Deletion of the N-terminal TPR domain had no effect on the disruptive activity of Unc-45b on myosin thick filament organization, whereas deletion of the C-terminal UCS domain abolished the disruptive effect of Unc-45b overexpression. A. DNA constructs, smyd1:unc-45bΔTPRflag and smyd1:unc-45bΔUCSflag, were generated as minigenes expressing Unc-45b mutant proteins without the N-terminal TPR domain (amino acids 1-119), or the C-terminal UCS domain (amino acids 537-934). A flag-tag was fused in frame at the C-terminus for monitoring gene expression. The DNA construct was injected into zebrafish embryos. The effect on thick, thin filament and Z-line organization was analyzed by double staining using anti-flag (red) and anti-myosin (F59, green) antibodies at 30 hpf. B-D. Expression of flag-tagged TPR deletion mutant of Unc-45b (B) in fiber disrupts thick filament organization (C). D represents the merged image of B and C. Forty two myofibers from 36 embryos were analyzed. 75% of the myofiber showed the disorganized thick filaments. E-G. Deletion of the C-terminal UCS domain abolished the disruptive effect of Unc-45b overexpression. Myofiber expressing the UCS deletion mutant (E) exhibited normal thick filament organization (F). G represents the merged image of E and F. Twenty nine myofibers from 25 embryos were analyzed. None of the myofiber showed the disorganized thick filaments. Scale bars = 25 μm.
Bernick et al. BMC Cell Biology 2010 11:70 doi:10.1186/1471-2121-11-70