Auswahl der wissenschaftlichen Literatur zum Thema „Division cellulaire orientée“

Development in multicellular organisms relies on cell proliferation and specialization. In plants, both these processes critically depend on the spatial organization of cells within a tissue. Owing to an absence of significant cellular migration, the relative position of plant cells is virtually made permanent at the moment of division. Therefore, in numerous plant developmental contexts, the (divergent) developmental trajectories of daughter cells are dependent on division plane positioning in the parental cell. Prior to and throughout division, specific cellular processes inform, establish and execute division plane control. For studying these facets of division plane control, the moss Physcomitrium (Physcomitrella) patens has emerged as a suitable model system. Developmental progression in this organism starts out simple and transitions towards a body plan with a three-dimensional structure. The transition is accompanied by a series of divisions where cell fate transitions and division plane positioning go hand in hand. These divisions are experimentally highly tractable and accessible. In this review, we will highlight recently uncovered mechanisms, including polarity protein complexes and cytoskeletal structures, and transcriptional regulators, that are required for 1D to 3D body plan formation.

We have taken advantage of the optical transparency of zebrafish embryos to investigate the patterns of cell division, movement and shape during early stages of development of the central nervous system. The surface-most epiblast cells of gastrula and neurula stage embryos were imaged and analysed using a computer-based, time-lapse acquisition system attached to a differential interference contrast (DIC) microscope. We find that the onset of gastrulation is accompanied by major changes in cell behaviour. Cells collect into a cohesive sheet, apparently losing independent motility and integrating their behaviour to move coherently over the yolk in a direction that is the result of two influences: towards the vegetal pole in the movements of epiboly and towards the dorsal midline in convergent movements that strengthen throughout gastrulation. Coincidentally, the plane of cell division becomes aligned to the surface plane of the embryo and oriented in the anterior-posterior (AP) direction. These behaviours begin at the blastoderm margin and propagate in a gradient towards the animal pole. Later in gastrulation, cells undergo increasingly mediolateral-directed elongation and autonomous convergence movements towards the dorsal midline leading to an enormous extension of the neural axis. Around the equator and along the dorsal midline of the gastrula, persistent AP orientation of divisions suggests that a common mechanism may be involved but that neither oriented cell movements nor shape can account for this alignment. When the neural plate begins to differentiate, there is a gradual transition in the direction of cell division from AP to the mediolateral circumference (ML). ML divisions occur in both the ventral epidermis and dorsal neural plate. In the neural plate, ML becomes the predominant orientation of division during neural keel and nerve rod stages and, from late neural keel stage, divisions are concentrated at the dorsal midline and generate bilateral progeny (C. Papan and J. A. Campos-Ortega (1994) Roux's Arch. Dev. Biol. 203, 178–186). Coincidentally, cells on the ventral surface also orient their divisions in the ML direction, cleaving perpendicular to the direction in which they are elongated. The ML alignment of epidermal divisions is well correlated with cell shape but ML divisions within the neuroepithelium appear to be better correlated with changes in tissue morphology associated with neurulation.

Tissue morphogenesis requires the coordinated regulation of cellular behavior, which includes the orientation of cell division that defines the position of daughter cells in the tissue. Cell division orientation is instructed by biochemical and mechanical signals from the local tissue environment, but how those signals control mitotic spindle orientation is not fully understood. Here, we tested how mechanical tension across an epithelial monolayer is sensed to orient cell divisions. Tension across Madin–Darby canine kidney cell monolayers was increased by a low level of uniaxial stretch, which oriented cell divisions with the stretch axis irrespective of the orientation of the cell long axis. We demonstrate that stretch-induced division orientation required mechanotransduction through E-cadherin cell–cell adhesions. Increased tension on the E-cadherin complex promoted the junctional recruitment of the protein LGN, a core component of the spindle orientation machinery that binds the cytosolic tail of E-cadherin. Consequently, uniaxial stretch triggered a polarized cortical distribution of LGN. Selective disruption of trans engagement of E-cadherin in an otherwise cohesive cell monolayer, or loss of LGN expression, resulted in randomly oriented cell divisions in the presence of uniaxial stretch. Our findings indicate that E-cadherin plays a key role in sensing polarized tensile forces across the tissue and transducing this information to the spindle orientation machinery to align cell divisions.

