My thesis, written in an alternative format, consists of three manuscripts. The first one is published in Journal of Cell Science and is entitled "GAS2-like proteins mediate communication between microtubules and actin through interaction with end-binding (EB) proteins." This article describes the mechanisms of how members of the GAS2 family of proteins mediate the crosstalk between actin and microtubules (MTs). We show that in particular GAS2-like 1 (G2L1) and GAS2-like 2 (G2L2) coordinate this cross-communication, as their exogenous expression leads to the stabilisation of MTs and guidance along actin stress fibres. We found that the association of GAS2-like members with MTs is mediated through their binding to EB proteins. The second article is a follow up story of the first article, in which we further elucidate the role of GAS2-like proteins during cell division. We show that G2L1 localises to the mitotic spindle and cleavage furrow during cell division. G2L1 knockdown leads to reduced cell division rates, multinucleation and nuclear deformation. As for MT guidance along actin filaments, we demonstrate that the binding of G2L1 to EB proteins plays an important role in cell division. Although overexpression of G2L1 had no effect, the expression of a mutant that blocks the association with EB proteins phenocopies the knockdown effect of G2L1 on cell division. Actin and MTs undergo major reorganisation during cell division. This reorganisation involves the fast remodelling of focal adhesions (FAs) but the mechanisms of this remodelling were not clear. In the third paper we demonstrate that the majority of FAs disassemble shortly before cell division and reassemble in newly formed daughter cells during cytokinesis. Interestingly, our data suggest that the regulation of FA disassembly during cell division differs from the disassembly processes during cell migration. While in migrating cells FAs can be stabilised by the expression of constitutively active vinculin (vinT12, known to circumvent the requirement forces for FA stability), this was not case for FAs during cell division. Further experiments using inhibitors suggested that calpain-driven cleavage of FA components but not endocytosis play a key role in FA disassembly during cell division. Altogether, the three manuscripts provide insight into important molecular aspects involved in the regulation of cell cytoskeletal networks and cell adhesion during cell division.

Le cancer du sein est une cause importante de mortalité féminine en France et l’émergence de chimiorésistance, en particulier avec les taxanes, dont le paclitaxel (Taxol®), est une limite importante à l’emploi de ces molécules. Comme de nombreux anticancéreux, les taxanes ciblent les microtubules (MTs), des polymères de tubuline intervenant dans de nombreuses fonctions cellulaires. Ces derniers alternent entre des phases de croissance et de désassemblage, leur conférant ainsi un caractère dynamique. En se liant à la beta-tubuline, le Taxol stabilise les MTs et bloque la mitose, conduisant la cellule vers l’apoptose. La résistance au Taxol est un processus multifactoriel impliquant des mécanismes tels que la surexpression de pompes d’efflux (P-glycoprotéine), des mutations des gènes d’alpha- et de beta-tubuline ou une expression altérée de protéines associées aux MTs (MAPs) telles que MAP4 (stabilisatrice des MTs) ou la stathmine (dépolymérisante), contribuant ainsi à la restauration de la dynamique microtubulaire.Le projet repose sur l’emploi de la lignée tumorale mammaire MDA-MB 231 rendue résistante par paliers à 25nM de Taxol dans des conditions de blocage des pompes d’efflux. L’objectif étant d’explorer les variations protéiques de l’environnement des MTs associées au phénotype chimiorésistant, nous avons comparé le protéome d’extraits microtubulaires de cellules sensibles (Tv) et résistantes au Taxol (T8). Parmi les 112 protéines statistiquement enrichies dans une des deux populations de MTs, on retrouve notamment une augmentation des tubulines beta III et IV, une augmentation et une diminution respectives des moteurs moléculaires kinésine-1 et dynéine ainsi que l’enrichissement de plusieurs septines (SEPT2, 8, 9 et 11) dans les fractions de MTs T8. Les septines sont des GTPases associées en complexes hétéro-oligomériques impliquées dans la cytocinèse et l’organisation du cytosquelette de MTs et d’actine. Le recrutement de ces protéines candidates sur les MTs des cellules