Microtubules are critically important cytoskeletal elements. Together with microtubule associated proteins (MAPs), they form the latticework on which eukaryotic life exists. Simply put, microtubules are polymers of tubulin heterodimers, which are composed of the globular proteins alpha and beta tubulin. In vivo, these monomers associate with one another to form heterodimers, which then polymerize to form microtubules. In mammals, microtubule polymerization is a temperature-dependent process with an optimum of 37°C (Detrich et al., 2000). If temperatures exceed this optimal temperature by even a few degrees, the microtubule will begin to dissemble due to denaturation of the tubulin subunit and permanent loss of both shape and function will occur. This thermal barrier seems to be consistent in most eukaryotic organisms. Two exceptions are the thermophilic red algae, Cyanidium caldarium and Cyanidioschyzon merolae. These thermophilic acidophiles have been discovered in volcanic vents around the globe from Yellow Stone Park to Italy and grow at optimal temperatures of around 55°C. These organisms have been primarily studied in the context of evolutionary biology because of their primitive characteristics. Very little is known about the molecular biology of these organisms, and certainly nothing is known about how the biochemistry of these organisms brings about the ability to survive the harsh conditions of their environment. Currently, my hypothesis concerning the thermostable tubulin expressed within these organisms is that there may be key amino acid differences in the tubulin's primary structure that confer enhanced thermostability. I am testing this hypothesis by sequencing the alpha and beta tubulin genes of Cyanidium caldarium and Cyanidioschyzon merolae, generating homology models of the tubulin dimers, and comparing these models to a known mesophilic tubulin heterodimer structure in order to identify potential structural differences. / Master of Science

Thesis (Ph. D.)--University of Virginia, 2008. / Includes bibliographical references. Also available online through Digital Dissertations.

L’émergence de phénomènes de résistance au paclitaxel (Taxol®), un agent stabilisateur de microtubules (MTs), est un obstacle majeur au succès de cette molécule dans les chimiothérapies anticancéreuses et limite son utilisation. Au laboratoire, un nouveau mécanisme de résistance au Taxol® a été mis en évidence dans les cellules tumorales mammaires MDA-MB 231. Il est basé sur une restauration de la dynamique microtubulaire et implique i) deux modifications post-traductionnelles de la tubuline (MPTs), la détyrosination/retyrosination et la polyglutamylation et ii) la surexpression et la relocalisation du cytosquelette d’actine sur les MTs de plusieurs septines, des GTPases filamenteuses impliquées dans la cytocinèse et la compartimentation membranaire. Plus précisément, une boucle fonctionnelle entre le recrutement des septines et la polyglutamylation des MTs a été démontrée : la polyglutamylation de la tubuline stimule le recrutement des septines sur les MTs et les septines jouent un rôle de protéine d’échafaudage pour les enzymes responsables de la polyglutamylation, favorisant l’élongation des chaines latérales de glutamate. Toutes ces modifications résultent en un recrutement accru sur les MTs de deux +TIPs, la kinésine dépolymérisante MCAK et le facteur de sauvetage CLIP-170, permettant ainsi de maintenir une dynamique microtubulaire malgré la présence du paclitaxel.De plus, l’étude de la contribution relative de chacun de ces acteurs dans ce mécanisme de chimiorésistance a permis de montrer que la stimulation de la polyglutamylation associée à la surexpression d’un ensemble de septines incluant la SEPT9_i1 est indispensable et suffisante pour induire une relocalisation des septines des microfilaments d’actine vers les MTs, une augmentation de la liaison de CLIP-170 et de MCAK aux MTs et une résistance au paclitaxel, non seulement dans les MDA-MB 231 mais aussi dans un certain nombre de lignées cellulaires sensibles (RPE-1, HeLa et CHO). L’analyse de ce phénomène a par ailleurs permis de montrer qu’à l’état basal, dans des cellules chimiosensibles, les MTs jouent un rôle essentiel dans l’organisation subcellulaire des filaments de septines sur l’actine, qu’un transport dépendant de la kinésine-1 était impliqué. / Acquired resistance to the microtubule (MT)-stabilizing agent paclitaxel (Taxol®) is a major obstacle for successful chemotherapy and limits its use as an anticancer drug. We evidenced a new mechanism of Taxol® resistance acquired by MDA-MB 231 breast cancer cells which is based on the restoration of MTs dynamics and involves i) two tubulin post-translational modifications (PTMs); detyrosination/retyrosination and polyglutamylation, and ii) overexpression and relocalization from the actin microfilaments to the MT network of several septins, a family of filamentous GTPases implicated in cytokinesis and membrane compartmentalization. More precisely, a functional loop between septin recruitment to MTs and tubulin polyglutamylation has been uncovered: tubulin polyglutamylation stimulates septin association with MTs, and septins act as scaffold proteins for tubulin polyglutamylation enzymes, thus promoting the elongation of lateral polyglutamate chains. Altogether, these modifications enhance the recruitment to MTs of two +TIPs, the MT-depolymerizing kinesin MCAK and the rescue factor CLIP-170, which would in turn compensate for paclitaxel-mediated inhibition of MT dynamics.Studying the relative contribution of each of these actors in this new chemoresistance mechanism further showed that stimulation of tubulin polyglutamylation together with the overexpression of a panel of septins that comprised the SEPT9_i1 isoform were necessary and sufficient to relocate septin filaments from actin microfilaments to MTs, to increase the binding of CLIP-170 and MCAK to MTs and to induce Taxol®-resistance, not only in MDA-MB 231but also in several other Taxol®-sensitive cell lines (RPE-1, HeLa and CHO). The analysis of this phenomenon also showed that, in Taxol®-sensitive cells, MTs play an essential role in the assembly and subcellular localization of septin filaments to actin microfilaments, and that a kinesin1-dependent transport is involved.

