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First published online 6 February 2008
doi: 10.1242/dev.018150

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The ATPase-dependent chaperoning activity of Hsp90a regulates thick filament formation and integration during skeletal muscle myofibrillogenesis

Thomas A. Hawkins¹, Anna-Pavlina Haramis^1,*, Christelle Etard², Chrisostomos Prodromou³, Cara K. Vaughan³, Rachel Ashworth^4,, Saikat Ray¹, Martine Behra^2,, Nigel Holder¹, William S. Talbot⁵, Laurence H. Pearl³, Uwe Strähle² and Stephen W. Wilson^1,

¹ Department of Anatomy and Developmental Biology, UCL, Gower Street, London WC1E 6BT, UK.
² Institute of Toxicology and Genetics, Forschungszentrum Karlsruhe, Postfach 3640, Karlsruhe, D-76021, Germany.
³ Section of Structural Biology, The Institute of Cancer Research, Chester Beatty Laboratories, 237 Fulham Road, London, SW3 6JB, UK.
⁴ Department of Physiology, UCL, Gower Street, London WC1E 6BT, UK.
⁵ Department of Developmental Biology, Stanford University School of Medicine, Beckman Center B315, 279 Campus Drive, Stanford, CA 94305-5329, USA.

View larger version (100K): [in this window] [in a new window]   Fig. 1. slou45 mutants have disorganised or missing expression of sarcomeric proteins. (A-J) Lateral views of muscle fibres in wild-type (Sib) (A,C,E,G,I) and slou45 (B,D,F,H,J) zebrafish embryos of ages shown bottom left and with reagents/antibodies shown bottom right. (A,B) F-Actin labelling with phalloidin showing a regular arrangement of fibrils in wild-type muscle fibres, whereas the slou45 mutant fibrillar organisation is disrupted. (C,D) Immunohistochemistry for -Actinin (green) marks the Z-disc, here combined with phalloidin (red); the merge appears yellow. Z-discs are present in the slou45 mutant (white arrowheads, D) but are disordered: the distance between Z-discs is irregular (blue arrowheads) and Z-discs from neighbouring fibrils are not in register with each other (inset, D). Z-discs are flanked by Actin filaments in both siblings and mutants. (E,F) Titin labelling (green) using an antibody that marks a region of the molecule around the Z-disc, counterstained with phalloidin (red). (G-J) Immunohistochemistry for MHC (slow muscle Myosin, F59, red in G,H; pan-Myosin A4.1025, red in I,J). In the slou45 muscle cells, anti-MHC staining is severely reduced and lacks organisation. (K) Western blots using anti-MHC antibody F59 and -Tubulin (-Tub) as a loading control on lysates of slou45 and slotu44c mutant and sibling embryos at 30 hpf and 48 hpf. MHC protein levels are reduced in mutants of both alleles. Scale bars: 20 µm in A,B,G,H; 10 µm in C-F; 8 µm in I,J.

View larger version (62K): [in this window] [in a new window]   Fig. 2. Sarcomeres in slou45 mutant myofibrils lack thick filaments. (A-H) Electron micrographs of sarcomeres from wild-type (Sib) (A,C,E,G) and slou45 mutant (B,D,F,H) myofibrils at the ages shown bottom left. (A,B) Caudal myotomes of 24 hpf zebrafish embryos. White arrowheads (A) show immature Z-discs with poorly positioned thick and thin filaments. In slou45 mutants, bundles of thin filaments are present with putative early Z-discs (B, arrowheads). Very rarely, structures of dimensions approximating to those of thick filaments were present (box, B). (C,D) Rostral myotomes of 24 hpf embryos. The wild type has recognisable sarcomeres with almost straight Z-discs (white arrowhead, C) and more fully formed sarcoplasmic reticulum with triads (black arrowhead, C). In the slou45 mutant, I-Z-I brushes are formed [Z-discs (white arrowheads, D) flanked on either side by thin filaments], and a few putative thick filaments are evident (box, D). (E-H) At 48 hpf, wild-type muscle fibres are packed with mature myofibrils (E,G), possessing mature triads at each Z-disc (black arrowhead, G). Mutant muscle fibres contain no thick filaments but numerous I-Z-I brushes are evident (F,H). The Z-lines are surrounded by an electron-dense region (bracket, H; F); beyond this region the thin filaments have light striations on them (see inset, H). Triads are present, although less common (black arrowhead, H). (I) Schematic summarising the principal differences between slou45 mutant and sibling sarcomere ultrastructure. At early stages of myofibrillogenesis (top), mutants have less numerous and malformed thick filaments (red), and I-Z-I brushes (green and blue) are present but less well aligned than siblings. By 48 hpf, wild-type sarcomeres are fully formed, whereas in mutants only misaligned and mis-spaced I-Z-I brushes with striated thin filaments are present (blue). T-tubules and sarcoplasmic reticulum are shown in yellow and purple, respectively. Scale bars: 500 nm in A-D,G,H; 2 µm in E,F; 100 nm in H inset.

