Rozprawy doktorskie na temat „Mitotic spindle orientations”

Robust control mechanisms ensure faithful inheritance of an intact genome through the processes of mitosis and cytokinesis. Different populations of the cytoplasmic dynein motor defined by specific dynein adaptor complexes are required for the formation of a stable bipolar mitotic spindle. This study analysed how different dynein subcomplexes contribute to spindle formation and orientation. Various dynein subpopulations were identified by mass spectrometry. I have shown that the dynein light chain 1 (DYNLL1) directly interacts with the kinetochore localised Astrin-Kinastrin complex as well as the spindle microtubule associated complex formed by CHICA and HMMR. I have characterised both complexes and identified unique functions in chromosome alignment and mitotic spindle orientation, respectively. I have found that Kinastrin (C15orf23) is the major Astrin-interacting protein in mitotic cells and is required for Astrin targeting to microtubule plus ends proximal to the plus tip tracking protein EB1. Fixed cell microscopy revealed that cells over-expressing or depleted of Kinastrin mislocalise Astrin. Additionally, depletion of the Astrin-Kinastrin complex delays chromosome alignment and causes the loss of normal spindle architecture and sister chromatid cohesion before anaphase onset (Dunsch et al., 2011). Using immunoprecipitation and microtubule binding assays, I have shown that CHICA and HMMR interact with one another, and target to the spindle by a microtubule-binding site in the amino-terminal region of HMMR. CHICA interacts with DYNLL1 by a series of conserved TQT motifs in the carboxy-terminal region. Depletion of DYNLL1, CHICA or HMMR causes a slight increase in mitotic index but has little effect on spindle formation or checkpoint function. Fixed and live cell microscopy reveal, however, that the asymmetric distribution of cor tical dynein is lost and the spindle in these cells fails to orient correctly in relation to the culture surface (Dunsch et al., 2012). These findings presented here suggest that the Astrin-Kinastrin complex is required for normal spindle architecture and chromosome alignment, and that per turbations of this pathway result in delayed mitosis and non-physiological separase activation, whereas HMMR and CHICA act as par t of a dynein-DYNLL1 complex with a specific function defining or controlling spindle orientation.

Proper orientation of the mitotic spindle is essential during animal development for the generation of cell diversity and organogenesis. To understand the molecular mechanisms regulating this process, genetic studies have implicated evolutionarily conserved proteins that function in diverse cell types to align the spindle along an intrinsic cellular polarity axis. This activity is achieved through physical contacts between astral microtubules of the spindle and a distinct domain of force generating proteins on the cell cortex. In this work, I shed light on how these proteins form distinct cortical domains, how their activity is coupled to their subcellular localization, and how they provide cytoskeletal and motor protein connections that are required to generate the forces necessary to position the mitotic spindle. I first discuss the mechanisms by which Mushroom body defect (Mud; NuMA in mammals), provides spindle orientation cues from various subcellular locations. Aside from its known role at the cortex as an adapter for the Dynein motor, I reveal novel isoform-dependent Mud functions at the spindle poles during assembly of the mitotic spindle and astral microtubules, thus implicating Mud in spindle orientation pathways away from the cell cortex. Moreover, through collaborative efforts with former lab members, I describe molecular regulation and assembly of two ‘accessory’ pathways that activate cortical Mud-Dynein, one through the tumor suppressor protein Discs large (Dlg), and another through the signaling protein Dishevelled (Dsh). I demonstrate that the Dlg pathway is spatially regulated by the polarity kinase atypical Protein Kinase C (aPKC) through direct phosphorylation of Dlg. This signal relieves Dlg autoinhibition to promote cortical recruitment of the Dlg-ligand Gukholder (Gukh), a novel microtubule-binding protein that provides an additional connection between astral microtubules and the cortex that is essential for activity of the Dlg pathway. Lastly, I determine that the Dsh accessory pathway provides an alternative cytoskeletal cue by recruiting Diaphanous (Dia), an actin nucleating protein. By demonstrating interchangeability between the two accessory pathways, we conclude that Mud-Dynein is activated by various cytoskeletal cues and that the mode of activation is cell-context dependent. This dissertation includes unpublished and previously published co-authored material.
