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Investigating how the Spindle Assembly Checkpoint inhibits the onset of anaphaseLara González, Pablo January 2013 (has links)
The Spindle Assembly Checkpoint (SAC) delays the onset of anaphase in response to unattached kinetochores. The mechanism by which the SAC works is by inhibiting the activity of the Anaphase-promoting complex/cyclosome (APC/C), a large E3 ubiquitin ligase that targets several anaphase inhibitors for proteasome-mediated degradation, including securin and cyclin B. When the SAC is satisfied, the APC/C becomes active and this allows progression through the cell cycle. Work from the last decade identified the mitotic checkpoint complex (MCC) as the main transducer of the SAC. The MCC is composed of BubR1, Bub3, Mad2 and Cdc20 and it is a very potent inhibitor of the APC/C. When the SAC is active, the MCC binds the APC/C and it inhibits its activity. Once the SAC is satisfied, the MCC becomes disassembled, which allows APC/C activation and mitotic progression. However, the mechanisms that dictate MCC assembly and how it inhibits the APC/C remain to be understood. Here, I used a combination of cell biology and in vitro biochemistry to investigate the mechanism by which the MCC component BubR1 participates in the SAC. My data shows that through its interaction with Bub3, BubR1 localises to kinetochores and this event greatly facilitates its assembly onto the MCC and its SAC function. On the other hand, MCC formation and APC/C binding were only dependent on BubR1's N-terminus, therefore questioning the existence of a second Cdc20 binding site. Within this region, TPR domains and an N-terminal motif known as the KEN box (KEN1) mediates these interactions. By contrast, BubR1's second KEN box (KEN2) does not participate in MCC assembly or APC/C binding. However, both in cells and in vitro, the KEN2 box is required for APC/C inhibition. Indeed, I show that this second KEN box promotes SAC function by blocking the interaction of the APC/C with its substrates. Thus, both KEN boxes in BubR1 participate differentially in the SAC, the first to promote MCC assembly and the second one to block substrate recruitment to the APC/C.In addition, I investigated the mechanisms that mediate MCC inactivation, following SAC silencing. I observed that p31comet and APC/C activity cooperate to promote MCC turnover. The implication of these observations in our understanding of the SAC is discussed.
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Fancc regulates the spindle assembly checkpoint to prevent tumorigenesis in vivoEdwards, Donna Marie 27 March 2017 (has links)
Indiana University-Purdue University Indianapolis (IUPUI) / The Fanconi anemia (FA) pathway consists of 21 genes that maintain genomic stability
and prevent cancer. Biallelic mutations within this network cause Fanconi anemia, an
inherited bone marrow failure and cancer predisposition syndrome. Heterozygous inborn
mutations in FA genes increase risk of breast/ovarian cancers, and somatic mutations
occur in malignancies in non-Fanconi patients. Understanding the tumor suppressive
functions of FA signaling is important for the study of Fanconi anemia, inherited cancers,
and sporadic cancers.
The FA network functions as a genome guardian throughout the cell cycle. In addition to
the well-established roles of FA proteins in interphase DNA replication/repair, the FA
pathway controls mitosis by regulating the spindle assembly checkpoint (SAC) to ensure
proper chromosome segregation. The SAC consists of several tumor suppressors,
including Mad2, and SAC impairment predisposes to aneuploidy and cancer. However,
the in vivo contribution of SAC dysfunction to malignant transformation of FA-deficient
cells remains unknown. Furthermore, the mechanisms by which FA proteins regulate the
SAC are unclear.
To test whether SAC dysfunction drives genomic instability and tumorigenesis in FA, we
generated a novel FA-SAC model by intercrossing Fancc-/- and Mad2+/- mice. The intercrossed mice displayed heightened aneuploidy secondary to exacerbated SAC
dysfunction. Importantly, these mice were prone to developing hematologic
malignancies, particularly leukemia, faithfully recapitulating the clinical phenotype of
Fanconi anemia.
Upon establishing SAC dysfunction as a driver of tumorigenesis in FA, we next explored
the mechanism by which FANCC regulates the SAC. We demonstrated that the mitotic
kinase CDK1 phosphorylates FANCC to regulate subcellular localization and SAC
function of FANCC during mitosis.