View larger version (21K): [in this window] [in a new window]   Fig. 3. The ATPase activity of Hsp90 is abrogated by the slou45 mutation. (A) Hsp90a domain structure (amino acid numbers of domain boundaries are shown beneath; known functions of regions are indicated by black bars). The positions and consequences of mutations in three slo alleles, u45, tu44c and tm201, are also illustrated beneath. The equivalent residue locations of yeast Hsp90 as represented in B are shown above. (B) Pymol diagram showing critical binding interactions of ADP with yeast Hsp90. Dotted blue lines are hydrogen bonds; amino acid residues (corresponding residue numbers in zebrafish are in parentheses and are also indicated in A) involved are in green; water molecules are cyan balls; residues packed against the C- atom of Gly83 are shown in cyan. Illustrated residues are conserved between yeast and zebrafish, except Ser138/Thr149 and Ser140/His151 (grey), which are conservative substitutions unlikely to cause major structural changes. Binding of ATP/ADP to the Hsp90 N-terminal domain involves highly conserved interactions including the carboxylate side-chain of Asp79 and main-chain carboxyl of Leu34, via a tightly bound water molecule, to the exocyclic N6 of adenine. The same Asp79 also interacts via another tightly bound water to the N1 imino-nitrogen of the adenine. This same water is bound by an interaction with the side-chain hydroxyl of Thr171 and main-chain amide of Gly83. An aspartic acid residue substitution at Gly83 (mimicking the u45 mutation) would lead to steric clashes likely to disrupt critical hydrogen bonding interactions with ADP/ATP. (C) ATPase activity of yeast Hsp90, human HSP90 and their u45-mimic mutants. ATPase activity of the yeast G83D and human G91D mutants is negligible relative to corresponding wild type.

View larger version (135K): [in this window] [in a new window]   Fig. 4. Wild-type Hsp90a rescues thick filament formation in slou45 muscle cells whereas Hsp90au45 does not. (A-D) Muscle fibres from a slou45 mutant (B,D) and wild-type (Sib) (A,C) injected with DNA encoding myc:WTHsp90a double stained for myc (red) and MHC (green). MHC staining in siblings decorates the cells with organised bands (C) indicative of mature A-bands. In the slou45 mutant, striations in the MHC staining are only evident in myc+ cells (D). (E,F) Muscle fibres in sibling embryos injected with myc-tagged hsp90au45 DNA show organised MHC staining of myc+ cells (E). Myc+ cells in slou45 mutants injected with the same construct do not show organised myofibrils (F). Scale bars: 100 µm in A,B; 10 µm in C-F.

View larger version (69K): [in this window] [in a new window]   Fig. 5. Abrogation of hsp90a but not hsp90a2 function leads to upregulation of genes encoding proteins likely to be involved in sarcomere assembly. (A-I) Lateral views, at the stage indicated bottom left, showing expression of the genes indicated bottom right in wild-type (Sib) and mutant zebrafish embryos. Note that although hspa8l and unc45b are upregulated in the slotu44c allele, hsp90a itself is not. (J) Western blots using lysates of slou45 and slotu44c mutant and sibling embryos. More Hsp90 protein was present in slou45 mutant lysates than in the corresponding siblings. By contrast, slotu44c mutant lysates contained less Hsp90 protein than siblings. (K,L) Quantitative PCR (qPCR) analyses of levels of hsp90a and hsp90a2 expression in wild type (Sibs), slou45 and slotu44c mutants (Muts). Absolute expression levels of hsp90a (normalised to µg of total RNA) were significantly increased (K; P=0.002, n=5) in slou45 mutants compared with siblings; levels of expression of hsp90a in slotu44c mutants were unchanged compared with siblings (K; P=0.75, n=3); and levels of expression of hsp90a2 were not significantly different between slou45mutants and siblings nor between slotu44c mutants and siblings (L; P=0.813, n=5 and P=0.074, n=3, respectively).

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