As part of a multi-subunit ring complex, γ-tubulin has been shown to promote microtubule nucleation both in vitro and in vivo, and the structural properties of the complex suggest that it also seals the minus ends of the polymers with a conical cap. Cells depleted of γ-tubulin, however, still display many microtubules that participate in mitotic spindle assembly, suggesting that γ-tubulin is not absolutely required for microtubule nucleation in vivo, and raising questions about the function of the minus end cap. Here, we assessed the role of γ-tubulin in centrosomal microtubule organisation using three-dimensional reconstructions of γ-tubulin-depleted C. elegans embryos. We found that microtubule minus-end capping and the PCM component SPD-5 are both essential for the proper placement of microtubules in the centrosome. Our results further suggest that γ-tubulin and SPD-5 limit microtubule polymerization within the centrosome core, and we propose a model for how abnormal microtubule organization at the centrosome could indirectly affect centriole structure and daughter centriole replication.
ABSTRACT Purpose Better methods are required to interpret the pathogenicity of disease-associated variants of uncertain significance (VUS), which cannot be actioned clinically. In this study, we explore the use of a tractable animal model ( Caenorhabditis elegans ) for in vivo interpretation of missense VUS alleles of TMEM67 , a cilia gene associated with ciliopathies. Methods CRISPR/Cas9 gene editing was used to generate homozygous knock-in C. elegans worm strains carrying TMEM67 patient variants engineered into the orthologous gene ( mks-3 ). Quantitative phenotypic assays of sensory cilia structure and function measured if the variants affect mks-3 gene function. Results from worms were validated by a genetic complementation assay in a human TMEM67 knock-out hTERT-RPE1 cell line that tests a TMEM67 signaling function. Results Assays in C. elegans accurately distinguished between known benign (Asp359Glu, Thr360Ala) and known pathogenic (Glu361Ter, Gln376Pro) variants. Analysis of eight missense VUS generated evidence that three are benign (Cys173Arg, Thr176Ile, Gly979Arg) and five are pathogenic (Cys170Tyr, His782Arg, Gly786Glu, His790Arg, Ser961Tyr). Conclusion Efficient genome editing and quantitative functional assays in C. elegans make it a tractable in vivo animal model that allows rapid, cost-effective interpretation of ciliopathy-associated missense VUS alleles.
Significance Mutations in complement factor H (CFH) binding sites for heparan sulfate (HS) increase risk for age-related macular degeneration (AMD), but the roles of CFH and HS in disease pathogenesis remain unclear. Here, we find that a CFH homolog, HS modification, and the HS proteoglycan syndecan/SDN-1 have roles in maintaining cilia compartment boundaries in the sensory neurons of aging C. elegans adults. The role of CFH in cilia compartment boundaries is conserved in vertebrate photoreceptors, suggesting that structural defects in photoreceptor cilia make a contribution to AMD progression in patients with CFH mutations that has not been appreciated previously.
Abstract During mitosis, kinetochore–microtubule interactions ensure that chromosomes are accurately segregated to daughter cells. RSA-1 (regulator of spindle assembly-1) is a regulatory B″ subunit of protein phosphatase 2A that was previously proposed to modulate microtubule dynamics during spindle assembly. We have identified a genetic interaction between the centrosomal protein, RSA-1, and the spindle- and kinetochore-associated (Ska) complex in Caenorhabditis elegans. In a forward genetic screen for suppressors of rsa-1(or598) embryonic lethality, we identified mutations in ska-1 and ska-3. Loss of SKA-1 and SKA-3, as well as components of the KMN (KNL-1/MIS-12/NDC-80) complex and the microtubule end-binding protein EBP-2, all suppressed the embryonic lethality of rsa-1(or598). These suppressors also disrupted the intracellular localization of the Ska complex, revealing a network of proteins that influence Ska function during mitosis. In rsa-1(or598) embryos, SKA-1 is excessively and prematurely recruited to kinetochores during spindle assembly, but SKA-1 levels return to normal just prior to anaphase onset. Loss of the TPX2 homolog, TPXL-1, also resulted in overrecruitment of SKA-1 to the kinetochores and this correlated with the loss of Aurora A kinase on the spindle microtubules. We propose that rsa-1 regulates the kinetochore localization of the Ska complex, with spindle-associated Aurora A acting as a potential mediator. These data reveal a novel mechanism of protein phosphatase 2A function during mitosis involving a centrosome-based regulatory mechanism for Ska complex recruitment to the kinetochore.
Abstract Better methods are required to interpret the pathogenicity of disease-associated variants of uncertain significance (VUS), which cannot be actioned clinically. In this study, we explore the use of an animal model (Caenorhabditis elegans) for in vivo interpretation of missense VUS alleles of TMEM67, a cilia gene associated with ciliopathies. CRISPR/Cas9 gene editing was used to generate homozygous knock-in C. elegans worm strains carrying TMEM67 patient variants engineered into the orthologous gene (mks-3). Quantitative phenotypic assays of sensory cilia structure and function (neuronal dye filling, roaming and chemotaxis assays) measured how the variants impacted mks-3 gene function. Effects of the variants on mks-3 function were further investigated by looking at MKS-3::GFP localization and cilia ultrastructure. The quantitative assays in C. elegans accurately distinguished between known benign (Asp359Glu, Thr360Ala) and known pathogenic (Glu361Ter, Gln376Pro) variants. Analysis of eight missense VUS generated evidence that three are benign (Cys173Arg, Thr176Ile and Gly979Arg) and five are pathogenic (Cys170Tyr, His782Arg, Gly786Glu, His790Arg and Ser961Tyr). Results from worms were validated by a genetic complementation assay in a human TMEM67 knock-out hTERT-RPE1 cell line that tests a TMEM67 signalling function. We conclude that efficient genome editing and quantitative functional assays in C. elegans make it a tractable in vivo animal model for rapid, cost-effective interpretation of ciliopathy-associated missense VUS alleles.