京都大学 / 0048 / 新制・課程博士 / 博士(工学) / 甲第19935号 / 工博第4218号 / 新制||工||1652(附属図書館) / 33021 / 京都大学大学院工学研究科マイクロエンジニアリング専攻 / (主査)教授 小寺 秀俊, 教授 中部 主敬, 准教授 横川 隆司 / 学位規則第4条第1項該当 / Doctor of Philosophy (Engineering) / Kyoto University / DGAM

Thesis advisor: Laura Anne Lowery / Thesis advisor: Eric Folker / In order to form the exact synaptic connection required for proper neurological function, the growing tip of the neuron hosts an orchestra of hundreds of different proteins interacting with extracellular cues to steer neuron growth in the right direction. The goal of our current research is to study several of the components of this pathway, known as the TACC family. Here, we present a detailed structure/function analysis of the TACC family in regards to binding and activity with other proteins in the growth cone. We investigate the function of TACC3 in mediating neuron outgrowth and guidance in vivo. We have found structural elements of the TACC family that enable their activity. Studying these conserved structures and functions of the TACC family will enable greater understanding of the entire process of cytoskeletal regulation and neurodevelopment. / Thesis (BS) — Boston College, 2015. / Submitted to: Boston College. College of Arts and Sciences. / Discipline: Scholar of the College. / Discipline: Biology.

Microtubules (MTs), whose basic units are a and ß tubulin proteins, are self-assembled proteinaceous filaments with nanometer scale diameters and micrometer scale lengths. Their aspect ratio, directionality, the reversibility of their assembly and their ability to be metallized by electroless plating make them good candidates to serve as templates for the fabrication of nanowires and other nanoscale devices. In addition, tubulin proteins can provide biological interactions with a naturally high specificity.Toward the goal of manufacturing MT-based metallic nanowires and networks of nanowires on a silicon wafer, I studied the influence of pH, temperature, and several biomolecules on the stability of MTs in solutions, as well as the surface effect on the dynamics of disassembly of microtubules. Secondly, I demonstrated the metallization of MTs by electroless nickel plating both in solution and on hydrophilic oxidized Si surface. After being activated by Pt, nickel coated MT surfaces during the electroless plating, with a thickness of several nanometers. Due to the different kinetics of the process, MTs metallized on the oxidized Si wafer are slightly different from MTs metallized in solutions. Finally, we explored controlled nucleation and growth of microtubules directly from a collection of g-tubulin monomers. g-tubulins bind to modified gold electrodes on a silicon wafer through an organic linker, Glutathione s-transferase, creating a g-tubulin layer for MT growth. MTs unambiguously originated from the surface-bound g-tubulin layer on the gold electrode, proving that the surface-bound g-tubulin retains its biological ability of nucleating MT growth.

In recent years, microlenses (ML), which are micro-scale spheres, have been used to overcome physical diffraction limit of optical microscopy (~200 nm). Although the use of such ML has provided highly resolved images of objects beyond the Abbe optical diffraction limit, the process needs to be refined before it can be applied widespread in materials, biological and clinical research. In this research work, we have implemented experiments on super-resolution imaging utilizing MLs of different refractive indices (n) and diameters to provide the scientific and engineering communities with practical guidelines for obtaining high resolution images with ease. With the support from experimental imaging data as well as FDTD simulations, we have shown that optimal super-resolution imaging with microspheres was accomplished under specific parameter range. We have identified ML with n=1.51 as a preferable choice over those MLs with n=1.4, 1.93, and 2.2, because of high reliability and high magnification for ML with n=1.51. With n=1.51 in mind, we have identified a diameter range from 15 μm to 50 μm provides high resolution and magnification for practical purposes. We show that other ML diameters provided high resolution as well; we believe that ML diameters between 15 μm and 50 μm are practically preferred. We were able to achieve <150 nm resolution and further refinement of this tool can potentially yield higher quality imaging results. Ideally, MLs will eventually be directly incorporated as a modular device in an optical microscope providing the researchers an effective, noninvasive, and economical alternative to complex super resolution microscopy techniques. To improve scanning efficiency, we also proposed microtubule (MT) based imaging. With the demonstration of theoretical optics, we conclude, at present time, that there are some practical concerns for MT-based imaging technique that may limit its application as super-resolution imaging technique. For example, MT-based imaging appears to possess a lower contrast than ML-based technique. Thus, although the concept of MT-based imaging is theoretically possible, we think that more work is needed to utilization of this tool for practical applications.