10000-01-01

Signaling systems regulate the flow of cellular information by organizing proteins in space and time to coordinate a variety of cellular activities that are critical for the proper development, function, and maintenance of cells. Signaling molecules can exhibit several levels of complexity through the utilization of modular protein interactions, which can generate simple linear behaviors or complex behaviors such as ultrasensitivity. Protein modularity also serves as the basis for the vast protein networks that form the regulatory networks that govern several biological activities. My work focuses on the importance of protein modularity in complex biological systems, in particular the regulatory pathways of spindle positioning. The first part of my work involves the construction of a synthetic regulatory network using modular protein interactions in an effort to understand the complex behavior of the natural spindle orientation regulator Pins. Utilizing well-characterized protein domains and their binding partners, I built an autoinhibited protein switch that can be activated by a small protein domain. We found that the input-output relationship of the synthetic protein switch could be tuned by the simple addition of "decoy" domains, domains that bind and sequester input signal, thereby impeding the onset of the output response to generate an input threshold. By varying the number and affinities of the decoy domains, we found that we could transform a simple linear response into a complex, ultrasensitive one. Thus, modular protein interactions can serve as a source of complex behaviors. The second part of my work focuses on elucidating the molecular mechanisms underlying spindle positioning in the Drosophila neuroblast. I found that Pins orients the mitotic spindle by coordinating two opposite-polarity microtubule motors Dynein and Kinesin-73 through its multiple domains. Kinesin-73 also relies on its modular domain architecture to perform its duties in Pins-mediated spindle positioning, where its N-terminal half functions in coordinating cortical-microtubule capture while its C-terminal half functions as a region necessary for the activation of Dynein. Thus, modular protein design allows for the organization of spindle orientation regulators in space to achieve the complex biological activity that is spindle positioning. This dissertation includes previously published and unpublished coauthored material.
10000-01-01

Les malformations du développement du cortex sont associées à des troubles de la prolifération des progéniteurs et de la migration neuronale. Les glies radiaires basales (bRGs), un type de progéniteur, sont limités dans les espèces lissencéphaliques mais abondants dans les cerveaux gyrencéphaliques. Le gène LIS1, codant pour un régulateur de la dynéine, est muté dans la lissencéphalie humaine. LIS1 a un rôle dans la division cellulaire et la migration neuronale. Dans cette étude, nous avons généré des cellules bRG-like dans le cerveau embryonnaire murin, pour étudier le rôle de Lis1 dans leur production. Ceci fut réalisé par électroporation in utero du gène hominoïde-spécifique TBC1D3 au jour embryonnaire (E) 14.5. Nous avons confirmé que l’expression de TBC1D3 dans des cerveaux WT induit un grand nombre de cellules bRG-like basales. Puis, nous avons étudié la production des bRGs-like dans des cerveaux murins hétérozygotes pour Lis1. Nos résultats novateurs montrent que la déplétion de Lis1 à partir de E9.5 empêche la production de cellules bRG-like induites par TBC1D3. La déplétion de Lis1 change l’orientation du fuseau mitotique, accroit le nombre de mitoses abventriculaires et altère l’expression de N-Cadhérine. Nous concluons que la perturbation du dosage de Lis1 pourrait perturber le nombre et la position corrects des progéniteurs, contribuant à la pathogenèse de Lis1
Human cortical malformations are associated with progenitor proliferation and neuronal migration abnormalities. Basal radial glia (bRGs), a type of progenitor cells, are limited in lissencephalic species (e.g. the mouse) but abundant in gyrencephalic brains. The LIS1 gene coding for a dynein regulator, is mutated in human lissencephaly, associated also in some cases with microcephaly. LIS1 was shown to be important during cell division and neuronal migration. Here, we generated bRG-like cells in the mouse embryonic brain, investigating the role of Lis1 in their formation. This was achieved by in utero electroporation of a hominoid-specific gene TBC1D3 at mouse embryonic day (E) 14.5. We first confirmed that TBC1D3 overexpression in WT brain generates numerous Pax6+ bRG-like cells that are basally localized. Second, we assessed the formation of these cells in heterozygote Lis1 mutant brains. Our novel results show that Lis1 depletion in the forebrain from E9.5 prevented subsequent TBC1D3-induced bRG-like cell amplification. Lis1 depletion changed mitotic spindle orientations at the ventricular surface, increased the proportion of abventricular mitoses, and altered N-Cadherin expression, altering TBC1D3 function. We conclude that perturbation of Lis1/LIS1 dosage is likely to be detrimental for appropriate progenitor number and position, contributing to lissencephaly pathogenesis