ABSTRACT Ciliopathies are inherited disorders caused by defects in motile and non-motile (primary) cilia. Ciliopathy syndromes and associated gene variants are often highly pleiotropic and represent exemplars for interrogating genotype-phenotype correlations. Towards understanding disease mechanisms in the context of ciliopathy mutations, we have used a leading model organism for cilia and ciliopathy research, Caenorhabditis elegans, together with gene editing, to characterise two missense variants (P74S and G155S) in mksr-2/B9D2 associated with Joubert syndrome (JBTS). B9D2 functions within the Meckel syndrome (MKS) module at the ciliary base transition zone (TZ) compartment and regulates the molecular composition and sensory/signalling functions of the cilium. Quantitative assays of cilium/TZ structure and function, together with knock-in reporters, confirm that both variant alleles are pathogenic in worms. G155S causes a more severe overall phenotype and disrupts endogenous MKSR-2 organisation at the TZ. Recapitulation of the patient biallelic genotype shows that compound heterozygous worms phenocopy worms homozygous for P74S. The P74S and G155S alleles also reveal evidence of a very close functional association between the B9D2-associated B9 complex and MKS-2/TMEM216. Together, these data establish C. elegans as a model for interpreting JBTS mutations and provide further insight into MKS module organisation. This article has an associated First Person interview with the first author of the paper.
Summary Protein phosphorylation and dephosphorylation is a key mechanism for the spatial and temporal regulation of many essential developmental processes and is especially prominent during mitosis. The multi-subunit protein phosphatase 2A (PP2A) enzyme plays an important, yet poorly characterized role in dephosphorylating proteins during mitosis. PP2As are heterotrimeric complexes comprising a catalytic, structural, and regulatory subunit. Regulatory subunits are mutually exclusive and determine subcellular localization and substrate specificity of PP2A. At least 3 different classes of regulatory subunits exist (termed B, B′, B″) but there is no obvious similarity in primary sequence between these classes. Therefore, it is not known how these diverse regulatory subunits interact with the same holoenzyme to facilitate specific PP2A functions in vivo. The B″ family of regulatory subunits is the least understood because these proteins lack conserved structural domains. RSA-1 (regulator of spindle assembly) is a regulatory B″ subunit required for mitotic spindle assembly in Caenorhabditis elegans. In order to address how B″ subunits interact with the PP2A core enzyme, we focused on a conditional allele, rsa-1(or598ts), and determined that this mutation specifically disrupts the protein interaction between RSA-1 and the PP2A structural subunit, PAA-1. Through genetic screening, we identified a putative interface on the PAA-1 structural subunit that interacts with a defined region of RSA-1/B″. In the context of previously published results, these data propose a mechanism of how different PP2A B-regulatory subunit families can bind the same holoenzyme in a mutually exclusive manner, to perform specific tasks in vivo.
ABSTRACT Ciliopathies are inherited disorders caused by cilia defects. Variants in ciliopathy genes are frequently pleiotropic and represent excellent case studies for interrogating genotype-phenotype correlation. We have employed Caenorhabditis elegans and gene editing to characterise two pathogenic biallelic missense variants (P74S, G155S) in B9D2/ mksr-2 associated with Joubert Syndrome (JBTS). B9D2 functions within the MKS module at the transition zone (TZ) ciliary subcompartment, and regulates the cilium’s molecular composition and signaling function. Quantitative assays of cilium/TZ structure and function, together with knock-in reporters, confirm both variant alleles are pathogenic. G155S causes a more severe overall phenotype and disrupts endogenous MKSR-2 organisation at the TZ. Recapitulation of the patient biallelic genotype shows that heterozygous worms phenocopy worms homozygous for P74S. This study also reveals a close functional association between the B9 complex and TMEM216/MKS-2. These data establish C. elegans as a paradigm for interpreting JBTS mutations, and provide insight into MKS module organisation.
As part of a multi-subunit ring complex, c-tubulin has been shown to promote microtubule nucleation both in vitro and in vivo, and the structural properties of the complex suggest that it also seals the minus ends of the polymers with a conical cap.Cells depleted of c-tubulin, however, still display many microtubules that participate in mitotic spindle assembly, suggesting that c-tubulin is not absolutely required for microtubule nucleation in vivo, and raising questions about the function of the minus end cap.Here, we assessed the role of c-tubulin in centrosomal microtubule organisation using threedimensional reconstructions of c-tubulin-depleted C. elegans embryos.We found that microtubule minus-end capping and the PCM component SPD-5 are both essential for the proper placement of microtubules in the centrosome.Our results further suggest that c-tubulin and SPD-5 limit microtubule polymerization within the centrosome core, and we propose a model for how abnormal microtubule organization at the centrosome could indirectly affect centriole structure and daughter centriole replication.
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