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The C-terminal IgI domains of myosin-binding proteins C and H (MyBP-C and MyBP-H) are both necessary and sufficient for the intracellular crosslinking of sarcomeric myosin in transfected non-muscle cells

Robert E. Welikson^* and Donald A. Fischman

Department of Cell Biology, Weill Medical College of Cornell University, 1300 York Avenue, York Avenue, New York, NY 10021, USA
^* Present address: Department of Biochemistry, University of Washington, Seattle, WA 98 195, USA

View larger version (19K): [in a new window]   Fig. 1. Cartoon representation of the MyBP expression constructs used in this study. GFP- and c-myc-tagged MyBP cDNAs were cloned into pEGFP-C1 and pJDp expression vectors and transiently expressed in COS cells. (A) GFP-tagged MyBPs. (B) Myc-tagged mutants of MyBP-C (white). (C) Myc-tagged mutants of MyBP- H (black). The positions of both the GFP epitope and myc at the N-termini are shown. Roman numerals denote the position of each IgI or FNIII-like domain. Each domain contains 100 amino acids.

View larger version (25K): [in a new window]   Fig. 2. Western blot of GFP-tagged MyBPs expressed in COS cells. Lysates from COS cells transfected with GFP-tagged full-length MyBP-C (GFP/C), the last four domains of MyBP-C (GFP/C7-10) and full-length MyBP-H (GFP/H) were analyzed on a 10% polyacrylamide SDS-PAGE gel, transferred to nitrocellulose for western blotting and probed with an anti-GFP-specific mAb. Mock-transfected COS cell lysate served as a negative control.

View larger version (66K): [in a new window]   Fig. 3. GFP-tagged MyBPs localize to the A-band of differentiating myotubes. Myoblasts from 11-day-old chick embryos were isolated, cultured and transfected the following day using lipofectamine with GFP/C, GFP/C7-10 or GFP/H. Five days after transfection, live cells expressing GFP-tagged MyBPs appeared striated and contractile when viewed with fluorescence optics. Cells were fixed and probed with F59, a mAb against sarcomeric myosin. GFP/C (B), GFP/C7-10 (E) and GFP/H (F) all incorporate into the A-bands of cultured embryonic muscle cells. F59 immunostaining revealed a similar myosin distribution to that seen with GFP/C (C). The overlap of GFP/C and myosin is shown in the D.

View larger version (61K): [in a new window]   Fig. 4. Cellular localization of GFP-tagged MyBPs and MyHC. COS cells were transiently transfected with GFP/C (A,D,G,J), GFP/C7-10 (B,E,H,K) and GFP/H (C,F,I,L). Cells were transfected either singly (A-C) or with MyHC (D-L). GFP/C (A,D) GFP/C7-10 (B,E) and GFP/H (C,F) protein expression was detected by direct fluorescence using fluorescein excitation and emission filters. Expression of MyHC was detected by indirect immunofluorescence using F59 (G-I). The overlap of GFP-tagged MyBPs and MyHC is shown in the bottom panels (J-L). Cells transfected singly with GFP-tagged MyBPs exhibit a diffuse distribution (A-C). Co-transfection of GFP/C (D, G and J) GFP/C7-10 (E, H and K) or GFP/H (F, I and L) and MyHC induce copolymer cable formation in the COS cells.

View larger version (8K): [in a new window]   Fig. 5. Western blot analysis of myc-tagged MyBP-C and MyBP-H recombinant proteins expressed in COS cells. (A) Cultured cells were transfected with pJDp-expression plasmids containing myc-tagged full-length MyBP-C, C7-10, C10 or C1-9, as described in Fig. 1. Two days after transfection whole cell lysates were prepared. The lysates were then subjected to SDS-PAGE, transferred electrophoretically to nitrocellulose and reacted with anti-c-myc mAb. Mock-transfected COS cell lysate served as a negative control. (B) COS cells were similarly transfected with myc-tagged MyBP-H constructs (full-length MyBP-H, HU, HU1 or H4) and analyzed on western blots with anti-c-myc antibody. (C) Myc-tagged MyBP-H recombinant proteins were further tested with a polyclonal antibody against chicken MyBP-H. A high salt crude extract of chicken skeletal muscle served as a positive control. The numbers to the left of each gel indicate relative molecular weights of a pre-stained protein standard (Life Technologies, Rockville, MD).

View larger version (79K): [in a new window]   Fig. 6. Effect of myc-tagged MyBP-C recombinant proteins on MyHC organization in transfected COS cells. COS cells were transiently transfected with full-length MyBP-C (A,E,I,M), C7-10 (B,F,J,N), C10 (C,G,K,O) and C1-9 (D,H,L,P) and analyzed by indirect immunofluorescence. Cells doubly transfected with MyHC are shown in E-P. Two days after transfection, the cells were fixed and immunoreacted with biotinylated anti-c-myc antibody, 9E10, and anti-myosin mAb F59. A-H show localization of the myc-tagged MyBP-C proteins. I-L reveal the expression and distribution of MyHC. The overlap of MyBP-Cs and MyHC is shown in M-P. All four MyBP-C recombinant proteins co-distributed with MyHC. Full-length MyBP-C, C7-10 and C10 induced cable formation when expressed with MyHC. By contrast, C1-9 did not form cables with MyHC.

View larger version (20K): [in a new window]   Fig. 8. Bar graph demonstrating the co-distribution and cable-forming capacity of various MyBP mutants with MyHC. COS cells were transfected with MyHC and MyBP-C (A) or MyBP-H (B) expression constructs. Two days after transfection the cells were fixed and immunostained with anti-MyHC and anti-c-myc antibody. Doubly transfected cells were then identified by indirect immunofluorescence and scored for the colocalization of MyBP and MyHC and cable formation. The number of cells demonstrating colocalization and/or cable formation is expressed as a percentage of the total number of doubly transfected cells. Data represent average values; bars represent one s.d.; ** indicates an average difference from full-length MyBP with a P<0.005.

View larger version (75K): [in a new window]   Fig. 7. Interaction of MyBP-H truncation mutants with MyHC when co-expressed in COS cells. Expression of MyBP-H constructs without MyHC (A-D) or with MyHC (E-P). Cells were probed with biotinylated 9E10 and F59 to reveal myc-tagged MyBP-H recombinant proteins (A-H) and MyHC (I-L), respectively. The overlap of MyBP-H and MyHC is shown in M-P. As described in Fig. 1, the following MyBP-H constructs were used: full-length MyBP-H (A,E,I,M), HU (B,F,J,N), HU1 (C,G,K,O) and H4 (D,H,L,P). Each of the MyBP-H fragments co-distributed with MyHC in the cell (M-P). H4, which lacks the major myosin rod-binding domain, did not induce MyHC to form cables.

View larger version (37K): [in a new window]   Fig. 9. Schematic representation of the myosin interaction sites within the C-terminal region of the MyBPs. The last four domains of MyBP-C and MyBP-H are represented in cartoon style with stippled squares (FnIII modules) and circles (IgI modules). The previously identified myosin interaction domains are listed above the cartoon of the MyBP C-terminus. Interaction sites identified in this study, myosin crosslinking and colocalization domains and the locations they map to are illustrated in the diagram.

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