Des mutations hétérozygotes faux-sens dans le gène de la tubuline gamma TUBG1, ont été identifiées dans le contexte des malformations du développement cortical, associées à une déficience intellectuelle et à l'épilepsie. Ici, nous avons étudié par la technique d’électroporation in-utero et par des études in vivo, l’effet de quatre de ces variantes sur le développement cortical. Nous montrons que les mutations dans TUBG1 affectent le positionnement neuronal dans la plaque corticale, en perturbant la locomotion des neurones nouvellement nés, mais sans affecter la neurogenèse. Nous proposons que la γ-tubuline mutante affecte le fonctionnement global de ses complexes, et en particulier leur rôle dans la régulation de la dynamique des microtubules. De plus, nous avons développé un modèle de souris knock-in Tubg1Y92C/+ et évalué les conséquences de la mutation sur le développement cortical, les caractéristiques neuroanatomiques et le comportement. Les souris mutantes présentent une microcéphalie globale, des anomalies du néocortex et de l'hippocampe, des altérations du comportement et une susceptibilité épileptique. Ainsi, nous montrons que les souris Tubg1Y92C/+ miment au moins partiellement le phénotype humain et représentent donc un modèle pertinent pour d'autres investigations de la physiopathologie des malformations du développement cortical. / Missense heterozygous variants in the gamma tubulin gene TUBG1 have been linked to malformations of cortical development, associated with intellectual disability and epilepsy. Here, we investigated through in-utero electroporation and in-vivo studies, how four of these variants affect cortical development. We show that TUBG1 mutants affect neuronal positioning within the cortical wall, by a disrupting the locomotion of newly born neurons but without affecting neurogenesis. We propose that mutant γ-tubulin affects overall functioning of γ-tubulin complexes, and in particular their role in the regulation of microtubule dynamics. Additionally, we developed a knock-in Tubg1Y92C/+ model and assessed consequences of the mutation on cortical development, neuroanatomical features and behaviour. Mutant mice present with global microcephaly, neocortical and hippocampal abnormalities, behavioural alterations and epileptic susceptibility. Thus, we show that Tubg1Y92C/+ mice partially mimic the human phenotype and therefore represent a relevant model for further investigations of the physiopathology of malformations of cortical development.

Semiconductor quantum dots (QDs) have emerged as a new class of fluorescent probes and labeling agents for biological samples. QDs are bright, highly photostable and allow simultaneous excitation of multiple emissions. Owing to these properties, QDs hold exceptional promise in enabling intracellular biochemical studies and diagnosis with unprecedented sensitivity and accuracy. However, use of QD probes inside living cells remains a challenge due to difficulties in delivery of nanoparticles without causing aggregation and imaging single nanoparticles inside living cells. In this dissertation, a systematic approach to deliver, image and locate single QDs inside living cells is presented and the properties of molecular motor protein driven QD transport are studied. First, spectroscopic and imaging methods capable of differentiating single nanoparticles from the aggregates were developed. These technologies were validated by differentiating surface protein expression on viral particles and by enabling rapid counting of single biomolecules. Second, controlled delivery of single QDs into living cells is demonstrated. A surprising finding is that single QDs associate non-specifically with the dynein motor protein complex and are transported to the microtubule organizing center. Accurate localization and tracking of QDs inside cell cytoplasm revealed multiple dynein motor protein attachment resulting in increased velocity of the QDs. Further, spectrin molecule which is known to recruit dynein motor protein complex to phospholipid micelles was found to associate with the QDs. These results may serve as a benchmark for developing new QD surface coatings suitable for intracellular applications. Since, nanoparticles are similar in size to viral pathogens; better understanding of nanoparticle-cell interactions should also help engineer nanoparticle models to study virus-host cell interactions. (Contains AVI format multimedia files)

