Knockdown and overexpression of Unc-45b result in defective myofibril organization in skeletal muscles of zebrafish embryos

Abstract

BackgroundUnc-45 is a myosin chaperone and a Hsp90 co-chaperone that plays a key role in muscle development. Genetic and biochemical studies in C. elegans have demonstrated that Unc-45 facilitates the process of myosin folding and assembly in body wall muscles. Loss or overexpression of Unc-45 in C. elegans results in defective myofibril organization. In the zebrafish Danio rerio, unc-45b, a homolog of C. elegans unc-45, is expressed in both skeletal and cardiac muscles. Earlier studies indicate that mutation or knockdown of unc-45b expression in zebrafish results in a phenotype characterized by a loss of both thick and thin filament organization in skeletal and cardiac muscle. The effects of unc-45b knockdown on other sarcomeric structures and the phenotype of Unc-45b overexpression, however, are poorly understood in vertebrates.ResultsBoth knockdown and overexpression provide useful tools to study gene function during animal development. Using such methods, we characterized the role of Unc-45b in myofibril assembly of skeletal muscle in Danio rerio. We showed that, in addition to thick and thin filament defects, knockdown of unc-45b expression disrupted sarcomere organization in M-lines and Z-lines of skeletal muscles in zebrafish embryos. Western blotting analysis showed that myosin protein levels were significantly decreased in unc-45b knockdown embryos. Similarly, embryos overexpressing Unc-45b also exhibited severely disorganized myosin thick filaments. Disruption of thick filament organization by Unc-45b overexpression depends on the C-terminal UCS domain in Unc-45b required for interaction with myosin. Deletion of the C-terminal UCS domain abolished the disruptive activity of Unc-45b in myosin thick filament organization. In contrast, deletion of the N-terminal TPR domain required for binding with Hsp90α had no effect.ConclusionCollectively, these studies indicate that the expression levels of Unc-45b must be precisely regulated to ensure normal myofibril organization. Loss or overexpression of Unc-45b leads to defective myofibril organization.

FRZB and melusin, overexpressed in LGMD2A, regulate integrin β1D isoform replacement altering myoblast fusion and the integrin-signalling pathway

“…There are similar biological systems responsible for reverse effects in the cell, such as the one reported by Bernick and colleagues in 2010 (Ref. 31), in which loss or overexpression of unc-45b leads to defective myofibril organisation; that is, unc-45b expression must be precisely regulated to ensure normal myofibril organisation. And there are other examples, for instance, abnormally high or low levels of IQGAP1 and SRPK1 expression reduce activation of MEK and ERK or promote cancer, respectively (Refs 32, 33).…”

The myosin-interacting protein SMYD1 is essential for sarcomere organization

“…UNC-45b-deficient zebrafish embryos and worms have strongly disorganized sarcomeres, although initial Zbody formation and the basal organization of thick filaments seems not to be altered, implicating a rather restricted role of UNC-45b in the integration of thick filaments into sarcomeres (Barral et al, 2002;Etard et al, 2007). Strikingly, not only the loss but also elevated levels of UNC-45b result in inhibition of sarcomere assembly in worms and zebrafish, indicating that muscle chaperone levels have to be precisely balanced for unconstrained sarcomerogenesis and myosin stability (Bernick et al, 2010;Hoppe et al, 2004;Janiesch et al, 2007;Landsverk et al, 2007). We find UNC-45b expression levels to be severely upregulated in fla. Interestingly, a mild knockdown of UNC-45b in fla mutant embryos does not reconstitute heart and skeletal muscle function, implying that overexpression of UNC-45b is not the molecular cause of the observed muscle phenotype in fla.…”

“…Very recently, it has been shown that overexpression of UNC-45b in zebrafish muscle cells leads to impaired sarcomere organization (Bernick et al, 2010). Thus, to investigate whether downregulation of UNC-45b in fla mutant embryos might rescue myofibrillogenesis, we injected 'subeffective' doses (i.e.…”

The myosin-binding UCS domain but not the Hsp90-binding TPR domain of the UNC-45 chaperone is essential for function in Caenorhabditis elegans

“…The Rng3 UCS domain restores myosin II motor Myo2 motility in vitro. In zebrafish, deletion of the N-terminal TPR domain has no effect on the disruptive activity of UNC-45b on myosin thick filament organization, whereas deletion of the C-terminal UCS domain abolishes the disruptive effect of UNC-45b overexpression (Bernick et al, 2010). UNC-45b and Apo2 proteins act in an Hsp90-independent pathway that is required for integrity of the myosepta and myofiber attachment (Etard et al, 2010).…”