Etard et al., 2007 - The UCS factor Steif/Unc-45b interacts with the heat shock protein Hsp90a during myofibrillogenesis. Developmental Biology   308(1):133-143 Full text @ Dev. Biol.

Fig. 1 Zebrafish embryos with a mutation in the steif gene fail to form myofibrils in the skeletal musculature and the heart. (A, B) 3-day-old steif mutants (B) are immotile and have smaller eyes than wild-type embryos (A). (C, D) electron microscopic analysis of parasagittal sections through a somitic fast muscle cell of a wild-type and a steif mutant embryo (48 hpf). In contrast to wild-type (C), steif mutant (D) skeletal fast muscle cells do not form ordered myofibrils. (E, F) electron micrographs of sections through cardiac muscle cells from 5-day-old wild-type (E) and mutant embryos (F). The mutant lacks myofibrils and does also not form proper intercalated disks (arrows indicate Z-lines of sarcomeres). (G, H) superficial wild-type and mutant slow muscle cells stained immunohistochemically with the antibody F59 directed against slow muscle myosin. Wild-type (G) but not the steif mutant cells (H) show the striped pattern of slow muscle myosin staining. (I, J) F-actin staining of wild-type (I) and steif (J) slow muscle cells. As in the case of the myosin-containing thick filaments, the thin filaments composed of F-actin are strongly disorganized in the mutant (J). Scale bars: 1 μm (C, D); 0,5 μm (E, F); 25 μm (G–J).

Fig. 2 The steif gene maps to a locus related to nematode unc-45. (A) the steif mutation was mapped to linkage group 8 (LG8) close to the marker fc15g07 and unc-45-related (now named unc-45b). (B) the point mutation in the unc-45b gene isolated from steif mutants changes a C to an A (arrow) leading to the formation of a stop codon. (C) in the steif mutant, the steif/unc-45b gene contains a point mutation that transforms the codon of cysteine 788 into a stop codon. The steif/unc-45b gene has the same domain structure as C. elegans unc-45 with N-terminal tricopeptide repeats (TPR), Central and the C-terminal UCS domain that is shared with the fungal proteins She4p and Cro1. The nonsense mutation in steif mutants leads to truncation of the UCS domain. (D–E) in situ hybridization to wild-type embryos showing steif/unc-45b mRNA localization in skeletal and cardiac muscles (arrow). (D) 24 hpf, lateral view; (E) 72 hpf, lateral view. am, adductor mandibulae; do, dilator operculi; hh, hyohyoideus; ih, interhyoideus; imp, intramandibular posterior; io, inferior oblique; ir, inferior rectus; lap, levator arcus palatini; lr, lateral rectus; so, superior oblique; tv, transversus ventralis. Arrow: heart. Scale bar: 120 μm (D); 60 μm (E).

Fig. 3 Expression of steif in the mutant restores myofibrils. (A) uninjected wild-type, (B) steif mutant injected with the BAC clone encoding the steif/unc-45b gene fused with CFP, (C) uninjected steif mutant. The color code on the inserted bar represents the intensity of birefringence covering a five fold range from red (high) to blue (low). (D) F59 staining of BAC-injected steif embryos showing rescue of single fibrils (arrowhead). (E) magnification of panel F. Muscle cells expressing Steif-CFP show normal striation (A-band, arrow head, vertical lines, Z-lines). (F–I) Steif-GFP plasmid injection into steif mutant embryos led to recovery of birefringence and rescue of myofibril organization (F, GFP fluorescence; G, birefringence; H, merge of panels F and G). (I) α-actinin staining showing a rescued fibril in steif mutant embryos (arrow indicates Z-line). Scale bars: 150 μm (A–C); 12 μm (D, F–H); 2 μm (E), 5 μm (I). (A–I) 3-day-old embryos.

Fig. 4 Knockdown of Steif phenocopies the mutant phenotype. Two-day-old wild-type (A, B), morpholino-injected (C, D) and steif mutant embryo (E, F). Embryos (A, C, E) were illuminated at 72 hpf with polarized light to visualize the birefringence in the somitic muscle (for color representation see Fig. 3). Knockdown of Steif/Unc-45b protein (C) by injection of antisense morpholino reduces the birefringence similarly to mutations in steif (E). (B, D, F) wild-type (B), morpholino injected (D) and steif mutant embryos (F) stained immunohistochemically at 48 hpf with the antibody F59 directed against slow muscle myosin. Knockdown embryos (D) and steif (F) mutants have similarly disorganized slow muscle myofibrils. Scale bar: 50 μm (A, C, E); 15 μm (B, D, F).

Fig. 5 Steif/Unc-45b interacts with Hsp90a in vitro. (A–C) in situ hybridization with hsp90b (A), hsp90a (B) and hsp90a2 (C) antisense probes on 1-day-old embryos. hsp90a and a2 are strongly expressed in skeletal and heart muscles (arrow), while hsp90b is ubiquitously expressed. (D) in vitro pull-down assay. Steif-GST interacts with Hsp90a-GFP, Hsp90a2-GFP and Hsp90b-GFP but not GFP alone (lanes 1 to 4). GST without fused Steif does not pull down Hsp90a-GFP, Hsp90a2-GFP, Hsp90b-GFP or GFP (lanes 5 to 8). The Western blots were developed with anti-GFP antibody. M: protein standards indicated in kDa.

Fig. 6 Knockdown of hsp90a phenocopies the steif phenotype. (A, B) uninjected, (C, D) hsp90a, (E, F) hsp90a2, and (G, H) hsp90b morpholino injected embryos show normal birefringence at 72 hpf while hsp90a morpholino (C) injected embryos lack birefringence similar to steif mutants. (B, D, F, H) immunostaining of slow muscle myosin of control (B), hsp90a-morpholino (D), hsp90a2-morpholino (F), and hsp90b-morpholino injected embryos. Scale bar: 120 μm (A, C, E, G); 50 μm (B, D, F, H).

Fig. 7 Hsp90a and Steif/unc-45b mRNA are up-regulated in steif mutant embryos. In situ hybridization with antisense probes for hsp90a (A–B), a2 (C, D) and steif/unc-45 (E, F). steif mutant embryos (B, D, F) show an up-regulation of hsp90a, a2 and steif/unc-45b transcripts in skeletal muscles. Only hsp90a and a2 expression levels were increased in the heart (arrow and insert in panels A to D). Note that for reasons of quantitative comparisons, the staining reactions had to be terminated before strong signals were detectable in the wild-type embryos. All embryos are 2 days old. Scale bar: 150 mm.

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Reprinted from Developmental Biology, 308(1), Etard, C., Behra, M., Fischer, N., Hutcheson, D., Geisler, R., and Strähle, U., The UCS factor Steif/Unc-45b interacts with the heat shock protein Hsp90a during myofibrillogenesis, 133-143, Copyright (2007) with permission from Elsevier. Full text @ Dev. Biol.