Nworu CU, Kraft R, Schnurr DC, Gregorio CC, Krieg PA.

HL083146 NHLBI NIH HHS , HL093694 NHLBI NIH HHS , HL108625 NHLBI NIH HHS , R01 HL108625 NHLBI NIH HHS , R01 HL093694 NHLBI NIH HHS , R01 HL083146 NHLBI NIH HHS

	Fig. 1. Structure and developmental expression of the leiomodin (Lmod) and tropomodulin (Tmod) gene families. (A) leiomodin (Lmod1–3) and tropomodulin (Tmod1–4) proteins are structurally related, yet possess unique domains. Lmods and Tmods share actin-binding (A1, A2), tropomyosin-binding (TM1) and leucine-rich-repeat (LRR) domains. A second tropomyosin-binding domain (TM2) is unique to the Tmods. Lmod proteins have a Cterminal extension that contains a third actin binding/WH2 domain (WH2/A3). (B–I) Lmod (C–E) and Tmod (F–I) family gene expression was analyzed during Xenopus embryonic development using in situ hybridization. All embryos are positioned with heads to the left. Expression patterns are shown at the early tailbud stage (st30), during the early stages of myofibrillogenesis. Earlier expression during the neurula stage (st15) is presented in supplementary material Fig. S1. Expression of the definitive striated muscle marker cardiac actin, actna1 (B), was used to indicate cardiac and skeletal muscle tissue in the embryo. Arrows indicate developing skeletal muscle and heart tissues. Only lmod3 (E) and tmod4 (I) are expressed at high levels in developing skeletal muscle.
	Fig. 2. Tmod4 and Lmod3 localize to the M-line during Xenopus skeletal myofibrillogenesis. The somite regions of Xenopus embryos were sectioned and protein distribution was examined using Texas-Red-conjugated phalloidin and antibodies against a-actinin, Tmod4 or Lmod3. (A–L) Localization of Tmod4 protein during skeletal muscle myofibrillogenesis. (A–D) At st20, continuous actin filament staining was visible (A), with no periodic localization of a-actinin (B) or Tmod4 staining (C). (E–H) At st24, diffuse striations were visible in a small number of actin filaments (E). In these striated regions, narrow bands of a-actinin (F, arrowheads) and Tmod4 staining (G, arrows) were visible. (I–L) At st34, broad sharply striated (i.e. highly organized) actin filaments (I) were observed throughout the skeletal muscle tissue. Sharp regions of a-actinin (J, arrowheads) and Tmod4 staining (K) were visible at the Z-disc and M-line regions, respectively. (M–X) Localization of Lmod3 protein during skeletal muscle myofibrillogenesis. (M–P) At st20, continuous actin filament staining was visible, but no localization of a-actinin (N) or Lmod3 (O) was observed. (Q–T) At st24, some regions of developing muscle showed narrow diffusely striated actin filaments (Q). In these regions, narrow bands of a-actinin (R, arrowheads) and Lmod3 staining (S, arrows) were visible. (U–X) At st34, broad sharply striated actin filaments were visible throughout the developing somites. Staining for a-actinin (V, arrowheads) and Lmod3 (W, arrows) was detected at the Z-line and M-line regions, respectively. We conclude that both Tmod4 and Lmod3 proteins are present at the M-line region of the sarcomere from the earliest stages of myofibril assembly. Scale bar: 5 mm.
	Fig. 3. Tmod4 is essential for skeletal muscle myofibril assembly. (A) Embryos treated with control MO (upper panel) and tmod4 MO (lower panel) develop normally. (B) Protein blots demonstrate that the anti-Tmod antibody specifically recognizes Xenopus Tmod4 protein (lane labeled GFP– Tmod4) and that the tmod4 MO effectively blocked translation of Tmod4 protein. (C–R) Loss of Tmod4 protein resulted in disruption of sarcomere assembly. (G–J) Sections through developing muscle tissue of embryos depleted of Tmod4 protein show severely disrupted sarcomeres compared with control embryos at the same developmental stage (C–F). (O–R) Localization of other sarcomeric components, including the N-terminal region of titin (P) and myosin heavy chain (Q) was also severely disorganized compared with that of controls (K–N). (S–Aa) The specificity of the Tmod4 knockdown was demonstrated by rescue experiments. In these studies, tmod4 MO was co-injected into the Xenopus embryo with mRNA encoding GFP–Tmod4. Organization of both thin filament (S–V) and other sarcomeric proteins (X,Y) was comparable to that of unmanipulated controls (C–F). All fluorescent images (C–Z) are presented at the same scale. Scale bar: 5 mm. Quantification of experimental results (Aa) showed that depletion of Tmod4 protein in st34 embryos resulted in a dramatic reduction in the percentage of correctly structured sarcomeres (,5.4% of fibers showing M-line gaps following phalloidin staining compared to 62.5% in controls). Addition of mRNA encoding GFP–Tmod4, together with the MO, resulted in rescue of sarcomere structure. Data show the mean6s.e.m.; ***P,0.001 (x2 analysis).
