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First published online 30 November 2004
doi: 10.1242/jcs.01574

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Myocyte differentiation generates nuclear invaginations traversed by myofibrils associating with sarcomeric protein mRNAs

¹ Department of Biology, Faculty of Science, and Graduate School of Science and Technology, Chiba University, Yayoicho, Inageku, Chiba, Chiba 263-8522, Japan
² CREST, Japan Science and Technology Agency (JST), Kawaguchi, Saitama 332-0012, Japan
³ Department of Anatomy, Chiba University School of Medicine, Chuoku, Chiba, Chiba 260-8670, Japan
⁴ Division of Biochemistry, Aichi Cancer Center Research Institute, Nagoya, Aichi 464-8681, Japan

View larger version (106K): [in a new window]   Fig. 1. Nuclear shape of C2C12 myoblasts and myotubes. C2C12 myoblasts were cultured in the growth medium for 1 day, and myotubes were formed by culturing for 4 days in the differentiation medium. (A) Phase-contrast images (a,c,e) and corresponding bisbenzimide H33258-stained images (b,d,f) of myoblasts (a,b) and myotubes (c-f). Arrowheads indicate the smooth nuclei of residual myoblasts in the myotube culture (c,d). (B) Electron micrographs of myotube nuclei. Bar, 20 µm (A); 5 µm (B).

View larger version (54K): [in a new window]   Fig. 2. Nuclear shape of 10T1/2 myotubes and biochemically differentiated C2C12 cells. (A) Phase-contrast image (a) and corresponding H33258-stained image (b) of a 10T1/2 myotube. 10T1/2 fibroblasts were transfected with MyoD and maintained for 4 days in the differentiation medium. Arrows indicate myotube nuclei with invaginations. Arrowheads indicate smooth nuclei in mononucleated cells. (B) Phase-contrast images (a,c) and corresponding immunofluorescent images detecting muscle-specific MyHC (b,d) of sodium butyrate-treated (a,b) or DMSO-treated (c,d) C2C12 cells. C2C12 myoblasts were cultured for 4 days in the differentiation medium containing either 5 mM sodium butyrate or 2% DMSO. Biochemically differentiated cells were detected by the staining with the mAb MF20 to muscle-specific MyHC. Arrows indicate biochemically differentiated cells with invaginated nuclei. (C) Ratio of smooth-surfaced and grooved or invaginated nuclei. C2C12 Mb, proliferating C2C12 myoblasts. C2C12 Mt, C2C12 myotubes cultured for 4 days in the differentiation medium. C2C12(DMSO), MyHC-positive biochemically differentiated C2C12 cells cultured for 4 days in the differentiation medium containing DMSO. C2Vm8 Mt, C2Vm8 myotubes cultured for 4 days in the differentiation medium. More than 200 nuclei were counted in each experiment. The values are the mean±s.d. of three experiments. Bar, 20 µm.

View larger version (88K): [in a new window]   Fig. 3. Downregulation of vimentin in C2C12 myotubes and biochemically differentiated cells. (A) Disappearance of vimentin filaments during terminal differentiation. Phase-contrast images (a,c) and corresponding immunofluorescent images detecting vimentin (b,d) in C2C12 myoblasts (a,b) and myotubes (c,d). (B) A decrease in vimentin (a) and an increase in desmin (b) during terminal differentiation detected by immunoblotting. (C) Disappearance of vimentin filaments in biochemically differentiated mononucleated cells. Phase-contrast images (a,c) and corresponding immunofluorescent images detecting vimentin (b,d) of sodium butyrate-treated (a,b) or DMSO-treated (c,d) C2C12 cells. Cells were treated as described for Fig. 2B. Arrows indicate a biochemically differentiated cell, which has a invaginated nucleus and a markedly reduced number of vimentin filaments (c,d). Bar, 20 µm.

View larger version (103K): [in a new window]   Fig. 4. Nuclear deformation in myotubes with forced expression of vimentin. (A) Northern blots detecting vimentin expression in C2C12 cells and the vimentintransfected clones C2Vm1-10 (top). Ethidium bromide staining pattern of cytoplasmic RNAs in agarose gel electrophoresis showing the 28S and 18S rRNAs (bottom). (B) Nuclear shapes of C2Vm8 myoblasts (a-c) and myotubes (d-f). Phase-contrast images (a,d) and corresponding immunofluorescent staining of vimentin (b,e) and H33258 staining (c,f). Although C2Vm8 myotubes contain adequate vimentin filament networks, their nuclei have grooves and invaginations. Bar, 20 µm.

