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First published online 13 May 2008
doi: 10.1242/jcs.028019

Journal of Cell Science 121, 1841-1851 (2008)
Published by The Company of Biologists 2008
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Interactions with titin and myomesin target obscurin and obscurin-like 1 to the M-band – implications for hereditary myopathies

Atsushi Fukuzawa1,*, Stephan Lange1,*,{ddagger}, Mark Holt1, Anna Vihola2, Virginie Carmignac3,4,§, Ana Ferreiro3,4, Bjarne Udd2,5 and Mathias Gautel1,¶

1 King's College London, The Randall Division for Cell and Molecular Biophysics, and Cardiovascular Division, New Hunt's House, London SE1 1UL, UK
2 The Folkhälsan Institute of Genetics and Department of Medical Genetics, University of Helsinki, Biomedicum, Helsinki, Finland
3 INSERM, U582, Institut de Myologie, Paris, France
4 Université Pierre et Marie Curie, Paris, France
5 Department of Neurology, Vasa Central Hospital, Vasa, Finland


Figure 1
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  		Fig. 1. Overview of the domain patterns of obscurin, Obsl1 and the C-terminal M-band-portion of titin. Key interaction sites between titin and obscurin/Obsl1 (above the domain pattern) and the positions of antibody epitopes (below the domain pattern) are marked. Also shown (in grey) are previously mapped interactions of obscurin with Z-disk titin and ankyrin, and M-band titin with MuRF, Nbr1, FHL2/DRAL and calpain-3 (CAPN3). The start of the alternative obscurin isoforms lacking the M-band-targeting motifs is shown. Signalling domains in obscurin include the Src homology domain (SH3), Dbl homology domain (DH) and pleckstrin homology domain (PH); DH and PH domains form a functional unit usually acting as a GDP-GTP exchange factor (GEF) of small G-proteins. Differentially spliced obscurin isoforms can also contain up to two protein kinase domains. The titin region deleted by the homozygous Mex3 mutation in Salih myopathy is marked.

 

Figure 2
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  		Fig. 2. Interaction of the N-terminal region of obscurin/Obsl1 with myomesin. (A) Sub-mapping of the minimal binding domain of obscurin/Obsl1 to My4-My5 and of myomesin to obscurin/Obsl1 Ig3 by successive deletion mutants of My2-My8 using Y2H assays identified Ig3 of obscurin/Obsl1 (top) and the linker between My4 and My5 of myomesin (bottom) as sufficient for binding. (B) These interactions were verified by pulldown assays using GST–My4-My5-linker bound to glutathione beads and GFP-tagged obscurin/Obsl1 Ig3 constructs. (B,C) Detection was performed by blotting equivalent amounts of input (I), unbound (U) and bound (P) fractions with anti-GFP antibody. (C) The interaction of obscurin/Obsl1 Ig3 with the linker between myomesin My4 and My5 is specific for myomesin and is not regulated by phosphorylation. (C, top) Predicted secondary structure of the My4-My5 linker with the protein kinase A (PKA) site. (C, middle) Alignment of the linker sequence of the three myomesin gene family members, MYOM1-MYOM3, with the phosphorylated serine is shown by a black arrow, the neighbouring serine 615 is shown by the grey arrow; red, residues conserved in all three genes; blue, residues conserved only in two MYOM genes. (C, bottom) Pulldown assay with mutation of the phosphorylated serine 618 in MYOM1 to alanine (SA) or aspartate (SD).

 

Figure 3
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  		Fig. 3. Interaction of the N-terminal region of obscurin/Obsl1 with titin. (A) Sub-mapping of the minimal binding domain of obscurin/Obsl1 to titin M10 and of titin to obscurin/Obsl1 Ig1 by Y2H assays identified that Ig1 of obscurin/Obsl1 (top) and M10 of titin (bottom) are sufficient for the binding. (B) These interactions were also verified by pulldown assays using GST-M10 bound to glutathione beads and GFP-tagged obscurin/Obsl1 Ig1 constructs. Detection was performed by blotting equivalent amounts of input (I), unbound (U) and bound (P) fractions with anti-GFP antibody. (C) Ternary-complex formation between obscurin, titin and myomesin in pulldown assays. GST–titin-M10 beads were incubated with GFP–My4-My5 alone (left) and in the presence of obscurin Ob-Ig1-Ig2 (middle) or Ob-Ig1-Ig3 (right). Ob-Ig1-Ig2 binds to titin M10, but myomesin My4-My5 is only bound when the binding site on Ob3 is present, demonstrating the formation of a ternary complex by independent binding motifs.