T8 a été validé par Western blot (Froidevaux-Klipfel et al., 2011) et immunofluorescence. Dans la littérature, les septines sont décrites comme étant localisées soit au niveau de l’actine soit au niveau des MTs dans les cellules de mammifères. De façon intéressante, dans nos cellules MDA-MB 231, nous observons un recrutement partiel de SEPT2 sur les fibres d’actine dans les cellules Tv alors qu’elle colocalise avec les MTs des cellules T8. Bien que la dépolymérisation de l’actine dans les cellules Tv n’entraine aucun déplacement des septines vers les MTs, cette localisation différentielle des septines sur les MTs des cellules résistantes pourrait néanmoins participer au phénotype chimiorésistant.Par ailleurs, il a été décrit dans la littérature que SEPT2 se lie aux MTs polyglutamylés afin de faciliter le transport vésiculaire à la membrane de protéines impliquées dans la polarisation cellulaire. La polyglutamylation est une modification post-traductionnelle permettant la formation de chaines latérales d’un à plusieurs résidus glutamate sur les tubulines alpha ou beta, régulant ainsi les interactions entre MTs et MAPs. Nos résultats montrent que l’accumulation des septines sur le réseau de MTs des cellules T8 s’accompagne d’une augmentation de la polyglutamylation mais aussi de la tyrosination de la tubuline. De plus, des tests de viabilité cellulaire ont mis en évidence que l’inhibition partielle par RNAi des septines ainsi que des polyglutamylases et de la tubuline tyrosine ligase, comme la surexpression d’enzymes responsables de la déglutamylation de la tubuline, permettent de restaurer une certaine sensibilité au Taxol des cellules T8. Inversement, la surexpression de certaines enzymes responsables de la polyglutamylation et de la tyrosination de la tubuline dans les cellules Tv permet d’instaurer une résistance au Taxol des cellules sensibles.La compilation de nos résultats permet de proposer un nouveau mécanisme de résistance au Taxol des cellules cancéreuses mammaires MDA-MB 231 : une augmentation du niveau de tyrosination de l’alpha-tubuline serait à l’origine de l’allongement des chaînes polyglutamylées sur le MT entraînant une diminution de la liaison de la protéine stabilisatrice MAP4 ainsi que le recrutement des septines sur les MTs. Ces modifications favoriseraient alors le recrutement du facteur de sauvetage CLIP-170 et de kinésines dépolymérisantes telles que MCAK à l’extrémité en croissance du MT des cellules résistantes T8, permettant une certaine restauration de la dynamique microtubulaire et contribuant ainsi à l’apparition du phénotype chimiorésistant.Ces études sont indispensables pour établir les bases d’un nouveau mécanisme de résistance au Taxol impliquant les septines, ainsi qu’un lien de causalité avec la tyrosination et la polyglutamylation. Au-delà, la recherche de ces modulations clés pourra être réalisée sur des cancers mammaires sensibles et résistants au Taxol, issus de biopsies de patients. Ainsi, il sera possible à terme, de déterminer si les septines, la tyrosination et la polyglutamylation de la tubuline ont une véritable importance fonctionnelle dans la résistance de cancers du sein au Taxol. / Breast cancer remains the leading cause of women mortality in France, and chemoresistance emergence, in particular to taxanes, including paclitaxel (Taxol®), is an important limitation to the use of these molecules. As do many anticancer drugs, taxanes target microtubules (MTs), tubulin polymers involved in many cellular functions. These alternate between growing and shrinking stages, thus providing dynamic instability. By binding to beta-tubulin, Taxol stabilizes MTs and prevents successful mitosis, leading to apoptosis. Taxol resistance is a multifactorial process including mechanisms such as overexpression of drug efflux pumps (P-glycoprotein), mutations in the genes for alpha- and beta-tubulin or altered expression of MT-associated proteins (MAPs) like the MT-stabilizing MAP4 or the depolymerizing stathmin, therefore contributing to MT dynamics restoration.The current project relies on the use of the breast carcinoma cell line MDA-MB 231 made gradually resistant to 25nM of Taxol under blocking conditions of efflux pumps. To get a broader insight into the protein modifications from the MT environment associated with the chemoresistant phenotype, we compared the proteomic profiles of total MT fractions from Taxol-sensitive (Tv) and Taxol-resistant (T8) cells. Among