QUICK SEARCH:   [advanced] Author: Keyword(s):
Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents

First published online 15 June 2004
doi: 10.1242/jcs.01158

This Article Summary Full Text Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by McElhinny, A. S. Articles by Gregorio, C. C. Search for Related Content PubMed PubMed Citation Articles by McElhinny, A. S. Articles by Gregorio, C. C. Social Bookmarking                         What's this?

Muscle-specific RING finger-2 (MURF-2) is important for microtubule, intermediate filament and sarcomeric M-line maintenance in striated muscle development

¹ Department of Cell Biology and Anatomy, University of Arizona, Tucson, AZ 85724, USA
² Department of Molecular and Cellular Biology, University of Arizona, Tucson, AZ 85724, USA
³ Institut für Anästhesiologie und Operative Intensivmedizin, Universitätsklinikum 68135 Mannheim, Germany

View larger version (85K): [in a new window]   Fig. 1. Characterization of MURF-2 expression patterns in striated muscle. (A) MURF-2 contains a RING-finger domain (green), the MURF family conserved region (red), a B-box domain (yellow), two coiled-coil domains (blue), and an acidic tail (orange). Previous mRNA studies predicted four potential MURF-2 isoforms generated by differential splicing events within the 3'-region: p60 (full-length, lower schematic), p50 (upper schematic), a p27 isoform (missing the N-terminal region; not shown) and p60B (containing an alternate C-terminus generated by translation of alternative frames in the terminal exon; not shown). Two different antibodies were generated against the C-terminal region of mouse MURF-2. Antibodies against a synthetic peptide (the extreme 17 C-terminal residues) are predicted to recognize the full-length p60 MURF-2, as well as the p50 and p27 forms. Antibodies also were generated against an internal 83-residue, recombinant fragment containing the acidic tail domain (residues 278-365) shared by the p60, the p50, and the potential p60B splice forms. For further description of the potential p27 and p60B isoforms (see Pizon et al., 2002). Positions corresponding to the antisense oligonucleotides (B, C, and D) for chick MURF-2 also are indicated. (B) Anti-C-terminal MURF-2 antibodies recognize a broad band (55-60 kDa) in fetal rat heart lysates (lane 1) and two distinct bands (55 and 60 kDa) in adult rat heart lysates (lane 2), in primary cultures of embryonic chick cardiac myocytes (lane 3), and in primary cultures of chick skeletal myocytes (lane 4: myoblasts; lane 5: myotubes at day 3 of culture). (C) Immunofluorescence staining revealed that MURF-2 is detected diffusely in the cytoplasm, and at the M-line region of a portion of myofibrils (a: MURF-2, red; b: -actinin, green; merged inset in b; double arrows point to striations). In the same culture, MURF-2 also was detected in a discontinuous, spotted pattern along a portion of microtubules in many chick cardiac myocytes (c: MURF-2, red; d: -tubulin, green; merged inset in d; arrow points to tubulin staining). MURF-2 was not detected in fibroblasts (data not shown). Bar, 10 µm.

View larger version (63K): [in a new window]   Fig. 2. (A) Antisense treatment of cardiac mycoytes knocks down MURF-2 mRNA and protein levels. Cardiac myocytes were treated with MURF-2 antisense or control oligonucleotides and RT-PCR was performed. MURF-2 mRNA transcript levels were significantly decreased in cultures treated with MURF-2 antisense oligonucleotides (lane 2) compared to control oligonucleotides (lane 1), although both GAPDH (lanes 5, 6) and MURF-3 (lanes 3, 4) mRNA transcript levels were unchanged. (B) Lysates of cardiac myocytes treated with control (C) or antisense (A) oligonucleotides were harvested 48 hours after treatment, protein assays were performed to ensure equal loading for SDS-PAGE, and transferred to nitrocellulose membranes. The strips were probed with anti-MURF-2 antibodies, revealing that both the 60 kDa and 55 kDa isoforms were significantly decreased in intensity (>50%) at the protein level in the antisense-treated lysates compared to control myocytes.