 

Figure 4
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  		Fig. 4. Localization of endogenous obscurin in NRCs transfected with components of the obscurin-Obsl1-titin complex. Overexpression of the titin-binding site in obscurin or Obsl1 (Ob-Ig1 or Obsl1-Ig1, respectively) leads to loss of endogenous obscurin (Ob58-59) from the M-band. Similarly, transfection of titin M10 reduces M-band-bound obscurin. Titin-normalized obscurin channel analysis of obscurin and/or titin, individual channels and overlay are shown. Range indicator indicates a range of 0 (black) to 3 (white). Scale bars: 10 µm.

 

Figure 5
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  		Fig. 5. Endogenous obscurin localization in NRCs transfected with fragments related to obscurin/Obsl1-myomesin interaction. (A) Overexpression of the myomesin-binding third Ig domains of obscurin (Ob-Ig3) or Obsl1 (Obsl1-Ig3) leads to loss of endogenous obscurin (Ob58-59) from the M-band. Note the strong reduction of obscurin M-band staining with diffuse localization in the transfected cells, visualized by GFP fluorescence of the tagged transfected constructs. Similarly, transfection of the myomesin My4-My5 linker leads to a reduction of M-band-bound obscurin with broad, fuzzy stripes. Overlay and individual channels are shown. (Inset) Twofold-magnified area of the same image showing broad doublet stripes (arrows). (B) Endogenous Obsl1 in NRCs transfected with fragments related to obscurin/Obsl1-myomesin interaction. Overexpression of the myomesin-binding third Ig domains of obscurin (Ob-Ig3) or Obsl1 (Obsl1-Ig3) leads to loss of endogenous Obsl1 from the M-band. Note the reduction of Obsl1 M-band staining with diffuse localization in the transfected cells, visualized by GFP fluorescence of the tagged transfected constructs. Similarly, transfection of the myomesin My4-My5 linker leads to a reduction of M-band-bound Obsl1, with broader, fuzzy stripes (arrows). Scale bars: 10 µm.

 

Figure 6
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  		Fig. 6. Downregulation of myomesin by siRNA interference disrupts M-band structure and leads to loss of obscurin striations in NRCs. NRCs transfected with myomesin siRNA, visualized by GFP fluorescence, show dramatically reduced myomesin levels with loss of M-band striations. Obscurin is diffusely localized in cells treated with siRNA, except for in areas of residual myofibrils, in which it colocalizes with myomesin (arrowheads in insets). Control cells transfected with unspecific siRNA vector show no change in the localization of obscurin or sarcomeric markers ({alpha}-actinin). (Inset) Twofold-magnified area of the same image showing residual primitive stripes. Scale bars: 10 µm.

 

Figure 7
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  		Fig. 7. Mutations in titin M10 that are associated with TMD and LGMD2J weaken the interaction with obscurin and Obsl1. (A) Y2H assay showed no reporter gene activity for either Finnish (Fin) or French (Fr) TMD M10 mutants. WT, wild type; Be, Belgian mutation; C, control. (B) The loss of binding was confirmed by GST pulldown assay. I, input; WT/Fin/Be/Fr, bound fraction on wild type M10 and on that containing the Finnish, Belgian and French mutation, respectively (see text for details). (C) Mutations in human M-band titin lead to disruptions in obscurin localization in diseased skeletal muscles. Obscurin localization (green) was compared with that of myomesin (red) in confocal analysis of cryosections of patient biopsies. In the presence of the Salih myopathy homozygous Mex3 deletion, general M-band morphology is disrupted, as shown by irregular myomesin striations. Even in areas with relatively normal-appearing myomesin stripes, obscurin is disrupted and localizes to split bands and longitudinal streaks, and diffusely. In skeletal muscles from LGMD2J patients, myomesin striations appear relatively normal, in agreement with the less-severe nature of the titin mutation. Obscurin is found in broad and diffuse stripes over the M-band, with loss of the tight colocalization with myomesin seen in normal control (ctrl) muscles. (Insets) Magnified 2x. Scale bars: 10 µm.

 

Figure 8
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  		Fig. 8. Model of the M-band incorporating the spatial constraints on the localization of titin, myomesin and obscurin on the basis of the protein interactions identified here and previously. The M-band lines are marked at the top of the figure. The path of titin is unknown outside the M-band, hence no precise arrangement is shown from the domains immediately N-terminal to the kinase domain. Ig and Fn3 domains are shown as ovals, the titin kinase domain (TK) as a circle and the interdomain-sequence insertions as lines. Titin M-band domains are numbered. Protein interactions are: (1) myomesin My1 and myosin; (2) obscurin/Obsl1 Ig1 with titin M10; (3) myomesin homodimerization via My13; and (4) obscurin/Obsl1 Ig3 with myomesin My4-My5. Note that this model makes no assumptions on whether titin crosses over between myosin filaments or is arranged in a strictly parallel fashion, but mechanical considerations make a crosslinking titin arrangement more plausible.

 

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© The Company of Biologists Ltd 2008