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doi: 10.1242/10.1242/jcs.00131

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Transient association of titin and myosin with microtubules in nascent myofibrils directed by the MURF2 RING-finger protein

¹ European Molecular Biology Laboratory, Cell Biology Division, Heidelberg, Germany
² Institut Jacques Monod, Paris, France
³ Max-Planck-Institut für molekulare Physiologie, Department of Physical Biochemistry, 44202 Dortmund, Germany
⁴ Potsdam University, Department of Cell Biology, Potsdam, Germany
⁵ King's College London, Muscle Cell Biology, The Randall Centre, New Hunt's House, London SE1 1UL, UK

View larger version (31K): [in a new window]   Fig. 1. Domain patterns and tissue-specific differential splicing of MURF2. (A) Tissue-specific differential splicing of MURF2. RT-PCR demonstrates tissue-specific co-expression of multiple MURF2 transcripts. The 0.75 kb band of the 27 kDa isoform is detected only in cardiac muscle (arrowhead) but not in cDNA from adult skeletal muscle. Faint bands above 2 kb may represent further, yet unidentified isoforms. H, adult cardiac muscle; Sk, adult skeletal muscle; M, marker ladder, sizes given in kb. (B) MURF2 is expressed in four isoforms derived from differential splicing events. MURF2 isoforms of 27, 50 and 60 kDa are generated by sequential internal expansion of a core molecule containing the Ring, B-Box and C-terminal domain present in MURF2p27. A third isoform, MURF2p60B, is generated by the splicing of an additional exon before that of the C-terminal domain; a frameshift in the reading frame of this exon leads to the generation of an alternative C-terminus. (C) Generation of alternative C-termini in MURF2. Alternative splicing of the MURF2 gene that maintains the last exon (highlighted in red in B and C) while splicing in an additional 60 bp exon is observed in skeletal muscle. This leads to a frameshift in the last exon, omitting its reading frame and creating an alternative C-terminus (purple in B and C) which is fused directly to the Ser/Ala rich domain (blue in B and C). Note the alternative reading frame use in the last exon. The three C-terminal exons localise to a 20409 bp genomic region on chromosome 8q12, separated by introns of 1285 bp to 18762 bp. The large stretch of sequence between the last two exons may suggest the presence of further, as yet unidentified differentially spliced exons. In this figure, only the 3' region of the p60B isoform was mapped. The start of a new exon is indicated (^). Reading frames are numbered a, b and c, and the MURF2p60B reading frame is shadowed in grey. Nucleotide numbering is according to our data library entry AJ431704.

View larger version (68K): [in a new window]   Fig. 2. Developmental expression of MURF2 and association with microtubules, myosin and titin. (A) Developmental time course of differentiating myoblasts probed with anti-MURF2 HPC. A single 60 kDa band is expressed upon myogenic differentiation. (B) Microtubule sedimentation assays were performed using protein extracts from undifferentiated (0 hours) and from differentiated C57 cells (48 hours, 120 hours) using nocodazole (n) or taxol (t). MURF2 was detected in the microtubule pellets of differentiated myotubes. (C-D) Localisation of endogenous MURF2 to microtubules in differentiated C57 cells. Double immunofluorescence was performed using anti-MURF2 and anti-tubulin antibodies. After 24 hours of differentiation (C), MURF2 directly colocalised with microtubules as seen in the overlay (yellow). In cells differentiated for 48 hours (D), some MURF2 protein (arrowhead) did not strictly associate with microtubules (green in overlay). (E) MURF2 interacts with sarcomeric myosin and P-zone titin. MURF2 was expressed in transfected HeLa cells and detected by the anti-MURF2-specific antibody HPC (lane 1). The myosin sedimentation assay demonstrates the presence of endogenous MURF2 in myosin preparations (lane 2); addition of transfected MURF2 increases the myosin-associated MURF2 fraction (lane 3). MURF2 associates with titin A164-169 (lane 5) but not with GST alone (lane 6). (Lane 4) Untransfected HeLa cell lysate control on titin A164-169 beads.

View larger version (127K): [in a new window]   Fig. 6. MURF2 associates with A-band titin in nascent myofibrils. C57 cells were stained with anti-titin A-band (T31, red) and anti-MURF2 (green) antibodies. After 24 hours of differentiation (A), titin aligned with MURF2 positive microtubules in a dotty pattern. After 48 hours of differentiation (B), titin A-band epitopes were organised in patches along MURF2 positive structures. At 120 hours of differentiation (C), MURF2 colocalises with titin A-band epitopes as longitudinally oriented dots (arrowhead). Some dots were observed at both sides of nascent striated myofibrils (arrow).