Our study highlights the essential role of compromised chromosome segregation in the
development of leukemia due to impaired FA signaling. This work furthers our
knowledge of FANCC signaling at the SAC, and has implications for future use of
mitotic-centered therapies for FA-associated tumors. / 2 years
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Using a novel small molecule inhibitor to investigate the role of Mps1 kinase activityHewitt, Laura January 2011 (has links)
During mitosis, accurate chromosome segregation is essential: gain or loss of genetic information can be detrimental to cell viability, or promote tumourigenesis. The mitotic checkpoint (also known as the spindle assembly checkpoint or SAC) ensures accurate chromosome segregation by delaying cell cycle progression until accuracy can be guaranteed. Mps1 is a protein kinase that is crucial for mitotic checkpoint signalling and also for proper chromosome alignment at metaphase. However, the precise role of Mps1’s catalytic activity is still unclear. Here, I present AZ3146, a novel small molecule inhibitor of Mps1. AZ3146 inhibits recombinant Mps1 in vitro with an IC50 of ~35 nM, and has low activity against a panel of 50 kinases, suggesting a reasonable degree of selectivity. As predicted for an Mps1 inhibitor, AZ1346 treatment led to spindle checkpoint malfunction in cells, accelerated mitotic timing, and perturbed the kinetochore localisation of the checkpoint effector Mad2. AZ3146 has a negative effect on cell viability, suggesting it leads to detrimental missegregations. Thus, the cellular effects of AZ3146 are consistent with Mps1 inhibition, and I was able to use the compound confidently as a tool to further probe the role of Mps1 activity in cells.Strikingly, levels of Mps1 increased at unattached kinetochores following inhibition of its kinase activity, suggesting Mps1’s kinetochore localisation is regulated by its own activity. A kinase-dead GFP-Mps1 fusion protein only accumulated at kinetochores in the absence of endogenous, active Mps1, implicating intra-molecular interactions in regulation of Mps1’s kinetochore localisation. I confirm a role for Mps1 in the mechanism of chromosome alignment, but in contrast to previous reports I did not detect a decrease in Aurora B activity following Mps1 inhibition. On the contrary, both Mps1’s phosphorylation status and its kinetochore localisation were affected by treatment with the Aurora B inhibitor ZM447439, placing Mps1 downstream of Aurora B. As an alternative explanation for the alignment defect in cells with reduced Mps1 activity, I found that levels of the plus-end directed kinesin Cenp-E were markedly decreased at unaligned kinetochores. I propose a model in which catalytically active Mps1’s auto-release from kinetochores simultaneously promotes both mitotic checkpoint signalling and chromosome alignment by facilitating Mad2 dimerisation and Cenp-E binding at unattached kinetochores.
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FANCA maintains genomic stability through regulating BUBR1 acetylationAbdul Sater, Zahi Abass 22 June 2017 (has links)
Indiana University-Purdue University Indianapolis (IUPUI) / Fanconi Anemia (FA), a chromosomal instability syndrome, is characterized by
bone marrow failure, genetic malformations, and predisposition to malignancies
like acute myeloid leukemia (AML) and solid tumors. FA is caused by germline
bi-allelic mutations in one of 21 known FA pathway genes and somatic mutations
in FA genes are also found in a variety of sporadic cancers. Recently, numerous
reports have discovered that the protective function of the FA pathway extends
beyond its canonical role in regulation of DNA repair in interphase. In particular,
the FA pathway has been shown to function in essential mitotic processes
including spindle assembly checkpoint (SAC), cytokinesis, and centrosome
maintenance.
Understanding of the mechanistic origins of genomic instability leading to
carcinogenesis and bone marrow failure has important scientific and clinical
implications. To this end, using a micronucleus assay, we showed that both
interphase DNA damage and mitotic errors contribute to genomic instability in FA
ex vivo and in vivo. Functional studies of primary FA patient cells coupled with
super-resolution microscopy revealed that FANCA is important for centrosome dependent spindle assembly supporting the protective role of FA pathway in
mitotic processes.