the 112 differentially enriched proteins found in one of the two populations, we evidenced increased levels of betaIII and betaIV-tubulins, a slight increase and a decrease of molecular motors kinesin-1 and dynein, respectively, and the enrichment of several septins (SEPT2, 8, 9 and 11) in MT fractions of T8 cells. Septins are GTPases that associate into hetero-oligomeric complexes involved in cytokinesis and in MT and actin cytoskeleton organization. Recruitment of these candidate proteins on MTs of T8 cells has been validated by Western blot analysis (Froidevaux-Klipfel et al., 2011) and immunofluorescence experiments.In the literature, septins localize either on actin or MTs in mammalian cells. Interestingly, in our MDA-MB 231 cells, SEPT2 is recruited partially on actin stress fibers in sensitive Tv cells, whereas it colocalizes with MTs in T8 ones. Although actin depolymerization in Tv cells does not induce any shift towards MTs, this differential localization of septins on MTs of resistant cells could nevertheless participate in the chemoresistant phenotype.Furthermore, it has been described in the literature that SEPT2 binds to polyglutamylated MTs to facilitate vesicular transport to the plasma membrane of proteins implicated in cell polarization. Polyglutamylation is a post-translational modification allowing the formation of side-chains of several glutamate residues on alpha- or beta-tubulins, thus regulating interactions between MTs and MAPs. Our results show that septin accumulation on the MT network of T8 cells is associated with an increase in polyglutamylation but also tyrosination of tubulin. In addition, cell viability assays showed that partial inhibition by RNAi of septins as well as polyglutamylases and tubulin tyrosine-ligase, but also overexpression of enzymes responsible for tubulin deglutamylation, could restore Taxol sensitivity of T8 cells. By contrast, overexpression of enzymes responsible for tubulin polyglutamylation and tyrosination in Tv cells could induce Taxol resistance of sensitive Tv cells.The compilation of our results enables us to provide a new mechanism of Taxol resistance of the breast cancer cells MDA-MB 231: an increased level of alpha-tubulin tyrosination would induce the lengthening of polyglutamylated chains on the MT, resulting in a reduced binding of the stabilizing protein MAP4 as well as the recruitment of septins on MTs. These modifications would promote the recruitment of the rescue factor CLIP-170 and that of depolymerizing kinesins such as MCAK to the growing end of the MT of resistant T8 cells, leading to a restoration of MT dynamics, thus contributing to the emergence of the chemoresistant phenotype.These studies are essential to lay the basis for a new mechanism of Taxol resistance involving septins, and a causal relationship to tyrosination and polyglutamylation. Beyond, the search for these key modulations will be performed on Taxol sensitive and resistant breast cancers, from patients’ biopsies. Thus, it will be eventually possible to determine whether septins, tubulin tyrosination and polyglutamylation have a real functional importance in breast cancer resistance to Taxol.

Cette thèse tente d'aborder, avec quelques détours, deux questions centrales concernant le<br />centrosome des vertébrés : son rôle dans la motilité cellulaire et son rôle dans le cycle de<br />division cellulaire.<br />Après une introduction en trois parties (un tour d'horizon dans une optique historique, un<br />exposé détaillé des connaissances actuelles, puis une réflexion plus générale sur des bases<br />phylogénétiques), deux travaux sont présentés : une étude des rôles respectifs des deux<br />centrioles du centrosome des cellules de vertébrés, puis une étude du comportement<br />particulier du centrosome dans ces cellules en sortie de mitose.<br />L'ensemble de ce travail se fonde sur un outil précieux : l'établissement de lignées cellulaires<br />qui expriment de manière stable la centrine 1 humaine couplée à la GFP, ce qui constitue un<br />excellent marqueur des centrioles et de leur stade de maturation. En effet, le centrosome des<br />vertébrés contient deux structures microtubulaires appelée centrioles qui se reproduisent en<br />synchronie avec le cycle de division cellulaire par un mécanisme de duplication, un nouveau<br />centriole étant assemblé à proximité de chaque centriole présent. Il y a donc dans chaque<br />cellule, après une mitose, un nouveau et un ancien centriole aussi appelés centriole parental<br />ou centriole père et centriole fils.