Cell lineage analysis of central nervous system progenitors during gastrulation and early segmentation in the zebrafish reveals consistent coupling of specific morphogenetic behaviors with particular cell cycles. Cells in single clones divide very synchronously. Cell divisions become progressively oriented, and act synergistically with oriented intercalations during the interphases of zygotic cell cycles 15 and 16 to extend a single lineage into a long, discontinuous string of cells aligned with the nascent embryonic axis. Dorsalwards convergence brings the string to the midline and, once there, cells enter division 16. This division, or sometimes the next one, and the following cell movement reorient to separate siblings across the midline. This change converts the single string into a bilateral pair of strings, one forming a part of each side of the neural tube. The stereotyped cellular behaviors appear to account for the previously reported clonal restriction in cell fate and to underlie morphogenesis of a midline organ of proper length and bilateral shape. Regulation of cellular morphogenesis could be cell-cycle dependent.

The pattern of cell division is very regular in Arabidopsis embryogenesis, enabling seedling structures to be traced back to groups of cells in the early embryo. Recessive mutations in the FASS gene alter the pattern of cell division from the zygote, without interfering with embryonic pattern formation: although no primordia of seedling structures can be recognised by morphological criteria at the early-heart stage, all elements of the body pattern are differentiated in the seedling. fass seedlings are strongly compressed in the apical-basal axis and enlarged circumferentially, notably in the hypocotyl. Depending on the width of the hypocotyl, fass seedlings may have up to three supernumerary cotyledons. fass mutants can develop into tiny adult plants with all parts, including floral organs, strongly compressed in their longitudinal axis. At the cellular level, fass mutations affect cell elongation and orientation of cell walls but do not interfere with cell polarity as evidenced by the unequal division of the zygote. The results suggest that the FASS gene is required for morphogenesis, i.e., oriented cell divisions and position-dependent cell shape changes generating body shape, but not for cell polarity which seems essential for pattern formation.

Cranial neural crest cells populate the future facial region and produce ectomesenchyme-derived tissues, such as cartilage, bone, dermis, smooth muscle, adipocytes, and many others. However, the contribution of individual neural crest cells to certain facial locations and the general spatial clonal organization of the ectomesenchyme have not been determined. We investigated how neural crest cells give rise to clonally organized ectomesenchyme and how this early ectomesenchyme behaves during the developmental processes that shape the face. Using a combination of mouse and zebrafish models, we analyzed individual migration, cell crowd movement, oriented cell division, clonal spatial overlapping, and multilineage differentiation. The early face appears to be built from multiple spatially defined overlapping ectomesenchymal clones. During early face development, these clones remain oligopotent and generate various tissues in a given location. By combining clonal analysis, computer simulations, mouse mutants, and live imaging, we show that facial shaping results from an array of local cellular activities in the ectomesenchyme. These activities mostly involve oriented divisions and crowd movements of cells during morphogenetic events. Cellular behavior that can be recognized as individual cell migration is very limited and short-ranged and likely results from cellular mixing due to the proliferation activity of the tissue. These cellular mechanisms resemble the strategy behind limb bud morphogenesis, suggesting the possibility of common principles and deep homology between facial and limb outgrowth.