Furthermore, we dissected the interactions between the FA pathway and cellular
kinase networks by employing a synthetic lethality sh-RNA screen targeting all
human kinases. We mapped kinases that were synthetically lethal upon loss of
FANCA, particularly those involved in highly conserved signal transduction
pathways governing proliferation and cell cycle homeostasis. We mechanistically
show that loss of FANCA, the most abundant FA subtype, results in in premature
degradation of the mitotic kinase BUBR1 and faster mitotic exit. We further
demonstrate that FANCA is important for PCAF-dependent acetylation of BUBR1
to prevent its premature degradation.
Our results deepen our understanding of the molecular functions of the FA
pathway in mitosis and uncover a mechanistic connection between FANCA and
SAC phosphosignaling networks. These findings support the notion that further
weakening the SAC through targeting kinases like BUBR1 in FA-deficient
cancers may prove to be a rational therapeutic strategy.
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Temporal Coordination Of Mitotic Chromosome Alignment And Segregation: Structural And Functional Studies Of Kif18aKim, Haein 01 January 2018 (has links)
Chromosome alignment is highly conserved in all eukaryotic cell divisions. Microtubule (MT) -based forces generated by the mitotic spindle are integral for proper chromosome alignment and equal chromosome segregation. The kinetochore is a multi-subunit protein complex that assembles on centromeric regions of chromosomes. Kinetochores tether chromosomes to MTs (K fibers) that emanate from opposite poles, in a process called biorientation. This linkage translates K fiber dynamics into chromosome movements during alignment and segregation. Stable, high-affinity kinetochore attachments promote spindle assembly checkpoint (SAC) silencing, which is active when unattached kinetochores are present. During chromosome alignment, 1) K fiber plus-end dynamics decrease, confining chromosome movements near the spindle equator, and 2) electrostatic interactions between kinetochore proteins and MTs increase. Chromosome segregation occurs as soon as all chromosomes are stably attached to microtubules and the SAC has been silenced. SAC silencing and chromosome alignment are temporally coordinated during normal divisions, implying that the mechanisms regulating K fiber dynamics and kinetochore affinity must be linked. Interestingly, HeLa cells depleted of a kinesin-8 motor Kif18A, known for its role in promoting chromosome alignment, display a SAC-dependent mitotic delay due to kinetochore-MT attachment defects. This is puzzling, as Kif18A's function in chromosome alignment is to suppress MT growth by stably associating with MT plus-ends. Whether Kif18A is required for attachment in all cells and how it promotes kinetochore microtubule linkages are not understood.
The work presented in this dissertation supports a model in which Kif18A functions as a molecular link that coordinates chromosome alignment and anaphase onset. We find that Kif18A is required to stabilize kinetochore-MT attachments during mammalian germline development, as germline precursor cells in Kif18A mutant mice are unable to divide during embryogenesis due to an active SAC. However, while all cell types require functional Kif18A for chromosome alignment, mouse primary somatic cells can still divide with normal timing. This finding indicates a cell-type specific dependence on Kif18A for stabilizing kinetochore-MT attachments, and provides evidence that this function might be separate from Kif18A's known role in chromosome alignment. Consistent with this idea, we find that an evolutionarily conserved binding motif for protein phosphatase 1 (PP1) is required for Kif18A's novel role in regulating kinetochore microtubule attachments. Kif18A-PP1 interaction is required for Kif18A-mediated dephosphorylation of the kinetochore protein Hec1, which enhances attachment. However, Kif18A's interaction with PP1 is dispensable for chromosome alignment. Thus, point mutations that disrupt PP1 binding separate Kif18A's role in stabilizing kinetochore attachments from its function in promoting chromosome alignment. Additionally, through structure function studies of the motor domain, we identified a long surface loop (Loop2) that is required for Kif18A's unique MT plus-end binding activity, which is essential for its function in confining chromosome movements. Taken together, we find that Kif18A is molecularly tuned to provide temporal control of chromosome alignment and anaphase entry.
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Initiating the Spindle Assembly Checkpoint Signal: Checkpoint Protein Mad1 Associates with Outer Kinetochore Protein Ndc80 in Budding YeastWeirich, Alexandra 14 June 2013 (has links)
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.
