Les microtubules sont des composants structuraux de cellules et gouvernent des fonctions cellulaires essentielles telles que les mitoses et le transport des vésicules. Ils sont composés de deux sous-unités non identiques (tubulines α et β), formant un dimère, et sont arrangés de sorte à former une structure tubulaire de 20nm de diamètre. Généralement, ils sont constitués de 13 ou 14 protofilaments arrangés en spirale. Les liaisons longitudinales entre dimères sont plus rigides et fortes que les liaisons latérales. Aussi, les microtubules sont des structures fortement anisotropes. Dans ces travaux de thèse, nous avons pour but de définir l'ensemble des coefficients élastique qui permet de reproduire leur comportement atomistique ainsi que de rendre compte de leur réponse mécanique selon des chemins de chargement variés. En négligeant la discontinuité hélicoïdale souvent observée, un microtubule est représenté par une structure triangulaire de dimères à partir desquels un volume élémentaire représentatif est défini. Un potentiel harmonique est utilisé pour décrire les interactions entre dimères voisins. A partir de l'estimation des constantes élastiques et de l'utilisation de la méthode proposée par Arslan et Boyce (2006) -alors pour analyser le comportement mécanique d'un réseau triangulaire de spectrines composant les membranes des globules rouges-, un modèle continu de comportement mécanique est présenté pour reproduire le comportement des parois des microtubules. Un modèle numérique éléments finis est ensuite créé pour modéliser le comportement d'un microtubule dans sa globalité. Des éléments coques sont utilisés pour reproduire les fines parois des microtubules. Les propriétés du modèle éléments finis sont ajustées à partir des résultats du modèle présenté ainsi qu'aux données expérimentales provenant de la littérature. La rigidité de flexion calculée au cours de simulation des tests de flexion 3 points est en accord avec les valeurs de la littérature. Ces tests révèlent les mécanismes de déformation en fonction de la longueur utile du tube utilisé: Flexion et cisaillement locaux de la paroi gouvernent la déformation pour de "petits" tubes. Pour des longueurs "moyennes" le cisaillement et la flexion du tube prédominent. Enfin, dans le cas de tubes "longs", la déformation est uniquement associée aux effets de flexion. Ces résultats témoignent de l'influence de l'anisotropie du tube sur la réponse observée selon différents mode de sollicitation. Ils permettent également d'expliquer l'évolution de la rigidité de flexion avec la longueur utile du tube, comme reportée dans la littérature. Enfin, des micrographes montrent la propension des extrémités des microtubules à diverger radialement -"à boucler"-. Une telle géométrie est causée par des instabilités propres aux microtubules et implique un état précontraint. Un «modèle d'interactions» est alors proposé de manière à considérer un état précontraint et ainsi reproduire la cinétique des instabilités des microtubules au cours de la polymérisation/dépolymérisation.

The microtubule cytoskeleton, which consists of dynamic polymers of alpha/beta tubulin heterodimers, organizes the cytoplasm and is essential for chromosome segregation during mitosis. My thesis addresses the significance and potential interplay between four distinct microtubule-regulatory proteins. The experimental approach included the development of a replicating vector system directing either constitutive expression of short hairpin RNAs or inducible ectopic expression, which allows stable depletion and/or conditional exchange of gene-products. Based on the originally observed activities in frog egg extracts, MCAK and TOGp have been viewed as major antagonistic proteins that regulate microtubule-dynamics throughout the cell cycle. Surprisingly, while my thesis work confirmed an essential role of these proteins to ensure mitotic fidelity, tubulin subunits partitioning is not controlled by the endogenous levels of MCAK and TOGp in human somatic cells. Our major discovery in these studies is that the activities of both CaMKII and TOGp are essential for spindle bipolarity through a mechanism involving protection of spindle microtubules against MCAK activity at the centrosome. In our search for the major antagonistic activities that regulates microtubule-dynamics in interphase cells, we found that the microtubule-destabilizing activity of Op18 is counteracted by MAP4. These studies also established Op18 and MAP4 as the predominant regulators of tubulin subunit partitioning in all three human cell model systems studied. Moreover, consistent with phosphorylation-inactivation of these two proteins during mitosis, we found that the microtubule-regulatory activities of both MAP4 and Op18 were only evident in interphase cells. Importantly, by employing a system for inducible gene product replacement, we found that site-specific phosphorylation-inactivation of Op18 is the direct cause of the demonstrated hyper-polymerization in response to T-cell antigen receptor triggering. This provides the first formally proven example of a signal transduction pathway for regulation of interphase microtubules. Op18 is frequently upregulated in various types of human malignancies. In addition, a somatic mutation of Op18 has recently been identified in an adenocarcinoma. This thesis work revealed that the mutant Op18 protein exerts increased microtubule-destabilizing activity. The mutant Op18 protein was also shown to be partially resistant to phosphorylation-inactivation during mitosis, which was associated with increased chromosome segregation aberrancies. Interestingly, we also observed the same phenotype by overexpressing the wild type Op18 protein. Thus, either excessive levels of wild type Op18 or normal levels of mutated hyper-active Op18 seems likely to contribute to tumor progression by exacerbating chromosomal instability.