Cellular self-organization is essential to physiological tissue and organ development. We previously observed that vascular mesenchymal cells, a multipotent subpopulation of aortic smooth muscle cells, self-organize into macroscopic, periodic patterns in culture. The patterns are produced by cells gathering into raised aggregates in the shape of nodules or ridges. To determine whether these patterns are accounted for by an oriented pattern of cell divisions or postmitotic relocation of cells, we acquired time-lapse, videomicrographic phase-contrast, and fluorescence images during self-organization. Cell division events were analyzed for orientation of daughter cells in mitoses during separation and their angle relative to local cell alignment, and frequency distribution of the mitotic angles was analyzed by both histographic and bin-free statistical methods. Results showed a statistically significant preferential orientation of daughter cells along the axis of local cell alignment as early as day 8, just before aggregate formation. This alignment of mitotic axes was also statistically significant at the time of aggregate development ( day 11) and after aggregate formation was complete ( day 15). Treatment with the nonmuscle myosin II inhibitor, blebbistatin, attenuated alignment of mitotic orientation, whereas Rho kinase inhibition eliminated local cell alignment, suggesting a role for stress fiber orientation in this self-organization. Inhibition of cell division using mitomycin C reduced the macroscopic pattern formation. Time-lapse monitoring of individual cells expressing green fluorescent protein showed postmitotic movement of cells into neighboring aggregates. These findings suggest that polarization of mitoses and postmitotic migration of cells both contribute to self-organization into periodic, macroscopic patterns in vascular stem cells.

Immunofluorescence microscopy has proven to be a valuable accompaniment to electron microscopy for study of the cytoskeleton of plant cells. Whereas electron microscopy provides greater resolution and details of the spatial relationships of the cytoskeleton to other cellular components, fluorescence visualization makes it possible to see the three-dimensional organization of cytoskeletal elements without laborious reconstruction of views from serial sections. An area in which immunofluorescence microscopy has been useful is the investigation of how plant cells organize and position the various microtubule arrays that are utilized during mitosis, cytokinesis and cell expansion phases. One of the earliest indications of an impending division in a meristematic plant cell is the formation of a preprophase band of microtubules in the cell cortex, at the site where the new wall will be placed at the subsequent cytokinesis. At its later stages, the band is narrower than when first identifiable. In most cells, preprophase band microtubules have the same general orientation as the preceding interphase microtubules, and so preprophase band formation here could, in theory, be achieved by lateral bundling of microtubules.Cells in which the division site and the preprophase band that marks it are not oriented parallel to interphase microtubules are found in stomatal complexes of grass leaves . Fig. 1 illustrates the arrangement of two such cell types: the guard mother cell, which divides lengthwise to form two guard cells, side-by-side, and the subsidiary mother cell, which undergoes a very asymmetric division to produce one of the pair of lens-shaped subsidiary cells that flank the guard cells. Interphase and preprophase arrangements of microtubules for each cell type are diagrammed in Figs. 2-4. In order to examine how these cell types achieve the reorientation of microtubules that is necessary to progress from interphase to preprophase, sheets of epidermis containing actively dividing stomatal complex cells were examined with immunofluorescence microscopy using antibodies to tubulin. Thin epidermal slices of leaves were fixed and glued down to a slide, whereupon cell walls were enzymatically weakened so that unwanted cell layers could be removed . Because waves of division pass along grass leaves, cells of the same type in a given file tend to be at similar stages, which facilitates deduction of the developmental pattern.

ABSTRACTProper epithelial development and homeostasis depends on strict control of oriented cell division. Current evidence shows that this process is regulated by intrinsic polarity factors and external spatial cues. Owing to the lack of an appropriate model system that can recapitulate the architecture of the skin, deregulation of spindle orientation in human epithelial carcinoma has never been investigated. Here, using an inducible model of human squamous cell carcinoma (SCC), we demonstrate that RAS-dependent suppression of PAR3 (encoded by PARD3) accelerates epithelial disorganization during early tumorigenesis. Diminished PAR3 led to loss of E-cadherin-mediated cell adhesion, which in turn contributed to misoriented cell division. Pharmacological inhibition of the MAPK pathway downstream of RAS activation reversed the defects in PAR3 expression, E-cadherin-mediated cell adhesion and mitotic spindle orientation. Thus, temporal analysis of human neoplasia provides a powerful approach to study cellular and molecular transformations during early oncogenesis, which allowed identification of PAR3 as a critical regulator of tissue architecture during initial human SCC development.