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Quels sont les signaux détectés par le point de contrôle du fuseau lors de la méiose dans l'ovocyte de souris ? / What are the signals detected by the spindle assembly checkpoint in mouse oocyte meiosis?Vallot, Antoine 08 September 2017 (has links)
Au cours de mon travail de doctorat, je me suis intéressé aux mécanismes qui contrôlent la séparation équitable du génome lors de la méiose dans l’ovocyte de souris.Le point de contrôle du fuseau contrôle la ségrégation des chromosomes en méiose : en cas d'attachement incorrect des chromosomes au fuseau, l'anaphase est retardée ce qui permet d'éviter les aneuploïdies. En métaphase, l’attachement des chromosomes homologues aux deux pôles opposés du fuseau, génère une force de tension au niveau des kinétochores. Mon travail de thèse a consisté à déterminer si la tension exercée sur les chromosomes est un signal qui permet de satisfaire le point du contrôle du fuseau en méiose I dans l'ovocyte de souris. Lorsque la tension exercée sur les chromosomes homologues par les microtubules est diminuée par un traitement pharmacologique, la dégradation de la sécurine, qui marque l’entrée en anaphase, est retardée. Si le point de contrôle du fuseau est inhibé en absence de tension, l’anaphase n’est pas retardée, ce qui indique que le point de contrôle du fuseau est sensible à la tension.Nous avons aussi montré que la kinase Aurora B/C n’est pas requise pour la réponse du point de contrôle du fuseau aux chromosomes non attachés, mais qu’elle est essentielle à la réponse du point de contrôle du fuseau à la baisse de tensionDans un contexte où les erreurs de ségrégation en méiose sont très fréquentes chez la femme et augmentent drastiquement avec l'âge, nos travaux pourraient permettre d'identifier si ces mécanismes de contrôle sont diminués et moins efficaces avec l'âge chez la femme. / At each cell division, chromosomes must be faithfully segregated so that exactly one set of chromosomes is passed on to the next generation. The spindle assembly checkpoint (SAC) ensures faithful chromosome segregation in meiosis: upon uncorrect attachment of the chromosome to the spindle, anaphase onset is delayed in order to avoid chromosome missegregation and aneuploidies. For my PhD thesis, I wanted to determine whether tension applied by the spindle microtubules on the chromosomes is itself a signal that satisfies the SAC in mouse oocyte meiosis I. When tension is decreased by small molecule inhibitors, securin degradation, which is a readout of anaphase onset, is delayed. If the SAC is inhibited, then tension defects cannot delay anaphase onset. This indicates that the SAC is able to delay anaphase onset upon tension defects.Furthermore, we showed that Aurora B/C kinase is not required for the SAC response to unattached chromosomes but that Aurora B/C is required for the SAC response to tension defects.Chromosome segregation errors are very common in women and increase with age. In that context, our work could help to identify whether these key control mechanisms are less efficient in the mammalian oocyte with age.
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Initiating the Spindle Assembly Checkpoint Signal: Checkpoint Protein Mad1 Associates with Outer Kinetochore Protein Ndc80 in Budding YeastWeirich, Alexandra January 2013 (has links)
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.
