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Targeting and functional role of N-RAP, a nebulin-related LIM protein, during myofibril assembly in cultured chick cardiomyocytes

Laboratory of Muscle Biology, National Institute of Arthritis and Musculoskeletal and Skin Diseases, National Institutes of Health, Bethesda, MD 20892, USA

View larger version (29K): [in a new window]   Fig. 1. (A) Regions of N-RAP expressed as GFP fusion proteins. GFP was fused to the N terminus of full-length N-RAP (1), the N-RAP super repeats (N-RAP-SR) (2), the region in between the super repeats and the LIM domain (N-RAP-IB) (3), and the N-RAP LIM domain (N-RAP-LIM) (4). Numbers above the diagram refer to amino acid residues from the full-length mouse N-RAP sequence, while Ab marks the position of a 30 residue peptide used as an antigen for the production of polyclonal antibodies. The predicted molecular weights of the N-RAP fusion proteins are indicated in parentheses, including the 28.6 kDa contributed by the N-terminal GFP. (B) Immunoblot analysis of chick cardiomyocytes transfected with GFP-N-RAP-SR (lane 1), GFP-N-RAP-IB (lane 2), GFP-N-RAP-LIM (lane 3) and GFP alone (lane 4). Equivalent volumes of lysate from cultured chick cardiomyocytes were loaded in each lane and probed with anti-GFP antibody. In each case, the detected band migrated near the position predicted from the sequence of the fusion plasmid. The detected bands are labeled (right), together with the position of the molecular weight markers (left). Note that GFP alone contains 4.7 kDa attributed to 46 C-terminal residues that are not present in the N-RAP fusion proteins, owing to the stop codons engineered into the fusion constructs at the precise end of the sequences derived from N-RAP (Table 1).

View larger version (16K): [in a new window]   Fig. 2. Expression patterns of GFP (A) and of -actinin in the same field (B). GFP is diffusely distributed in the single transfected cardiomyocyte (arrow, A). -actinin (B) is clearly localized in striated myofibrils in both the GFP transfected cell (arrow) and the neighboring untransfected cell (asterisk). (C) Myofibril content versus days in culture. The open symbols show results for mock transfected cardiomyocytes, and the closed symbols show results for cardiomyocytes transfected with GFP alone. Cells were transfected after 1 day (circles) or 2 days (squares) in culture and viewed 1 day or 3 days post-transfection. Each point represents the mean±s.e.m. from 4 to 27 transfected cells. No significant difference was observed between the mock-transfected cardiomyocytes and cardiomyocytes expressing GFP.

View larger version (101K): [in a new window]   Fig. 3. Expression patterns of GFP-N-RAP (A,C,E) and of -actinin in the same fields (B,D,F). Cardiomyocytes were transfected with GFP-N-RAP at 2 days and viewed 1 day post-transfection (A-B,E-F) or transfected at 4 days and viewed 3 days post-transfection (C-D). In cells containing mature sarcomeres linked by immature areas, GFP-N-RAP is localized only in the myofibril precursors (A,B, arrows), as well as at cell to cell contacts (A,B, arrowheads). In more mature cardiomyocytes that had well-established intercalated disks, GFP-N-RAP was highly concentrated at these sites, and often exhibited a doublet band of expression that appeared to bracket the -actinin band (C,D, double arrows). Note that GFP-N-RAP was not expressed within mature myofibrils detected by -actinin staining (C,D, arrowheads). Some cells exhibit specific localization of GFP-N-RAP along fibrillar structures that were not detected by -actinin staining (E,F, arrows). Note the absence of myofibrillar -actinin striations in this transfected cardiomyocyte compared with the surrounding untransfected cells (E,F). (B) The method for quantitating myofibril content in individual cardiomyocytes: the periphery of the transfected cardiomyocyte stained with -actinin is outlined by the unbroken white line; areas containing mature myofibrils are outlined by broken lines. The area of each enclosed region was measured, and the percentage of the total area taken up by mature myofibrils was calculated.

View larger version (96K): [in a new window]   Fig. 4. Expression patterns of GFP-N-RAP-LIM (A,C) and of -actinin in the same fields (B,D). Cardiomyocytes were transfected with GFP-N-RAP-LIM at 4 days and viewed 3 days post transfection (A,B) or transfected at 2 days and viewed 1 day post transfection (C,D). In cardiomyocytes transfected after 4 days in culture, GFP-N-RAP-LIM was targeted to the cell periphery and sites of cell to cell contact (arrowheads, A,B). Punctate or filamentous -actinin staining revealed that myofibril precursors were present in these cardiomyocytes, but did not contain GFP-N-RAP-LIM (arrows, A,B). In cardiomyocytes transfected after 1 or 2 days in culture, both GFP-N-RAP-LIM and -actinin were diffusely distributed (arrows, C,D), in contrast to neighboring untransfected cells exhibiting clear striations when stained for -actinin (asterisks, C,D).