The actin and microtubule cytoskeleton plays a critical role in the establishment of cell polarity. Cell processes like mitosis and migration rely on the reorganization of the cytoskeleton to properly function. One driver of cell polarity is the formin, Inverted Formin-1 (INF1). INF1 is able to induce F-actin formation, activate the Serum Response Factor (SRF) pathway, stabilize microtubules, associate with microtubules, and disperse the Golgi body. Regulation of INF1 is unique, since it does not possess conserved formin regulatory domains. However, INF1 does possess many potential phosphorylation sites. In this study, we demonstrate that INF1’s ability to induce F-actin stress fibers and activate SRF is inhibited by Microtubule-Affinity Regulating Kinase 2 (MARK2). Inhibition of INF1’s actin polymerization activity by MARK2 likely occurs near INF1’s C-terminus. However, MARK2 was unable to inhibit INF1’s ability to stabilize microtubules, associate with microtubules, and disperse the Golgi. Furthermore, we show that INF1 overexpression is associated with primary cilium absence and in some cases, the presence of long cilia, suggesting that INF1 plays a role in primary cilium formation.

A major challenge in nanotechnology is the spatially controlled transport of cargo on the nanometer scale. The use of a nanoscale transport system based on molecular motors and filaments of the cytoskeleton proved as a promising approach to this problem. Therefore, the objective of this work was to pattern planar surfaces with motor proteins in a way that allows controlled and guided movement of microtubule-shuttles. The first part of the work was in particular focused on generating nanometer–sized tracks of motor proteins on unstructured surfaces. Specifically, microtubules themselves were used as biological templates for the stamping and alignment of motor proteins. Compared to other soft lithography techniques like microcontact printing this approach circumvented protein denaturation due to drying and conformational changes caused by mechanical stress. Given the large persistence length of microtubules their encounters with the boundaries of the nanotracks are limited to shallow approach angles. This way, the generated structures proved very efficient for the guiding of microtubules without topographical barriers. Topography-free guiding, as demonstrated in this work, is expected to significantly ease the design and fabrication of microtubule-transport systems and opens up the possibility to transport cargo of unlimited size, i.e. without any constraints by the dimensions of topographic guiding channels. Moreover, the biotemplated patterning is a promising tool for in vitro studies on the individual and cooperative action of motor proteins. In particular it might be helpful for the reconstitution of complex subcellular machineries in synthetic environments. As an example, microtubule-microtubule sliding by the biomolecular motor ncd has been shown to induce directional sliding between antiparallel microtubules and static cross-linking between parallel ones. The second part of the work explored an in-situ patterning technique for motor proteins to enable user-defined pattern designs, and investigated the achievable resolution. Photothermal patterning, based on localized light-to-heat conversion combined with a thermoresponsive polymer layer, was presented as a novel method. Specifically, the conformation of poly(N-isopropylacrylamide) (PNIPAM) molecules in aqueous solution was switched between the swollen state at T < 30°C (protein-repelling conformation) to the collapsed state at T > 33°C (protein-binding conformation) by optical signals of visible light to generate heat in a highly-localized manner. Upon heating of a light-absorbing layer on the substrate, the surface-grafted PNIPAM molecules collapsed locally and allowed motor proteins in solution to bind in the illuminated areas. To confirm the successful patterning of kinesin-1 molecules and their functionality microtubule-based gliding motility assays were performed. It was shown that the microtubules bind to the patterned kinesin-1 molecules and are transported exclusively in the patterned areas. While the achieved pattern