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In vitro effects of 2-methoxyestradiol, an endogenous estrogen, on MCF-12A and MCF-7 cell cycle progressionVan Zijl, Magdalena Catherina 24 July 2007 (has links)
2-Methoxyestradiol (2ME) is an endogenous estrogen metabolite with antiproliferative and antiangiogenic properties. 2ME also plays an active role in the induction of apoptosis, especially in cancerous cells. These properties have been confirmed by various in vitro and in vivo studies and render 2ME a potential antitumor agent. The mechanism of action of 2ME, however, is not yet fully elucidated and it is believed that multiple mechanisms are involved that may be dependent on cell type. The aim of this study was to investigate the differential effects of 2ME on cell growth, morphology and spindle formation in the non-tumorigenic MCF-12A breast cell line and the tumorigenic MCF-7 breast cell line. In dose-dependent studies, cell growth was determined spectrophotometrically. Light microscopy was used to investigate the morphological changes induced by 2ME and its effect on spindle formation was investigated by means of indirect immunofluorescence. The estrogen receptor status of the MCF-12A cells was confirmed with immunocytochemistry. In order to investigate the effect of 2ME on the length of the cell cycle, cells were blocked in early S-phase with hydroxyurea, then allowed to continue through the cell cycle and mitotic indices determined at regular time intervals. Checkpoint kinase and Cdc2 kinase assays were used to determine the effect of 2ME on relevant cell cycle kinases. Although 2ME inhibited cell growth in both cell lines, the MCF-7 cells were inhibited from much lower concentrations and growth inhibition was more pronounced than in the MCF-12A cells. Treated MCF-7 cells showed abnormal metaphase cells, membrane blebbing, apoptotic cells and disrupted spindle formation. These observations were either absent, or not as prominent in the MCF-12A cells. Therefore, differential mechanism(s) of growth inhibition are evident between the normal and tumorigenic cells. Although the two cell lines differ in their estrogen receptor status, this could not explain the differential effects, for 2ME has a very low affinity for the estrogen receptor. 2ME had no effect on the length of the cell cycle, but blocked MCF-7 cells in mitosis. There were no significant alterations in the phosphorylation status of Cdc25C after 2ME treatment. However, Cdc2 activity was increased to a greater extend in the MCF-7 cells than in the MCF-12A cells. Therefore, it is suggested that exposure to 2ME disrupts mitotic spindle formation and enhances Cdc2 kinase activity, leading to persistence of the spindle checkpoint and thus prolonged metaphase arrest, which may result in the induction of apoptosis. The tumorigenic MCF-7 cells are especially sensitive to 2ME treatment compared to the normal MCF-12A cells. 2ME shows potential for the treatment of breast cancer. Selecting the concentration of 2ME that has maximum inhibitory effect on tumorigenic, but minimal effect on normal cells is crucial in its possible application as antitumor agent. Furthermore, research concerning the differential action mechanisms of 2ME is essential to create a better understanding regarding the treatment of cancer and may possibly contribute to the development and/or improvement of novel chemotherapeutic agents. / Dissertation (MSc (Physiology))--University of Pretoria, 2008. / Physiology / unrestricted
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The nuclear pore protein Nup153: Dissecting its role in nuclear envelope and nuclear pore complex architecture and its interaction with the spindle assembly checkpoint protein Mad1Mossaid, Ikram 04 August 2016 (has links) (PDF)
Nuclear pore complexes (NPCs) are embedded in the nuclear envelope (NE) and composed of proteins called nucleoporins. NPCs as such control the bidirectional traffic of proteins and RNAs between the nucleus and the cytoplasm in eukaryotic cells whereas individual nucleoporins were found to be implicated in other cellular processes such as, cell division, kinetochore assembly, gene expression and cell migration. A prime example for nucleoporin functional versatility can be seen in Nup153. Nup153 is since its discovery known to be a central player in nucleocytoplasmic transport, but additionally participates directly or indirectly, for example, in gene expression and cell cycle control. In this context, it was previously shown that altered levels of Nup153 led to mitotic abnormalities, particularly in cytokinesis and in the spindle assembly checkpoint (SAC). The SAC promotes accurate chromosome separation to ensure the faithful segregation of genetic material to daughter cells. Nup153 was found to interact with the SAC protein Mad1. In the present study, we have further dissected the interaction between Nup153 and Mad1 and investigated the function of the Nup153-Mad1 complex in human cells. By using the high resolution imaging technique “in situ proximity ligation assay”, we found that Nup153 and Mad1 interact with each other exclusively in the presence of a NE, from late mitosis to prophase. By in vitro binding assays, we have confirmed