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Mad1 est une protéine clé du point de contrôle du fuseau en mitose. Associée à Mad2,elle est recrutée aux kinétochores non-attachés où elle y catalyse la production du complexe inhibiteur d’anaphase. La protéine Mad1 a longtemps été décrite comme étant un simple récepteur de Mad2 aux kinétochores. Certaines études laissaient toutefois entrevoir des rôles additionnels de cette protéine en mitose comme en interphase.Afin d’explorer ces fonctions additionnelles de Mad1, j’ai étudié le phénotype mitotique associé à une déplétion de la protéine par ARN interférence dans des lignées cellulaires S2 de drosophile. J’ai également analysé des mutations et délétions du domaine N-terminal de Mad1, celui-ci présentant certaines particularités de structures primaires et secondaires tels que des sites de phosphorylation, un putatif NLS et des hélices amphipathiques. J’ai ainsi montré que le recrutement de Mad1 aux kinétochores en mitose nécessitait la phosphorylation de son domaine N-terminal. Mes analyses cytologiques ont de plus permis de déterminer que le NLS, situé dans ce même domaine N-terminal, est non seulement fonctionnel mais également essentiel à la localisation de Mad1 à l’enveloppe nucléaire et dans le nucléoplasme en interphase.J’ai finalement étudié une protéine Mad1 déplétée de son domaine N-terminal (Mad1Δ71) en spermatocytes de drosophile. Notre laboratoire a récemment montré que dans ces cellules, la protéine Mad1 fait partie d’un territoire nucléaire associé à la chromatine, appelé MINT (Mad1 containing Intranuclear Territory). Ce nouveau territoire, comportant au moins quatre autres protéines (Mad2, Mtor/Tpr, Ulp1 et Raf2), est impliqué dans la régulation de la conformation de la chromatine. Mes analyses ont révélé que la protéine Mad1 Δ71 se localisait anormalement dans le noyau, restant accolée à l’enveloppe nucléaire, et entraînait avec elle l’ensemble de ses partenaires. Ceci suggère que Mad1 est essentielle à l’organisation de ces protéines dans le nucléoplasme, mais également qu’elle pilote la mise en place du territoire MINT. / Mad1 is a key component of the spindle assembly checkpoint in mitosis. Recruitedwith Mad2 to unattached kinetochores, they catalyze the formation of the anaphase inhibitor.Mad1 has long been described as a simple receptor for Mad2 at kinetochores. However,studies are pointing toward additional roles of this protein in mitosis as well as in interphase. To explore these additional functions of Mad1, I studied the mitotic phenotypeassociated with a depletion of the protein by RNA interference in Drosophia S2 cell lines. Ialso analyzed mutations and deletions of the N-terminal domain of Mad1, this one havinginteresting features in its primary and secondary structures, namely phosphorylation sites, aputative NLS and amphipathic helices. I have shown that Mad1 recruitment to kinetochore inmitosis depends on phosphorylations of its N-terminal domain. Moreover, my cytologicalanalyses allowed me to determine that the N-terminal NLS was not only functional but alsoessential for the localization of Mad1 into the nucleus in interphase. Finally, I studied a Mad1 mutant depleted for its N-terminal region (Mad1 Δ71) indrosophila spermatocytes. Our laboratory recently showed that in these cells, Mad1 is part ofa nuclear territory associated with chromatin, named MINT (Mad1 containg IntranuclearTerritory). This new territory, composed of at least four other proteins (Mad2, Mtor/Tpr,Ulp1, Raf2), is involved in chromatin conformation regulation. My studies revealed that inthese cells, Mad1 Δ71 is abnormally localized in the nucleus, staying closed to the nuclearenvelope, and carry with it all its partners. This suggests that Mad1 is essential for the nuclearorganization of these proteins, but also that it pilots the establishment of the MINT territory.

The spindle assembly checkpoint (SAC) is an evolutionarily conserved mechanism that delays the initiation of anaphase by inhibiting the Anaphase Promoting Complex (APC) until all kinetochores have achieved bipolar attachment on the mitotic spindle. Mad1-3, Bub1, and Bub3, components of the SAC, are conserved from yeast to humans. These proteins localize to unattached kinetochores, though it is unknown with which kinetochore proteins they interact and how these interactions transduce information about microtubule attachement. Here, purification of the checkpoint proteins from Saccharomyces cerevisiae suggests that Mad1 interacts with the outer kinetochore protein Ndc80 in a SAC, cell cycle, and DNA dependent manner. Ndc80 is thought to mediate attachment of kinetochores to microtubules so the interaction between Mad1 and Ndc80 suggests a mechanism by which cells sense kinetochore-microtubule attachment. The SAC is of special importance in some types of cancer where genetic damage and aneuploidy is correlated with mutated SAC genes. A better understanding of the SAC mechanism will aid in the development of targetted cancer therpeutics.