	Fig. 4. Lmod3 is essential for skeletal myofibril assembly. (A) Embryos treated with control MO (upper panel) and lmod3 MO (lower panel) develop normally. (B) Protein blots demonstrate that the anti-Lmod antibody specifically recognizes Xenopus Lmod3 protein (lane labeled Myc– Lmod3) and that the lmod3 MO effectively blocked translation of Lmod3 protein. (C–R) Loss of Lmod3 protein resulted in disruption of sarcomere assembly. (G–J) Sections through developing muscle tissue in embryos depleted of Lmod3 protein show severely disrupted sarcomere structure compared with that of control embryos of the same developmental stage (C–F). (O–R) The localization of other sarcomeric components, including titin (P) and myosin (Q) was also severely disorganized compared with that of controls (K–N). (S–Aa) The specificity of the Lmod3 knockdown was demonstrated by rescue experiments. lmod3 MO was coinjected into the Xenopus embryo with mRNA encoding Myc– Lmod3. Organization of thin filament (S–V) and other sarcomeric proteins (X,Y) was comparable to that of unmanipulated controls (C–F). Scale bar: 5 mm. Quantification of results (Aa) showed that depletion of Lmod3 protein in st34 embryos resulted in a dramatic reduction in the percentage of correctly structured sarcomeres (70.2% in control MO-treated compared to 23.6% in lmod3 MO-treated). The addition of mRNA encoding Myc–Lmod3 together with the lmod3 MO restored sarcomere structure to control levels. Data show the mean6s.e.m.; ***P,0.001 (x2 analysis).
	Fig. 5. Lmod3 and Tmod4 display largely equivalent functions during skeletal myofibril assembly. (A–L) In these studies, tmod4 MO was co-injected into the Xenopus embryo along with mRNA encoding Myc–Lmod3. (E–L) When Myc–Lmod3 was added to embryos depleted of Tmod4, sarcomere assembly was significantly rescued compared with that of embryos depleted of Tmod4 alone (A–D). Note that in regions showing clear striated organization, Myc–Lmod3 was abundant at the M-line (F,J), whereas Tmod4 was not detected (G). The structure of other sarcomeric proteins, assayed by localization of myosin (K) was also restored. (M–X) In these studies, lmod3 MO was co-injected into the Xenopus embryo along with mRNA encoding GFP–Tmod4 (Q–X). When GFP–Tmod4 was added to embryos depleted of Lmod3, sarcomere assembly was significantly rescued compared with that of embryos depleted of Lmod3 alone (M– P). Note that in regions showing mature striated organization, GFP–Tmod4 was abundant at the M-line (R,V), whereas Lmod3 was not detected (S). The structure of other sarcomeric proteins, assayed by the localization of myosin (W) was also restored. Scale bar: 5 mm. (Y,Z) Quantification of experimental results showed that the addition of Myc–Lmod3 to embryos depleted of Tmod4 resulted in a significant increase in the percentage of sarcomeres displaying normal structure compared with that of Tmod4-depleted embryos (5.6-fold increase). Similarly, addition of GFP–Tmod4 to embryos depleted of Lmod3 resulted in a dramatic rescue of sarcomere structure compared with that observed in Lmod3- depleted muscle (2.9-fold). Data show the mean6s.e.m.; ***P,0.001 (x2 analysis).
	Fig. S1. Developmental expression of Tmod and Lmod family proteins at the neurula stage prior to skeletal muscle assembly. Tmod (B-D) and Lmod (E-H) family gene expression was analyzed using in situ hybridization. All embryos are positioned with heads to the left. Expression patterns were analyzed at the neurula stage (st15), before the onset of myofibrillogenesis. Expression of the striated muscle marker, cardiac actin, actna1 (A) was used to indicate skeletal muscle tissue in the embryo. Arrows indicate developing skeletal muscle in the somites. Only lmod3 (D) and tmod4 (H) are expressed at high levels in developing skeletal muscle.