AtMAP65-4 est une protéine associée aux microtubules appartenant à la famille des AtMAP65s qui compte 9 membres identifiés chez Arabidopsis thaliana. Ces protéines appartiennent à une famille conservée au cours de l'évolution, les MAP65s. Ainsi, des protéines homologues sont présentes chez de mammifères (PRC1), chez la levure (Ase1p) ou chez la drosophile (FEO). Jusqu'ici l'étude des propriétés moléculaires et fonctionnelles des AtMAP65s s'est portée essentiellement sur l'étude d'AtMAP65-1 et AtMAP65-5. La principale caractéristique de ces protéines est d'induire la formation de faisceaux de microtubules in vitro. La distribution des AtMAP65s in vivo est très régulée, celle-ci sont localisées avec des réseaux des microtubules bien définis. Ainsi, leur rôle supposé est de mettre en place ces réseaux puis de participer à leur maintient. La localisation d'AtMAP65-4 apparait comme très intéressante car elle est strictement associée avec les microtubules du fuseau mitotique. D'après les résultats obtenus au cours de ce travail, nous avons suggéré que la fonction in vivo d'AtMAP65-4 est de participer à la mise en place et au maintient des microtubules en faisceaux dans les fibres kinétochoriennes lors de la division cellulaire. Lors d'une étude in vitro nous avons montré qu'AtMAP65-4 modifie les paramètres dynamiques de polymérisation des microtubules. Outre sa capacité à former des faisceaux, AtMAP65-4 permet une croissance régulière des microtubules au sein des faisceaux qu'elle induit. Le mécanisme d'action de la MAP à l'échelle moléculaire a été analysé à travers une étude bioinformatique où nous avons modélisé l'activité d'AtMAP65-4. Les données obtenues montrent qu'AtMAP65-4 peut bloquer les évènements de dépolymérisation des microtubules. Par ailleurs, l'activité d'AtMAP65-4 pourrait être régulée in vivo par des modifications post traductionnelles. En effet, nous avons montré et étudié l'effet de la phosphorylation d'AtMAP65-4 par les kinases Auroras. Cette phosphorylation pourrait être impliquée dans la régulation de l'activité d'AtMAP65-4 au cours de la mitose. / AtMAP65-4 is a microtubule-associated protein belonging to the AtMAP65s family that comprises 9 members identified in Arabidopsis thaliana. These proteins belong to a family conserved during evolution, MAP65s. Thus, homologous proteins are present in mammals (PRC1), in yeast (Ase1p) or Drosophila (FEO). So far the study of molecular properties and functional AtMAP65s has focused mainly on AtMAP65-1 and AtMAP65-5. The main feature of these proteins is to induce the formation of microtubule bundles in vitro. In vivo, these AtMAP65s are localized with subsets of microtubule bundles as they are suggested to play a role in establishing and maintaining these networks. From the results we obtained on AtMAP65-4 properties during this work such as the in vivo localization, biochemical properties and functional effetc on the MT polymerization, we suggested that the in vivo function of AtMAP65-4 is involved in setting up and maintaining microtubule bundles within kinetochore fibers during cell division. In vitro studies allowed us to show that AtMAP65-4 changes the dynamic parameters of microtubule. In addition to its ability to form bundles, AtMAP65-4 allows steady growth of microtubules in bundles it induces. The mechanism of action of the MAP at the molecular level was analyzed through a bioinformatics study where we modeled the activity of AtMAP65-4 and concluded that it could block the depolymerization events. Moreover, the activity of AtMAP65-4 could be regulated in vivo by post-translational modifications. Indeed, we have shown that AtMAP65-4 is phosphorylated by Aurora kinases in vitro. The effect of this phosphorylation during mitosis is under investigation.