the direct interaction between Nup153 and Mad1 and furthermore identified two independent Nup153-binding sites in Mad1. We have also provided some evidence that Nup153 interacts also with SUMO-modified Mad1.It was previously shown that depletion of Nup153 had no obvious effect on Mad1 and SAC activity. In the present study, we have shown by time-lapse imaging microscopy that the depletion of Mad1 led to a delayed recruitment of Nup153 at the reforming NE during anaphase in living cells, which was often accompanied by a prolongation of anaphase. Furthermore, Mad1 depletion led to alterations in the NE architecture, which were characterized by a change of the membrane curvature at the NPC-NE interface. This was followed by an expansion of the spacing between the inner and outer membranes as seen by electron microscopic and three-dimensional structured illumination investigations. This suggests an implication of Mad1 in a mechanism related to the NE reformation and stability independent of the SAC. Mad1 depletion also resulted in redistribution of the ER network and mitochondria throughout the cell as seen by fluorescence microscopy. Nup153 depletion coincided with the NE abnormalities and alteration of these organelles similar to that seen in Mad1-depleted cells. Further, by fluorescence microscopy, we have shown that Nup153 depletion, but not of Mad1, partially affected the localization of the cytoplasmic nucleoporins in human and in mouse cells and thus the NPC integrity. In conclusion, altogether, our results suggest that Nup153 is essential for NE and NPC integrity. Nup153 has likely separable roles in this context: one in post-mitotic NE reformation with Mad1 and one in interphase in NPC assembly. Nup153-Mad1 complex has a function independent of the spindle checkpoint, but important for the establishment of an intact NE architecture. / Les pores nucléaires sont des structures enchâssées dans l’enveloppe nucléaire et composées de protéines appelées les nucléoporines. Ces pores nucléaires contrôlent le trafic bidirectionnel des protéines et des ARNs entre le noyau et le cytoplasme dans les cellules eucaryotes tandis que les nucléoporines individuelles sont également impliquées dans d’autres processus cellulaires tels que la division cellulaire, l’assemblage des kinétochores, l’expression génétique et la migration cellulaire. Un exemple primordial de la versatilité fonctionnelle des nucléoporines peut être observé à travers Nup153. Depuis sa découverte, Nup153 est connue pour être un élément clé dans le transport nucléo-cytoplasmique, mais il a également été démontré qu’elle participait directement ou indirectement à l’expression génétique et au contrôle du cycle cellulaire. Dans ce contexte, nous avons montrés précédemment que des niveaux altérés de Nup153 menaient à des anomalies mitotiques, particulièrement en cytokinèse et dans le point de contrôle de l’assemblage du fuseau mitotique (SAC). Le SAC assure la ségrégation correcte du matériel génétique entre les cellules filles. Il a été montré que Nup153 interagit avec la protéine du SAC Mad1. Dans cette étude, nous avons utilisé une technique d’imagerie de haute résolution, « in situ proximity ligation assay » pour disséquer davantage l’interaction entre Nup153 et Mad1 dans les cellules humaines. Nous avons montré que ces deux protéines interagissent exclusivement au niveau de l’enveloppe nucléaire, depuis les dernières phases de la mitose jusqu’à la prophase. Par des expériences d’interaction in vitro, nous avons également identifiés sur Mad1 deux sites de liaison indépendants pour Nup153. Nous avons également fourni des indications que Nup153 interagit aussi avec une forme SUMOylée de Mad1. La déplétion de Mad1 menait à un recrutement tardif de Nup153 au niveau de l’enveloppe nucléaire en cours de reformation en anaphase dans les cellules vivantes et à des altérations de l’architecture de l’enveloppe nucléaire, caractérisées par un changement de la courbure membranaire au niveau de l’interface pore nucléaire-enveloppe nucléaire. Suite à cela, une expansion de l’espace entre les membranes nucléaires internes et externes a été observée par microscopie électronique. Ceci suggère une implication de Mad1 dans un mécanisme lié à la stabilité de l’enveloppe nucléaire indépendant du SAC. La déplétion de Mad1 résultait également en une redistribution du RE et des mitochondries à travers la cellule. La déplétion de Nup153 coïncidait avec des anomalies similaires au niveau de l’enveloppe nucléaire et des organelles. De plus, la déplétion de Nup153 affectait partiellement la localisation des nucléoporines cytoplasmiques, contrairement à la déplétion de Mad1. Ensemble, nos résultats suggèrent que Nup153 est essentielle pour l’intégrité des pores nucléaires et de l’enveloppe nucléaire. Nup153 semble avoir deux rôles, un au niveau de la formation de l’enveloppe nucléaire en fin de mitose, en complexe avec Mad1 et un autre rôle au niveau de l’assemblage des pores nucléaires. Le complexe Nup153-Mad1 a une fonction indépendante du SAC, mais importante pour l’établissement d’une enveloppe nucléaire intacte. / Doctorat en Sciences / info:eu-repo/semantics/nonPublished
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