The Drosophila neuroblast undergoes repeated asymmetric cell divisions that produce one daughter cell that assumes a neuronal fate and another that remains a neuroblast. During mitosis, the neuroblast polarizes the conserved Par polarity complex to the apical cortex, which is responsible for segregating fate determinants to the basal cell cortex. Polarity is accompanied by orientation of the mitotic spindle through the proteins Pins, Mud, and Dlg to ensure that the cleavage furrow properly segregates the fate determinants. The adaptor protein Inscuteable coordinates these two pathways. In my work, I have addressed how asymmetrically dividing cells are dynamically polarized during the cell cycle and how the resulting polarity is coupled to spindle position. To address how neuroblast polarity is dynamically controlled, I identified the protein Inscuteable as a continuously polarized cue for Par complex localization during mitosis. Inscuteable and Bazooka, a member of the Par complex, interact directly and form a complex that is regulated by the mitotic kinase Aurora A. Regulating this interaction allows for cell-cycle dependent establishment of polarity and for the subsequent loss of polarity after the cell divides. To investigate how Par complex directed polarity is connected to spindle position, I investigated the effect of Inscuteable binding on the spindle orientation ability of the protein Pins. When bound to Inscuteable, Pins' spindle orientation activity becomes repressed. Inscuteable competes with Mud for Pins binding and represses the Gai-Pins-Mud signaling pathway. Function of the parallel Pins-Dlg pathway remains unaffected. This repression behavior may allow differential timing of spindle attachment (through Dlg) and spindle shortening (through Mud) pathways that ensures correct alignment of the mitotic spindle. I was able to model the spindle orientation behavior of Pins using a synthetic protein containing activation sites that have different affinities for the activator. Changing the number and affinities of these activation sites leads to different response profiles that mimic the ultrasensitive behavior of Pins using a non-cooperative mechanism. Together, these regulatory mechanisms cooperate to allow for spatial and temporal control of polarity and for physical connection of polarity to the mitotic spindle. This dissertation includes previously published and unpublished co-authored material.

Correct spindle positioning is essential for tissue morphogenesis and homeostasis. The orientation of the mitotic spindle is determined by cortical force generators formed on NuMA:LGN:Gαi complexes, which anchor astral microtubules emanating from the spindle poles at specialized domains of the plasma membrane via direct interaction with the motor proteins Dynein/Dynactin. Cortical polarity cues and actin-associated proteins synergize with extrinsic signals (such as cell-to-cell and cell-to-extracellular matrix contacts) in recruiting NuMA:LGN:Gαi complexes at the cell cortex. In addition, spindle placement is coordinated with mitotic progression by mitotic kinases regulating the timely cortical recruitment of NuMA:LGN:Gαi above the spindle poles. My PhD project focused on the study of the molecular mechanisms accounting for the spindle orientation functions of the Aurora-A kinase, the polarity protein Lgl2, and the junctional protein Afadin. The Aurora-A kinase is known for being implicated in spindle alignment, however the molecular events underlying this function remain to date unclear. To study the spindle orientation functions of Aurora-A, I developed protocols for the partial inhibition of its activity in transformed and non-transformed cells in culture. Under these conditions, in metaphase NuMA and Dynactin accumulate abnormally at the spindle poles without reaching the cortex, while the cortical distribution of LGN remains unperturbed. Fluorescence Recovery After Photobleaching (FRAP) experiments conducted on GFP-NuMA revealed that Aurora-A governs the dynamic exchange between the cytoplasmic and the spindle-pole-localized pools of NuMA. Molecularly, Aurora-A phosphorylates directly the C-terminus of NuMA on three serine residues, among which Ser-1969 