Abstract Frontotemporal lobar degeneration (FTLD) is the second most common neurodegenerative disease leading to early-onset dementia (< 65 years), next to Alzheimer’s disease. FTLD is substantially a genetic disorder with up to 50% of cases having a positive family history. Mutations in the genes microtubule-associated protein tau (MAPT) and progranulin (PGRN) account for about 10–20% of all cases of FTLD. Hexanucleotide repeat expansion mutation within the gene C9ORF72 has recently been identified as the major cause of FTLD, FTLD with amyotrophic lateral sclerosis (ALS) and pure ALS. During this study, hexanucleotide repeat expansion within the C9ORF72 gene was shown to explain nearly 50% of familial and 30% of all FTLD cases in the Finnish population. Otherwise, the genetic background of Finnish FTLD is largely unknown. The object of the present work was to disentangle the genetic aetiology of FTLD in the Finnish population. We studied a cohort of patients with a clinical diagnosis of FTLD from the province of Northern Ostrobothnia, Finland. Sequencing analysis of the genes MAPT, charged multi-vesicular body protein 2B (CHMP2B) and TAR DNA binding protein (TARDBP) were performed and the MAPT haplotypes were analysed. Correlations between genotype and phenotype were studied in patients with C9ORF72 repeat expansion mutation. C9ORF72 expansion mutation explained nearly 30% of cases of FTLD in our cohort. Concomitant ALS and positive family history of the disease increased the possibility of carrying expanded C9ORF72. The clinical phenotype of C9ORF72 expansion carriers varied at presentation: both behavioural and language variants were detected with or without ALS. The behavioural presentations included prominent psychotic features, although psychiatric presentations were not overrepresented in expansion carriers. No pathogenic mutations were identified in the MAPT, CHMP2B and TARDBP genes in our series of FTLD patients. The H2 MAPT haplotype was associated with FTLD in the series. Our findings emphasise the importance of C9ORF72 expansion mutation in FTLD. While mutations in MAPT and PGRN cause a significant proportion of cases of FTLD worldwide, they seem to be rare causes of FTLD in the Finnish population. Besides being infrequent in other populations, mutations in CHMP2B and TARDBP are rare causes of FTLD in the Finnish population as well. Our findings have clinical implications for recognising phenotypic features characteristic of expanded C9ORF72 as well as for genetic counselling of Finnish patients with FTLD. Even though a considerable proportion of our cases of familial FTLD is caused by the C9ORF72 expansion, over 50 % of our familial cases are without a molecular genetic diagnosis, suggesting that there are other unidentified causal genes to be found. / Tiivistelmä Otsa-ohimolohkorappeumat on toiseksi yleisin työikäisten dementiaa aiheuttava etenevä aivojen rappeumasairaus. Toisinaan otsa-ohimolohkorappeumat esiintyvät yhdessä liikehermorappeuman, amyotrofisen lateraaliskleroosin (ALS), kanssa. Perinnöllisillä tekijöillä on todennäköisesti keskeinen merkitys taudin taustalla. Mutaatiot microtubule-associated protein tau (MAPT)- ja progranulin (PGRN) geeneissä aiheuttavat yhteensä 10–20 % otsa-ohimolohkorappeumista maailmalla. C9ORF72-geenissä sijaitsevan toistojaksomonistuman on vastikään todettu olevan yleisin otsa-ohimolohkorappeumia ja ALS:a aiheuttava mutaatio. Mutaatio on erityisen yleinen suomalaisessa väestössä selittäen lähes 50 % suvuittaisista ja 30 % kaikista otsa-ohimolohkorappeumista. Oireyhtymän perinnöllisyys on muutoin huonosti tunnettu suomalaisessa väestössä. Tutkimuksen tavoitteena oli selvittää otsa-ohimolohkorappeumien geneettisiä syitä aineistossa, joka koostui vuosina 1999–2010 Oulun yliopistollisessa sairaalassa tutkituista potilaista. Tutkimuksessa selvitettiin MAPT-, charged multi-vesicular body protein 2B (CHMP2B)- ja TAR DNA-binding protein (TARDBP) geenien mutaatioiden esiintyvyyttä ja määritettiin MAPT-geenin haplotyypit. Lisäksi tutkittiin taudin kliinisiä erityispiirteitä C9ORF72-mutaation kantajilla. C9ORF72-mutaatio selitti lähes 30 % otsa-ohimolohkorappeumista aineistossamme. Tutkimuksessa havaittiin, että suvuittain esiintyvä