Mutations in TUBG1, DYNC1H1, KIF5C and KIF2A cause malformations of cortical development and microcephaly

The genetic causes of malformations of cortical development (MCD) remain largely unknown. Here we report the discovery of multiple pathogenic missense mutations in TUBG1, DYNC1H1 and KIF2A, as well as a single germline mosaic mutation in KIF5C, in subjects with MCD. We found a frequent recurrence of mutations in DYNC1H1, implying that this gene is a major locus for unexplained MCD. We further show that the mutations in KIF5C, KIF2A and DYNC1H1 affect ATP hydrolysis, productive protein folding and microtubule binding, respectively. In addition, we show that suppression of mouse Tubg1 expression in vivo interferes with proper neuronal migration, whereas expression of altered gamma-tubulin proteins in Saccharomyces cerevisiae disrupts normal microtubule behavior. Our data reinforce the importance of centrosomal and microtubule-related proteins in cortical development and strongly suggest that microtubule-dependent mitotic and postmitotic processes are major contributors to the pathogenesis of MCD

Mutations in TUBG1, DYNC1H1, KIF5C and KIF2A cause malformations of cortical development and microcephaly.

International audienceThe genetic causes of malformations of cortical development (MCD) remain largely unknown. Here we report the discovery of multiple pathogenic missense mutations in TUBG1, DYNC1H1 and KIF2A, as well as a single germline mosaic mutation in KIF5C, in subjects with MCD. We found a frequent recurrence of mutations in DYNC1H1, implying that this gene is a major locus for unexplained MCD. We further show that the mutations in KIF5C, KIF2A and DYNC1H1 affect ATP hydrolysis, productive protein folding and microtubule binding, respectively. In addition, we show that suppression of mouse Tubg1 expression in vivo interferes with proper neuronal migration, whereas expression of altered γ-tubulin proteins in Saccharomyces cerevisiae disrupts normal microtubule behavior. Our data reinforce the importance of centrosomal and microtubule-related proteins in cortical development and strongly suggest that microtubule-dependent mitotic and postmitotic processes are major contributors to the pathogenesis of MCD

L'ENIGME DU CYCLE DE TYROSINATION: DES REPONSES DANS LA LEVURE

Les travaux et les projets que je compte mener utilisent les levures produisant une tubuline alpha sans le dernier acide aminé (tubuline Glu) pour comprendre pourquoi les microtubules Glu lient moins bien la protéine Biklp/CLIPl70, en quoi ce défaut affecte les relations entre microtubules et cortex et entre microtubules et polarisation cellulaire. Les études chez les mammifères ont permis de mettre en avant l'importance du cycle de tyrosination dans la différentiation neuronale (Erck et a1.,2005). Il est reconnu que la cellule nerveuse est une cellule hautement polarisée, nous espérons que nos études chez la levure permettront d'apporter des informations sur le fonctionnement des microtubules Glu lors de la polarisation cellulaire. Par ailleurs il a aussi été montré que la TTL est un suppresseur de tumeur. Nous souhaitons comprendre le fonctionnement de Biklp/CLIPl70 dans le déroulement d'une mitose dans les levures Glu et les relations entre protéines desextrémités + des microtubules et le kinétochore. Enfin, une dernière approche plus technologique consistera en la recherche de drogues spécifiques des protéines Biklp/CLIPl70 pax une méthode originale

L'ENIGME DU CYCLE DE TYROSINATION: DES REPONSES DANS LA LEVURE

Les travaux et les projets que je compte mener utilisent les levures produisant une tubuline alpha sans le dernier acide aminé (tubuline Glu) pour comprendre pourquoi les microtubules Glu lient moins bien la protéine Biklp/CLIPl70, en quoi ce défaut affecte les relations entre microtubules et cortex et entre microtubules et polarisation cellulaire. Les études chez les mammifères ont permis de mettre en avant l'importance du cycle de tyrosination dans la différentiation neuronale (Erck et a1.,2005). Il est reconnu que la cellule nerveuse est une cellule hautement polarisée, nous espérons que nos études chez la levure permettront d'apporter des informations sur le fonctionnement des microtubules Glu lors de la polarisation cellulaire. Par ailleurs il a aussi été montré que la TTL est un suppresseur de tumeur. Nous souhaitons comprendre le fonctionnement de Biklp/CLIPl70 dans le déroulement d'une mitose dans les levures Glu et les relations entre protéines desextrémités + des microtubules et le kinétochore. Enfin, une dernière approche plus technologique consistera en la recherche de drogues spécifiques des protéines Biklp/CLIPl70 pax une méthode originale

Tubulin mutations in neurodevelopmental disorders as a tool to decipher microtubule function

International audienceMalformations of Cortical Development (MCDs) are a group of severe brain malformations associated with intellectual disability and refractory childhood epilepsy. Human missense heterozygous mutations in the 9 α-tubulin and 10 β-tubulin isoforms forming the heterodimers that assemble into microtubules (MTs) were found to cause MCDs. However, how a single mutated residue in a given tubulin isoform can perturb the entire microtubule population in a neuronal cell remains a crucial question. Here, we examined 85 MCD-associated tubulin mutations occurring in TUBA1A, TUBB2 and TUBB3 and their location in a three-dimensional (3D) microtubule cylinder. Mutations hitting residues exposed on the outer microtubule surface are likely to alter microtubule association with partners, while alteration of intra-dimer contacts may impair dimer stability and straightness. Other types of mutations are predicted to alter inter-dimer and lateral contacts, which are responsible for microtubule cohesion, rigidity and dynamics. MCD-associated tubulin mutations surprisingly fall into all categories, thus providing unexpected insights into how a single mutation may impair microtubule function and elicit dominant effects in neurons

CAP-Gly proteins contribute to microtubule-dependent trafficking via interactions with the C-terminal aromatic residue of α-tubulin

International audienc

A new role for kinesin-directed transport of Bik1p (CLIP-170) in Saccharomyces cerevisiae.