is the major determinant for the dynamic behaviour of NuMA at the spindle poles. Most interestingly, we identify a new microtubule-binding domain of NuMA, which does not overlap with the LGN-binding motif, thus suggesting that NuMA can associate concomitantly with LGN and microtubules. This finding indicates that the microtubule-binding activity of NuMA might contribute to anchor microtubule +TIPs at cortical sites with LGN. Collectively, my studies demonstrate that in metaphase the direct phosphorylation of NuMA by Aurora-A controls its cortical enrichment, and that this is the major event underlying the spindle orientation functions of Aurora-A in cultured cells. Phosphorylation of NuMA by Aurora-A does not affect its affinity for microtubules nor for LGN, but rather determines the mobility of the protein at the spindle-poles. Biochemical studies suggested that Lgl2 can associate with LGN, hinting at a possible role of this protein in spindle orientation in mammalian system. On these premises, I found that depletion of Lgl2 misorients the spindle in HeLa cells plated on fibronectin. However, I could not reproduce the Lgl2:LGN interaction in vitro nor ex vivo. Interestingly, by immunoprecipitation experiments I detected an interaction between NuMA and Lgl2, which could explain the phenotype of spindle misorientation resulting from the silencing of Lgl2 in HeLa cells. Further studies will be required to gain a molecular understanding of the relevance Lgl2:NuMA interaction in oriented divisions. Part of my PhD studies addressed the role of Afadin in spindle orientation; I demonstrated that Afadin is required for spindle positioning, and correct epithelial morphogenesis of Caco-2 three-dimensional cysts. At a molecular level, Afadin binds directly and concomitantly to F-actin and to LGN. Indeed, in mitotic HeLa cells, Afadin is required for cortical accumulation of LGN, NuMA and Dynein above the spindle poles, in a F-actin dependent manner. Collectively, these results uncovered a pivotal role of Afadin in governing the enrichment of LGN and NuMA at the lateral cortex of polarized epithelia. They also depict that Afadin as the first mechanical anchor between Dynein and cortical F-actin.

Carriers of mutations in the Breast Cancer 1 (BRCA1) gene have an increased risk to develop breast cancer, which tend to be early-onset, lack expression of estrogen receptor, progesterone receptor and human epidermal growth factor receptor 2, and resemble basal epithelia by gene expression. The phenotypic resemblance of these tumours to normal stem/progenitor cells suggests that the loss of BRCA1 function may dysregulate stem cell maintenance and differentiation. In model organisms, a mechanism that promotes the generation of daughter cells different from a mother stem cell is the asymmetric segregation of non-genetic factors through mitotic spindle orientation. BRCA1 regulates mitotic spindle assembly through the post-transcriptional degradation of the low-penetrance breast cancer susceptibility gene product RHAMM and the abundance of RHAMM influences mitotic spindle orientation by regulating its movement along the cell cortex. This led to the hypothesis that BRCA1 is necessary for the correct orientation of the mitotic spindle in mammary epithelial cells, which controls their proliferation, polarization and growth arrest. To address this hypothesis, I studied non-malignant human mammary cell-lines and primary human progenitor cell-enriched populations. BRCA1 was silenced by shRNA introduced through lentiviral transduction. Silencing of BRCA1 in cell-lines increased both mitotic and post-mitotic abnormalities, including the loss of spindle orientation with subsequent lagging chromosomes and micronucleus formation in 2D cultures. The consequence of these defects included a significant decrease in colony-forming capacity. I then enquired whether BRCA1 is necessary for MCF10A cells to proliferate, form polarized acini, and growth arrest in 3D cultures. Control cells underwent planar division to form polarized, growth arrested acini, while BRCA1 silenced structures were larger, less polarized and more proliferative. Loss of correct spindle orientation was also observed. These results indicate that BRCA1 plays a role in maintaining the integrity of human mammary cell division. Loss of BRCA1 induces mitotic and post-mitotic consequences that impair cellular proliferative capacity and abolish ability to undergo directional division, polarization and arrest growth. These findings thus raise the possibility that breast cancer treatments aimed at counteracting the BRCA1-mediated loss of polarity may complement drugs that combat the diminished DNA repair characteristic of BRCA1-associated tumours.
