Gene organization, evolution and expression of the microtubule-associated protein ASAP (MAP9)

International audienceBackground : ASAP is a newly characterized microtubule-associated protein (MAP) essential for propercell-cycling. We have previously shown that expression deregulation of human ASAP results in profounddefects in mitotic spindle formation and mitotic progression leading to aneuploidy, cytokinesis defects and/or cell death. In the present work we analyze the structure and evolution of the ASAP gene, as well as thedomain composition of the encoded protein. Mouse and Xenopus cDNAs were cloned, the tissueexpression characterized and the overexpression profile analyzed. Results : Bona fide ASAP orthologs are found in vertebrates with more distantly related potentialorthologs in invertebrates. This single-copy gene is conserved in mammals where it maps to syntenicchromosomal regions, but is also clearly identified in bird, fish and frog. The human gene is stronglyexpressed in brain and testis as a 2.6 Kb transcript encoding a ~110 KDa protein. The protein containsMAP, MIT-like and THY domains in the C-terminal part indicative of microtubule interaction, while the N-terminal part is more divergent. ASAP is composed of ~42% alpha helical structures, and two main coiled-coil regions have been identified. Different sequence features may suggest a role in DNA damage response. As with human ASAP, the mouse and Xenopus proteins localize to the microtubule network in interphaseand to the mitotic spindle during mitosis. Overexpression of the mouse protein induces mitotic defectssimilar to those observed in human. In situ hybridization in testis localized ASAP to the germ cells, whereasin culture neurons ASAP localized to the cell body and growing neurites. Conclusion : The conservation of ASAP indicated in our results reflects an essential function invertebrates. We have cloned the ASAP orthologs in mouse and Xenopus, two valuable models to studythe function of ASAP. Tissue expression of ASAP revealed a high expression in brain and testis, two tissuesrich in microtubules. ASAP associates to the mitotic spindle and cytoplasmic microtubules, and representsa key factor of mitosis with possible involvement in other cell cycle processes. It may have a role inspermatogenesis and also represents a potential new target for antitumoral drugs. Possible involvement inneuron dynamics also highlights ASAP as a candidate target in neurodegenerative disease

ASAP, une nouvelle protèine du fuseau mitotique (étude d'un partenaire, la kinase Aurora-A et implication durant le cycle cellulaire)

Le laboratoire a caractérisé une nouvelle protéine associée aux microtubules (MAP) interphasique et au fuseau mitotique, appelée ASAP (ASter Associated Protein) ou MAP9. La dérégulation de son expression entraîne de sévères défauts mitotiques : fuseaux anormaux, retard de la progression mitotique avec des défauts de congression et de ségrégation des chromosomes aboutissant à une cytokinèse défectueuse, des cellules aneuploïdes et à la mort cellulaire. ASAP est donc nécessaire à l assemblage du fuseau et au bon déroulement de la mitose. Les mécanismes contrôlant la mitose et l assemblage du fuseau sont très finement régulés par phosphorylation assurées par plusieurs familles de kinases parmi lesquelles la kinase Aurora-A qui recrute et phosphoryle de nombreuses MAPs. Mon projet principal de recherche consistait à déterminer le lien fonctionnel entre ASAP et Aurora-A. J ai montré qu ASAP était phosphorylée par Aurora-A, caractérisé le site majeur de cette phosphorylation in vivo sur la sérine 625 et montré que cette phosphorylation était essentielle à l assemblage du fuseau mitotique et à la progression correcte de la mitose. Par ailleurs, la déplétion d Aurora-A induit la dégradation d ASAP par le protéasome suggérant que cette interaction et/ou phosphorylation est nécessaire à la stabilisation d ASAP. Ce travail a ainsi permis de caractériser le premier partenaire d ASAP, de confirmer son rôle crucial au cours de la mitose et d en déterminer les premiers mécanismes moléculaires. Enfin, la caractérisation de l orthologue de souris et de son expression tissulaire m a permis de valider le modèle murin comme modèle d étude de différentes fonctions biologiques d ASAP.MONTPELLIER-BU Pharmacie (341722105) / SudocSudocFranceF

ASAP (Identification et caractérisation d'une nouvelle protéine associée aux microtubules)

Au cours de la division cellulaire, la mise en place d un fuseau bipolaire correct est nécessaire au maintien de la stabilité génomique, en permettant une ségrégation égale des chromosomes dans les deux cellules filles. Les microtubules, éléments constitués d un assemblage de tubuline sous une forme cylindrique et creuse, sont les constituants de base de ce fuseau mitotique. Lors de l assemblage du fuseau, les centrosomes se séparent, et des changements surviennent au niveau de la dynamique des microtubules. Cependant, sans les propriétés fonctionnelles d autres protéines venant réguler cette dynamique, les microtubules ne seraient pas capables d accomplir seuls les tâches qui leurs sont attribuées. Les protéines responsables de cette régulation sont les Protéines associées aux Microtubules ou MAPs. Des variations de la stabilité et de la dynamique des microtubules sont susceptibles d atteindre la formation et l organisation du fuseau mitotique ainsi que l attachement des chromosomes, et peuvent provoquer des problèmes d instabilité génétique et la cancérogenèse. Nous avons identifié au laboratoire une nouvelle protéine humaine associée aux microtubules que nous avons appelé ASAP pour ASter-Associated Protein. La protéine ASAP est associée au fuseau mitotique. Sa surexpression entraîne des mitoses aberrantes et abortives (fuseaux multipolaires et cellules multinucléées avec une amplification des centrosomes, fuseaux monopolaires avec non séparation/migration des centrosomes). Ces phénotypes sont fréquemment observés dans des cancers. Sa déplétion provoque des fuseaux anormaux, retarde la mitose en entraînant des défauts de congression et de ségrégation des chromosomes et aboutit à des cytokinèses abortives ou à la mort cellulaire. ASAP est donc une nouvelle MAP jouant un rôle crucial au cours du cycle cellulaire. Une étude plus ciblée sur son implication dans la régulation de la dynamique et de l organisation des microtubules a ensuite été entreprise afin de mieux comprendre l impact d ASAP au niveau du réseau de microtubules. Nous avons ainsi pu mettre en évidence une implication étendue de la protéine, tant au niveau des différentes phases dynamiques de la polymérisation, qu au niveau de l assemblage et de l organisation des microtubules. Nous avons également clairement mis en évidence la présence d ASAP aux centrosomes, avec la possibilité pour cette protéine de participer à la nucléation des microtubules, un rôle qu il reste encore cependant à démontrer in vivo.MONTPELLIER-BU Pharmacie (341722105) / SudocPARIS-BIUP (751062107) / SudocSudocFranceF