The Caenorhabditis elegans germ line provides a model for understanding how signaling from a stem cell niche promotes continued mitotic divisions at the expense of differentiation. Here we report cellular analyses designed to identify germline stem cells within the germline mitotic region of adult hermaphrodites. Our results support several conclusions. First, all germ cells within the mitotic region are actively cycling, as visualized by bromodeoxyuridine (BrdU) labeling. No quiescent cells were found. Second, germ cells in the mitotic region lose BrdU label uniformly, either by movement of labeled cells into the meiotic region or by dilution, probably due to replication. No label-retaining cells were found in the mitotic region. Third, the distal tip cell niche extends processes that nearly encircle adjacent germ cells, a phenomenon that is likely to anchor the distal-most germ cells within the niche. Fourth, germline mitoses are not oriented reproducibly, even within the immediate confines of the niche. We propose that germ cells in the distal-most rows of the mitotic region serve as stem cells and more proximal germ cells embark on the path to differentiation. We also propose that C. elegans adult germline stem cells are maintained by proximity to the niche rather than by programmed asymmetric divisions.

La morphogenèse est un processus qui nécessite une régulation à plusieurs niveaux à la fois physique et génétique. Les perturbations de ce programme dans le contexte du cœur ont souvent des conséquences importantes sur l'organe, comme le prouve l’incidence de 1% des cardiopathies congénitales à la naissance. Les cardiopathies congénitales telles que les cardiomyopathies, affectent l’architecture du muscle cardiaque essentielle à sa fonction contractile. Les parois ventriculaires sont particulièrement importantes, à la fois pour définir la taille des lumières ventriculaires et pour établir une architecture de myofibre orientée, renforçant l’efficacité de la contraction. Des travaux antérieurs dans le laboratoire ont permis de mettre en évidence l’émergence de l’orientation du myocarde. L'analyse clonale a révélé que la croissance tissulaire orientée corrélait avec l'expansion des ventricules (Sigolène M. Meilhac et al., 2004) et préfigurait l’architecture des myofibres du cœur nouveau-né (Meilhac et al., 2003). L'analyse de l’architecture cellulaire a révélé une coordination locale des divisions cellulaires indiquant une orientation des divisions cellulaires (Le Garrec et al., 2013). Ces études suggèrent que la division cellulaire orientée joue un rôle important dans la formation du cœur. Cependant, les mécanismes par lesquels cela est régulé doivent encore être identifiés dans les ventricules embryonnaires. Dans ce projet, nous utilisons une combinaison d'approches transcriptomique, segmentation cellulaire 3D, traitements chimiques en culture d'embryons et interférence moléculaire pour, étudier un mécanisme de division cellulaire orientée. Par séquençage ARN des ventricules, nous avons identifié l’expression de composants de l’appareil NuMA: GPSM, de la voie de la polarité cellulaire planaire et de la voie de mécano-détection des intégrines, qui sont des voies candidates pour réguler l’orientation de la division cellulaire. En parallèle, nous avons voulu déterminer si les cellules des ventricules en expansion se comportaient conformément à la règle de Hertwig. Pour ce faire, nous avons mis en place une méthode d’imagerie de l’architecture cellulaire dans le cœur entier par transparisation CUBIC et microscopie tridimensionnelle à feuille de lumière. Nous avons également amorcé le développement d’une méthode automatique de segmentation pour quantifier les axes de division cellulaire dans les ventricules. En comparant l’axe d'élongation des cellules aux axes de division les outils et les approches décrite ci-dessus permettront de conclure s'il existe une coordination entre les deux. Pour analyser l'importance des contractions cardiaques sur la croissance orientée des ventricules, nous avons établi des conditions de culture d'embryons traités avec des perturbateurs pharmaceutiques de la contraction cardiaque. Les résultats préliminaires indiquent qu'une augmentation et une diminution du taux de contraction affectent la forme du cœur. Enfin, nous avons conçu des vecteurs pour cibler les trois voies mentionnées ci-dessus avec des protéines dominant négatives. Les résultats de cette recherche pourraient avoir des applications en ingénierie tissulaire pour la réparation du cœur
The development of the heart is an intricate process both physically and genetically which requires regulation on many levels. Perturbations of this cardiogenic programme often has potent consequence on the organ and this is evident from the 1% incidence in births which are affected by a congenital heart disease (CHD). CHDs, such as cardiomyopathies, affect the architecture of the cardiac muscle, which is vital to the heartsfunction. The shape of the ventricular walls is particularly important to their function in terms of both defining the shape of the ventricular chambers and in establishing an appropriate myofiber architecture for efficient contractions (Meilhac et al., 2003). Previous work in the lab has provided insight into how this is achieved in the ventricles. It was found, through clonal analysis, that oriented tissue growth underlies cardiac chamber expansion (Meilhac et al., 2004). Analysis of earlier stages of the embryonic heart found regional coordination of cell divisions which preconfigured the myofiber architecture of the adult heart (Le Garrec et al., 2013). These studies suggest that oriented cell division plays an important role in sculpting the heart. However a mechanism by which this is regulated has yet to be established in the expanding ventricular chambers. In this project we use a combination of transcriptomic analysis, 3D cell segmentation, embryo culture experiments and molecular interference to investigate a mechanism for oriented cell division. Using bulk RNAseq we identified the NuMA:GPSM apparatus, the Planar Cell Polarity pathway and the integrin mechano-sensing pathway as candidates for further analysis. In combination with the transcriptomic analysis we wanted to identify if cells in the expanding ventricles were behaving according to Hertwig’s rule. To do this we have established CUBIC clearing and three dimensional lightsheet microscopy along with an automatic cell segmentation method to quantify cell elongations in the cardiac chambers. By comparing the elongation ratio of the cell to the detected axes of division the tools and approaches described above will enable us to identify if coordination existed between the two and if this was regionally specific. To analyse the impact of cardiac contractions on oriented cell division we established embryo culture experiment conditions paired with pharmaceutical interference of contractions. Preliminary results indicate that both an increase and decrease of contraction rate affects the shape of the heart. Finally, we will target the three pathways mentioned above with dominant negative proteins in chimeric hearts to dissect the molecular pathways. The outcome of this research will have potential applications in tissue engineering therapies targeting the heart