tautimuoto ja ALS yhdistyneenä otsa-ohimolohkorappeumaan liittyivät merkittävästi C9ORF72-mutaatioon. Monistuman kantajien fenotyyppi oli moninainen – ensioireina oli sekä käytösongelmia että kielellisiä vaikeuksia. Vaikka C9ORF72-mutaation kantajilla on kuvattu runsaasti psykoottisia oireita, psykoottiset oireet eivät olleet selvästi yliedustettuna mutaation kantajilla aineistossamme. Tutkimuksessa ei löydetty tautia aiheuttavia mutaatioita MAPT-, CHMP2B- tai TARDBP-geeneistä. Havaitsimme kuitenkin tilastollisesti merkittävän yhteyden MAPT-geenin H2-haplotyypin ja otsa-ohimolohkorappeumien välillä. Tuloksemme antavat uutta tietoa C9ORF72-mutaation kantajien kliinisistä erityispiirteistä. MAPT-geenin mutaatioiden merkitys otsa-ohimolohkorappeumien synnyssä ei näyttäisi olevan suomalaisessa väestössä niin merkittävä kuin muissa väestöissä. CHMP2B- ja TARDBP-mutaatiot ovat harvinainen oireyhtymän syy myös suomalaisessa väestössä. Tuloksiamme voidaan hyödyntää suomalaisten otsa-ohimolohkorappeumapotilaiden perinnöllisessä neuvonnassa. Huomattavista edistysaskelista huolimatta yli puolet suvuittain esiintyvistä tautitapauksistamme on vailla geneettistä diagnoosia, mikä antaa aihetta jatkotutkimuksille.

Hereditary spastic paraplegia type 54 is a rare autosomal recessive neurological gait disorder characterized by paraplegia, muscle spasticity, and intellectual disability. This length-dependent distal axonopathy is caused by mutations in the DDHD2 gene, which encodes the intracellular phospholipase A1 DDHD2. Little is known about the molecular function of the DDHD2 protein, especially in the context of HSP54. Thus, there is a need to further investigate its molecular functions and investigate the impact of DDHD2 deficiency in disease-relevant cells. Here, lipidomic profiling of dermal fibroblasts derived from three unrelated patients has revealed 19 glycerophosphoethanolamine species at differential levels in patients relative to unaffected controls. However, patient cells appear to have an unaffected Golgi apparatus morphology and lipid droplet formation, despite DDHD2’s proposed roles in these processes. To study the gene function in neuronal cells, I transdifferentiated the fibroblasts into induced neuronal precursor cells and found all the patient cells arrested in the G0/G1 phase of upon conversion. Given that these cell lines are unsustainable, I generated a stable knockdown cell line in the highly proliferative HEK293A to study the molecular biology of DDHD2. The knockdown cells had a reduced growth, were delayed in the G2/M phase of the cell cycle, and became multinucleated. I then treated the cells with antineoplastic compounds paclitaxel and nocodazole and found more knockdown cells in G0/G1 than controls, suggesting the possible occurrence of mitotic slippage. Lastly, I report a novel subcellular localization for DDHD2 at the microtubule organization center.

Proteins of DREPP family (20-25 kDa, syn. PCaP1 in Arabidopsis thaliana) first appeared in ferns and we have shown that several independent duplications of DREPP protein occurred during evolution of large families (Poaceae, Brassicaceae, Solanaceae and Asteraceae) and in group Coniferophyta. Secondary losses of one paralogue occurred in subfamilies Pooideae and Solanoideae.We have also detected two large-scale modification of DREEP protein in Asparagales and Brassicaceae (this divergent paralogue was previously described as MAP18 protein). We have examined colinearity of chromosome fragments in vicinity both PCaP1 and MAP18 paralogues in Arabidopsis thaliana and we hypothesize that MAP18 gene arose during genome duplication on the origin of Brassicaceae family. DREPP protein was previously identified in detergent-resistant membrane microdomain fraction and a myristyl anchor was shown to be necessary for their membrane localization. Membrane association was shown to be modified by the interaction of unique N-terminal domain with PtdInsPs, which was inhibited by binding of Ca-calmodulin (Nagasaki et al., 2008). The mutation of Gly2 by Ala in the myristilation site, or C-terminal GFP-fusion (GFP-DREPP), affect membrane association in Arabidopsis thaliana (Nagasaki et al., 2008). Several DREPP paralogues in...