International audienceBik1p is the budding yeast counterpart of the CLIP-170 family of microtubule plus-end tracking proteins, which are required for dynein localization at plus ends and dynein-dependent spindle positioning. CLIP-170 proteins make up a CAP-Gly microtubule-binding domain, which sustains their microtubule plus-end tracking behaviour. However, in yeast, Bik1p travels towards plus ends as a cargo of the plus-end-directed kinesin Kip2p. Additionally, Kip2p behaves as a plus-end-tracking protein; hence, it has been proposed that Bik1p might track plus ends principally as a cargo of Kip2p. Here, we examined Bik1p localization in yeast strains expressing mutant tubulin lacking the C-terminal amino acid (Glu tubulin; lacking Phe), the interaction of which with Bik1p is severely impaired compared with wild type. In Glu-tubulin strains, despite the presence of robust Kip2p comets at microtubule plus ends, Bik1p failed to track plus ends. Despite Bik1p depletion at plus ends, dynein positioning at the same plus ends was unperturbed. Video microscopy and genetic evidence indicated that dynein was transported at plus ends in a Kip2p-Bik1p-dependent manner, and was then capable of tracking Bik1p-depleted plus ends. These results indicate that Bik1p interactions with tubulin are important for Bik1p plus-end tracking, and suggest alternative pathways for Bik1p-Kip2p-dependent dynein localization at plus ends

A neurodevelopmental TUBB2B β-tubulin mutation impairs Bim1 (yeast EB1)-dependent spindle positioning

Malformations of the human cerebral cortex can be caused by mutations in tubulins that associate to compose microtubules. Cerebral cortical folding relies on neuronal migration and on progenitor proliferation partly dictated by microtubule-dependent mitotic spindle positioning. A single amino acid change, F265L, in the conserved TUBB2B β-tubulin gene has been identified in patients with abnormal cortex formation. A caveat for studying this mutation in mammalian cells is that nine genes encode β-tubulin in human. Here, we generate a yeast strain expressing F265L tubulin mutant as the sole source of β-tubulin. The F265L mutation does not preclude expression of a stable β-tubulin protein which is incorporated into microtubules. However, impaired cell growth was observed at high temperatures along with altered microtubule dynamics and stability. In addition, F265L mutation produces a highly specific mitotic spindle positioning defect related to Bim1 (yeast EB1) dysfunction. Indeed, F265L cells display an abnormal Bim1 recruitment profile at microtubule plus-ends. These results indicate that the F265L β-tubulin mutation affects microtubule plus-end complexes known to be important for microtubule dynamics and for microtubule function during mitotic spindle positioning

A novel mass assay to quantify the bioactive lipid PtdIns3P in various biological samples.

International audiencePtdIns3P is recognized as an important player in the control of the endocytotic pathway and in autophagy. Recent data also suggest that PtdIns3P contributes to molecular mechanisms taking place at the plasma membrane and at the midbody during cytokinesis. This lipid is present in low amounts in mammalian cells and remains difficult to quantify either by traditional techniques based on radiolabelling followed by HPLC to separate the different phosphatidylinositol monophosphates, or by high-sensitive liquid chromatography coupled to MS, which is still under development. In the present study, we describe a mass assay to quantify this lipid from various biological samples using the recombinant PtdIns3P 5-kinase, PIKfyve. Using this assay, we show an increase in the mass level of PtdIns3P in mouse and human platelets following stimulation, loss of this lipid in Vps34-deficient yeasts and its relative enrichment in early endosomes isolated from BHK cells

An inducible ESCRT-III inhibition tool to control HIV-1 budding

Abstract HIV-1 budding as well as many other cellular processes require the Endosomal Sorting Complex Required for Transport (ESCRT) machinery. Understanding the architecture of the native ESCRT-III complex at HIV-1 budding sites is limited due to spatial resolution and transient ESCRT-III recruitment. Here, we developed a drug-inducible transient HIV-1 budding inhibitory tool to enhance the ESCRT-III lifetime at budding sites. We generated auto-cleavable CHMP2A, CHMP3, and CHMP4B fusion proteins with the hepatitis C virus NS3 protease. We characterized the CHMP-NS3 fusion proteins in the absence and presence of protease inhibitor Glecaprevir with regard to expression, stability, localization and HIV-1 Gag VLP budding. Immunoblotting experiments revealed rapid and stable accumulation of CHMP-NS3 fusion proteins with variable modification of Gag VLP budding upon drug administration. Notably, CHMP2A-NS3 and CHMP4B-NS3 fusion proteins substantially decrease VLP release while CHMP3-NS3 exerted a minor effect and synergized with CHMP2A-NS3. Localization studies demonstrated the re-localization of CHMP-NS3 fusion proteins to the plasma membrane, endosomes, and Gag VLP budding sites. Through the combined use of transmission electron microscopy and video-microscopy, we unveiled drug-dependent accumulation of CHMP2A-NS3 and CHMP4B-NS3, causing a delay in HIV-1 Gag-VLP release. Our findings provide novel insight into the functional consequences of inhibiting ESCRT-III during HIV-1 budding and establish new tools to decipher the role of ESCRT-III at HIV-1 budding sites and other ESCRT-catalyzed cellular processes