Medicine, Faculty of
Medicine, Department of
Experimental Medicine, Division of
Graduate

Mitotic spindle orientation is a prerequisite for the correct completion of mitosis, and is essential for tissue morphogenesis and maintenance. Divisions occurring within the plane of epithelia, or planar divisions, shape the architecture of epithelial sheets, whereas vertical divisions along the apicobasal axis are associated with asymmetric fate specification and stratification. Several studies described the evolutionary conserved trimeric Gi:LGN:NuMA complex as the core constituent of the spindle orientation machinery. In mitosis Gi:LGN:NuMA complexes localize at the cortex and orient the spindle by generating pulling forces on astral microtubules emanating from the spindle poles, via interaction of NuMA with the minus-end directed motor proteins dynein/dynactin. Biochemical and structural studies identified the minimal binding domains of the NuMA:LGN interaction, showing that a 30-residues stretch in the C-terminal part of NuMA binds to the inner groove formed by the eight TPR repeats at the N-terminus of LGN. However, how such interaction is organized at the cell cortex and triggers microtubules-motor activation still remains largely unclear. My PhD project focused on the characterization of the NuMA:LGN interaction and on the analysis of the role of the microtubule-binding domain of NuMA. Studies conducted during this thesis revealed that NuMA and LGN assemble in hetero-hexameric structures organized in a donut-shape architecture. In such arrangement, the LGN helices preceding and following the TPR repeats, and a NuMA motif preceding the shortest LGN-binding motif, are essential for the interaction. Consistently, an LGN oligomerization-deficient mutant cannot rescue misorientation defects caused in HeLa cells and Caco-2 three-dimensional cysts by endogenous LGN ablation. Importantly, in cells expressing the oligomerization-deficient mutant, force generators are correctly localized at the cell cortex. We provided evidence that LGN and NuMA assemble high-order oligomers in cells, and that the 3:3 stoichiometry of the NuMA:LGN complex combined with the dimeric state of NuMA coiled-coils promote the formation of a large proteins network. We also showed that ectopic targeting of an oligomerization-deficient NuMA mutant at the cortex is not sufficient to orient the spindle, indicating that the molecular organization of NuMA in complex with LGN is required to orient the spindle in metaphase. Furthermore, we provided evidence that the NuMA:LGN oligomers are compatible with the direct association of NuMA to microtubules, and that the microtubules-binding domain of NuMA is required to correctly localize NuMA at the poles and at the cortex, and to orient the spindle. Collectively, our findings suggest a model whereby cortical LGN:NuMA hetero-hexamers favor the accumulation of dynein motors at cortical sites. We speculate that direct binding of NuMA to astral microtubule plus-tips assists the processive movement of dynein along the depolymerizing astral microtubules to promote spindle placement.

