Christelle Etard

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

The mechanisms that regulate sarcomere assembly during myofibril formation are poorly understood. In this study, we characterise the zebrafish slothu45 mutant, in which the initial steps in sarcomere assembly take place, but thick filaments are absent and filamentous I-Z-I brushes fail to align or adopt correct spacing. The mutation only affects skeletal muscle and mutant embryos show no other obvious phenotypes. Surprisingly, we find that the phenotype is due to mutation in one copy of a tandemly duplicated hsp90a gene. The mutation disrupts the chaperoning function of Hsp90a through interference with ATPase activity. Despite being located only 2 kb from hsp90a, hsp90a2 has no obvious role in sarcomere assembly. Loss of Hsp90a function leads to the downregulation of genes encoding sarcomeric proteins and upregulation of hsp90a and several other genes encoding proteins that may act with Hsp90a during sarcomere assembly. Our studies reveal a surprisingly specific developmental role for a single Hsp90 gene in a regulatory pathway controlling late steps in sarcomere assembly.

Exome sequencing of Bardet–Biedl syndrome patient identifies a null mutation in the BBSome subunit BBIP1 (BBS18)

Background Bardet–Biedl syndrome (BBS) is a recessive and genetically heterogeneous ciliopathy characterised by retinitis pigmentosa, obesity, kidney dysfunction, postaxial polydactyly, behavioural dysfunction and hypogonadism. 7 of the 17 BBS gene products identified to date assemble together with the protein BBIP1/BBIP10 into the BBSome, a protein complex that ferries signalling receptors to and from cilia. Methods and results Exome sequencing performed on a sporadic BBS case revealed for the first time a homozygous stop mutation (NM_001195306: c.173T>G, p.Leu58*) in the BBIP1 gene. This mutation is pathogenic since no BBIP1 protein could be detected in fibroblasts from the patient, and BBIP1[Leu58*] is unable to associate with the BBSome subunit BBS4. Conclusions These findings identify BBIP1 as the 18th BBS gene (BBS18) and suggest that BBSome assembly may represent a unifying pathomechanism for BBS.

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

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.

Pontin and Reptin regulate cell proliferation in early Xenopus embryos in collaboration with c-Myc and Miz-1

Pontin (Tip49) and Reptin (Tip48) are highly conserved components of multimeric protein complexes important for chromatin remodelling and transcription. They interact with many different proteins including TATA box binding protein (TBP), beta-catenin and c-Myc and thus, potentially modulate different pathways. As antagonistic regulators of Wnt-signalling, they control wing development in Drosophila and heart growth in zebrafish. Here we show that the Xenopus xPontin and xReptin in conjunction with c-Myc regulate cell proliferation in early development. Overexpression of xPontin or xReptin results in increased mitoses and bending of embryos, which is mimicked by c-Myc overexpression. Furthermore, the knockdown of either xPontin or xReptin resulted in embryonic lethality at late gastrula stage, which is abrogated by the injection of c-Myc-RNA. The N-termini of xPontin and xReptin, which mediate the mitogenic effect were mapped to contain c-Myc interaction domains. c-Myc protein promotes cell cycle progression either by transcriptional activation through the c-Myc/Max complex or by repression of cyclin dependent kinase inhibitors (p21, p15) through c-Myc/Miz-1 interaction. Importantly, xPontin and xReptin exert their mitogenic effect through the c-Myc/Miz-1 pathway as dominant negative Miz-1 and wild-type c-Myc but not a c-Myc mutant deficient in Miz-1 binding could rescue embryonic lethality. Finally, promoter reporter studies revealed that xPontin and xReptin but not the N-terminal deletion mutants enhance p21 repression by c-Myc. We conclude that xPontin and xReptin are essential genes regulating cell proliferation in early Xenopus embryogenesis through interaction with c-Myc. We propose a novel function of xPontin and xReptin as co-repressors in the c-Myc/Miz-1 pathway.

Lack of Apobec2-related proteins causes a dystrophic muscle phenotype in zebrafish embryos

Apo2 proteins interact with chaperone Unc45b (but not Hsp90) and are required for correct zebrafish skeletal musculature and heart function.

Mutation in the δ-subunit of the nAChR suppresses the muscle defects caused by lack of Dystrophin

Normal motility of the zebrafish embryo requires a large number of gene loci, many of which have human orthologues implicated in myasthenias and other myopathies. We have identified a mutation in the zebrafish that abolishes body motility. Embryos have narrower myofibrils and lack clusters of nicotinic acetylcholine receptors (nAChRs) on the surface of the somitic muscle. We mapped the mutation to the δ‐subunit of the nAChR, showing this mutant to be a new allele of the previously named sofa potato (sop). The mutant allele carries a missense mutation in the extracellular domain altering the cysteine at position 150 to an arginine. The δ‐subunit is expressed in all striated muscles in embryonic and early larval stages together with the α1, β1, ϵ, and γ‐subunits of nAChR. In contrast to mammals that show switching from the γ embryonic to the adult ϵ‐subunit, the two subunits are coexpressed in zebrafish embryos. We, furthermore, demonstrated that the sop/δ‐nAChR mutation is a suppressor of the myopathy caused by lack of Dystrophin. The myofiber detachment phenotype of Dystroglycan‐deficient embryos was not suppressed, suggesting that Dystrophin and Dystroglycan play distinct roles in muscle formation and maintenance of muscle integrity. Developmental Dynamics 234:1016–1025, 2005.

A mutation in VPS15 (PIK3R4) causes a ciliopathy and affects IFT20 release from the cis-Golgi.