<br />La première étude, après avoir succinctement défini le comportement des centrioles dans les<br />différentes phases du cycle, se concentre plus précisément sur la phase G1 pendant laquelle il<br />a pu être observé que les deux centrioles peuvent transitoirement se séparer de plus de dix<br />microns. L'un des deux centrioles, qui a pu être identifié comme le centriole le plus jeune, a<br />une mobilité parfois importante, alors que le plus ancien, qui est associé à l'aster de<br />microtubules par des appendices qui sont caractéristiques de son ancienneté reste près du<br />centroïde de la cellule. La différence d'abondance des microtubules à proximité des deux<br />centrioles a pu être attribuée à une régulation différentielle de l'ancrage : le centriole le plus<br />ancien capture les microtubules qui sont nucléés dans un rayon de quelques microns autours<br />de lui, alors que le centriole fils, qui a une capacité de nucléation équivalente, a une capacité<br />d'ancrage des microtubules réduite. Ainsi, quand les deux centrioles sont éloignés l'un de<br />l'autre, de nombreux microtubules libres peuvent être observés dans la cellule, au contraire,<br />quand ils sont proches, la plupart des microtubules cellulaires sont ancrés sur le centrosome<br />(et en particulier sur le centriole père). Nous avons donc proposé que la cellule puisse<br />modifier son réseau microtubulaire en modulant la distance intercentriolaire.<br />La deuxième étude présentée porte sur un comportement particulier du centriole parental en<br />fin de mitose : après que les cellules se sont étalées, mais alors qu'elles sont encore reliées par<br />un pont cytoplasmique, les deux centrioles, dans chaque cellule fille, se séparent puis le<br />centriole parental quitte sa position centrale et stationne pendant 10 à 30 minutes à proximité<br />du pont cytoplasmique intercellulaire. Le pont se pince alors de chaque côté de la pièce<br />intermédiaire puis se rompt lorsque le centriole parental regagne sa position près du noyau.<br />Nous avons pu déterminer que le pincement du pont correspondait au détachement des<br />faisceaux de microtubules qu'il contient. Nous avons ensuite, à l'aide de drogue qui<br />dépolymérisent les microtubules, suggéré l'existence d'un contrôle de la présence du centriole<br />parental dans le pont. Nous avons étudié des cas de cellules acentriolaires et pu mettre en<br />évidence des défauts liés à la cytocinèse. Enfin, il nous est apparu, à la suite d'expériences sur<br />des substrats plus ou moins adhésifs que l'adhésion de la cellule à son substrat est un des<br />paramètres clé de la régulation de cet événement de fin de mitose.<br />Des interprétations plus spéculatives sont proposées dans les discussions qui suivent l'exposé<br />des résultats, ainsi que des expériences pour les tester.

Cilia and flagella are sensory organelles, found in the majority of eukaryotic organisms that play a vital role in the general physiology, health and early development of humans. Intraflagellar transport (IFT) is tasked with building and maintaining the entire ciliary structure by facilitating the transport of axonemal precursors, trafficking of ciliary membrane proteins and turnover products. Currently, there are no complete models detailing how ciliated organisms regulate the entry and exit of IFT particles, a multi-meric adaptor complex that ferries flagellar proteins. In this thesis, I focus on small Rab-like protein IFT22, an IFT-particle integrated protein with predicted GTPase activity, as a potential regulatory component of IFT particle trafficking in Chlamydomonas. Using an artificial microRNAs strategy, I show that IFT22 regulates the available cellular pool of IFT particles and the distribution profile of the IFT particles between the cytoplasm and the flagellar compartment. Additionally, I demonstrate how the putative constitutive active mutant of IFT22 is able properly localize to the peri-basal body and enter the flagellar compartment using immunofluorescence and immunoblot analysis of flagella extracts. Finally, preliminary RNAi data suggests IFT25 the IFT particle/motor/BBSome assembly downstream of IFT22 regulation, evident from the depletion of kinesin-2 subunit FLA10, IFT-dynein-2 subunit D1bLIC and BBsome component BBS3from whole cell extracts of IFT25 knockdown transformants.