Pour maintenir l'architecture du tissue, les cellules épithéliales se divisent de manière planaire, perpendiculaire à leur axe principal de polarité. Du fait que le centrosome retrouve sa localisation apicale à l'interphase l'orientation du fuseau mitotique est réinitialisée à chaque cycle cellulaire. Nous utilisons de l'imagerie live en trois dimensions de centrosome marqués en GFP pour investiguer la dynamique de l'orientation du fuseau mitotique des cellules neuroépithéliales de l'embryon de poulet. Le fuseau mitotique présente des mouvements stéréotypiques pendant la métaphase, avec dans un premier temps une phase active de d'orientation planaire suivie par une phase de maintenance planaire jusqu'à l'anaphase. Nous décrivons la localisation des protéines NuMA et LGN formant un anneau au niveau du cortex latéral cellulaire au moment de l'orientation du fuseau. Enfin, nous montrons que le complexe protéique formé par LGN, NuMA et par la sous unité Gai localisé au cortex est nécessaire pour les mouvements du fuseau et pour réguler la dynamique de l'orientation du fuseau. La localisation restreinte de LGN et NuMA en anneau cortical est instructive pour l'alignement planaire du fuseau mitotique et est également requise pour sa maintenance planaire
To maintain tissue architecture, epithelial cells divide in a planar fashion, perpendicular to their main polarity axis. As the centrosome resumes an apical localization in interphase, planar spindle orientation is reset at each cell cycle. We used three-dimensional live imaging of GFP-labeled centrosomes to investigate the dynamics of spindle orientation in chick neuroepithelial cells. The mitotic spindle displays stereotypic movements during metaphase, with an active phase of planar orientation and a subsequent phase of planar maintenance before anaphase. We describe the localization of the NuMA and LGN proteins in a belt at the lateral cell cortex during spindle orientation. Finally, we show that the complex formed of LGN, NuMA, and of cortically located Gái subunits is necessary for spindle movements and regulates the dynamics of spindle orientation. The restricted localization of LGN and NuMA in the lateral belt is instructive for the planar alignment of the mitotic spindle, and required for its planar maintenance

La mutation à l’origine de la maladie de Huntington (MH) correspond à une expansion anormale de glutamines sur la protéine huntingtine (HTT). La MH est caractérisée par des symptômes moteurs et cognitifs mais également des troubles psychiatriques tels que l’anxiété et la dépression.Au cours de ma thèse, j’ai montré que la HTT module le statut anxio-dépressif de la souris via ses phosphorylations aux sérines 1181/1201. En effet, l’ablation des phosphorylations sur la HTT endogène améliore significativement le phénotype anxio-dépressif de la souris. Chez la souris, cette modulation dépend d’une augmentation de la maturation et de la survie des nouveaux neurones dans l’hippocampe. En effet, l’irradiation focale de l’hippocampe, dans un contexte où les phosphorylations sont absentes, supprime la neurogenèse et la réduction du statut anxio-dépressif observée en l’absence de phosphorylations. Au niveau moléculaire, la HTT non phosphorylée accroît l’association des moteurs moléculaires et des vésicules de BDNF sur les microtubules, ce qui augmente les dynamiques et la libération du BDNF. Ceci active la voie de signalisation MAPK/CREB dans l’hippocampe, cette voie pouvant ainsi stimuler la neurogenèse.J’ai ensuite étudié le rôle de ces phosphorylations dans un contexte MH et j’ai démontré l’effet anxiolytique/antidépresseur de l’absence de ces phosphorylations.J’ai également montré le rôle de ces phosphorylations de la HTT au cours du développement du cortex embryonnaire.Les résultats obtenus au cours de ma thèse suggèrent que les mécanismes fondamentaux de neurogenèse sont régulés par la HTT et ses phosphorylations. De plus, ils identifient une nouvelle voie de modulation de l’anxiété/dépression faisant intervenir la HTT
Huntington disease (HD) is a fatal neurodegenerative disorder associated with early psychiatric symptoms including anxiety and depression.During my thesis, I have demonstrated that huntingtin, the protein mutated in HD, modulates anxiety/depression-related behaviors through its phosphorylations at serines 1181 and 1201. Indeed, genetic phospho-ablation at serines 1181 and 1201 in mouse reduces basal levels of anxiety/depression-like behaviors in mouse. Suppression of neurogenesis by focal hippocampal irradiation abolishes this reduction of basal levels of anxiety/depression on some behavioral test demonstrating that neurogenesis is involved in this process. Ablation of HTT phosphorylations may stimulate neurogenesis through BDNF transport, release and signaling.I have also shown that ablation of phosphorylations on HTT is sufficient to ameliorate the anxiety/depression-like behavior of a mouse model of HD, which develops a behavior indicative of depression–like state.I have finally explored the role of HTT phosphorylation at serines 1181 and 1201 during brain development. During early steps of cortical neurogenesis, I have shown that ablation of HTT phosphorylations affects the mitosis of cortical progenitors, the fate of newly generated cells and the migration of new neurons.The results obtained during my thesis support the notion that HTT regulates key molecular mechanisms during neurogenesis both in adult and embryo. It also supports the notion that huntingtin participates to anxiety and depression-like behavior with potential consequences for the etiology of mood disorders and anxiety/depression in HD