The spindle assembly checkpoint (SAC) is an evolutionarily conserved mechanism that delays the initiation of anaphase by inhibiting the Anaphase Promoting Complex (APC) until all kinetochores have achieved bipolar attachment on the mitotic spindle. Mad1-3, Bub1, and Bub3, components of the SAC, are conserved from yeast to humans. These proteins localize to unattached kinetochores, though it is unknown with which kinetochore proteins they interact and how these interactions transduce information about microtubule attachement. Here, purification of the checkpoint proteins from Saccharomyces cerevisiae suggests that Mad1 interacts with the outer kinetochore protein Ndc80 in a SAC, cell cycle, and DNA dependent manner. Ndc80 is thought to mediate attachment of kinetochores to microtubules so the interaction between Mad1 and Ndc80 suggests a mechanism by which cells sense kinetochore-microtubule attachment. The SAC is of special importance in some types of cancer where genetic damage and aneuploidy is correlated with mutated SAC genes. A better understanding of the SAC mechanism will aid in the development of targetted cancer therpeutics.

<p>The Mad/Max/Myc transcription factor network takes part in the control of vital cellular functions such as growth, proliferation, differentiation and apoptosis. Dimerization with the protein Max is necessary for the Myc-family of oncoproteins and their antagonists, the Mad-family proteins, to regulate target genes and carry out their intended functions. Myc functions as a positive regulator of proliferation, antagonized by the growth inhibitory Mad-proteins that potentially functions as tumor supprerssors. Deregulated Myc expression is found in a variety of tumors and signals negatively regulating Myc expression and/or activity could therefore be of potential use in treating tumors with deregulated Myc.</p><p>Our aim was to therefore to investigate possible negative effects on Myc expression and activity by growth inhibitory cytokines and by the Myc antagonists, the Mad-family proteins.Two different cellular model systems of neuronal and hematopoietic origin have been utilized for these studies.</p><p>Our results show that Mad1 is upregulated during induced neuronal differentiation of SH-SY5Y cells. Further, the growth inhibitory cytokine interferon-g (IFN-g) was shown to cooperate with retinoic acid (RA) and the phorbol ester TPA in inducing growth arrest and differentiation in N-<i>myc</i> amplified neuroblastoma cell lines. In contrast to treatment with either agent alone, the combined treatment of TPA+IFN-g and RA+IFN-g led to upregulation of Mad1 and to downregulation of N-Myc, respectively, thus correlating with the enhanced growth inhibition and differentiation observed after combination treatment. Ectopic expression of an inducible Mad1 in monoblastic U-937 cells led to growth inhibition but did not lead to differentiation or enhancement of differentiation induced by RA, vitamin D3 or TPA. In v-Myc transformed U-937 cells Mad1 expression reestablished the TPA-induced G1 cell cycle arrest, but did not restore differentiation, blocked by v-Myc. The growth inhibitory cytokine TGF-b was found to induce Mad1 expression and Mad1:Max complex formation in v-Myc transformed U-937 cells correlating with reduced Myc activity and G1 arrest. </p><p>In conclusion, our results show that the Myc-antagonist Mad1 is upregulated by growth inhibitory cytokines and/or differentiation signals in neuronal and hematopoietic cells and that enforced Mad1 expression in hematopoietic cells results in growth inhibition and increased sensitivity to anti-proliferative cytokines. Mad1 and cytokine-induced signals therefore seem to cooperate in counteracting Myc activity.</p>

The Mad/Max/Myc transcription factor network takes part in the control of vital cellular functions such as growth, proliferation, differentiation and apoptosis. Dimerization with the protein Max is necessary for the Myc-family of oncoproteins and their antagonists, the Mad-family proteins, to regulate target genes and carry out their intended functions. Myc functions as a positive regulator of proliferation, antagonized by the growth inhibitory Mad-proteins that potentially functions as tumor supprerssors. Deregulated Myc expression is found in a variety of tumors and signals negatively regulating Myc expression and/or activity could therefore be of potential use in treating tumors with deregulated Myc. Our aim was to therefore to investigate possible negative effects on Myc expression and activity by growth inhibitory cytokines and by the Myc antagonists, the Mad-family proteins.Two different cellular model systems of neuronal and hematopoietic origin have been utilized for these studies. Our results show that Mad1 is upregulated during induced neuronal differentiation of SH-SY5Y cells. Further, the growth inhibitory cytokine interferon-g (IFN-g) was shown to cooperate with retinoic acid (RA) and the phorbol ester TPA in inducing growth arrest and differentiation in N-myc amplified neuroblastoma cell lines. In contrast to treatment with either agent alone, the combined treatment of TPA+IFN-g and RA+IFN-g led to upregulation of Mad1 and to downregulation of N-Myc, respectively, thus correlating with the enhanced growth inhibition and differentiation observed after combination treatment. Ectopic expression of an inducible Mad1 in monoblastic U-937 cells led to growth inhibition but did not lead to differentiation or enhancement of differentiation induced by RA, vitamin D3 or TPA. In v-Myc transformed U-937 cells Mad1 expression reestablished the TPA-induced G1 cell cycle arrest, but did not restore differentiation, blocked by v-Myc. The growth inhibitory cytokine TGF-b was found to induce Mad1 expression and Mad1:Max complex formation in v-Myc transformed U-937 cells correlating with reduced Myc activity and G1 arrest. In conclusion, our results show that the Myc-antagonist Mad1 is upregulated by growth inhibitory cytokines and/or differentiation signals in neuronal and hematopoietic cells and that enforced Mad1 expression in hematopoietic cells results in growth inhibition and increased sensitivity to anti-proliferative cytokines. Mad1 and cytokine-induced signals therefore seem to cooperate in counteracting Myc activity.