Sadikot T et al. (2010), Distinct roles for telethonin N-versus C-termin...

XB-ART-41293

Dev Dyn 2010 Apr 01;2394:1124-35. doi: 10.1002/dvdy.22263.

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Distinct roles for telethonin N-versus C-terminus in sarcomere assembly and maintenance.

Sadikot T , Hammond CR , Ferrari MB .

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The N-terminus of telethonin forms a unique structure linking two titin N-termini at the Z-disc. While a specific role for the C-terminus has not been established, several studies indicate it may have a regulatory function. Using a morpholino approach in Xenopus, we show that telethonin knockdown leads to embryonic paralysis, myocyte defects, and sarcomeric disruption. These myopathic defects can be rescued by expressing full-length telethonin mRNA in morpholino background, indicating that telethonin is required for myofibrillogenesis. However, a construct missing C-terminal residues is incapable of rescuing motility or sarcomere assembly in cultured myocytes. We, therefore, tested two additional constructs: one where four C-terminal phosphorylatable residues were mutated to alanines and another where terminal residues were randomly replaced. Data from these experiments support that the telethonin C-terminus is required for assembly, but in a context-dependent manner, indicating that factors and forces present in vivo can compensate for C-terminal truncation or mutation.

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Species referenced: Xenopus
Genes referenced: acta1 actl6a actn1 actn2 myc tcap ttn
???displayArticle.antibodies??? Actn2 Ab1 Fluro-phalloidin Ab Myc Ab3 Myh1 Ab1 Ttn Ab1
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	Figure 4. Organization of sarcomeric proteins actin, myosin, titin, and alpha -actinin in WT versus TEL MO+ myocytes. A-D: Sarcomeric staining for actin, myosin, titin, and alpha -actinin in WT myocytes fixed at 42 hr in culture is highly periodic as compared to immunostained myocytes from TEL MO+ embryos (E-H). Note the distinct titin PEVK doublets visible in WT (C) but not TEL MO+ (G) myocytes. I: Qualitative scoring of WT and TEL MO+ myocytes stained for titin and alpha -actinin; sarcomeric organization of WT cells is significantly better as compared to TEL MO+ myocytes (**P < 0.001). Scale bars = 10 mu m (A, B ,E, F), 2 mu m (C, D, G, H).
	Fig. 2. Telethonin knockdown results in paralysis and kyphosis. A: Bright-field images of uninjected WT (left), TEL MO� (middle), and SCM (right) stage-42 embryos. WT, TEL MO�, and SCM embryos are from the same clutch. Note the tail curvature in TEL MO� embryo compared to WT and SCM embryos. B: Summary data for motility and curvature scores of WT, TEL MO�, and SCM embryos analyzed at stage 42. Unless stated otherwise, stage-42 embryos are analyzed for motility and curvature throughout this study. Note the marked decrease in motility and increased tail curvature in TEL MO� embryos compared to WTand SCM embryos (**P < 0.001).
	Fig. 3. Embryonic and sarcomeric defects in TEL MO� embryos. Somite and myocyte organization in WT (A�C, J), TEL MO� (D�F, K), and SCM-injected embryos (G�I, L). The columns from left to right are anterior somites (A, D, G), mid somites (B, E, H), and posterior somites (C, F, I). Note decreased sarcomere formation and appearance of wavy and disorganized myofibrils in TEL MO� somites compared to WT and SCM. Scale bar � 10 mm. Primary myocyte cultures obtained from WT embryos (J) and embryos injected with TEL MO� (K) or SCM (L). Cell cultures were fixed at 42 hr after plating and stained for actin using Alexa-Fluor phalloidin 488. Note the lack of sarcomeres in TEL MO� myocyte (K) as compared to highly organized sarcomere structure in WT (J) or SCM (L). Scale bar � 10 mm. Unless otherwise noted, images in this and subsequent figures are maximum projections of Z-series (see Experimental Procedures section).