The mitotic spindle is a bipolar microtubule-based structure that mediates proper cell division by segregating the genetic material and by positioning the cytokinesis cleavage plane. Spindle assembly is a complex process, involving the modulation of microtubule dynamics, microtubule focusing at spindle poles and the formation of stable microtubule attachments to chromosomes. The cellular events leading to spindle formation are highly regulated, and mitotic kinases have been implicated in many aspects of this process. However, little is known about their counteracting phosphatases. A screen for genes required for early embryonic cell divisions in C. elegans identified rsa-1 (for regulator of spindle assembly 1), a putative Protein Phosphatase 2A (PP2A) regulatory subunit whose silencing causes defects in spindle formation. Upon rsa-1(RNAi), spindle poles collapse onto each other and microtubule amounts are strongly reduced. My thesis work demonstrates that RSA-1 indeed functions as a PP2A regulatory subunit. RSA-1 associates with the PP2A enzyme and recruits it to centrosomes. The centrosome binding of PP2A furthermore requires the new protein RSA-2 as well as the core centrosomal protein SPD-5 and is based on a hierarchical protein-protein interaction pathway. When PP2A is lacking at centrosomes after rsa-1(RNAi), the centrosomal amounts of two critical mitotic effectors, the microtubule destabilizer KLP-7 and the kinetochore microtubule stabilizer TPXL-1, are altered. KLP-7 is increased, which may account for the reduction of microtubule outgrowth from centrosomes in rsa-1(RNAi) embryos. TPXL-1 is lost from centrosomes, which may explain why spindle poles collapse in the absence of RSA-1. TPXL-1 physically associates with RSA-1 and RSA-2, suggesting that it is a direct target of PP2A. In summary, this work defines the role of a novel PP2A complex in mitotic spindle assembly and suggests a model for how different microtubule re-organization steps might be coordinated during spindle formation.

Neurons exhibit highly polarized structures, including two morphologically and functionally distinct domains, axons and dendrites. Dendrites and axons receive versus send information, and proper execution of each requires different sets of molecules. Differential distribution of membrane proteins in distinct neuronal compartments plays essential roles in neuronal functions. The major goal of my doctoral thesis was to study the molecular mechanisms that govern the differential distribution of membrane proteins in neurons, using the Drosophilalarval mushroom body (MB) as a model system. My work was initiated by an observation of differential distribution of distinct Dscam isoforms in neurons. Dscam stands for Down Syndrome Cell Adhesion Molecule, which is a Drosophila homolog of human DSCAM. According to genomic analysis, DrosophilaDscam gene can generate more than 38,000 isoforms through alternative splicing in its exons 4, 6, 9 and 17. All Dscam isoforms share similar domain structures, with 10 immunoglobulin domains and 6 fibronectin type III repeats in the ectodomain, a single transmembrane domain and a cytoplasmic endodomain. There are two alternative exons in exon 17 (17.1 and 17.2), which encodes Dscam’s transmembrane domain. Interestingly, in ectopic expression, Dscam isoforms carrying exon 17.1 (Dscam[TM1]) can be preferentially localized to dendrites and cell bodies, while Dscam isoforms carrying exon 17.2 (Dscam[TM2]) are distributed throughout the entire neuron including axons and dendrites. To unravel the mechanisms involved in the differential distribution of Dscam[TM1] versus Dscam[TM2], I conducted a mosaic genetic screening to identify the possible factors affecting dendritic distribution of Dscam[TM1], established an in vivoTARGET system to better distinguish the differential distribution of Dscam, identified the axonal and dendritic targeting motifs of Dscam molecules and further showed that Dscam’s