Drosophila UNC-45 accumulates in embryonic blastoderm and in muscles, and is essential for muscle myosin stability

Abstract

UNC-45 is a chaperone that facilitates folding of myosin motor domains. We have used Drosophila melanogaster to investigate the role of UNC-45 in muscle development and function. Drosophila UNC-45 (dUNC-45) is expressed at all developmental stages. It colocalizes with non-muscle myosin in embryonic blastoderm of 2-hour-old embryos. At 14 hours, it accumulates most strongly in embryonic striated muscles, similarly to muscle myosin. dUNC-45 localizes to the Z-discs of sarcomeres in third instar larval body-wall muscles. We produced a dunc-45 mutant in which zygotic expression is disrupted. This results in nearly undetectable dUNC-45 levels in maturing embryos as well as late embryonic lethality. Muscle myosin accumulation is robust in dunc-45 mutant embryos at 14 hours. However, myosin is dramatically decreased in the body-wall muscles of 22-hour-old mutant embryos. Furthermore, electron microscopy showed only a few thick filaments and irregular thick-thin filament lattice spacing. The lethality, defective protein accumulation, and ultrastructural abnormalities are rescued with a wild-type dunc-45 transgene, indicating that the mutant phenotypes arise from the dUNC-45 deficiency. Overall, our data indicate that dUNC-45 is important for myosin accumulation and muscle function. Furthermore, our results suggest that dUNC-45 acts post-translationally for proper myosin folding and maturation.

Fig. 1.

Drosophila unc-45 genomic region in three fly lines. (A) The wild-type gene consists of three exons and two introns, with the translation start site located 14 bp downstream from the beginning of the second exon. The gene encodes a three-domain protein of approximately 105 kDa with an N-terminal TPR domain, a central domain and a C-terminal UCS domain. (B) The dunc-45 mutant Tom34^EY03034 contains a P element insertion in the first exon, upstream of the translation start site. (C) The T-33 dunc-45 knockout mutant contains a 1304 bp deletion that removes the majority of both the first and second exons.

Fig. 2.

Western blot analysis showing dUNC-45 antibody specificity and dUNC-45 expression at all stages of development in wild-type Drosophila. (A) Western blot of 2-hour-old adult (2hA) and 1-day-old adult (1dA) whole fly lysates. The third lane is bacterially expressed recombinant dUNC-45. It contains a His tag, which accounts for its slightly larger size. The protein size ladder is given on the right. (B–D) Developmental expression of dUNC-45. (B) Early (EE) and late stage embryos (LE). (C) First instar (1L), second instar (2L), and third instar larvae (3L). (D) Early pupae (EP), late pupae (LP), 2-hour-old adult (2hA), and 1-day-old adult (1dA). Equivalent amounts of protein were loaded in each lane.

Fig. 3.

Immunofluorescence confocal micrographs showing protein localization in wild-type yw embryos. (A–G) Embryos were probed with non-muscle myosin (A,D), muscle myosin (B,E), and dUNC-45 (C,F,G) antibodies. (A–C) 2-hour-old embryos. At this stage, both non-muscle myosin and dUNC-45 localized to the embryonic blastoderm (yellow stars). Furthermore, UNC-45 can be seen in the pole cells (upper left of C). Muscle myosin is not detected in 2-hour-old embryos (B). (D–F) Ventral views of 14-hour-old embryos. At 14 hours, dUNC-45 localization is more similar to that of muscle myosin, which can be found in the body wall (pink arrowheads) and the pharyngeal muscles (white diamonds). G gives a lateral view of an embryo, confirming dUNC-45 localization in the body-wall muscle. Scale bars: 75 μm.

Fig. 4.

Immunofluorescence confocal micrographs showing subcellular dUNC-45 localization in the body-wall muscle of third instar wild-type larvae. (A) α-Actinin staining highlights the location of the Z-discs. (B) dUNC-45 is discretely localized in the sarcomere. (C) Actin staining with phalloidin shows the location of I bands in the sarcomeres. (D) dUNC-45 colocalizes with α-actinin (bright pink in the overlay panel) and bisects the actin-containing I bands, which supports dUNC-45 location in the Z-discs of the sarcomeres.

Fig. 5.

Western blot analysis of dUNC-45 and myosin expression in wild-type (yw), Tom34^EY03034 and T-33 22-hour-old embryos. (A) Western blot analysis of dUNC-45 expression. Control is bacterially expressed recombinant dUNC-45. (B) Western blot analysis of myosin expression. Control is indirect flight muscle lysate. Results of the analysis show that dUNC-45 can still be detected in the Tom34^EY03034 line but not in the T-33 line. Muscle myosin heavy chain is somewhat reduced in Tom34^EY03034 homozygotes, but is nearly absent from T-33 embryos. 10 μg of protein were loaded for each sample.

Fig. 6.

Immunofluorescence confocal micrographs of 14-hour-old T-33/lacZ or lacZ/lacZ embryos and homozygous T-33 embryos. (A–F) Embryos were probed with antibodies to lacZ (A,D), muscle myosin (B,E) and dUNC-45 (C,F). T-33/lacZ or lacZ/lacZ embryos were identified by lacZ expression in the hind-gut (A). Homozygous T-33 embryos do not show a detectable expression of lacZ (D). dUNC-45 cannot be detected in the body-wall muscle of the homozygous T-33 embryos (F). Muscle patterning and muscle myosin expression are not affected in the absence of dUNC-45 at this stage of embryogenesis (E). Scale bar: 75 μm.

Fig. 7.

Electron micrographs showing body-wall muscle ultrastructure in 22-hour-old embryos. (A,B) yw control line, (C,D) T-33 dUNC-45 knockout embryos, and (E,F) T-33 rescued embryos with a wild-type transgene. Electron micrographs show that T-33 embryos exhibit a reduced level of thick filaments and a loss of the thick–thin filament lattice spacing (C,D). This phenotype can be rescued with a wild-type dunc-45 transgene, which confirms the effects of dUNC-45 knockout in the muscles. Transverse orientation is shown in A, C and E. Longitudinal orientation is shown in B, D and F. Scale bars: 0.25 μm (A,C,E); 1.0 μm (B,D inset,F); 1.0 μm (D).