View larger version (84K): [in a new window]   Fig. 3. Stable microtubule populations are specifically disrupted when MURF-2 levels are knocked down in cardiac myocytes. (A) Day 3 cardiac myocytes were treated with control oligos (a-e) or MURF-2 antisense oligos (f-j) and incubated for 48 hours before fixation. MURF-2 staining was detected in control myocytes (a) but was nearly undetectable in antisense-treated myocytes (f). Staining for -tubulin appeared in a complex, radial web-like array in control myocytes (b), but appeared somewhat sparser in MURF-2 antisense treated cells (g). Strikingly, staining for Glu-tubulin appeared dim in antisense-treated myocytes (h), but bright and filamentous in control cells (c). Staining for Ac-tubulin also appeared dimmer in MURF-2 antisense treated myocytes compared to control cells (d,i). Finally, staining for the dynamic Tyr-MT populations did not appear to differ between control (e) and MURF-2 antisense treated cells (j). (B) Human GFP-MURF-2 expression rescues Glu-MTs in antisense-treated chick myocytes. Chick cardiac myocytes co-transfected with MURF-2 antisense oligos and human GFP-MURF-2 also were stained for Glu-tubulin. Myocytes expressing GFP-hMURF-2 (k), often detected in aggregates in the cytoplasm, contained bright, complex arrays of Glu-MTs (l) compared to antisense-treated myocytes not expressing GFP-h-MURF-2 (Fig. 3A,h). Bar, 10 µm.

View larger version (117K): [in a new window]   Fig. 4. Staining for the intermediate filament proteins, desmin and vimentin, is also perturbed in MURF-2 antisense-treated cardiac myocytes. (A) Chick cardiac myocytes treated with control (a,b,d,e,g,h) or MURF-2 antisense oligonucleotides (c,f) were co-stained for desmin (b,c,g), vimentin (e,f,h) and Glu-tubulin (a,d). Desmin and vimentin staining was detected as filaments (b,e; double arrows) that colocalized with a portion of Glu-tubulin staining (a,d; merged insets in b, e; red is Glu-tubulin staining and green is desmin or vimentin), as well as striated at the Z-line regions (g,h) and at cell junctions (data not shown). In MURF-2 antisense treated myocytes, staining for desmin and vimentin was dim and diffuse (c,f). (B) Human GFP-MURF-2 expression rescues desmin staining in antisense-treated chick myocytes. Chick cardiac myocytes co-transfected with MURF-2 antisense oligos and human GFP-MURF-2 also were stained for desmin. Myocytes expressing GFP-hMURF-2 (i) contained bright filamentous desmin staining (j), compared to antisense-treated myocytes not expressing GFP-h-MURF-2 (Fig. 4A,c). Bar, 10 µm.

View larger version (121K): [in a new window]   Fig. 5. Knock-down of MURF-2 levels specifically disrupts M-line region components of myofibrils, but not Z-lines or thick filament components. Chick cardiac myocytes were treated with antisense oligonucleotides to MURF-2 or control oligonucleotides, fixed 48 hours later, and stained for various sarcomeric components. Control myocytes exhibited bright staining for MURF-2 and staining for -actinin, the peripheral Z-line region of titin (T11 epitope), myosin, and the M-line components myomesin and the M-line region of titin (AB5) in regular, striated patterns (a-c, g-i). Myocytes that had knocked-down levels of MURF-2 (d) exhibited staining for -actinin, titin T11, and myosin in regular, striated patterns (e,f,j). However, in 80% of the antisense-treated myocytes, staining for the M-line components myomesin and the C-terminal region of titin was strikingly disrupted (k,l). Double arrows point to regular striations and single arrowheads point to disrupted myofibrils. (B) Human GFP-MURF-2 expression rescues myomesin staining in antisense-treated chick myocytes. Chick cardiac myocytes co-transfected with MURF-2 antisense oligos and human GFP-MURF-2 were co-stained for myomesin. Myocytes expressing GFP-hMURF-2 (m) contained regular, striated myomesin staining (n), compared to antisense-treated myocytes not expressing GFP-h-MURF-2 (k). Bar, 10 µm.

View larger version (84K): [in a new window]   Fig. 6. MURF-2 colocalizes with a portion of microtubules and with assembling myofibrils in primary cultures of skeletal myocytes. Myofibrillogenesis in skeletal myogenic cells was analyzed by costaining myotubes for -actinin (d,f and data not shown). MURF-2 staining was mainly diffuse in the cytoplasm throughout the culture period (a,c,e). In addition, some MURF-2 staining was detected colocalized with -tubulin staining early in culture (a,b; merged inset in b; red is MURF-2 staining and green is tubulin; double arrows point to colocalization). Later during myofibrillogenesis, MURF-2 staining was detected at the M-line region, although in relatively few myofibrils (c,d; merged image in d; MURF-2 staining in red, -actinin staining is green; double arrows point to myofibrils). In mature myotubes, MURF-2 staining also could be detected along the length of some myofibrils (e,f; double arrowheads). Bar, 10 µm.