sizes were currently in the range of ten micrometers, finite element modeling (implemented in COMSOL) showed that increased optical intensities possibly combined with cooling of the sample allow to significantly scale down the pattern dimensions. The produced patterns can be reversibly activated and deactivated at high and low temperature, respectively. Moreover, sequential patterning of multiple kinds of proteins on the same surface will be possible in a similar way without the need for specific linker molecules or elaborate surface preparation. Another advantage of the method is the use of visible light, which is versatile as any wavelength can be applied. In addition visible light is in comparison to other UV-based photopatterning techniques non-damaging to proteins. / Der räumlich kontrollierte Transport von nanoskaligen Objekten ist eine große Herausforderung auf dem Gebiet der Nanotechnologie. Ein auf molekularen Motoren und Filamenten des Zellskeletts basierendes Nanotransportsystem hat sich dabei als ein viel versprechender Ansatz erwiesen. Das Ziel der vorgelegten Arbeit war es daher, ebene Oberflächen so mit Motorproteinen zu strukturieren, dass eine kontrollierte und geführte Bewegung von Mikrotubuli-Transportern ermöglicht wird. Der erste Teil der Arbeit war insbesondere darauf fokussiert, Motorprotein-Spuren im Nanometerbereich zu erzeugen. Im zweiten Teil der Arbeit wurde eine Strukturierungsmethode zur Realisierung von benutzerdefinierten Musterdesigns untersucht und die erreichbare Auflösung bestimmt. Für die Nanometerstrukturierung von Oberflächen mit funktionalen Motorproteinen wurde ein neuer Ansatz demonstriert. Mit der Anwendung von Biotemplaten, wie hier der Mikrotubuli, konnte ein hoch-lokalisiertes und orientiertes Anbinden von Proteinen an Oberflächen sowie gleichzeitig geringer Proteindenaturierung erreicht werden. Durch spezifisches Stempeln beziehungsweise Binden von Motoren wurden Muster aus funktionellen Proteinen mit hoher Oberflächendichte hergestellt. Die erzeugten Motor-Spuren haben gezeigt, dass Nanometerstrukturierungen möglich sind und ohne topographische Barrieren zu zuverlässiger Führung von Mikrotubuli führen können. Bisher konnte die nicht-topographische Strukturierung von Oberflächen mit Kinesin-1-Motoren nur im Mikrometerbereich demonstriert werden. Wegen der hohen Steifigkeit der Mikrotubuli war die thermische Energie des Systems in diesen Fällen nicht ausreichend, um die führende Spitze der Mikrotubuli zurück auf das Gebiet mit den strukturierten Motoren zu biegen. Dieses Problem wird durch die kleine Breite der hier demonstrierten Motor-Nanospuren verhindert, da das Auftreffen der Mikrotubuli mit den Grenzlinien auf extrem flache Winkel begrenzt ist. Interessanterweise haben sich Spuren des nicht-prozessiven Motors Kinesin-14 für das Führen und den Transport im Nanometerbereich als noch zuverlässiger herausgestellt als Kinesin-1-Spuren. Es ist zu erwarten, dass nicht-topographisches Führen, wie es in dieser Arbeit gezeigt wurde, das Design und die Herstellung von Mikrotubuli-Transportsystemen deutlich vereinfacht und die Möglichkeit eröffnet, Cargo mit unlimitierter Größe, d.h. ohne Einschränkungen durch die Abmessungen der topographischen Führungskanäle, zu transportieren. Zusätzlich ist die biotemplierte Strukturierung ein viel versprechendes Werkzeug um das individuelle und das kooperative Arbeiten von Motorproteinen in vitro untersuchen und komplexe subzelluläre Maschinerien in synthetischer Umgebung rekonstituieren zu können. Dies wurde am Beispiel des gerichteten Gleitens des biomolekularen Motors Kinesin-14 gezeigt, der ein gerichtetes Gleiten zwischen antiparallelen Mikrotubuli und statisches Vernetzen zwischen parallelen Mikrotubuli hervorruft. Mit dem Ansatz des biotemplierten Strukturierens ist es jedoch nicht einfach möglich, benutzerdefinierte Spuren zu erzeugen. Daher wurde die photothermische Proteinstrukturierung als eine neue Methode für die frei programmierbare, hochauflösende und schnelle Herstellung von strukturierten Proteinoberflächen eingeführt. Auf diese Weise wurden Kinesin-1-Muster durch licht-induziertes Heizen einer licht-absorbierenden Substratschicht erzeugt. Die thermisch schaltbaren