Dynamics of Multiple Nuclei in Ashbya gossypii Hyphae Depend on the Control of Cytoplasmic Microtubules Length by Bik1, Kip2, Kip3, and Not on a Capture/Shrinkage Mechanism

The multinucleate growth mode of A. gossypii has resulted in a unique control of cytoplasmic MT dynamics. Our analyses of MT +tips behavior and cMT–cell cortex interactions show the necessity of A. gossypii to produce very long cMTs for nuclear migration to compensate the lack of MT capture/shrinkage mechanisms important in budding yeast

ETUDE FONCTIONNELLE DE LA PROTEINE APPARENTEE A L'ACTINE ARP5 ET D'UN DE SES PARTENAIRES CELLULAIRES AFILP, DANS LA LEVURE SACCHAROMYCES CEREVISIAE

CE TRAVAIL DE THESE PORTE SUR LA CARACTERISATION FONCTIONNELLE DE LA PROTEINE ARP5. LE GENE ARP5 (YNL059C) A ETE IDENTIFIE AU COURS DU SEQUENCAGE SYSTEMATIQUE DU GENOME DE LA LEVURE S. CEREVISIAE EN 1995. IL CODE POUR UNE PROTEINE HYPOTHETIQUE DE 755 AA QUI PRESENTE 26% D'IDENTITE ET 51% DE SIMILARITE A L'ACTINE. DES SOUCHES DE LEVURE DELETEES DU GENE ARP5 MONTRENT QU'IL EST TRES IMPORTANT VOIRE ESSENTIELLE POUR LEUR CROISSANCE CELLULAIRE (SELON LEUR CONTEXTE GENETIQUE). LES MUTANT ARP5-D, AINSI QUE LES MUTANTS PONCTUELS ARP5-TS, PRESENTENT DES PHENOTYPES PLEIOTROPES (INCAPACITE A METABOLISER DIFFERENTES SOURCES DE CARBONE, FRAGILITE DE PAROI CELLULAIRE, ETC). L'INTEGRATION D'UNE FUSION ARP5-GFP (GREEN FLUORESCENT PROTEIN) AU LOCUS CHROMOSOMIQUE ARP5 A PERMIS DE MONTRER QUE LA PROTEINE ARP5 EST NUCLEAIRE TOUT AU LONG DU CYCLE CELLULAIRE. AFIN DE MIEUX APPREHENDER SES FONCTIONS NUCLEAIRES, NOUS AVONS RECHERCHE DES PARTENAIRES FONCTIONNELS OU PHYSIQUES D'ARP5P PAR L'INTERMEDIAIRE DE CRIBLES DE SUPPRESSEURS-MULTICOPIES ET DE DOUBLE-HYBRIDES RESPECTIVEMENT. LA PROTEINE JUSQU'ALORS INCONNUE AFILP (ARP-FIVE-INTERACTING) EST LA SEULE A AVOIR ETE SELECTIONNEE DANS LES DEUX CRIBLES. LA PROTEINE DE FUSION AFIL-GFP EST LOCALISEE DANS LE NOYAU TOUT AU LONG DU CYCLE. LA DIVERSITE DES PHENOTYPES OBSERVES POUR LES MUTANTS ARP5 ET AFIL NOUS A SUGGERE L'IMPLICATION D'ARP5P ET AFILP DANS UNE FONCTION NUCLEAIRE GENERALE TELLE QUE LA TRANSCRIPTION OU LA MATURATION DES ARNM. DES MARQUAGES IMMUNOLOGIQUES SUR CHROMOSOMES ONT MONTRE QU'ARP5P ET AFILP SE FIXENT A LA CHROMATINE SOUS FORME DE POINTS BIEN PRECIS (ENV 15 A 60 PAR NOYAU). PAR AILLEURS, LA CARACTERISATION D'UN PHENOTYPE SPT (SUPPRESSOR OF TY) DANS LES MUTANTS ARP5-D SUGGERE QU'ARP5P EST IMPLIQUEE PLUS PRECISEMENT DANS LA REGULATION DE LA STRUCTURE CHROMATINIENNE. DE PLUS, LA SENSIBILITE DES MUTANTS ARP5 ET AFIL A DES AGENTS DOMMAGEABLES POUR L'ADN INDIQUE QU'ARP5P ET AFILP POURRAIENT ETRE IMPLIQUEES DANS LA REPARATION DE L'ADN. LES RESULTATS DECRITS DANS CE TRAVAIL MONTRE QU'ARP5P ET AFILP INTERAGISSENT PHYSIQUEMENT POUR REMPLIR UNE FONCTION COMMUNE. ILS CONVERGENT VERS L'IMPLICATION D'ARP5P DANS LE CONTROLE TRANSCRIPTIONNEL OU LA REPARATION DE L'ADN VIA LE REMODELAGE DE LA CHROMATINE.STRASBOURG-Sc. et Techniques (674822102) / SudocSudocFranceF

Clustering of Nuclei in Multinucleated Hyphae Is Prevented by Dynein-Driven Bidirectional Nuclear Movements and Microtubule Growth Control in Ashbya gossypii ▿ †

During filamentous fungus development, multinucleated hyphae employ a system for long-range nuclear migration to maintain an equal nuclear density. A decade ago the microtubule motor dynein was shown to play a central role in this process. Previous studies with Ashbya gossypii revealed extensive bidirectional movements and bypassings of nuclei, an autonomous cytoplasmic microtubule (cMT) cytoskeleton emanating from each nucleus, and pulling of nuclei by sliding of cMTs along the cortex. Here, we show that dynein is the sole motor for bidirectional movements and bypassing because these movements are concomitantly decreased in mutants carrying truncations of the dynein heavy-chain DYN1 promoter. The dynactin component Jnm1, the accessory proteins Dyn2 and Ndl1, and the potential dynein cortical anchor Num1 are also involved in the dynamic distribution of nuclei. In their absence, nuclei aggregate to different degrees, whereby the mutants with dense nuclear clusters grow extremely long cMTs. As in budding yeast, we found that dynein is delivered to cMT plus ends, and its activity or processivity is probably controlled by dynactin and Num1. Together with its role in powering nuclear movements, we propose that dynein also plays (directly or indirectly) a role in the control of cMT length. Those combined dynein actions prevent nuclear clustering in A. gossypii and thus reveal a novel cellular role for dynein

Structural Mutants of the Spindle Pole Body Cause Distinct Alteration of Cytoplasmic Microtubules and Nuclear Dynamics in Multinucleated Hyphae

To determine how microtubule (MT) nucleation and nuclear migration are controlled in multinucleated hyphae we deleted genes encoding MTOC subunits and AgStu2. The novel phenotypes we observed in these mutants compared with analogous deletions in budding yeast allowed us to assign functions to the two types of cMTs that we observe in A. gossypii

Mobility, Microtubule Nucleation and Structure of Microtubule-organizing Centers in Multinucleated Hyphae of Ashbya gossypii

We used live imaging and EM to study migration of multiple nuclei in A. gossypii. Three types of nuclear movements, oscillation, rotation, and bypassing, depend on cytoplasmic microtubules while a fourth type, co-transport with the cytoplasmic stream, does not. Nuclear MTOCs emanating perpendicular and tangential cMTs lead cMT-dependent movement