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First published online December 1, 2003
doi: 10.1242/10.1242/jcs.00847

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N-RAP scaffolds I-Z-I assembly during myofibrillogenesis in cultured chick cardiomyocytes

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

View larger version (39K): [in a new window]   Fig. 1. (A) Schematic diagram of N-RAP domain organization (Mohiddin et al., 2003) and regions of N-RAP expressed as GFP fusion proteins. An alternatively spliced single module that is skeletal muscle-specific is shown as a filled box. The position of a 30-residue peptide used as an antigen for the production of polyclonal antibodies is marked as `ab'. Previously described single region constructs contained GFP fused to the N terminus of the LIM, IB and SR regions. The new double region constructs contain each combination of the single regions; these include the LIM + IB construct (LIM-IB), the IB + SR construct (IB-SR) and the LIM + SR construct (LIM-SR). (B) Immunoblot analysis of chick cardiomyocytes transfected with constructs encoding GFP tagged N-RAP constructs. The cells were expressing unfused GFP (lane 1), cardiac IB (lane 2), cardiac IB-SR (lane 3), cardiac LIM-IB (lane 4), LIM-SR (lane 5) and skeletal muscle IB (lane 6). Equivalent volumes of lysate from cultured chick cardiomyocytes were loaded in each lane and probed with anti-GFP or anti-N-RAP antibodies, as indicated. In each case a band was detected migrating near the size predicted from the sequence of the fusion plasmid, which is indicated below each lane. A prominent extraneous band was sometimes also detected at 65 kDa, but did not interfere with the identification of the intact fusion proteins.

View larger version (82K): [in a new window]   Fig. 2. Expression patterns of GFP-tagged LIM-SR (A,D), -actinin in the same fields (B,E), and the merged images with LIM-SR and -actinin in the green and red channels, respectively (C, F). In some cells LIM-SR is diffusely distributed (A), but it is most often concentrated at the cell periphery (arrows) and along fibers continuously stained for -actinin (arrowheads). The transfected cells in A and D exhibit LIM-SR expression levels that do not differ by more than a factor of two when normalized to cell area. LIM-SR expression appears to have disrupted -actinin organization into periodic striations when compared with neighboring untransfected cells.

View larger version (77K): [in a new window]   Fig. 3. Expression patterns of GFP-tagged LIM-IB (A,D), -actinin in the same fields (B,E) and the merged images with LIM-IB and -actinin in the green and red channels, respectively (C,F). LIM-IB was co-localized with -actinin at the cell periphery (asterisks), in myofibril precursors (arrows), and at Z-lines (arrowheads), with no apparent disruption of -actinin organization. Summing the striated areas visualized by -actinin staining (areas within dashed magenta lines in B) and dividing by the total cell area visualized by GFP fluorescence (area within the solid magenta line in A) yields a morphometric measure of myofibril content (Carroll et al., 2001).

View larger version (83K): [in a new window]   Fig. 4. Expression patterns of GFP-tagged IB-SR (A,D), -actinin in the same fields (B,E), and the merged images with IB-SR and -actinin in the green and red channels, respectively (C,F). In many areas IB-SR was co-localized with -actinin at Z-lines (arrowheads), but in some areas IB-SR appeared to be excluded from mature Z-lines (D-F, asterisk). IB-SR fluorescence often extended beyond the Z-lines, marking the entire I-bands (A-C, inset). In addition, IB-SR fluorescence appeared continuous in immature regions where myofibrils appeared to be fusing laterally (arrows).

View larger version (13K): [in a new window]   Fig. 5. Effect of GFP-tagged N-RAP constructs on Z-disk content as indicated by morphometric measurements of -actinin organization into broad striations. Results for both cardiac and skeletal muscle (S) isoforms of the IB construct are shown; LIM-IB and IB-SR, the other two constructs containing the IB region, are both cardiac isoforms. Each bar represents the mean ± s.e.m. of 12-53 transfected cells. *Significant difference from GFP alone (P<0.05); previously published data (Carroll et al., 2001).

View larger version (102K): [in a new window]   Fig. 6. Expression patterns of GFP-tagged IB-SR (A,D), actin in the same fields (B,E), and the merged images with IB-SR and actin in the green and red channels, respectively (C,F). In some cases IB-SR was localized in narrow bands at apparent Z-lines associated with broad-banded actin staining in the I-bands (A-C, arrowheads). In other cases IB-SR colocalized with actin in the I-bands (D-F). In addition, IB-SR colocalized with actin in nonstriated fibers (arrows).

View larger version (105K): [in a new window]   Fig. 7. Expression patterns of GFP-tagged LIM-IB (A), actin in the same fields (B), and the merged images with LIM-IB and actin in the green and red channels, respectively (C). Actin staining in an untransfected cell is shown for comparison (D). LIM-IB was consistently found in appropriately spaced Z-lines (arrowheads). Actin in normal sarcomeres is organized into well-defined broad bands (D), but in sarcomeres incorporating LIM-IB at the Z-line, actin staining is more amorphous or severely reduced (arrowheads and arrow, respectively).

View larger version (42K): [in a new window]   Fig. 8. (A-C) A cardiomyocyte with sarcomeres containing varying amounts of GFP-tagged LIM-IB at the Z-lines. (A) GFP-LIM-IB fluorescence appears to increase from left to right in this field. (B) Actin staining is strong in the sarcomeres at the left side of the field (arrowheads), but decreases as GFP fluorescence increases (asterisk). (C) The merged LIM-IB fluorescence and actin staining are shown in the green and red channels, respectively. (D) Line plots along the long axis of the myofibril were used to quantitate fluorescence intensity of GFP incorporation and actin staining. Plots from the region marked by a white line in C are shown in D. The peak GFP intensity at the Z-line was measured, and the actin peaks in the associated half sarcomeres around the Z-line were measured along with the actin background staining intensity. The specific actin staining was taken as the difference between the peak and background levels. (E) Specific actin staining intensity is plotted versus the peak GFP-LIM-IB fluorescence at the Z-line for sarcomeres in the cell in A-C. There is a significant negative correlation between LIM-IB incorporation at the Z-line and actin content in the associated half sarcomeres (P<0.05).

View larger version (35K): [in a new window]   Fig. 9. Schematic model illustrating N-RAP scaffolding during assembly of the I-Z-I complex. (1) N-RAP binds to a membrane-associated complex containing integral membrane and linker components. (2) -actinin is recruited to the complex. (3) Actin polymerizes along the N-RAP super repeats, with the barbed end of the actin filament integrating with -actinin at the Z-body. The orientation of the actin filament is indicated by an arrowhead at the pointed end. (4) The premyofibril I-Z-I complex is released from the membrane, and N-RAP dissociates as the Z-bodies fuse laterally to form mature Z-lines. Krp1 is hypothesized to play a role in N-RAP dissociation from the nascent myofibril.