poly(N-isopropylacrylamid) (PNIPAM) Moleküle auf der Oberfläche kollabierten lokal und erlaubten es den Motorproteinen, in den beleuchteten Gebieten aus der Lösung an die Oberfläche zu binden. Die Bewegung gleitender Mikrotubuli bestätigte anschließend die erfolgreiche Strukturierung der Kinesin-1-Motoren und deren Funktionalität, da die Mikrotubuli an die strukturierten Motoren banden und ausschließlich in den strukturierten Gebieten transportiert wurden. Neben der Proteinstrukturierung wurde die lokalisierte Licht-zu-Wärme-Umwandlung kombiniert mit einer thermisch schaltbaren Polymerschicht auch für die lokale Aktivierung von Kinesin-1-Motoren auf der Oberfläche genutzt. Ein Vorteil der photothermischen Proteinstrukturierung ist die Möglichkeit, sichtbares Licht zu verwenden, da jede beliebige Wellenlänge angewendet werden kann und sichtbares Licht, im Vergleich zu anderen UV-basierten Photostrukturierungsmethoden, Proteine nicht schädigt. Modellierungen mit Hilfe der Finite-Elemente-Methode (implementiert in der Software COMSOL) haben gezeigt, dass die Lichtintensität und die Oberflächentemperatur speziell eingestellt werden müssen, um definierte Strukturgrößen zu erzielen. Während die derzeitig erzeugten Muster Größen im Bereich von zehn Mikrometern hatten, könnten durch höhere optische Intensitäten kombiniert mit Kühlung der Probe die Größenordnungen signifikant reduziert werden. Die reale experimentelle Auflösung wird jedoch auch von der Schaltcharakteristik des Polymers und der Proteinbindungsdynamik abhängen. Die hergestellten Muster können reversibel bei hohen beziehungsweise niedrigen Temperaturen aktiviert und deaktiviert werden. Zusätzlich können auf die gleiche Weise verschiedene Proteinsorten sequentiell auf einer Oberfläche strukturiert werden, ohne dass spezifische Bindungsmoleküle oder aufwändige Oberflächenpräparationen notwendig wären. Die Möglichkeit, Proteine reversibel an die Oberfläche zu binden, um geschriebene Muster wieder löschen zu können, wäre eine Weiterentwicklung und würde die Anwendungsmöglichkeiten der photothermischen Strukturierungsmethode erweitern. Außerdem würden optisch schaltbare Polymere das direkte Strukturieren von Motoren mit Licht ermöglichen und daher die Methode vereinfachen.

The growth of a neuronal dendritic tree depends on the neuron’s internal state and the environment within which it is situated. Different types of neuron develop dendritic trees with specific characteristics, such as the average number of terminal branches and the average length of terminal and intermediate segments. A key aspect of the growth process is the construction of the microtubule cytoskeleton within the dendritic tree. Neurite elongation requires assembly of microtubules from free tubulin at the growth cone. The stability of microtubule bundles is an important factor in determining how likely it is for a growth cone to split to form new daughter branches. Microtubule assembly rates and bundle stability are controlled by microtubule-associated proteins, principally MAP2 in dendrites. Extending previous work (Hely et al, J. Theor. Biol. 210:375-384, 2001) I have developed a mathematical model of neurite outgrowth in which elongation and branching rates are determined by the phosphorylation state of MAP2 at the tips of each terminal branch. Tubulin and MAP2 are produced in the cell body and transported along the neurite by a combination of diffusion and active transport. Microtubule (dis)assembly at neurite tips is a function of tubulin concentration. The rate of assembly depends on the amount of unphosphorylated MAP2 bound to the microtubules and linking them together. Phosphorylation of MAP2 destroys its linking capability and destabilises the microtubule bundles. Each terminal has a probability of branching that depends on the phosphorylation of MAP2 which, in turn, is a function of calcium concentration. Results from this model show that changes in the (de)phosphorylation rates of MAP2 affect the topology of the final dendritic tree. Higher phosphorylation promotes branching and results in trees with many short terminal branches and relatively long intermediate segments. Reducing phosphorylation promotes elongation and inhibits branching.