L'homéostasie de l'épithélium de la muqueuse colique est régie par un ensemble de voies de signalisation, au rang desquelles la voie Wnt. Le gène suppresseur de tumeur Apc (Adenomatous Polyposis Coli) joue un rôle majeur dans la régulation de la voie Wnt. Des mutations germinales hétérozygotes prédisposent fortement au cancer du côlon, et les premières structures aberrantes visibles à l'échelle tissulaire, ACF (Aberrant Crypt Foci), apparaissent suite à la perte de l'allèle sauvage. Ainsi un des premiers signes de la perte d'APC est une modification morphologique de la crypte. Pour autant, pratiquement rien n'est connu sur la morphogenèse de ce tissu à l'âge adulte, en particulier quel est le rôle des divisions orientées dans ce processus, et l'implication potentielle des mutations dans Apc dans ces mécanismes. Nous avons développé une technique d'observation de cryptes entières fixées et intactes pour la mesure d'orientation des fuseaux mitotiques avec l'axe long de la crypte. Nos résultats montrent qu'il existe une orientation préferentielle, avec 80% des fuseaux mitotiques formant un angle de moins de 30° avec l'axe de la crypte. La perte totale d'APC perturbe profondément cette orientation, et est corrélée à une augmentation du diamètre de la crypte. Une mutation hétérozygote dans Apc n'a pas d'effet sur l'orientation, et une seconde mutation (situation rencontrée dans les ACF) est nécessaire pour perturber l'orientation. Ces données démontrent l'existence d'une division orientée, ce qui suppose un mécanisme général d'établissement de la polarité à l'échelle tissulaire, la polarité planaire. Nous nous sommes ensuite attaché à comprendre le rôle d'APC dans la polarité apico-basale et dans la régulation des générateurs de force impliqués dans l'orientation du fuseau. Nous avons utilisé un modèle cellulaire de gain de fonction pour la protéine APC, composé des lignées SW480 (qui n'expriment que la protéine tronquée) et SW480-APC qui exprime en plus la protéine entière. L'expression d'APC induit un renforcement de la polarité apico-basale dans les cellules SW480-APC, et modifie le comportement du fuseau. J'ai développé un programme d'analyse du comportement du fuseau pour quantifier ces modifications, basé sur l'analyse d'un grand nombre d'évènements. L'expression d'APC entraîne une augmentation de la dynamique du fuseau, des réorientations, et des déplacements du fuseau loin du centroïde de la cellule. APC joue donc potentiellement dans la polarité apico-basale et ainsi dans le contrôle de l'orientation du fuseau
Colonic epithelium homeostasis rely on signaling pathways among wich the Wnt pathway. Heterozygous germ cell mutations of the Apc tumour suppressor gene are highly penetrant in predisposing to colorectal cancer. Loss of Heterozygosity (LOH) at the Apc locus is believed to initiate the tumourigenic pathway, typically with the appearance of potentially pre-cancerous lesions: Aberrant Crypt Foci (ACF). Little is known about the morphogenesis of this tissue in adult, in particular what is the role of oriented cell division in this mechanism, and if an Apc mutation could affect those mechanisms. We have developed a technique to isolate and observe intact entire fixed crypts. Our data indicates that there is a preferential axis of orientation, with 80% of cells being oriented with less than 30° along the crypt axis. Total loss of APC triggers profound perturbation of orientation, correlated with crypt width increase. A single mutation has no effect, and a second mutation is needed to perturb cell orientation. We then analysed the effect of Apc on apico-basal polarity and force generators regulation using a gain of function for Apc. This model composed of SW480 expressing a truncated fragment and SW480-APC expressing full length APC allow us to demonstrate reinforcement of apico-basal polarity, and spindle behavior changes. Our data show that APC expression triggers an increase of spindle dynamic, with spindle undergoing more reorientation and more displacements. APC could play a role in apico-basal cell polarity and hence in spindle orientation

