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First published online 6 January 2004
doi: 10.1242/jcs.00899

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Chaperone-mediated folding and assembly of myosin in striated muscle

Department of Pathology and Laboratory Medicine, Robert Wood Johnson Medical School, University of Medicine and Dentistry of New Jersey, Piscataway, NJ 08854, USA

View larger version (52K): [in a new window]   Fig. 1. Fluorescence microscopy of postmitotic C2C12 myocytes, infected with recombinant adenovirus, AdGFP-MHC, reveals the distribution of the expressed GFP-myosin. (A,B) GFP-myosin expression is readily apparent within 18-24 hours of infection. The GFP-myosin first appears in either fluorescent globular foci (A) or short filamentous structures (B). These globular and filamentous myosin intermediates are found distributed throughout the cytoplasm of young multi-nucleated syncitia as well as mono-nucleated myocytes. (C) As the differentiation of the C2C12 cells proceeds, large multinucleated myotubes develop (72-120 hours post infection) and the distribution of the GFP-myosin shifts to a pattern characteristic of striated myofibrils.

View larger version (55K): [in a new window]   Fig. 2. Time-lapse fluorescent microscopy reveals the accumulation and dynamics of GFP-myosin in post-mitotic C2C12 myocytes. Fluorescent images of two different cells (A,B) from the same field are shown at various times after starting observation. Cells were infected 24 hours before the start of the time-lapse sequence. (A) The GFP-myosin is first detected near the periphery of the cell in globular intermediates (the position of the nucleus, N, is indicated). The fluorescence intensity and number of globules increases with time and the globules move about the cytoplasm as this mono-nucleate myocyte elongates. In this example, the globular intermediate is pulled along with the elongating cell, and stretches into a 25-30 µm long fiber that is labeled with arrowheads in the 16 hour 30 minutes frame. (B) Another cell elongates (arrowhead marks the end of the cell) and with the elongation there is a redistribution of the globular intermediates to the very tip of the cytoplasmic extension. Cells were maintained at 34°C with a heated microscope stage and perfused with heated media. A time-lapse movie of this experiment can be found in Supplemental Data (http://jcs.biologists.org/supplemental).

View larger version (145K): [in a new window]   Fig. 3. Immunofluorescence microscopy of C2C12 cells expressing GFP-myosin (green channel in A,D,G) after fixation and staining (red channel) with either an Hsp90 antibody (B,E) or an Hsc70 antibody (H). In cells expressing GFP-myosin the filamentous and globular early intermediates co-localize with Hsp90, and Hsc70. The GFP-myosin assembled in striated myofibrils does not co-localize with either Hsp90 or Hsc70 (inset is an enlargement of the boxed regions in G-I showing a small region of a striated myofibril). Most uninfected cells show diffuse staining of Hsp90 and Hsc70 in the cytosol. In addition, anti-Hsp90 and anti-Hsc70 staining reveals structures similar to the early myosin intermediates in an uninfected population of cells that lack GFP-myosin (e.g. the cell marked by an arrow in E and F). In the color merge panels (C,F,I) the structures in which Hsp90 and Hsc70 (red) co-localize with GFP-myosin (green) are yellow.

View larger version (29K): [in a new window]   Fig. 4. Characterization of the conformation-sensitive anti-myosin mAb F59. (A) Stained 6% SDS-PAGE pattern of purified chicken pectoralis myosin (1), myosin from infected (2), and uninfected (3) C2C12 cells. The expressed GFP-myosin and the endogenous C2C12 myosin are resolved and migrate at 250 kDa and 220 kDa respectively. (B) Western blot of the same samples probed with anti-S1 mAb F59. F59 reacts with all three striated muscle myosin types. (C) Native myosin binding assay with anti-myosin mAbs F59 and F18. Synthetic myosin filaments (circles) or a rigor complex of acto-S1 (triangles) were incubated with Fab fragments of F59 (open symbols) and F18 (filled symbols). The free and bound Fab fragments were separated by sedimentation of the myosin-containing filaments and quantitative SDS-PAGE and densitometry. The smooth curve is the fit to a standard binding isotherm (Winkelmann and Lowey, 1986).

