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Shuttling of the chaperones Unc45b and Hsp90a between the A band and the Z line of the myofibril.

Etard C, Roostalu U, Strähle U - J. Cell Biol. (2008)

Bottom Line: We show that Unc45b and Hsp90a, two zebrafish orthologues, colocalize with myosin during myofibrillogenesis and associate with the Z line when myofibril assembly is completed.Although chaperone activity of Unc45b requires the full-length protein, only the central and Unc45-Cro1p-She4p domains are required to anchor it to the Z line, and multiple subdomains mediate association with nascent myosin.Our data are consistent with a differential affinity model as an explanation for the shuttling of the chaperones between the Z line and myosin.

View Article: PubMed Central - PubMed

Affiliation: Institute for Toxicology and Genetics, Forschungszentrum Karlsruhe, 76021 Karlsruhe, Germany.

ABSTRACT
The formation of thick filaments in striated muscle involves the chaperones Hsp90a and Unc45. We show that Unc45b and Hsp90a, two zebrafish orthologues, colocalize with myosin during myofibrillogenesis and associate with the Z line when myofibril assembly is completed. In response to stress or damage to the myofiber, Unc45b and Hsp90a dissociate from the Z line and transiently associate with myosin. Although chaperone activity of Unc45b requires the full-length protein, only the central and Unc45-Cro1p-She4p domains are required to anchor it to the Z line, and multiple subdomains mediate association with nascent myosin. We propose that the Z line serves as a reservoir for chaperones, allowing a rapid mobilization in response to muscle damage. Our data are consistent with a differential affinity model as an explanation for the shuttling of the chaperones between the Z line and myosin.

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Unc45b-GFP associates with nascent myosin. (A–C) Wild-type embryo expressing Unc45b-GFP. At 24 hpf, Unc45b-GFP was ubiquitously localized in the cytoplasm (A). In later developmental stages, Unc45b-GFP localized to the Z line (B, 48 hpf; and C, 72 hpf). (D–G) unc45b mutant rescued to varying degrees by expression of Unc45b-GFP. At 24 hpf, Unc45b-GFP was distributed ubiquitously (D). From 30 hpf, it started to aggregate in patches (E) and to form A-band–like structures (F). From 72 hpf, Unc45b-GFP localized to the Z line in rescued embryos (G). (H–J) Wild-type embryos were coinjected with unc45b-gfp plasmid and morpholinos against hsp90a mRNA (hsp90a-Mo). At 30 hpf, Unc45b-GFP aggregated in patches and a striation started to appear (H). At 48 hpf, Unc45b-GFP was localized in A-band–like structures (I) and remained associated with those at 96 hpf (J). (K and L) unc45b mutant expressing Unc45b-GFP (K) in A-band–like patterns. Embryos were fixed with A/PFA, and stained with anti–slow muscle myosin antibody F59 (L). (M) Merge. Unc45b-GFP colocalizes with myosin. Bars, 4 μm. Arrowheads indicate the Z line and white boxes outline an A-band–like pattern. Stars indicate the nucleus and arrows indicate the perinuclear area. (N) Unc45b-GST protein interacts with myosin from rabbit muscle in an in vitro pulldown assay (2). GST protein alone used as a control did not bind myosin (1). Anti-myosin MF20 antibody was used to develop the Western blot (250 kD).
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fig2: Unc45b-GFP associates with nascent myosin. (A–C) Wild-type embryo expressing Unc45b-GFP. At 24 hpf, Unc45b-GFP was ubiquitously localized in the cytoplasm (A). In later developmental stages, Unc45b-GFP localized to the Z line (B, 48 hpf; and C, 72 hpf). (D–G) unc45b mutant rescued to varying degrees by expression of Unc45b-GFP. At 24 hpf, Unc45b-GFP was distributed ubiquitously (D). From 30 hpf, it started to aggregate in patches (E) and to form A-band–like structures (F). From 72 hpf, Unc45b-GFP localized to the Z line in rescued embryos (G). (H–J) Wild-type embryos were coinjected with unc45b-gfp plasmid and morpholinos against hsp90a mRNA (hsp90a-Mo). At 30 hpf, Unc45b-GFP aggregated in patches and a striation started to appear (H). At 48 hpf, Unc45b-GFP was localized in A-band–like structures (I) and remained associated with those at 96 hpf (J). (K and L) unc45b mutant expressing Unc45b-GFP (K) in A-band–like patterns. Embryos were fixed with A/PFA, and stained with anti–slow muscle myosin antibody F59 (L). (M) Merge. Unc45b-GFP colocalizes with myosin. Bars, 4 μm. Arrowheads indicate the Z line and white boxes outline an A-band–like pattern. Stars indicate the nucleus and arrows indicate the perinuclear area. (N) Unc45b-GST protein interacts with myosin from rabbit muscle in an in vitro pulldown assay (2). GST protein alone used as a control did not bind myosin (1). Anti-myosin MF20 antibody was used to develop the Western blot (250 kD).

Mentions: To study the intracellular location of Unc45b, we constructed an unc45b-gfp chimeric gene by fusing the unc45b cDNA to gfp at the C terminus. We injected the construct into fertilized zebrafish eggs and followed the location of the fusion protein by confocal microscopy. The Unc45b-GFP protein accumulated at high concentrations at reiterative transverse bands along the myofibril in 72-h postfertilization (hpf) embryos, which correspond to the Z lines (Fig. 1 A, arrowhead). Although the nucleus was free of Unc45b-GFP, the perinuclear area had frequently increased GFP fluorescence relative to the rest of the myofibril-free cytoplasm and the nucleus (Fig. 1 A, arrow; Fig. 2 C).