View larger version (127K): [in a new window]   Fig. 7. Treatment of skeletal myoblasts with MURF-2 antisense oligonucleotides delays fusion events. Primary cultures of chick myoblasts were treated with control or MURF-2 antisense oligonucleotides and observed by phase microscopy at different stages of differentiation. Within 24 hours of treatment, control myoblasts were elongating, aligning, and beginning to fuse (a, arrows). In contrast, myoblasts treated with MURF-2 antisense oligonucleotides were elongated, but were not fusing (b, arrows). Within 48 hours of treatment, control myotubes were fusing extensively, although MURF-2 antisense-treated cells appeared only to be beginning to fuse (c,d, arrows). Control myotubes were thick and branched within 72 hours of treatment (e, arrow) and were often twitching, although antisense-treated myotubes appeared thinner, much less branched (f, arrow), and were not observed to twitch. Within 96 hours of treatment, control and antisense-treated myotubes appeared indistinguishable by light microscopy (g,h, arrows), and both populations of myotubes were observed to twitch.

View larger version (44K): [in a new window]   Fig. 8. Knock-down of MURF-2 levels in chick skeletal myoblasts perturbs -tubulin and desmin staining and significantly inhibits fusion. (A) Skeletal myoblasts were treated with antisense or control oligonucleotides and fixed at 24 or 48 hours. The cells were stained with antibodies to -tubulin (b,f,j,n) and with DAPI (c,g,k,o) to quantify the number of nuclei/myotube as a measure of fusion. Within 24 hours of treatment, thick control myotubes contained diffuse MURF-2 staining, bright filamentous microtubules parallel to the longitudinal axis, and bright, diffuse desmin staining (a,b,d). Control myotubes also had a mean of 16 nuclei (c,B) (note: the myotube in c also had nuclei outside the field of view). In contrast, antisense-treated myotubes were strikingly thin, had low levels of MURF-2 staining (e), low -tubulin and desmin staining (f,h) and significantly fewer nuclei (g,B). Within 48 hours of treatment, MURF-2 levels were comparable in antisense-treated myotubes (m) and control myotubes (i), and -tubulin staining in both populations of myotubes appeared in bright filaments (j,n). Desmin staining appeared bright and diffuse in both populations of myotubes (l,p). However, the MURF-2 antisense treated myotubes remained thinner and qualitatively contained fewer nuclei/myotube compared to controls (k,o). Note that the large number of overlapping nuclei/myotube could not be quantified accurately at 48 hours in control myotubes. Bar, 10 µm. (C) Human GFP-MURF-2 expression rescues fusion events in antisense-treated chick skeletal myotubes. Chick skeletal myoblasts were co-transfected with MURF-2 antisense oligos and human GFP-MURF-2, fixed 24 hours after treatment, and stained with DAPI to quantify the number of nuclei per myotube. Myotubes expressing GFP-hMURF-2 (n=15 from two different cultures) contained similar numbers of nuclei/myotube compared to myotubes treated with control oligonucleotides; both populations contained significantly greater numbers of nuclei/myotube compared with antisense-treated myotubes not expressing human MURF-2-GFP.

View larger version (72K): [in a new window]   Fig. 9. Myofibrillogenesis is delayed in chick skeletal myotubes after treatment with MURF-2 antisense oligonucleotides. Control or MURF-2 antisense-treated myotubes were fixed after 48 or 72 hours of treatment and stained for various sarcomeric components to analyze myofibril assembly. Within 48 hours of treatment, the majority of control myotubes were beginning to assemble -actinin, the M-line region of titin (A168-170), and myosin into regular, striated patterns (a,b,c, double arrows). In contrast, myotubes treated with MURF-2 antisense oligonucleotides were thin and exhibited no myofibril components in striated patterns (d,e,f). Within 72 hours of treatment, both control and MURF-2 antisense treated myotubes contained -actinin, the M-line region of titin, and myosin in regular, striated patterns (g-l, double arrows), although the antisense-treated myotubes still appeared thinner than controls (j-l). Bar,10 µm.

CiteULike

Complore

Connotea

Del.icio.us

Digg

Reddit

Technorati

Twitter