Ciliopathies are a group of diseases that affect kidney and retina among other organs. Here, we identify a missense mutation in PIK3R4 (phosphoinositide 3-kinase regulatory subunit 4, named VPS15) in a family with a ciliopathy phenotype. Besides being required for trafficking and autophagy, we show that VPS15 regulates primary cilium length in human fibroblasts, as well as ciliary processes in zebrafish. Furthermore, we demonstrate its interaction with the golgin GM130 and its localization to the Golgi. The VPS15-R998Q patient mutation impairs Golgi trafficking functions in humanized yeast cells. Moreover, in VPS15-R998Q patient fibroblasts, the intraflagellar transport protein IFT20 is not localized to vesicles trafficking to the cilium but is restricted to the Golgi. Our findings suggest that at the Golgi, VPS15 and GM130 form a protein complex devoid of VPS34 to ensure the IFT20-dependent sorting and transport of membrane proteins from the cis-Golgi to the primary cilium.

Mutations in TUBGCP4 Alter Microtubule Organization via the γ-Tubulin Ring Complex in Autosomal-Recessive Microcephaly with Chorioretinopathy

We have identified TUBGCP4 variants in individuals with autosomal-recessive microcephaly and chorioretinopathy. Whole-exome sequencing performed on one family with two affected siblings and independently on another family with one affected child revealed compound-heterozygous mutations in TUBGCP4. Subsequent Sanger sequencing was performed on a panel of individuals from 12 French families affected by microcephaly and ophthalmic manifestations, and one other individual was identified with compound-heterozygous mutations in TUBGCP4. One synonymous variant was common to all three families and was shown to induce exon skipping; the other mutations were frameshift mutations and a deletion. TUBGCP4 encodes γ-tubulin complex protein 4, a component belonging to the γ-tubulin ring complex (γ-TuRC) and known to regulate the nucleation and organization of microtubules. Functional analysis of individual fibroblasts disclosed reduced levels of the γ-TuRC, altered nucleation and organization of microtubules, abnormal nuclear shape, and aneuploidy. Moreover, zebrafish treated with morpholinos against tubgcp4 were found to have reduced head volume and eye developmental anomalies with chorioretinal dysplasia. In summary, the identification of TUBGCP4 mutations in individuals with microcephaly and a spectrum of anomalies in eye development, particularly photoreceptor anomalies, provides evidence of an important role for the γ-TuRC in brain and eye development.

Glycolysis supports embryonic muscle growth by promoting myoblast fusion

Significance We report that glycolytic genes are synchronously activated in midembryogenesis in developing Drosophila muscles and that muscle-targeted attenuation of their expression leads to reduced muscle size and the presence of unfused myoblasts. Importantly, a “thin muscle” phenotype is also observed in fast muscle fibers of pgam2 zebrafish morphants, strongly suggesting a conserved role of glycolysis in promoting myoblast fusion-based muscle growth. We also show that insulin positively regulates glycolysis and that blocking the insulin pathway phenocopies the loss of function phenotypes of glycolytic genes, leading to myoblast fusion arrest and reduced muscle size. These findings led us to propose that setting metabolism to glycolysis-based high-rate biomass production is part of a core myogenic program that promotes formation of syncytial muscles. Muscles ensure locomotion behavior of invertebrate and vertebrate organisms. They are highly specialized and form using conserved developmental programs. To identify new players in muscle development we screened Drosophila and zebrafish gene expression databases for orthologous genes expressed in embryonic muscles. We selected more than 100 candidates. Among them is the glycolysis gene Pglym78/pgam2, the attenuated expression of which results in the formation of thinner muscles in Drosophila embryos. This phenotype is also observed in fast muscle fibers of pgam2 zebrafish morphants, suggesting affected myoblast fusion. Indeed, a detailed analysis of developing muscles in Pglym78 RNAi embryos reveals loss of fusion-associated actin foci and an inefficient Notch decay in fusion competent myoblasts, both known to be required for fusion. In addition to Pglym78, our screen identifies six other genes involved in glycolysis or in pyruvate metabolism (Pfk, Tpi, Gapdh, Pgk, Pyk, and Impl3). They are synchronously activated in embryonic muscles and attenuation of their expression leads to similar muscle phenotypes, which are characterized by fibers with reduced size and the presence of unfused myoblasts. Our data also show that the cell size triggering insulin pathway positively regulates glycolysis in developing muscles and that blocking the insulin or target of rapamycin pathways phenocopies the loss of function phenotypes of glycolytic genes, leading to myoblast fusion arrest and reduced muscle size. Collectively, these data suggest that setting metabolism to glycolysis-stimulated biomass production is part of a core myogenic program that operates in both invertebrate and vertebrate embryos and promotes formation of syncytial muscles.

Loss of function of myosin chaperones triggers Hsf1-mediated transcriptional response in skeletal muscle cells

BackgroundMutations in myosin chaperones Unc45b and Hsp90aa1.1 as well as in the Unc45b-binding protein Smyd1b impair formation of myofibrils in skeletal muscle and lead to the accumulation of misfolded myosin. The concomitant transcriptional response involves up-regulation of the three genes encoding these proteins, as well as genes involved in muscle development. The transcriptional up-regulation of unc45b, hsp90aa1.1 and smyd1b is specific to zebrafish mutants with myosin folding defects, and is not triggered in other zebrafish myopathy models.ResultsBy dissecting the promoter of unc45b, we identify a Heat shock factor 1 (Hsf1) binding element as a mediator of unc45b up-regulation in myofibers lacking myosin folding proteins. Loss-of-function of Hsf1 abolishes unc45b up-regulation in mutants with defects in myosin folding.ConclusionsTaken together, our data show that skeletal muscle cells respond to defective myosin chaperones with a complex gene program and suggest that this response is mediated by Hsf1 activation.