Olfactory receptors

Olfaction is an ancient sensory system allowing an organism to detect chemicals in its environment. The first step in odor transduction is mediated by binding odorants to olfactory receptors (ORs) which belong to the heptahelical G-protein-coupled receptor (GPCR) superfamily. Mammalian ORs are disposed in clusters on virtually all chromosomes. They are encoded by the largest multigene family (∼1000 members) in the genome of mammals and Caenorhabditis elegans, whereas Drosophila contains only 60 genes. Each OR specifically recognizes a set of odorous molecules that share common molecular features. In mammals, signal transduces through the G-protein-dependent signal pathway in the olfactory sensory neurons that synapse ultimately in the glomeruli of the olfactory bulb, and is finally processed in higher brain structures. The expression of a given OR conditions neuron and glomerulus choices. To date, the processes which monitor OR expression and axon wiring have emerged but are not completely elucidated

Microtubule-associated protein 9 (Map9/Asap) is required for the early steps of zebrafishdevelopment

International audienceMicrotubules are structural components of the cell cytoskeleton and key factors for mitosis and ciliogenesis in eukaryotes. The regulation of MT dynamics requires non-motor MAPs. We previously showed that, in human cells in culture, MAP9 (also named ASAP) is involved in MT dynamics and is essential for mitotic spindle formation and mitosis progression. Indeed, misexpression of MAP9 leads to severe mitotic defects and cell death. Here, we investigated the in vivo role of map9 during zebrafish development. Map9 is expressed mainly as a maternal gene. Within cells, Map9 is associated with the MT network of the mitotic spindle and with centrosomes. Morpholino-mediated depletion of map9 leads to early development arrest before completion of epiboly. Map9 localizes to the MT array of the YSL. This MT network is destroyed in Map9-depleted embryos, and injection of anti-map9 morpholinos directly in the nascent YSL leads to arrest of epiboly/gastrulation. Finally, map9 knockdown deregulates the expression of genes involved in endodermal differentiation, dorso–ventral and left–right patterning, and other MT-based functions. At low morpholino doses, the surviving embryos show dramatic developmental defects, spindle and mitotic defects, and increased apoptosis. Our findings suggest that map9 is a crucial factor in early zebrafish development by regulating different MT-based processes

Expansion of the BPI family by duplication on human chromosome 20: characterization of the RY gene cluster in 20q11.21 encoding olfactory transporters/antimicrobial-like peptides☆

Antimicrobial peptides provide a defense system against microorganisms. One class of these molecules binds lipophilic substrates and is therefore directed against gram-negative bacteria. This family includes proteins related to bactericidal/permeability-increasing protein (BPI). We characterized an approximately 100-kb cluster of three human genes named RYSR, RYA3, and RY2G5 that are related to the BPI family. The RY cluster maps to 20q11.21, >5 Mb upstream of the BPI cluster. The RY and BPI genes have similar exon structures, indicating that they were derived by duplication from a common ancestor. We identified mouse BPI-related and RY orthologues in syntenic regions, indicating that the gene family expanded before mouse and human diverged. Expression analyses show that RYs are strongly expressed in the olfactory epithelium, suggesting that they also could act as odorant transporters or detoxification agents in the olfactory system. Together, these data show how mammals diversified their antimicrobial defenses/olfactory pathways through a duplication-driven adaptive selection process

Expression of the Microtubule-Associated Protein MAP9/ASAP and Its Partners AURKA and PLK1 in Colorectal and Breast Cancers

Background. Colorectal and breast cancers are among the most common cancers worldwide. They result from a conjugated deficiency of gene maintenance and cell cycle control. Objective. We investigate the expression of the microtubule-associated protein MAP9/ASAP and its two partners AURKA and PLK1 in colorectal tumors as well as in ductal breast cancers. Materials and Methods. 26 colorectal cancer samples and adjacent normal tissues and 77 ductal breast cancer samples from grade I to grade III were collected. Real-time quantitative PCR was used to analyse the expression of MAP9, AURKA, and PLK1. Results. Expression of MAP9 is downregulated in colorectal cancer compared to normal tissues (P>10-3), whereas those of AURKA and PLK1 are upregulated (P>10-4). In ductal breast cancer, we found a grade-dependent increase of AURKA expression (P>10-3), while the variations of expression of MAP9 and PLK1 are not significant (P>0.2). Conclusions. MAP9 downregulation is associated with colorectal malignancy and could be used as a disease marker and a new drug target, while AURKA and PLK1 are upregulated. In ductal breast cancer, AURKA overexpression is strongly associated with the tumor grade and is therefore of prognostic value for the progression of the disease