L'orientation du fuseau mitotique joue un rôle essentiel dans le choix du destin cellulaire et dans l'homéostasie des tissus. Dans certains contextes, l'orientation du fuseau est contrôlée par le complexe moléculaire LGN, dont la localisation sous-corticale détermine le site de recrutement du moteur dyneine, lequel exerce des forces sur les microtubules astraux pour orienter le fuseau. Chez les vertébrés la régulation moléculaire de ce processus est cependant peu caractérisée. Nous avons décidé de chercher de nouveaux régulateurs de l'orientation du fuseau chez les vertébrés. Avec cet objectif, j'ai développé un modèle d'orientation du fuseau spécifiquement contrôlé par le complexe LGN. Avec ce modèle, j'ai réalisé un crible RNAi en évaluant 110 candidats incluant des moteurs moléculaires pour leur fonction dans l'orientation du fuseau. Notamment, ce crible a révélé que les régulateurs de la dyneine sont inégalement requis pour orienter le fuseau. De plus, entre les sous-unités de la dynactine, j'ai trouvé que la protéine du capping de l'actine, CAPZ-B, est un régulateur majeur de l'orientation du fuseau. La caractérisation de la fonction de CAPZ-B in vitro a révélé que CAPZ-B contrôle l'orientation du fuseau en régulant les complexes dyneine et dynactine ainsi que la dynamique des microtubules du fuseau, indépendamment de son rôle comme modulateur de l'actine. Finalement, nous avons démontré que CAPZ-B régule l'orientation planaire du fuseau in vivo dans le neuroépithelium. Je pense que mes travaux vont contribuer à la compréhension de la fonction de la dyneine dans l'orientation du fuseau chez les vertébrés, ouvrant la voie pour de nouvelles recherches dans le domaine
Mitotic spindle orientation is involved in cell fate decisions, tissue homeostasis and morphogenesis. In many contexts, spindle orientation is controlled by the LGN molecular complex, whose subcortical localization determines the site of recruitment of the dynein motor which exerts forces on astral microtubules orienting the spindle. In vertebrates, there is missing information about the molecules regulating the formation of the complex and those working downstream of it. This prompted us to screen for new regulators of vertebrate spindle orientation. For this, I developed a novel model of spindle orientation specifically controlled by the LGN complex. Using this model, I performed a live siRNA screen testing 110 candidates including molecular motors for their function in spindle orientation. Remarkably, this screen revealed that specific dynein regulators contribute differentially to spindle orientation. Moreover, I found that an uncharacterized member of the dynactin complex, the actin capping protein CAPZ-B, is a strong regulator of spindle orientation. Analyses of CAPZ-B function in cultured cells showed that CAPZ-B regulates spindle orientation independently of its classical role in modulating actin dynamics. Instead, CAPZ-B controls spindle orientation by modulating the localization/activity of the dynein/dynactin complexes and the dynamics of spindle microtubules. Finally, we demonstrated that CAPZ-B regulates planar spindle orientation in vivo in the chick embryonic neuroepithelium. I expect that my work will contribute to the understanding of dynein function during vertebrate spindle orientation and will open the path for new investigations in the field

