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. 2012 Feb 3;415(5):807-18.
doi: 10.1016/j.jmb.2011.11.042. Epub 2011 Dec 6.

Effects of pathogenic proline mutations on myosin assembly

Massimo Buvoli  1 , Ada Buvoli, Leslie A Leinwand
Affiliations

Effects of pathogenic proline mutations on myosin assembly

Massimo Buvoli et al. J Mol Biol. .

Abstract

Laing distal myopathy (MPD1) is a genetically dominant myopathy characterized by early and selective weakness of the distal muscles. Mutations in the MYH7 gene encoding for the β-myosin heavy chain are the underlying genetic cause of MPD1. However, their pathogenic mechanisms are currently unknown. Here, we measure the biological effects of the R1500P and L1706P MPD1 mutations in different cellular systems. We show that, while the two mutations inhibit myosin self-assembly in non-muscle cells, they do not prevent incorporation of the mutant myosin into sarcomeres. Nevertheless, we find that the L1706P mutation affects proper antiparallel myosin association by accumulating in the bare zone of the sarcomere. Furthermore, bimolecular fluorescence complementation assay shows that the α-helix containing the R1500P mutation folds into homodimeric (mutant/mutant) and heterodimeric [mutant/wild type (WT)] myosin molecules that are competent for sarcomere incorporation. Both mutations also form aggregates consisting of cytoplasmic vacuoles surrounding paracrystalline arrays and amorphous rod-like inclusions that sequester WT myosin. Myosin aggregates were also detected in transgenic nematodes expressing the R1500P mutation. By showing that the two MPD1 mutations can have dominant effects on distinct components of the contractile apparatus, our data provide the first insights into the pathogenesis of the disease.

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Figures

Figure 1
Figure 1
The R1500P and L1706P mutations alter the formation of myosin ordered structures in COS-7 cells. COS-7 cells were transfected with GFP-tagged myosin rod constructs as indicated. 12 h later, cells were imaged by confocal microscopy. While micrographs of cells transfected with GFP-tagged constructs alone (WT, R1500P, R1500W, L1706) are shown in Panel A, co-transfections with mCherry-tagged WT myosin rod are shown in Panel B. Bar, 10 μm.
Figure 2
Figure 2
Analysis of mutants in C2C12 cells. Cells transfected with the WT and mutant GFP-tagged myosin rod constructs as depicted, were imaged 24 h (D0) or 14 days (D14) after transfection. Pictures shown in D0 and D14 do not correspond to the same cell followed over time. Bar, 10 μm. D0 arrows indicate myosin aggregates; D14 white arrow indicates sarcomere bare zone, red arrow indicates mutant accumulation in the bare zone.
Figure 3
Figure 3
Analysis of mutants in NRVMs. (A) NRVMs were electroporated with the WT/mutant GFP-tagged and WT mCherry-tagged myosin rod constructs as indicated. Cells were imaged by confocal microscopy 96 h later. HMV: High Magnification View of the merged images; I, I-Band; H, H-zone (bare zone); the red arrow in the L1706P HMV panel indicates the presence of fluorescence in the sarcomere bare zone. Bar, 10 μm. Linescan panels show a typical graphical representation of the fluorescence intensity values of myosin constructs expressing GFP-tagged WT, R1500P, R1500W, L1706P myosin (green line) and mCherry-tagged WT myosin (red line) measured along 4 sarcomeres; x-axis, distance (μm); y-axis, gray levels (avg). (B) The percentage of transfected NRVMs showing organized sarcomeres containing GFP-myosin was scored 96 h after electroporation. Data were obtained from 3 independent transfections and a total of ~ 4000 cells/construct scored blind.
Figure 4
Figure 4
The R1500P mutation causes myosin cytoplasmic accumulation. (A) A representative cardiomyocyte co-transfected with the GFP-tagged R1500P and WT-mCherry myosin constructs showing myosin aggregates 96 h after electroporation. (B) Top panels: NRVMs were plated on gridded coverslips and 3 individual cells containing myosin cytoplasmic accumulations were identified and then retrieved for electron microscopy analysis. Bar, 10 μm. Middle and bottom panels show respectively the sarcomere organization and myosin cytoplasmic accumulations of the first cell shown in the top left panel analyzed by electron microscopy. Bars, as indicated. White arrows indicate myosin aggregates, black arrows indicate amorphous matrix with disorganized rod shaped structures.
Figure 5
Figure 5
Bimolecular fluorescence complementation (BiFC) assay detects R1500P homo/heterodimeric myosin molecules in live cells. COS-7 cells (Panels A and B) and NRVMs (Panels C and D) were co-transfected with the combinations of myosin rods fused to the amino (NGFP, 1-157) or carboxyl (CGFP158-230) portions of GFP as depicted in the top panels. A, and C: formation of R1500P homodimers in COS-7 and NRVMs cells respectively; B and D formation of R1500P/WT heterodimers in COS-7 and NRVMs cells respectively. Cells were imaged by confocal microscopy 24 h (COS-7) and 48 h (NRVMs) after tranfection. Bars, 10 μm.
Figure 6
Figure 6
Analysis of mutations in transgenic C. elegans. The motor domain of C. elegans myosin heavy chain B (MHC B) was tagged with GFP and human mutations* were introduced in the corresponding positions of the rod: R1500P*/W* →R1512P/W; L1706P* →A1718P. Constructs were injected in the unc-54 null worms lacking MHC B (CB 190, unc-54 (e190)). (A) Fluorescence images of body wall muscles of transgenic worms injected with the indicated constructs. Nematodes expressing the R1500P mutation displayed normal sarcomere organization; a representative picture of the R1500P mutant aggregation (indicated by arrow) is shown. Top bar, 100 μm, bottom bar, 10 μm. (B) Sinusoidal movement of WT, unc-54 null, unc-54 null rescued with the unc-54 gene and the mutants as indicated (R1500P*, R1500W*, and L1706P*). (C) Histogram showing the distance covered by the WT, unc-54 null, and rescued transgenic worms. Error bars indicate the standard deviations of the means (n=20 for each group). * P <0.01, one-way analysis of variance (ANOVA) with Tukey post hoc analysis.
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