differential roles in dendrites versus axons are correlated with its localization. Several mutants affecting dendritic distribution of Dscam[TM1] have been identified using a MARCM genetic screen. Three of these mutants (Dlis1, Dmn and p24) are components of the dynein/dynactin complex. Silencing of other dynein/dynactin subunits and blocking dynein function with a dominant-negative Glued mutant also resulted in mislocalization of Dscam[TM1] from dendrites to axons. However, microtubule polarity in the mutant axons was maintained. Taken together, this was the first demonstration that the dynein/dynactin complex is involved in the polarized distribution of membrane proteins in neurons. To further examine how dynein/dynactin is involved in the dendritic distribution of Dscam[TM1], I compromised dynenin/dynactin function with dominant-negative Glued and transiently induced Dscam[TM1] expression. The results suggested that dynein/dynactin may not be directly involved in the targeting of newly synthesized Dscam[TM1] to dendrites. Instead, it plays a role in maintaining dendritic restriction of Dscam[TM1]. Notably, dynein/dynactin dysfunction did not alter distribution of another dendritic transmembrane protein Rdl (Resistant to Dieldrin), supporting involvement of diverse mechanisms in distributing distinct molecules to the dendritic membrane. To identify the targeting motifs of Dscam, I incorporated the TARGET (Temporal and regional gene expression targeting) system into mushroom body (MB) neurons, and this allowed the demonstration of the differential distribution of Dscam[TM1] and Dscam[TM2] with more clarity than conventional overexpression techniques. Using the TARGET system, I identified an axonal targeting motif located in the cytoplasmic juxtamemebrane domain of Dscam[TM2]. This axonal targeting motif is dominant over the dendritic targeting motif located in Dscam’s ectodomain. Scanning alanine mutagenesis demonstrated that two amino acids in the axonal targeting motif were essential for Dscam’s axonal distribution. Interestingly, swapping the cytoplasmic juxtamembrane portions between TM1 and TM2 not only reversed TM1’s and TM2’s differential distribution patterns but also their functional properties in dendrites versus axons. My thesis research also involved studying endodomain diversity of Dscam isoforms. Besides the diversity originally found in the ectodomain and transmembrane domain of Dscam, my colleagues and I further demonstrated the existence of four additional endodomain variants. These four variants are generated by skipping or retaining exon 19 or exon 23 through independent alternative splicing. Interestingly, different Dscam endodomain isoforms are expressed at different developmental stages and in different areas of the nervous system. Through isoform-specific RNA interference, we showed the differential involvement of distinct Dscam endodomains in specific neuronal morphogenetic processes. Analysis of the primary sequence of the Dscam endodomain indicated that endodomain variants may confer activation of different signaling pathways and functional roles in neuronal morphogenesis. In Summary, my thesis work identified and characterized several previously unknown mechanisms related to the differential distribution of membrane proteins in neurons. I showed that there may be a dynein/dynactin-independent mechanism for selective transport of dendritic membrane proteins to dendrites. Second, dynein/dynactin plays a maintenance role in dendritic restriction of Dscam[TM1]. Third, different membrane proteins may require distinct combinations of mechanisms to be properly targeted and maintained in certain neuronal compartments. Further analysis of the mutants indentified from my genetic screen will definitely help to resolve the missing pieces of the puzzle. These findings provide novel mechanistic insight into the differential distribution of membrane proteins in polarized neurons.

No description available.