L'orientation du fuseau mitotique joue un rôle essentiel dans le choix du destin cellulaire et dans l'homéostasie des tissus. Dans certains contextes, l'orientation du fuseau est contrôlée par le complexe moléculaire LGN, dont la localisation sous-corticale détermine le site de recrutement du moteur dyneine, lequel exerce des forces sur les microtubules astraux pour orienter le fuseau. Chez les vertébrés la régulation moléculaire de ce processus est cependant peu caractérisée. Nous avons décidé de chercher de nouveaux régulateurs de l'orientation du fuseau chez les vertébrés. Avec cet objectif, j'ai développé un modèle d'orientation du fuseau spécifiquement contrôlé par le complexe LGN. Avec ce modèle, j'ai réalisé un crible RNAi en évaluant 110 candidats incluant des moteurs moléculaires pour leur fonction dans l'orientation du fuseau. Notamment, ce crible a révélé que les régulateurs de la dyneine sont inégalement requis pour orienter le fuseau. De plus, entre les sous-unités de la dynactine, j'ai trouvé que la protéine du capping de l'actine, CAPZ-B, est un régulateur majeur de l'orientation du fuseau. La caractérisation de la fonction de CAPZ-B in vitro a révélé que CAPZ-B contrôle l'orientation du fuseau en régulant les complexes dyneine et dynactine ainsi que la dynamique des microtubules du fuseau, indépendamment de son rôle comme modulateur de l'actine. Finalement, nous avons démontré que CAPZ-B régule l'orientation planaire du fuseau in vivo dans le neuroépithelium. Je pense que mes travaux vont contribuer à la compréhension de la fonction de la dyneine dans l'orientation du fuseau chez les vertébrés, ouvrant la voie pour de nouvelles recherches dans le domaine
Mitotic spindle orientation is involved in cell fate decisions, tissue homeostasis and morphogenesis. In many contexts, spindle orientation is controlled by the LGN molecular complex, whose subcortical localization determines the site of recruitment of the dynein motor which exerts forces on astral microtubules orienting the spindle. In vertebrates, there is missing information about the molecules regulating the formation of the complex and those working downstream of it. This prompted us to screen for new regulators of vertebrate spindle orientation. For this, I developed a novel model of spindle orientation specifically controlled by the LGN complex. Using this model, I performed a live siRNA screen testing 110 candidates including molecular motors for their function in spindle orientation. Remarkably, this screen revealed that specific dynein regulators contribute differentially to spindle orientation. Moreover, I found that an uncharacterized member of the dynactin complex, the actin capping protein CAPZ-B, is a strong regulator of spindle orientation. Analyses of CAPZ-B function in cultured cells showed that CAPZ-B regulates spindle orientation independently of its classical role in modulating actin dynamics. Instead, CAPZ-B controls spindle orientation by modulating the localization/activity of the dynein/dynactin complexes and the dynamics of spindle microtubules. Finally, we demonstrated that CAPZ-B regulates planar spindle orientation in vivo in the chick embryonic neuroepithelium. I expect that my work will contribute to the understanding of dynein function during vertebrate spindle orientation and will open the path for new investigations in the field