Microtubules are the most rigid element of the cytoskeleton. They are responsible for the structure of cells and make up the tracks for intracellular cargo transport. Interactions between microtubules, motor proteins, and microtubule-associated proteins drive important mechanisms in the cell, such as cell division, cell motility, cell homeostasis, and cell signaling. I seek to understand how such complex, energy-consuming non-equilibrium biological networks self-organize by studying in vitro microtubules bundled by microtubule-associated protein 65 (MAP65), in kinesin-1 gliding assays. I found that large networks can break into smaller, cell-like networks that can mimic types of cell motility. Dynamics of these networks change with varying concentrations of MAP65 and microtubules.

A mathematical approach to the concepts of copolymerization of microtubules in the presence of anti-mitotic drugs is presented in this work. A general feature of the mathematical equations is presented. The possibility of having analytical steady state solutions of dynamic equations is investigated. The structure of equations is narrowed down for the specific brand of anti-mitotic drug, colchicine. The behavior of total T-tubulin concentration in the steady state in a regeneration system is investigated and analyzed through the numerical calculations. / Ph. D.

Dans l’arsenal thérapeutique anticancéreux, les agents anti-microtubules (MTA) occupent une place essentielle dans le traitement de tumeurs solides et d’hémopathies malignes. Néanmoins, leur utilisation est limitée par l’induction d’une toxicité neurologique qui affecte la qualité de vie des patients et dont les mécanismes d’action demeurent peu compris. L’absence de solutions préventives ou curatives réellement efficaces, reflète la complexité des mécanismes d’action des MTA. Dans le cadre du projet « Mitotarget » (7ème PCRD) porté par le partenaire industriel Trophos, notre objectif était de préciser le mécanisme à l’origine de la neurotoxicité des MTA et d’évaluer le potentiel neuroprotecteur de l’olesoxime, composé ayant fait la preuve de son efficacité neuroprotectrice dans différents modèles de pathologies neurodégénératives. Nous montrons ici que les réseaux microtubulaire (dynamique des microtubules, localisation de la protéine EB1) et mitochondrial (motilité des mitochondries), cibles des MTA dans les cellules cancéreuses, sont aussi affectés dans les cellules de type neuronal. Leur préservation par l’olesoxime est nécessaire à l’établissement d’une neuroprotection. Ce travail met en évidence l’originalité du mécanisme d’action de l’olesoxime, premier neuroprotecteur capable d’agir tout à la fois sur les microtubules et les mitochondries, et souligne l’importance des liens étroits existant entre ces deux compartiments. Deux axes d’étude ont été initiés à la suite de ce projet afin de (i) déchiffrer les interconnexions microtubules-mitochondries dans la réponse des cellules cancéreuses aux MTA; (ii) préciser l’importance et la régulation post-traductionnelle de la protéine EB1 dans l’efficacité anti-migratoire des MTA. L’ensemble des données obtenues appelle à poursuivre la caractérisation des mécanismes de réponse aux agents anti-microtubules afin d’optimiser les stratégies thérapeutiques existantes. / Nowadays, the so-called Microtubule Targeting Agents (MTAs) remain benchmark clinical treatments displaying high efficiency and are still widely used against a broad spectrum of tumors and hemopathies. The new compounds in clinical development and the discovery of their anti-angiogenic properties make them a family booming. However, MTAs treatment is limited by the occurrence of neurological toxicities that greatly impair patients quality of life and which mechanisms of action are still poorly understood. The current absence of really efficient curative of preventive strategies underline the complexity of MTA mechanisms of action. In the framework of the “MitoTarget” project from the 7th PCRD,lead by the industrial partner Trophos, we aimed to precise MTA neurotoxic mechanisms and to evaluate neuroprotective potential of olesoxime, a compound that already showed to be efficient in various models of neurodegenerative diseases. Our data show that microtubular (microtubule dynamics parameters, EB1 protein localization) and mitochondria (mitochondria) networks, MTA targeted compartments in cancer cells, are damaged in neuronal-like cells. Interestingly, olesoxime neuroprotective activity implies preservation of both microtubule and mitochondria from MTA-induced damages. This work highlights the original mechanism of action of olesoxime as the first neuroprotective agent able to act on both microtubule and mitochondria and underlines the strengthened link existing between these compartments. It thus gave rise to two side projects with the aim to (i) decipher microtubule-mitochondria interconnections in response to MTA treatment; (ii) precise the importance and regulation of EB1 in the anti-migratory efficacy of MTA by looking at EB1 post-translational modifications. Altogether, the data obtained incite to keep on characterizing mechanisms involved in response to MTA in order to optimize the existing therapeutic strategies.