View larger version (75K): [in a new window]   Fig. 5. Expression patterns of GFP-N-RAP-SR (A,C) and of -actinin in the same fields (B,D). Cardiomyocytes were transfected with GFP-N-RAP-SR at 2 days and viewed 1 day post transfection (A,B) or transfected at 1 day and viewed 3 days post transfection (C,D). In cardiomyocytes transfected after 2 days in culture, GFP-N-RAP-SR is concentrated in myofibril precursors (arrows, A,B), as well as in mature sarcomeres (arrowheads, A,B). The inset (A,B) shows a magnified view of the -actinin striations marked with arrowheads, showing that N-RAP-SR is concentrated within the myofilament-containing region of the sarcomere, but not in the Z-lines marked by -actinin staining. The sarcomeres shown in the inset have an average length of 1.4 µm. Cardiomyocytes transfected with GFP-N-RAP-SR after 1 day in culture rarely form mature myofibrils (C,D).

View larger version (52K): [in a new window]   Fig. 6. Expression patterns of GFP-N-RAP-IB (A,C) and of -actinin in the same fields (B,D). Cardiomyocytes were transfected with GFP-N-RAP-IB at 2 days and viewed 1 day post-transfection. GFP-N-RAP-IB was concentrated in myofibril precursors (arrows, A,B) as well as in Z-lines of mature sarcomeres (arrowheads, A,B). GFP-N-RAP-IB also colocalized with -actinin at the cell periphery (asterisks, A,B; arrows, C,D) and at cell to cell contacts (arrowheads, C,D).

View larger version (93K): [in a new window]   Fig. 7. Expression patterns of GFP-N-RAP-IB (A,C) and of -actinin in the same fields (B,D). Cardiomyocytes were transfected with GFP-N-RAP-IB at 2 days and viewed 1 day post transfection. In some cardiomyocytes, GFP-N-RAP-IB is organized in striations resembling the Z-lines of mature sarcomeres (A), even though -actinin is diffusely distributed (B). Note the presence of normal -actinin striations in the neighboring untransfected cell (B). In addition, N-RAP-IB often inhibits myofibril formation (C,D). In this example, N-RAP-IB is localized at cell-cell contacts and cytoskeletal structures resembling myofibril precursors (C), but the transfected cell exhibits far fewer striations than neighboring cells (D).

View larger version (17K): [in a new window]   Fig. 8. Myofibril content versus days in culture. The open symbols show results for cardiomyocytes transfected with GFP-N-RAP (A), GFP-N-RAP-LIM (B), GFP-N-RAP-SR (C) and GFP-N-RAP-IB (D); in each case the results from cardiomyocytes transfected with GFP alone are shown for comparison (filled symbols). Cells were transfected after 1 day (circles) or 2 days (squares) in culture and viewed 1 day or 3 days post-transfection. Significant differences (P<0.05) between the N-RAP constructs and GFP alone were determined by unpaired Student’s t-tests and are indicated by asterisks. Each point represents the mean±s.e.m. from 3 to 40 transfected cells.

View larger version (15K): [in a new window]   Fig. 9. -actinin (filled symbols) and N-RAP (open symbols) staining intensity versus days in culture. Results are for cardiomyocytes transfected with GFP-N-RAP (A), GFP-N-RAP-IB (B), GFP-N-RAP-LIM (C) and GFP-N-RAP-SR (D). In each case the results were normalized to values obtained from untransfected cardiomyocytes on the same slides. Cells were transfected after 1 day (circles) or 2 days (squares) in culture and viewed 1 day or 3 days post-transfection. Each point represents the mean±s.e.m. from 2 to 14 transfected cells.

View larger version (28K): [in a new window]   Fig. 10. The putative first steps of myofibrillogenesis. (1) N-RAP binds to a membrane-associated complex containing ß-integrin, talin and vinculin. (2) Z-line proteins are recruited to the complex. (3) Actin polymerizes along the N-RAP super repeats, with the barbed end of the actin filament integrating with the Z-line components. The orientation of the actin filament is indicated by an arrowhead at the pointed end. (4) Post-translational modification of the N-RAP LIM domain accompanies release of the premyofibril complex from the membrane. (5) The Z-bodies fuse laterally to form Z-lines, and the modified N-RAP is removed.

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