View larger version (89K): [in a new window]   Fig. 5. Penetration of premyofibrils and myofibrils into the nuclear grooves and invaginations of myotubes. (A) Penetration of both muscle- and non-muscle-type actin-containing premyofibrils. (B) Penetration of muscle-type actin containing premyofibrils. Non-muscle-type actin is not present in the premyofibrils. (C) Penetration of actin- and myosin-containing premyofibrils. (D) Penetration of actin- and myosin-containing myofibrils with crossstriations. PC, phase-contrast images; H33258, nuclear staining with H33258; actin, total actin staining with rhodamine-phalloidin; NM actin, non-muscle actin staining with absorbed antigizzard actin pAb; MyHC, sarcomeric MyHC staining with the mAb A4.1025; H33258 + actin, H33258 + NM actin, H33258 + MyHC, doubly merged images; Merge, triply merged images. Bar, 10 µm.

View larger version (104K): [in a new window]   Fig. 6. Termination of myofibrillar tips within the nuclei and existence of microtubules in the nuclear invaginations. (A) Termination of myofibrillar tips within the nuclei. A differential interference contrast (DIC) image (a), corresponding rhodamine-phalloidin staining (b), and an immunofluorescent image detecting sarcomeric MyHC (c). Arrows indicate the tip of a premyofibril within the nucleus. (B) Existence of microtubules in the nuclear invaginations. A DIC image (a), corresponding rhodamine-phalloidin staining (b), and an immunofluorescent image detecting ß-tubulin (c). Arrows indicate the nuclear invaginations containing premyofibrils and microtubules. Bar, 20 µm.

View larger version (57K): [in a new window]   Fig. 7. Traversal of premyofibrils and myofibrils through the nuclear grooves and invaginations. Confocal optical sections (XY planes) and three-dimensional reconstitutions of the orthogonal planes derived from these sections (XZ and YZ planes). (A-C) Rhodamine-phalloidin (red) and anti-sarcomeric MyHC (green) staining of myofibrils passing through the nuclear grooves (A) and the nuclear invaginations (B,C). Arrows point to the traversing myofibrils. (D) Rhodamine-phalloidin (red) and anti-nucleoporin p62 (green) staining of myofibrillar structures and nuclear pore complexes, respectively. Boxed area in YZ plane is magnified three times. (E) Rhodamine-phalloidin (red) and DiOC6(3) (green) staining of myofibrillar structures and the ER together with the nuclear envelope, respectively. Bar, 10 µm.

View larger version (37K): [in a new window]   Fig. 8. Formation of the nuclear grooves and invaginations by myofibrillar structures horizontally cutting into the nuclei. (A-C) The nuclei of living C2C12 myotubes expressing EGFP-tagged -actin were stained with H33342. The fluorescence of EGFP and H33342 was recorded at intervals of 20-30 minutes at 37°C. Shown are selected images of three cells by time-lapse microscopy. The numbers indicate minutes after starting the observation. Arrows point to the positions of nuclei, where myofibrillar structures enter horizontally. Bar, 10 µm.

View larger version (47K): [in a new window]   Fig. 9. Reduction of straight and longitudinal nuclear grooves and invaginations by preventing myofibrillogenesis. C2C12 myoblasts were cultured for 4 days in the differentiation medium (control) or the medium containing 10 mM BDM or 12 mM KCl to prevent myofibril assembly. (A) Staining of the myotubes with rhodamine-phalloidin (actin), anti-MyHC mAb, and H33258 along with their merged images. (B) Ratio of all the nuclei in myotubes with grooves or invaginations and that of the nuclei with straight and longitudinal grooves or invaginations. More than 200 nuclei were counted in each experiment. The values are mean±s.d. of three experiments. Bar, 10 µm.

View larger version (74K): [in a new window]   Fig. 10. Association of sarcomeric protein mRNAs with myofibrillar structures traversing through the nuclear invaginations. Confocal optical sections of FISH detecting sarcomeric protein mRNAs (red) and Alexa 488-phalloidin (green) staining (XY planes) and three-dimensional reconstitutions of the orthogonal planes derived from these sections (XZ and YZ planes). (A) FISH of embryonic fast skeletal muscle MyHC mRNA. Different focal planes (XY1 and 2; XZ1-4) of a myotube are shown. Association of the mRNA with the myofibrillar structures is more evident on the XZ planes. Penetration of myofibrils into the nuclear invaginations is evident on the YZ plane. (B) FISH of skeletal muscle -actin mRNA. Different focal planes (XY1 and 2; XZ1 and 2) of a myotube are shown. Arrowheads on the XZ planes indicate association of the mRNA with the myofibrillar structures. Bar, 10 µm.

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