	Fig. 5. FL telethonin rescues MO� knockdown both in vivo and in culture. A�C: Somites from stage-42 embryos co-injected with telethonin mRNA encoding full-length telethonin protein (MO�FL) show restored sarcomere organization and regular myofibrillar bundling in all regions. Scale bar � 20 mm. D�F: Primary myocytes in culture from WT-, TEL MO�-, and MO�FLinjected embryos, fixed at 42 hr after plating. Scale bar � 10 mm.
	Fig. 6. C�del telethonin does not effect rescue of MO� cellular phenotype. A�C: Actin staining of MO�C�del (C�del: 13 residue truncation at telethonin C-terminal) embryos. Note lack of sarcomeres and myofibrillar organization in each of the somite regions. Scale bar � 20 mm. D�F: MO�C�del myocytes fixed at 17, 25, and 42 hr post-plating, respectively. Myocytes have significantly decreased bundling and sarcomere formation compared to WT (see Fig. 5D) or MO�FL rescue (see Fig. 6F). Scale � 10 mm.
	Fig. 7. Summary data for MO�FL and MO�C�del rescue experiments. A: Curvature and motility scores for WT, TEL MO�, MO�FL, and MO�C�del embryos. Note the significant degree of recovery in morphology and motility after FL rescue compared to TEL MO� and MO�C�del. B: Sarcomere organization in cell cultures fixed at 17, 25, and 42 hr post-plating. Note that sarcomere organization is severely disrupted in both TEL MO� and MO�C�del myocytes (**P < 0.001).
	Fig. 8. C-terminal telethonin mutants in MO� background produce defects in bundling and sarcomere organization. A�C: Images from embryos rescued with C�Ala mRNA in MO� background. Anterior (A) and mid somites (B) have largely intact sarcomeres but note the wavy unbundled myofibrils. The posterior somites have fewer sarcomeres and significant actin aggregation at the somite borders. Scale bar � 20 mm. D�F: In contrast, anterior (D), mid (E), and posterior (F) somite regions from MO�C�mis (FL residues 1�163, 164 LSRTIVSRIT 173) embryos have striations but also display actin aggregation and defects in bundling (D�F). Scale bar � 20 mm. G�I, J�L: Myocytes from MO�C�Ala and MO�C�mis fixed at 17, 25, and 42 hr in culture. G�I: Cultures from MO�C�Ala embryos have few mature sarcomeres and lack myofibrillar organization. Cells from MO�C�mis fixed at 42 hr (L) have fewer sarcomeres and appear degraded compared to 17-hr (J) and 25-hr (K) time points. Note the significant difference in striations between C�Ala and C�mis rescues at the 17- and 25-hr time points. Scale bar � 10 mm.
	Fig. 9. Summary data of MO�C�Ala and MO�C�mis rescue experiments. A: WT, TEL MO�, MO�C�Ala, and MO�C�mis stage-42 embryos scored for motility and curvature. Note significant decrease in motility in TEL MO�, MO�C�Ala, and MO�C�mis embryos compared to WT embryos (**P < 0.001). B,C: Sarcomere organization in MO�C�Ala and MO�C�mis myocytes fixed at 17, 25, and 42 hr in culture. Note that the decrease in sarcomeric organization in MO�C�Ala myocytes at each time point is comparable to TEL MO� cells and significantly different than WT myocytes (B). Sarcomere organization is distinctly decreased in myocytes from MO�C�mis embryos fixed at 42 hr in culture compared to 17- and 25-hr time points.
	Fig. 10. Myc-telethonin fusion proteins express and localize to the Z-disc. A: Western data for myc-telethonin overexpression. Lanes 1�5 represent WT, FL, C�del, C�Ala, and C�mis. Note the expected slight difference in molecular weight for myc-C�del (13 residue truncation, lane 3) compared to other myc-fusions. B: Localization of myc-telethonin protein at the Z-disk. a�e: Myocytes from embryos overexpressing myc-telethonin fusion proteins fixed at 42 hr post-plating. Anti-c-myc staining for each myc-fusion overlaps with the middle of the actin arrays indicating localization at the Z-disc. Note the diffuse myc staining in WT myocytes (a) compared to myocytes from embryos injected with the myc-telethonin constructs (rows b�e).