Thesis (Ph.D.)--University of Alabama at Birmingham, 2009. / Title from PDF title page (viewed on Feb. 19, 2010). Includes bibliographical references.

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Internal and external signaling dependent changes in cell behavior are directly linked to force-generating remodeling of the actin microfilament system which is juxtaposed to the inside of the plasma membrane. This dynamic filament system is involved in many processes in the cytoplasm and the nucleus of eukaryotic cells.   This thesis studies profilin, a regulator of actin filament dynamics which functions during incorporation of new actin molecules at growing filament ends at the cell periphery. Profilin is also present in the nucleus but its function is less well understood in this compartment. Here I present results concerning profilin and the activity of the transcription factor SRF, which is known to control the expression of actin and many actin-binding proteins in a process requiring the MRTF-A co-factor. MRTF-A binds monomeric actin and is released upon receptor mediated actin polymerization. Depletion of the two profilin isoforms I and IIa reduced MRTF-A/SRF-dependent transcription, most likely since the lack of profilin enable more MRTF-A to bind actin monomers and thereby prevent SRF-transcription. Interestingly profilin depletion also seemed to affect general transcription in the two cell lines investigated. In a separate study, a close connection between profilin, and possibly also profilin:actin, with microtubules was revealed. Microtubules are important for intracellular trafficking of vesicles as well as directional cell migration and the observation made here suggests the existence of a microtubule-associated platform for actin filaments formation. In congruence, the microtubule-associated actin nucleation promoting factor WHAMM was found to interact with profilin. Finally, the intracellular distribution of profilin was investigated by fluorescence microscopy using different peptide specific antibodies. Since these antibodies showed unique but varying results our work emphasizes common problems connected with this technique. / <p>At the time of the doctoral defence the following papers were unpublished and had a status as follows: Paper1: Manuscript; Paper 2: Manuscript; Paper 3: Manuscript</p>

The cancer antigen 125 (CA125) is a cell-surface mucin which is over-expressed by the majority of ovarian cancers. However, its biology and the role it plays in ovarian cancer is largely unknown, although other cell-surface mucins have been shown to play a role in apoptosis, cell growth and tumour immune evasion. To analyse the function of CA125 in ovarian cancer, we initially knocked down the expression of CA125 using RNA interference. Knocking down CA125 expression using in vitro transcribed short interfering RNAs (siRNAs) induced a potent cell death response, which has been well characterised in the literature as an induction of an interferon response and resulting in cell apoptosis. Subsequently, using the short hairpin RNA expression vector, pSUPER, which has been shown to knock down genes with high efficiency with reduced off-target affects, we generated stable sub-lines of the ovarian cancer cell line, OVCAR-3, which had been transfected with pSUPER constructs targeting CA125. Intriguingly, these sub-lines had a range of abnormal mitotic events and nuclear defects. However, there was no clear association with the level of CA125 knock down. This could be either due to clonal selection from the parent OVCAR-3 cell line or in addition to CA125 knock down, additional genetic changes are required to occur to favour a state of survival. Similar to the in vitro data, xenografts of the sub-clones into SCID mice generated inconclusive results as to whether CA125 knock down contributes to tumour growth, invasion and metastasis in vivo. More recently, we have been able to achieve high levels of short-term CA125 knock down using synthetic siRNAs designed to reduce off-target affects. These preliminary in vitro and in vivo experiments conducted with pSUPER sub-lines should be repeated using synthetic siRNAs to confirm the role of CA125 in this context. Given the role which the cytoplasmic tail of cell-surface mucins plays in its function, we generated a polyclonal antibody recognising the CA125 cytoplasmic tail, designated M16.1. Immunofluorescence imaging of CA125 in ovarian cancer cell lines, OVCAR-3 and PEO-1, using the OC125 extracellular domain antibody indicated cell-surface localisaton of CA125. However, in addition to the cell-surface localisation, the M16.1 antibody localised to the cell cytoplasm, indicating cleavage and release of the CA125 cytoplasmic tail into the cytosol. Additionally, M16.1 co-localised with α-tubulin at perinuclear sites and to areas resembling microtubule organising centres. However, M16.1 did not