Cytotoxicity refers to the ability of a molecule or a compound to cause some type of cellular damage, of which some of the adverse effects that can occur include injuries to some structures or the fundamental processes involved in cell maintenance, such as survival, cell division, cell biochemistry, and the normal cell physiology. The potential for cytotoxicity is one of the first tests that must be performed to determine the effects of drugs, biomolecules, nanomaterials, medical devices, pesticides, heavy metals, and solvents, among others. This potential may be oriented in the mechanism under which it generates cell death, the dose, and the target cells that generate the response. The evaluation of the toxicologic and cytotoxic properties of the chemical substances through in vitro tests has become a competitive alternative to in vivo experimentation as a consequence of ethical considerations. Presently, there are numerous tests conducted to evaluate the cytotoxicity of a certain agent, the selection of which depends on the purpose of the study. In this sense, the present review provides a general overview of the different responses of a cell to xenobiotic agents and the different test that can be useful for evaluation of these responses.

The original objectives of the approved proposal were: 1. To construct a YFP fused Arabidopsis cDNA library in a mammalian expression vector. 2. To infect the library into a host fibroblast cell line and to screen for new cytoskeletal associated proteins using an automated microscope. 3. Isolate the new genes. 4. Characterize their role in plants. The project was approved as a feasibility study to allow proof of concept that would entail building the YFP library and picking up a couple of positive clones using the fluorescent screen. We report here on the construction of the YFP library, the development of the automatic microscope, the establishment of the screen and the isolation of positive clones that are plant cDNAs encoding cytoskeleton associated proteins. The rational underling a screen of plant library in fibroblasts is based on the high conservation of the cytoskeleton building blocks, actin and tubulin, between the two kingdoms (80-90% homology at the level of amino acids sequence). In addition, several publications demonstrated the recognition of mammalian cytoskeleton by plant cytoskeletal binding proteins and vice versa. The major achievements described here are: 1. The development of an automated microscope equipped with fast laser auto-focusing for high magnification and a software controlling 6 dimensions; X, Y position, auto focus, time, color, and the distribution and density of the fields acquired. This system is essential for the high throughput screen. 2. The construction of an extremely competent YFP library efficiently cloned (tens of thousands of clones collected, no empty vectors detected) with all inserts oriented 5't03'. These parameters render it well representative of the whole transcriptome and efficient in "in-frame" fusion to YFP. 3. The strategy developed for the screen allowing the isolation of individual positive cDNA clones following three rounds of microscopic scans. The major conclusion accomplished from the work described here is that the concept of using mammalian host cells for fishing new plant cytoskeletal proteins is feasible and that screening system developed is complete for addressing one of the major bottlenecks of the plant cytoskeleton field: the need for high throughput identification of functionally active cytoskeletal proteins. The new identified plant cytoskeletal proteins isolated in the pilot screen and additional new proteins which will be isolated in a comprehensive screen will shed light on cytoskeletal mediated processes playing a major role in cellular activities such as cell division, morphogenesis, and functioning such as chloroplast positioning, pollen tube and root hair elongation and the movement of guard cells. Therefore, in the long run the screen described here has clear agricultural implications.