This thesis uses methods and models from non-equilibrium statistical physics to describe intracellular processes. Bidirectional microtubule-based transport within axons is modeled as a quasi-one-dimensional stochastic lattice gas with two particle species moving in opposite directions under mutual exclusion interaction. Generically occurring clusters of particles in current models for intracellular transport can be dissolved by additionally considering the dynamics of the transport lattice, i.e., the microtubule. An idealized model for the lattice dynamics is used to create a phase transition toward a homogenous state with efficient transport in both directions. In the thermodynamic limit, a steady state property of the dynamic lattice limits the maximal size of clusters. Lane formation mechanisms which are due to specific particle-particle interactions turn out to be very sensitive to the model assumptions. Furthermore, even if some particle-particle interaction is considered, taking the lattice dynamics into account almost always improves transport. Thus the lattice dynamics seems to be the key aspect in understanding how nature regulates intracellular traffic. The last part introduces a model for the dynamics of a microtubule which is limited in its growth by the cell boundary. The action of a rescue-enhancing protein which is added to the growing tip of a microtubule and then slowly dissociates leads to interesting aging effects which should be experimentally observable.

No description available.

Microtubules are tubular polymers that are involved in a variety of cellular processes such as cell movement, mitosis and intracellular transport. The dynamic behavior of microtubules makes this possible because all of these processes require quick responses. Embryonic stem (ES) cells were first isolated from mouse embryos and they have two unique characteristics; they can be kept undifferentiated for many passages with a stable karyotype and they can be differentiated into any type of cells under appropriate conditions. The pluripotency of ES cells, their ease of manipulation in culture, and their ability to contribute to the mouse germ-line provides us a model of differentiation both in vitro and in vivo. In my thesis I focused on the cell division and neuronal differentiation of ES cells and developed two methods to understand the effects of microtubule dynamics in spindle assembly and chromosome segregation and to reveal the roles of different Microtubule Associated Proteins (MAPs) in the neuronal morphology formation. In the first part, we developed a live-cell imaging method for ES cells to visualize, track and analyze the single cell behavior in a cell population over a time period. So far many techniques have been adapted and combined for imaging of cell lines, mainly for the cancer or immortalized ones. However, because ES cells are very prone to apoptosis, tend to form spheres and hard to stably label, it is quite tricky to image them in culture conditions. In our system, we combined the BAC-based gene expression with wide-field deconvolution microscopy for ES cells that are plated onto the laminin-511 coated surface and kept in CO2 independent culture conditions. This combined technique does not interfere with the growth of cells and keeps them healthy up to 24 hours on the microscope stage. In the second part, we analyzed the effects of MAPs chTOG, EB1, Kif18A and MCAK in the overall spindle morphology and mitotic progression in mES cells. For this purpose, we utilized our stable TUBB-GFP and H2A-GFP cell lines along with our live-cell imaging set-up to reveal the effects of the above-mentioned proteins and the interplay among each other. By using RNAi method we either single or co-depleted the genes by siRNAs and measured the spindle length and width in RNAi conditions. We further analyzed the mitotic progression in H2A-GFP cell line in terms of the metaphase timing and the percentage of chromosome segregation errors. Our results showed that, EB1 depletion did not cause any significant changes in the overall spindle morphology or in the metaphase timing. However, the co-depletion of EB1 with chTOG partially rescued the sichTOG specific mini-spindle phenotype. siKif18A produced longer spindles without any change in the spindle width. Surprisingly, the co-depletion of antagonistic chTOG and Kif18A proteins had additive effects on the spindle dynamics and on mitotic progression in a way that spindle assembly was severely disrupted by the absence of these two proteins and as a result of this, both metaphase timing and chromosome missegregation levels increased significantly. These results overall indicate that MAPs have important roles in the regulation of dynamic instability and these proteins have an interplay among each other to be able to control the morphology of the spindle as well as the correct segregation of chromosomes into daughter cells. In the last part, I will introduce you a new ES cell based differentiation and morphology model, which brings the advantages of high resolution imaging capacity, control over development and easy genetic manipulation and culturing. We have generated Tet-induced shRNA cell lines against chTOG, Kif18A and MCAK, which are also stably expressing TUBB-GFP. These labeled cells were mixed with unlabeled wild-type mES cells before differentiation at 1:1000 ratio and then they were differentiated into mouse cortical cells and spinal motor neurons. Our results showed that, all of the three genes could be successfully knocked-down by shRNA after 48 hours of Tet induction. After mixing the labeled and unlabeled cells, single neurons could be imaged at high resolution and their skeletons could be generated afterwards. The RNAi studies in shchTOG cell line showed that, the knock-down of this gene in early differentiation interferes with the neuronal differentiation.