co-localise with γ-tubulin at the centrosome, indicating association with non-centrosomal microtubules. Furthermore, depolymerisation of microtubules on ice for 1 hour resulted in loss of diffuse cytoplasmic M16.1 staining but co-localisation between M16.1 and α-tubulin at non-centrosomal sites remained. Intriguingly, when microtubules were allowed to reform at 37oC in PEO-1 cells which had CA125 knocked down by synthetic siRNAs, the ability to reform radial asters was impaired, possibly indicating the CA125 cytoplasmic tail involvement in anchoring microtubules to non-centrosomal sites. Furthermore, we also cloned a portion of CA125 encompassing the cytoplasmic tail, transmembrane domain and 9 tandem repeats. When this construct was transfected into COS-1 cells, the CA125 cytoplasmic tail localised to microtubule bundles during metaphase. Mitotic involvement of the endogenous CA125 cytoplasmic tail was confrmed in OVCAR-3 and PEO-1 cells using M16.1. Given this association and also the results from the pSUPER sub-lines with CA125 knockdown, CA125 may be involved in controlling the fidelity of mitosis, which is grossly altered during tumourigenesis. More recently, it was identified that galectin-1 (Gal-1), an S-type lectin, is a ligand for CA125. Gal-1 is a potent inducer of T cell apoptosis and has been implicated as playing a major role in immune evasion for cancer cells. Consequently, we analysed the expression of CA125 and Gal-1 in ovarian cancer and confirmed the two molecules were expressed concurrently at the mRNA level by RT-PCR. Moreover, immunofluorescence studies also confirmed that CA125 and Gal-1 interacted with each other at the cell-surface of 27/87 cells, an ovarian cancer cell-line. Therefore, we hypothesised that CA125 presents Gal-1 to the immune system, which then induces T cell apoptosis and allows the tumour to escape the immune system. However, CA125 did not protect tumour cells from recognition or killing by T cells, which was shown by no differences in IFN-γ secretion or tumour lysis by cytotoxic T cells using influenza peptide pulsed pSUPER sub-lines with CA125 knockdown. The work described in this thesis suggests that CA125 plays a major role in the aetiology and progression of ovarian cancer through its actions on mitosis, microtubule organisation and immune evasion.

Pinus radiata is by far the dominant species grown in New Zealand plantations as a renewable source of wood. Several wood quality issues have been identified in the material produced, including the high incidence of compression wood, which is undesirable for end users. At present our understanding of the complex array of developmental processes involved in wood formation (which has a direct bearing on wood quality) is limited. Hence, the forest industry is interested in attaining a better understanding of the processes involved. Towards this goal, and for reasons of biological curiosity, the experiments described in this thesis were carried out to investigate several aspects of xylem cell development. In an in arbor study, changes in the orientation of cortical microtubules and cellulose microfibrils were observed in developing tracheids. Results obtained provide evidence that cortical microtubules act to guide cellulose synthase complexes during secondary wall formation in tracheids. The mechanisms involved in controlling cell wall deposition in wood cells are poorly understood, and are difficult to study, especially in arbor. A major part of this thesis involved the development of an in vitro method for culturing radiata pine wood in which hormone levels, nutrients, sugars and other factors, could be controlled without confounding influences from other parts of the tree. The method developed was used in subsequent parts of this thesis to study compression wood development, and the influence of the hormone gibberellin on cellulose microfibril organisation in the cell wall. Results from the in vitro compression wood experiments suggested that: 1. when a tree is growing at a lean, the developing cell wall was able to perceive compressive forces generated by the weight of the rest of the tree, rather than perceive the lean per se. 2. ethylene, rather than auxin, was involved in the induction of compression wood. Culture of stem explants with gibberellin resulted in wider cells, with steeper cortical microtubules, and correspondingly steeper cellulose microfibrils in the S2 layer of developing wood cells. This observation provides further evidence that the orientation of microtubules guides the orientation of cellulose microfibrils. Overall, the work described in this thesis furthers our knowledge in the field of xylem cell development. The stem culture protocol developed will undoubtedly provide a valuable tool for future studies to be carried out.

Thesis (Ph. D.)--University of Washington, 1996. / Vita. Includes bibliographical references (leaves [69]-76).