myosin heavy chain from the C2C12 myosin heavy chain: lane 1-Purified adult chicken pectoralis muscle myosin; lane 2-myosin isolated from infected C2C12 cells; lane 3 - Myosin from uninfected C2C12 myotubes. A western blot of the same samples developed with mAb 12C5.3 that selectively reacts with the chicken muscle myosin and labels the 250 kDa GFP-myosin heavy chain, but does not detect the C2C12 myosin. (C) SDS-PAGE analysis of myosin isolated from C2C12 myotubes of uninfected cells (Lane 1), and cells expressing WT GFP-myosin (Lane 2), R403Q (Lane 3), R453C (Lane 4) and G584R (Lane 5). These cells were harvested 96 hours post-infection. Densitometry of the stained gel reveals that the GFP-myosin amounts to 25-40% of the total myosin from the infected myotubes. (D) Immunoprecipitation of the GFP-myosin under native conditions with two anti-myosin mAbs (12C5.3 and 10F12.3) that uniquely recognize the chimeric GFP-myosin reveals that the chimeric protein is predominantly a homodimer.

View larger version (19K): [in a new window]   Fig. 8. The myosin concentration dependence of the actin filament velocity measured in an in vitro motility assay. (A) The velocity of actin filament was measured on antibody capture surfaces incubated with various concentrations of WT and G584R myosin. The velocity of sliding movement at saturating myosin surface density is indicated. (B) The R403Q and R453C data are plotted and the actin filament velocity is indicated. The data points correspond to the mean (±s.d.) for a Gaussian distribution of the actin filament velocity at each myosin concentration tested (Kinose et al., 1996). The myosin concentration dependence of the actin filament sliding velocity was fit to the empirical equation: v=vmax(1-e-m(-onset)). The GFP-myosin concentration was determined by densitometry of stained SDS gels and fluorescence spectroscopy. Actin filament motility was measured at 30°C.

View larger version (16K): [in a new window]   Fig. 9. Myosin surface density determination and actin filament length dependence of unloaded shortening velocity. (A) Fluorescent quantitation of the binding of GFP-myosin to mAb-coated glass coverslips. The fluorescence from GFP-myosin bound to the surface was measured by placing the coverslip at a 45° angle to the light path in motility buffer in a standard 1 cm cuvette. The solid line is a fit of the myosin concentration dependence of iodinated adult skeletal muscle myosin binding to the mAb surfaces (Winkelmann et al., 1995). This provides a rapid method for determining the myosin surface density used in the in vitro motility assay. (B) Actin filament length dependence of the unloaded shortening velocity was used to estimate the duty ratio for WT and mutant GFP-myosin. The sliding filament velocity (vexp) and actin filament length (l) data were fit to the equation for each myosin surface density () (Harada et al., 1990; Uyeda et al., 1990). The data is weighted by the number of filaments (n) in each length bin. This is indicated for each data point by the vertical bars that are equal to 1/n1/2. These data are for WT GFP-myosin at a myosin surface density of 830 mol/µm2 and the G584R mutant at 414 mol/µm2.

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