Résumé Les protéines XMAP215/ch-TOG appartiennent à une famille de protéines associées aux microtubules (MAPs), bien conservée tout au long de l'évolution, la famille XMAP215/Dis1. Cette famille joue un rôle dans la régulation du cytosquelette des microtubules (MT), en particulier pendant la division cellulaire. Chez l'humain, ch-TOG est la protéine surexprimée dans les tumeurs du colon et du foie, une protéine qui provient de cellules blastiques et de plusieurs formes de cancer. Certaines protéines XMAP215/ch-TOG ont été retrouvées dans différentes localisations cellulaires, toujours reliées aux MTs, donnant origine à une activité spécifique. Cependant, la localisation exacte de XMAP215/ch-TOG ainsi que son activité restait à être déterminées. Dans ce contexte scientifique, nous avons développé une série d'anticorps monoclonaux (mcAB) qui nous ont permis d'identifier deux populations différentes de la famille des protéines XMAP215/Dis1. Les images de microscopie confocale des cellules fixées ont montré une première localisation, la colocalisation bien connue XMAP215-microtubulaire (MT-XMAP215) qui s'observe pendant l'interphase et pendant la mitose (fuseau mitotique). Une deuxième localisation a été identifiée sur le bout plus des MTs, donnant XMAP215/ch-TOG comme faisant parti de la famille des protéines de bout plus (+TIPs). Cette deuxième colocalisation a été identifiée comme +TIP XMAP215/ch-TOG. La +TIP XMAP215 est la protéine la plus distale du bout des MTs. La hiérarchie a été établie en faisant la comparaison de la localisation de XMAP215/ch-TOG avec les protéines les plus connues du bout plus, telles qu'EB1, CLIP170 et p150Glued. Dans l'extrait mitotique de Xenopus laevis, les images obtenues in vivo par la microscopie de fluorescence par réflexion totale interne (TIRF) ont permis d'identifier une +TIP XMAP215 présente au bout des MTs qui polymérisent et dépolymérisent. Les images de microscopie cryo-électronique (Cryo-EM) ont montré une activité spécifique de la population +TIP XMAP215. Dans les solutions de tubuline pure, XMAP215 induit la formation de structures au bout des MTs, cette activité est compatible avec les mécanismes de croissance des MTs. Sur la base de nos résultats, nous proposons un modèle où XMAP215 se charge des dimères de tubuline en devenant une structure de type protofilament. Cette structure se lie au bout du MT en utilisant son domaine C-terminal, en rajoutant les dimères de tubuline et aussi certainement en participant à la fermeture de la structure microtubulaire même. La protéine interviendrait donc dans la dépolymérisation et aurait un rôle dans le mécanisme de dépolymérisation contrôlée. Une fois que l'addition de tubuline a eu lieu, la +TIP XMAP215 pourrait évoluer en MT-XMAP215, la forme la plus connue de la protéine qui a été associée au trafic des granules d'ARN. Mots cles : XMAP215, ch-TOG, microtubules, anticorps monoclonaux.