View larger version (125K): [in a new window]   Fig. 5. The myosin folding intermediates exhibit partially folded motor domains. C2C12 cells expressing GFP-myosin were fixed either in 4% paraformaldehyde (A-C,G-I), or 100% methanol (D-F), and labeled with anti-myosin mAb, F59. (A) In paraformaldehyde-fixed cells the GFP-myosin is found in striated myofibrils and in folding intermediates (green). (B) The mAb F59 labels the intermediates, but not the mature myofibrils of infected or uninfected myotubes (red). (C) The color merged image shows a green myotube surrounded by yellow and red labeled cells containing the myosin folding intermediate. The arrow in B and C marks an uninfected cell that contains F59-stained folding intermediates. Methanol fixation partially denatures cellular proteins and exposes the F59 epitope in the striated myofibrils. (D) The GFP-myosin is assembled predominantly in striated myofibrils in the myotubes of this 96-hour culture. (E) F59 uniformly labels all the myosin-containing structures in the methanol fixed sample, resulting in a complete overlap with the GFP-myosin as seen in the color merged image (F). The arrow in E and F mark an uninfected cell that contains an F59 stained folding intermediate. (G-I) Uninfected C2C12 myocytes, 48 hour after transfer to differentiation medium, were paraformaldehyde fixed and stained with mAb F59 (green), anti-Hsp90 (red) and DAPI (blue). The filamentous and globular folding intermediates stain with both F59 and anti-Hsp90 in this projection of a 3D confocal microscopy volume dataset. The magnification in A-F is the same.

View larger version (160K): [in a new window]   Fig. 6. The rod myosin domain is folded and supports filament formation. (A) The GFP-myosin in infected C2C12 cells fixed with paraformaldehyde. (B) mAb 10F12.3 selectively binds the native coiled-coil conformation of S2 region of the rod domain and labels the GFP-myosin in the folding intermediates as well as in the striated myofibrils. (C) Areas of infected C2C12 cells containing the myosin maturation intermediates were marked during processing for electron microscopy and sections of these regions examined. The electron micrograph of an area of one cell reveals unusual cytoplasmic structures that contain loosely packed fibrils enclosed in a protein dense matrix that is devoid of polysomes (white arrows). Individual filaments in these structures are just discernable (black arrows). These intermediates range from 1-9 µm in length, and 0.5-3.5 µm in diameter and contain filamentous material organized in loose parallel or cross-hatched patterns. The boxed region is enlarged in the upper right corner. The filaments are best seen when viewed at a glancing angle.

View larger version (171K): [in a new window]   Fig. 7. Geldanamycin treatment of C2C12 myocytes triggers accumulation of myosin maturation intermediates. (A) Control, untreated C2C12 myocytes fixed with cold methanol 52 hour after induction of differentiation and stained with mAb F59. Under these conditions, F59 stains all the muscle myosin, folded and unfolded. (B) Accumulation of myosin trapped in maturation intermediates in myocytes that were treated for 22 hour with geldanamycin (GA) is revealed after methanol fixation and F59 staining. Myosin maturation intermediates accumulate in all myosin-expressing cells. (C,D) Paraformaldehyde fixation of cells treated for 22 hour with geldanamycin reveals the association of the partially folded myosin (F59 staining) with Hsp90 (anti-Hsp90 staining). (E) Control C2C12 myocytes 92 hour post-induction fixed with cold methanol and stained with mAb F59. The large, well differentiated myotubes are filled with striated myofibrils. (F) Geldanamycin-treated cells 40 hour after washing out the drug show reversal of drug effects and recovery of the myofibril formation. These cells were also fixed with methanol and stained with F59 to detect both the folded and unfolded myosin.

View larger version (86K): [in a new window]   Fig. 8. Association of Hsp90 and Hsc70 with denatured myosin S1. The myosin S1 proteolytic fragment of pectoralis muscle myosin was denatured with 6.5 M guanidine HCl and rapidly diluted into C2C12 S30 fraction. After a short incubation at room temperature, the S1 and associated S30 proteins were immunoadsorbed to beads coated with an anti-S1 mAb. The immunopellets were resolved on 10% SDS-PAGE and stained or immunoblotted with Hsp90 and Hsc70 specific antibodies. The Hsp90 antibody detects an 86 kDa band in the complete reaction containing denatured myosin S1. Similarly, the Hsc70 antibody detects a 70 kDa band in this reaction. The Hsp90, and Hsc70 antibodies identify single bands of sizes 86 kDa, and 70 kDa respectively in the S30 used here as positive control.

View larger version (54K): [in a new window]   Fig. 9. A model summarizing a proposed myosin folding pathway in striated muscle cells. The molecular chaperones in Step 1 interact with nascent and partially folded myosin molecules. The light chains are not depicted in this drawing but are assumed to associate with the light chain binding region of the myosin head soon after synthesis (Srikakulam and Winkelmann, 1999). This first step involves the initial folding and dimerization of the myosin molecule. Step 2 involves the formation of the myosin maturation complexes containing partially folded myosin and the molecular chaperones Hsc70 and Hsp90. Myosin transit through this intermediate is normally rapid. However, these intermediates accumulate if myofibril assembly is delayed or if Hsp90 activity is inhibited with geldanamycin (GA). Myosin filaments emerge from the maturation complexes containing folded native myosin and are then incorporated into nascent myofibrils in the final step (3) of the pathway.

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