View larger version (66K): [in a new window]   Fig. 3. Selective association of MURF2 with post-translationally modified microtubules. (A) Transfection of full-length T7-tagged MURF2 in the myogenic cell line BHK-21 shows that microtubule-associated MURF2 (green, arrows) is excluded specifically from tyrosinated microtubules, which are stained by the YL1/2 antibody (red, arrowheads). (B) Transfected MURF2 (green) is localised on polyglutaminated microtubules in HeLa labelled by the monoclonal antibody ID5 (red; overlay yellow).

View larger version (101K): [in a new window]   Fig. 4. MURF2 and sacrcomeric actin are localized in different cell regions. Cells stained with anti-MURF2 (green) and with anti-sarcomeric actin (red) antibodies did not show significant colocalisation, neither after 48 hours (A) nor after 120 hours of differentiation (B). When cross-striated actin begins to be organised (B), MURF2 decorated microtubules align laterally to these structures (arrowheads in B).

View larger version (72K): [in a new window]   Fig. 5. Localisation of endogenous MURF2 relative to titin Z-disk and I-band epitopes. At different stages of differentiation, double immunofluorescence experiments were performed with anti-MURF2 (green), and either anti-titin I-band (A,B) or anti-titin Z-disk (C-E) antibodies (red). At 24 hours of differentiation (A) MURF2 did not decorate the stress fibre like structures revealed with titin I antibody. After 120 hours of differentiation (B), MURF2 and I-band titin epitopes showed occasional transversal colocalisation (arrowhead) near mature cross-striated regions of the myofibril (arrow). In young myotubes (24 hours of differentiation) Z-disk titin can align along MURF2 positive microtubules (C,D). When sarcomeres started to be organised (E), structures with MURF2 punctate staining (arrowhead) co-aligned with maturing Z-disk titin (arrow).

View larger version (107K): [in a new window]   Fig. 7. MURF2 and sarcomeric myosin show colocalisation in premyofibrils. C57 cells were stained with anti-MURF2 (green) and with anti sarcomeric myosin heavy chain antibodies (red). Double immunofluorescence experiments revealed that MURF2 stained filamentous structures containing sarcomeric myosin at early stages of differentiation (A, arrowheads). Later, MURF2 and sarcomeric myosin colocalise in non-striated regions parallel to striated myofibrils (B, arrowheads). Upon sarcomere formation, MURF2 and sarcomeric myosin antibodies labelled identical rod-like structures (C, arrowheads). Mature skeletal sarcomeres (arrow) did not show colocalisation of MURF2 and sarcomeric myosin.

View larger version (74K): [in a new window]   Fig. 8. MURF2 is translocated from the sarcomeric M-band to the nucleus in stressed cardiomyocytes. (A) In neonatal cardiomyocytes cultured for 72 hours in high-serum conditions, MURF2 (green) is found at the sarcomeric M-band (arrows) and excluded from the nucleus. These cells show a large myofibrillar area with mature, parallel alignment of sarcomeres visualised by the Z-disk titin antibody T12 (red). MURF2 is arranged centrally between Z-disks, at the M-band. (B) Cells cultured in minimal medium for 36 hours show few, poorly arranged and non-parallel myofibrils, stained by titin T12 (red). MURF2 (green) is diffusely distributed in the cytosol, and in a speckled pattern in the nucleus (arrow). MURF2 at the nuclear envelope is marked by an arrowhead. Bar, 20 µm.

View larger version (18K): [in a new window]   Fig. 9. MURF2 in relation to titin, myosin and the filamentous systems of actin and microtubules during sarcomere formation. (A) At initial stages of myofibril assembly, MURF2 (red ovals) is microtubule associated. Myosin (dark green rods) colocalises early with MURF-decorated microtubules (light-green). (B) Titin (red chains) localises in dot-like aggregates on actin SFLS (blue) with a spacing of around 1 µm. Black links, -actinin crosslinks. These structures co-align with MURF-decorated microtubules. (C) MURF2 and myosin colocalise strictly in nascent striated myofibrils, when the cross-striated pattern of myosin begins to form and titin Z-Z staining increases to the mature approximately 2 µm pattern. Transient colocalisation with A-band titin is observed at this stage. MURF-MURF heteromultimeres could link different sarcomeric components at this stage and result in the exact alignment of titin and myosin. (D) In mature myofibrils, myosin and actin are arranged in highly ordered cross-striated patterns with ordered polarity (actin pointed ends marked by arrows), and the titin molecule is extended, with the N-terminal portion remaining in the Z-disk and the C-terminus integrated into the M-band. Depending on differentiation state and/or muscle type, MURF2 can be present in the M-band or the nucleus. Bar, 1 µm.