Coordinated polarity and differentiation of cells in the plane of a tissue layer are essential to the development of multicellular organisms. Arabidopsis thaliana root hairs and trichomes provide model systems to study the pathways that control planar polarity and cell fate specification in plants. A concentration gradient of the plant hormone auxin provides an instructive cue that coordinates polar assembly of signalling complexes at plasma membranes of root epidermal cells; however, knowledge about additional players and cytoskeletal effectors driving cell polarization prior to hair emergence remains limited. On the other hand, epidermal cell fate specification is controlled by a well-characterized gene network of transcription factors that translate positional signals and cell-to-cell communication into tissue-wide patterning. Yet, new components are continuously found to interact with the patterning pathway, shedding light on its connections with diverse developmental processes. This thesis presents the SABRE (SAB) gene as a novel player in planar polarity establishment and root epidermal patterning. SAB is a large protein with sequence similarity to proteins present in all eukaryotes and affects planar polarity as well as orientation of cell divisions and cortical microtubules. Genetic interaction with the microtubule-associated protein gene CLASP further supports involvement of SAB in microtubule arrangement, suggesting a role for this gene in cytoskeletal organisation. Strikingly, SAB also interacts genetically with ACTIN7 (ACT7), and both ACT7 and its modulator ACTIN INTERACTING PROTEIN 1-2 (AIP1-2) contribute to planar polarity of root hair positioning. Cell-file specific expression of AIP1-2 depends on the epidermal-patterning regulator WEREWOLF (WER), revealing a connection between actin organization, planar polarity and cell fate specification. Consistent with this finding, SAB also functions in patterning of the root epidermis by stabilizing cell fate acquisition upstream of the core patterning pathway. These results unveil new roles for SAB in planar polarity and epidermal patterning and suggest that organization of the microtubule and the actin cytoskeleton are important to both planar polarity establishment and cell fate specification. / Samordning av polaritet och differentiering av celler inom ett vävnadslager är avgörande för utvecklingen av multicellulära organismer. Rothår och bladhår hos Arabidopsis thaliana utgör modellsystem för att studera signalvägar som kontrollerar planpolaritet och specifikation av cellers öde hos växter. En koncentrationsgradient av växthormonet auxin ger en instruktiv signal som koordinerar polär hopsättning av signalkomplex vid plasmamembranet i rotepidermisceller; dock är kunskapen om ytterligare aktörer och hur cytoskelettets aktörer påverkar cellpolaritet innan rothår bildas begränsad. Vad gäller differentieringen av epidermala cellers öde kontrolleras dessa genom ett väl karakteriserat nätverk av transkriptionsfaktorer som överför positionssignaler och cell-till-cell kommunikation till vävnadsomfattande mönsterbildning. Fortfarande hittas dock nya komponenter som interagerar med signalvägarna för mönsterbildning, vilket ger nya insikter om dess förbindelser med diverse utvecklingsprocesser. Denna avhandling presenterar genen SABRE (SAB) som en ny aktör i etableringen av planpolaritet och mönsterbildning av rotepidermis. SAB är ett stort protein som har sekvenslikhet med proteiner som finns i alla eukaryoter och det påverkar planpolaritet, orientering av celldelning och kortikala mikrotubler. Genetisk interaktion med genen för det mikrotubuli-associerade proteinet CLASP stärker ytterligare inblandningen av SAB i organiserandet av mikrotubler och antyder att denna gen har en roll i organiserandet av cytoskelettet. Slående är att SAB även interagerar genetiskt med ACTIN7 (ACT7) och att både ACT7 och dess modulator ACTIN-INTERACTING PROTEIN1-2 (AIP1-2) bidrar till planpolaritet vid positionering av rothår. Cellfils-specifikt uttryck av AIP1-2 beror på den epidermala mönsterbildande genen WEREWOLF (WER), vilket påvisar ett samband mellan organisationen av aktin, planpolaritet och specifikationen av cellers öde. SAB fungerar även i mönsterbildning av rotens epidermis och stabiliserar förvärvet av cellöde uppströms av den centrala signalvägen för mönsterbildning. Dessa resultat visar på nya roller för SAB i planpolaritet och mönsterbildning av epidermis och indikerar att organiseringen av mikrotubler och aktin-cytoskelettet är viktiga både för etablerandet av planpolaritet och för specificeringen av cellers öde.