Shuttling of the chaperones Unc45b and Hsp90a between the A band and the Z line of the myofibril.

Etard C, Roostalu U, Strähle U - J. Cell Biol. (2008)

Unc45b-GFP associates with nascent myosin. (A–C) Wild-type embryo expressing Unc45b-GFP. At 24 hpf, Unc45b-GFP was ubiquitously localized in the cytoplasm (A). In later developmental stages, Unc45b-GFP localized to the Z line (B, 48 hpf; and C, 72 hpf). (D–G) unc45b mutant rescued to varying degrees by expression of Unc45b-GFP. At 24 hpf, Unc45b-GFP was distributed ubiquitously (D). From 30 hpf, it started to aggregate in patches (E) and to form A-band–like structures (F). From 72 hpf, Unc45b-GFP localized to the Z line in rescued embryos (G). (H–J) Wild-type embryos were coinjected with unc45b-gfp plasmid and morpholinos against hsp90a mRNA (hsp90a-Mo). At 30 hpf, Unc45b-GFP aggregated in patches and a striation started to appear (H). At 48 hpf, Unc45b-GFP was localized in A-band–like structures (I) and remained associated with those at 96 hpf (J). (K and L) unc45b mutant expressing Unc45b-GFP (K) in A-band–like patterns. Embryos were fixed with A/PFA, and stained with anti–slow muscle myosin antibody F59 (L). (M) Merge. Unc45b-GFP colocalizes with myosin. Bars, 4 μm. Arrowheads indicate the Z line and white boxes outline an A-band–like pattern. Stars indicate the nucleus and arrows indicate the perinuclear area. (N) Unc45b-GST protein interacts with myosin from rabbit muscle in an in vitro pulldown assay (2). GST protein alone used as a control did not bind myosin (1). Anti-myosin MF20 antibody was used to develop the Western blot (250 kD).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2290844&req=5

fig2: Unc45b-GFP associates with nascent myosin. (A–C) Wild-type embryo expressing Unc45b-GFP. At 24 hpf, Unc45b-GFP was ubiquitously localized in the cytoplasm (A). In later developmental stages, Unc45b-GFP localized to the Z line (B, 48 hpf; and C, 72 hpf). (D–G) unc45b mutant rescued to varying degrees by expression of Unc45b-GFP. At 24 hpf, Unc45b-GFP was distributed ubiquitously (D). From 30 hpf, it started to aggregate in patches (E) and to form A-band–like structures (F). From 72 hpf, Unc45b-GFP localized to the Z line in rescued embryos (G). (H–J) Wild-type embryos were coinjected with unc45b-gfp plasmid and morpholinos against hsp90a mRNA (hsp90a-Mo). At 30 hpf, Unc45b-GFP aggregated in patches and a striation started to appear (H). At 48 hpf, Unc45b-GFP was localized in A-band–like structures (I) and remained associated with those at 96 hpf (J). (K and L) unc45b mutant expressing Unc45b-GFP (K) in A-band–like patterns. Embryos were fixed with A/PFA, and stained with anti–slow muscle myosin antibody F59 (L). (M) Merge. Unc45b-GFP colocalizes with myosin. Bars, 4 μm. Arrowheads indicate the Z line and white boxes outline an A-band–like pattern. Stars indicate the nucleus and arrows indicate the perinuclear area. (N) Unc45b-GST protein interacts with myosin from rabbit muscle in an in vitro pulldown assay (2). GST protein alone used as a control did not bind myosin (1). Anti-myosin MF20 antibody was used to develop the Western blot (250 kD).
Mentions: To study the intracellular location of Unc45b, we constructed an unc45b-gfp chimeric gene by fusing the unc45b cDNA to gfp at the C terminus. We injected the construct into fertilized zebrafish eggs and followed the location of the fusion protein by confocal microscopy. The Unc45b-GFP protein accumulated at high concentrations at reiterative transverse bands along the myofibril in 72-h postfertilization (hpf) embryos, which correspond to the Z lines (Fig. 1 A, arrowhead). Although the nucleus was free of Unc45b-GFP, the perinuclear area had frequently increased GFP fluorescence relative to the rest of the myofibril-free cytoplasm and the nucleus (Fig. 1 A, arrow; Fig. 2 C).

Bottom Line: We show that Unc45b and Hsp90a, two zebrafish orthologues, colocalize with myosin during myofibrillogenesis and associate with the Z line when myofibril assembly is completed.Although chaperone activity of Unc45b requires the full-length protein, only the central and Unc45-Cro1p-She4p domains are required to anchor it to the Z line, and multiple subdomains mediate association with nascent myosin.Our data are consistent with a differential affinity model as an explanation for the shuttling of the chaperones between the Z line and myosin.

View Article: PubMed Central - PubMed

Affiliation: Institute for Toxicology and Genetics, Forschungszentrum Karlsruhe, 76021 Karlsruhe, Germany.

ABSTRACT
The formation of thick filaments in striated muscle involves the chaperones Hsp90a and Unc45. We show that Unc45b and Hsp90a, two zebrafish orthologues, colocalize with myosin during myofibrillogenesis and associate with the Z line when myofibril assembly is completed. In response to stress or damage to the myofiber, Unc45b and Hsp90a dissociate from the Z line and transiently associate with myosin. Although chaperone activity of Unc45b requires the full-length protein, only the central and Unc45-Cro1p-She4p domains are required to anchor it to the Z line, and multiple subdomains mediate association with nascent myosin. We propose that the Z line serves as a reservoir for chaperones, allowing a rapid mobilization in response to muscle damage. Our data are consistent with a differential affinity model as an explanation for the shuttling of the chaperones between the Z line and myosin.

Show MeSH
Related in: MedlinePlus