Effect of S225A, S225T, S225D, F223A, H224A and Q226A substitution on the sarcomeric localization of Smyd1_tv1-EGFP in zebrafish embryos.

<p>DNA construct expressing Smyd1_tv1-EGFP or its derived mutants of S225A, S225T, S225D, F223A, H224A and Q226A was injected into zebrafish embryos. Their localization was analyzed in myofibers of the injected embryos at 48 hpf. A, Smyd1_tv1-EGFP; B, Smyd1_tv1-EGFP(S225A); C, Smyd1_tv1-EGFP(S225T); D, Smyd1_tv1-EGFP(S225D), E, Smyd1_tv1-EGFP(F223A), F, Smyd1_tv1-EGFP(H224A), and Smyd1_tv1-EGFP(Q226A).</p

Rescue of myofibril organization defect in smyd1b knockdown embryos by expression of Smyd1b_tv1-EGFP or Smyd1b_tv2-EGFP fusion protein.

<p><b>A.</b> DNA constructs encoding Smyd1b_tv1-EGFP, or Smyd1b_tv2-EGFP fusion proteins or EGFP control were generated and injected into zebrafish embryos. <b>B and C.</b> Myofibers expressing Smyd1_tv1-EGFP (B) or Smyd1_tv2-EGFP (C) was directly observed by GFP. <b>D and E.</b> Myosin thick filaments organization was determined by F59 antibody staining in Smyd1_tv1-EGFP (D) or Smyd1_tv2-EGFP (E) co-injected embryos. <b>F and G.</b> Double staining shows the colocalization of normal fibers with Smyd1_tv1-EGFP (F) or Smyd1_tv2-EGFP (G) expression. Scale bars: 20 µm.</p

The Serine 225 is required for the enhanced sarcomeric localization of Smyd1b_tv1.

<p><b>A.</b> Sequence comparison shows that the alternative splicing of smyd1b in various vertebrates and the conserved serine and threonine residues within the 13 aa insertion. <b>B and C.</b> Immunostaining using anti-myc antibody shows the sarcomeric localization of Smyd1b_tv1^myc in myofibers of zebrafish embryos at 38 hpf. C represents the highlighted box area in A. <b>D–G.</b> Immunostaining using anti-myc antibody shows the sarcomeric localization of Smyd1b_tv1^myc mutant proteins that carry substitutions at S217A and T221A (D), S225A (E), S225T (F), S225D (G). Scale bars: B = 40 µm; C = 20 µm.</p

Knockdown of endogenous Smyd1b advances the timing of sarcomeric localization of Smyd1b_tv1^myc in zebrafish embryos.

<p><b>A.</b> Smyd1b E9I9-MO was injected into Smyd1b_tv1^myc or Smyd1b_tv1^myc transgenic zebrafish embryos at 1–2 cell stages. Western blot analysis shows the expression of myc-tagged Smyd1b_tv1^myc and Smyd1b_tv1^myc in un-injected control or E9I9-MO injected transgenic zebrafish embryos at 24 hpf. γ-Tubulin was used as loading control. <b>B and C.</b> Immunostaining using anti-myc antibody shows the cytoplasmic (B) or sarcomeric localization (C) of <i>smyd1b_tv1^myc</i> in control (B) or E9I9-MO injected (C) transgenic zebrafish embryos at 24 hpf. <b>D and E.</b> Immunostaining using anti-myc antibody shows the cytoplasmic localization of <i>smyd1b_tv2^myc</i> in control or E9I9-MO injected transgenic zebrafish embryos at 24 hpf. Scale bar: 30 µm.</p

The effect of <i>hsp90α1</i> mutation or knockdown on Smyd1b_tv1 sarcomeric localization.

<p><b>A and D.</b> Immunostaining using anti-myomesin antibody shows the organization of myomesin in <i>hsp90α1</i> mutant (A), or control (D) <i>smyd1b_tv1^myc</i> transgenic embryos at 72 hpf. <b>B and E.</b> Immunostaining using anti-myc antibody shows the localization of Smyd1b_tv1^myc in <i>hsp90α1</i> mutant (B), or control (E) <i>smyd1b_tv1^myc</i> transgenic embryos at 72 hpf. <b>C and F.</b> F59 staining shows the organization of slow muscle myosin in <i>hsp90α1</i> mutant (C), or control (F) <i>smyd1b_tv1^myc</i> transgenic embryos at 72 hpf. <b>G and H.</b> DNA construct <i>Smyd1_tv1-EGFP</i> was injected alone or together with <i>hsp90α1</i> ATG-MO into zebrafish embryos. Smyd1_tv1-EGFP localization was determined in myofibers of the control (G), or <i>hsp90α1</i> knockdown (H) at 48 hpf. Scale bars: D = 40 µm; G = 15 µm.</p

Smyd1b_tv1-EGFP is localized on the M-line of zebrafish skeletal muscles.

<p>Smyd1b_tv1-EGFP construct was injected into Myomesin-RFP (A, C, E) or wild type (B, D, F) zebrafish embryos at 1–2 cell stages. Smyd1b_tv1-EGFP localization was determined together with M-line marker (Myomesin-RFP) and A-band marker (Myosin heavy chain) at 96 hpf. <b>A, B and C.</b> Co-localization of Smyd1b_tv1-EGFP and Myomesin-RFP was observed in myofibers of the injected embryos. <b>D, E and F.</b> Immunostaining with anti-MyHC antibody (F59) shows the localization of Smyd1b_tv1-EGFP in the middle of the A-bands in myofibers of zebrafish embryos. Scale bar: 12 µm.</p

The sarcomeric localization of Smyd1b_tv1 occurs after the sarcomere formation in myofibers of zebrafish embryos.

<p><b>A–C.</b> Immunostaining using sarcomeric specific antibodies against MyHC (A), α-actin (B), and α-actinin (C) in the trunk muscles of zebrafish embryos at 24 hpf. <b>D.</b> Immunostaining using anti-myc antibody shows the primary cytoplasmic localization of Smyd1b_tv1 in the trunk muscles of <i>smyd1b_tv1^myc</i> transgenic fish embryos at 24 hpf. Muscle pioneer cells with the sarcomeric localization are indicated by arrows. Scale bar: 30 µm.</p

Generation of Smyd1b_tv1 and Smyd1b_tv2 by alternative splicing and construction of the Smyd1b_tv1^myc and Smyd1b_tv2^myc transgenes.

<p><i>Smyd1b_tv1</i> and <i>Smyd1b_tv2</i> transcripts are generated by alternative splicing. Their cDNA sequences are identical, with the exception of the 39 bp insertion encoded by exon 5. It translates into a 13 amino acids insertion in Smyd1b_tv1. Smyd1b_tv1^myc and Smyd1b_tv2^myc transgenes are constructed by fusing with an in frame myc-tag at the N-terminus. Expression of the transgenes are directed by its own promoter. A SV40 PolyA signal was included at the 3′ end.</p

Characterization of the sarcomeric localization using Smyd1b_tv1-EGFP and Smyd1b_tv2-EGFP fusion proteins.

<p>DNA constructs encoding Smyd1b_tv1-EGFP, or Smyd1b_tv2-EGFP fusion proteins or EGFP control injected into zebrafish embryos. Their expression and localization was determined in myofibers of the injected zebrafish embryos at 48 (A–C) and 96 (D–F) hpf. A and D, Smyd1b_tv1-EGFP; B and E, Smyd1b_tv2-EGFP; C and F, EGFP control. Scale bar: 8 µm.</p