	Fig. S2. Expression of endogenous lmod and tmod family genes is not altered by tmod4 MO treatment. tmod (A-H) and lmod (I-N) family gene expression was analyzed in control MO- (left panels) and tmod4 MO- (right panels) treated embryos using in situ hybridization. All embryos were assayed at tailbud stage (st34), the same stage that myofibril structure was analyzed. No appreciable alteration of gene expression is observed following treatment with tmod4 MO.
	Fig. S3. Localization of tagged Tmod4 and Lmod3 proteins. (A-D) At the levels used for rescue studies, GFP-Tmod4 did not alter sarcomere or thin filament dimensions or localization of Lmod3 at the M-line. Embryos were microinjected with 900pg of GFP-Tmod4 mRNA and assayed for myofibril organization at stage 34. Endogenous Lmod3 localized to the M-line in the presence of additional GFP-Tmod4 protein. (E-H) At the levels used for rescue studies, myc-Lmod3 did not alter sarcomere or thin filament dimensions or localization of Tmod4 at the M-line. Embryos were microinjected with 900pg of mRNA encoding myc-Lmod3 and assayed at stage 34. Endogenous Tmod4 localized to the M-line in the presence of additional myc-Lmod3 protein. In all panels, arrows indicate localization of Tmod4 at the M-line, and arrowheads indicate Lmod3 localization, co-localizing with Tmod4 at the M-line. Scale bar is 5 microns. Journal
	Fig. S4. Truncated versions of Lmod3 protein, lacking the C-terminal extension, do not incorporate into developing myofibrils and cannot rescue sarcomere structure in knockdown experiments. Two separate Lmod3 truncations (Lmod3d1 and Lmod3d2) were generated. Lmod3d1 is truncated at a position exactly equivalent to the C-terminus of Tmod4 and Lmod3d2 is 11 aa longer. (A-H). In contrast to full length Lmod3 (Fig. S4), truncated forms of Lmod3 did not detectably incorporate into developing sarcomeres. (I-T). Myofibrillogenesis was severely disrupted by depletion of Tmod4 protein synthesis using tmod4 MO, but this could be rescued by addition of either GFP-Tmod4 or GFP-Lmod3 proteins. (U-Ab). In contrast to full-length GFP-Lmod3 (Q-T) neither of the truncated forms of Lmod3 was sufficient to rescue sarcomere structure. (Ac). Western blot, using anti-GFP antibody, showing equivalent expression of GFP-Lmod3 and GFP-Lmod3 truncations in injected embryos.
	lmod1 (leiomodin 1 (smooth muscle)) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 15, lateral view, anterior left, dorsal up.
	lmod1 (leiomodin 1 (smooth muscle)) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 34, lateral view, anterior left, dorsal up.
	lmod2 (leiomodin 2 (cardiac)) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 15, lateral view, anterior left, dorsal up.
	lmod2 (leiomodin 2 (cardiac)) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 34, lateral view, anterior left, dorsal up.
	lmod3 (leiomodin 3 (fetal)) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 15, lateral view, anterior left, dorsal up.
	lmod3 (leiomodin 3 (fetal)) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 34, lateral view, anterior left, dorsal up.
	tmod1 (tropomodulin 1) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 15, lateral view, anterior left, dorsal up.
	tmod1 (tropomodulin 1) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 34, lateral view, anterior left, dorsal up.
	tmod2 (tropomodulin 2 (neuronal)) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 15, lateral view, anterior left, dorsal up.
	tmod2 (tropomodulin 2 (neuronal)) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 34, lateral view, anterior left, dorsal up.
	tmod3 (tropomodulin 3 (ubiquitous)) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 15, lateral view, anterior left, dorsal up.
	tmod3 (tropomodulin 3 (ubiquitous)) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 34, lateral view, anterior left, dorsal up.
	tmod4 (tropomodulin 4 (muscle)) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 15, lateral view, anterior left, dorsal up.
	actc1 (actin, alpha, cardiac muscle 1) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 34, lateral view, anterior left, dorsal up.
	actc1 (actin, alpha, cardiac muscle 1) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 15, lateral view, anterior left, dorsal up.

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