Les microtubules sont des éléments dynamiques du cytosquelette qui contrôlent à la fois l’organisation du cytoplasme, la polarité, la migration et la division cellulaire. Notre laboratoire a précédemment montré que la kinase de stress JNK (c-Jun NH2-terminal Kinase) régule la dynamique des microtubules dans les cellules épithéliales de mammifères, en augmentant les vitesses de polymérisation, ainsi que les fréquences de sauvetage (transition vers une phase de repolymérisation). Alors que certaines protéines neuronales capables de réguler la dynamique des microtubules ont été identifiées comme des substrats de JNK, leurs équivalents dans les cellules épithéliales sont largement méconnus. Dans le but de comprendre comment JNK module la dynamique des microtubules dans les cellules épithéliales de mammifère, nous avons étudié deux substrats potentiels de JNK : la -tubuline et le facteur de sauvetage CLIP-170. Nous avons bien mis en évidence in vitro, une phosphorylation de la -tubuline par JNK sur une thréonine non-consensus, mais cette phosphorylation n’a pas été retrouvée dans les cellules HeLa, suggérant que la -tubuline n’est pas un substrat naturel de JNK in vivo. Nous avons mis en évidence par ailleurs que CLIP-170 est un nouveau substrat de JNK. Dans les cellules épithéliales, JNK activée phosphoryle trois résidus (Thr25, Thr45 et Ser147) situés dans la partie N-terminale de CLIP-170 de part et d’autre du premier domaine CAP-Gly qui est nécessaire pour l’interaction avec les microtubules. Ces acides aminés présentent des différences aussi bien dans leur phosphorylation basale que dans leurs cinétiques de phosphorylation par JNK sous divers stress. De plus, nous avons trouvé que dans différentes cellules épithéliales, la phosphorylation de ces sites est conservée. In vitro, ces résidus sont directement phosphorylés par JNK, préférentiellement quand le domaine N-terminal de CLIP-170 lie la tubuline. De plus, l’expression de mutants de CLIP-170 phospho-mimétiques et non-phosphorylables a montré que la phosphorylation de chaque site augmente la fréquence des sauvetages microtubulaires. Cette modulation n’est pas corrélée à une augmentation de la capacité de CLIP-170 à former des comètes aux extrémités plus en croissance ou à être retenue aux croissements microtubulaires, qui sont des sites de sauvetage potentiels.Ce travail a permis de décrire les premières phosphorylations de CLIP-170 qui stimulent sa fonction de sauvetage in vivo. Il souligne par ailleurs la complexité des mécanismes de sauvetage, qui demeurent un aspect encore énigmatique de l’instabilité dynamique des microtubules. L’activité de JNK sur CLIP-170 ne permet d’expliquer qu’une partie des effets de la kinase sur la dynamique des microtubules, aussi la recherche d’autres protéines cibles de JNK pouvant réguler notamment leur vitesse de polymérisation, reste à entreprendre. / Microtubules are dynamic cytoskeleton elements, which control cytoplasm organization, cell polarity, migration and division. Our laboratory has previously shown that the stress kinase JNK (c-Jun NH2-terminal Kinase) regulates microtubule dynamics in mammalian epithelial cells, by increasing their growth rates, and their rescue frequencies (transition towards phases of repolymerization). While several neuronal proteins regulating microtubule dynamics have been identified as JNK substrates, their counterparts in epithelial cells are largely unknown. With the aim to understand how JNK modulates microtubule dynamics in mammalian epithelial cells, we studied two putative substrates of JNK: -tubulin and the rescue factor CLIP-170. Regarding -tubulin, using an in vitro kinase assay, we found that a non-consensus threonine is actually phosphorylated by JNK, but we were not able to find this phosphorylation in HeLa cells, suggesting that -tubulin is not a natural JNK substrate. In parallel, we found that CLIP-170 is a new substrate of JNK in epithelial cells. Activated JNK phosphorylates three residues (Thr25, Thr45 and Ser147) located in the N-terminal part of CLIP-170, on each side of the first CAP-Gly domain, which is required for CLIP-170 interaction with microtubules. These residues exhibit differences in their level of basal phosphorylation and their kinetics of phosphorylation by JNK under various stresses. Moreover, we found that in different epithelial cells, the phosphorylation of these sites is conserved. Using an in vitro kinase assay, we found that all these residues are directly phosphorylated by JNK, preferentially when the N-terminal domain of CLIP-170 binds tubulin. Furthermore, using phospho-mimetic and non-phosphorylatable CLIP-170 mutants in epithelial cells, we revealed that the phosphorylation of each site increases microtubule rescues. Such modulation operates without increasing CLIP-170 capability to form comets at the microtubule growing plus ends or to accumulate at microtubule crossings, which are potential rescue sites.This work described the first phosphorylations that enhance CLIP-170 rescue factor function in vivo. It also points out to which extent rescue mechanisms are complex and remain an elusive aspect of dynamic instability. JNK-mediated phosphorylation of CLIP-170 only partly explains the kinase effects on microtubule dynamics. Therefore, identifying other JNK targets that may regulate microtubule polymerization rate, remains to be addressed.