The eyespot of the biflagellate unicellular green alga Chlamydomonas reinhardtii is a complex organelle that facilitates directional responses of the cell to environmental light stimuli. The eyespot, which assembles de novo after every cell division and retains a distinctive association with the microtubule cytoskeleton, comprises an elliptical patch of rhodopsin photoreceptors in the plasma membrane and stacks of carotenoid-rich pigment granule arrays in the chloroplast and serves as a model for understanding how organelles are formed and placed asymmetrically in the cell. This study describes the roles of several factors in the assembly and positioning of the eyespot. Two loci, EYE2 and EYE3, define factors involved in the formation and organization of the eyespot pigment granule arrays. Whereas EYE3, a serine/threonine kinase of the ABC1 family, localizes to pigment granules, EYE2 localization corresponds to an area of the chloroplast envelope in the eyespot. These proteins play interdependent roles: EYE2 and the ChR1 photoreceptor co-position in the absence of pigment granules, and the pigment granules are required to maintain the shape and integrity of the EYE2/ChR1 patch. The miniature-eyespot locus MIN2 affects eyespot size and likely regulates the amount of material available for eyespot assembly. The MLT2 locus regulates eyespot size, number, and asymmetry. A novel locus, PEY1, modulates the position of the eyespot on the anterior-posterior axis by affecting microtubule rootlet length. A working model is developed wherein rootlet microtubule-directed photoreceptor localization establishes connections in the chloroplast envelope with EYE2, which directs the site for pigment granule array assembly, and MLT2 is proposed to negatively regulate the levels of eyespot proteins.

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No description available.

An introduction, background and recent advances in the areas of microtubule-binding agents and heat shock protein 90 (HSP90) inhibitors as anticancer agents are briefly reviewed. The work in this dissertation is centered on the synthesis of substituted pyrrolo[2,3-<italic>d</italic>]pyrimidines as potential anticancer agents that act via microtubule inhibition or HSP90 inhibition.<br>Microtubule-binding agents are effective against a broad range of tumors and lymphomas and have been common components of combination cancer-chemotherapy in the clinic. Despite the unparalleled success, drawbacks among microtubule-binding agents such as multi-drug resistance, dose-limiting toxicity, poor pharmacokinetic profile and high cost have supported the sustaining momentum in searching for novel agents of this class.<br>The research on microtubule-binding agents in this dissertation was initiated by an unexpected discovery. The lead compound, a 4-<italic>N</italic>-methyl-4'-methoxyaniline-substituted pyrrolo[2,3-<italic>d</italic>]pyrimidine, was found to inhibit the majority cancer cell lines in the NCI-60 panel at sub-micromolar concentration. The COMPARE analysis based on the activity profile indicated microtubule inhibition as the main mechanism of action of this compound, and was later confirmed through multiple assays. Further, the lead compound displaced 70% of [<super>3</super>H]colchicine from tubulin at a concentration of 5 μM, and was identified as a colchicine-site binder. The compound has also shown unabated or even increased activities against several drug-resistant cancer cell lines, especially the cell lines overexpressing P-glycoprotein or βIII-tubulin. In addition, the compound has favorable physicochemical properties such as high water solubility as its hydrochloride salt.<br>Based on the preliminary data and molecular modeling, a hypothesis on the relationship between binding affinity and the lowest-energy conformation of pyrrolo[2,3-<italic>d</italic>]pyrimidines was proposed. To test the hypothesis and search for compounds with improved potency, 38 pyrrolo[2,3-<italic>d</italic>]pyrimidine analogs in six series were designed and synthesized. The biological evaluations of these compounds are currently in progress at the time this dissertation is submitted.<br>HSP90 is one the molecular chaperones that assist the proper folding of the newly synthesized polypeptides and proteins. The majority of its client proteins are signal transducers with unstable conformations, which play critical roles in growth control, cell survival and development. The expressions of these proteins in normal cells were much less than cancer cell, making HSP90 a viable target for cancer chemotherapy. As of 2012, there are 16 HSP90 inhibitors in clinical trial, among which four are based on the purine-scaffold. All the compounds in clinical trials bind to or overlap with the ATP site on the N-terminal of HSP90.<br>The pyrrolo[2,3-<italic>d</italic>]pyrimidine scaffold is structurally close to purines. In the design of receptor tyrosine kinase (RTK) inhibitors, Gangjee et al. have shown that properly functionalized pyrrolo[2,3-<italic>d</italic>]pyrimidines bind to the ATP site and achieve high degrees of selectivity. This was partly attributed to the incorporation of substitution patterns that are impossible on the purine scaffold. Based on these previous findings and the established SAR of the two purine derivatives in clinical trials (<bold>PU-H71</bold> and <bold>BIIB021</bold>), 18 substituted pyrrolo[2,3-<italic>d</italic>]pyrimidines in three series (in connection with this dissertation) were designed and synthesized. The biological evaluations of these compounds are currently in progress. / Mylan School of Pharmacy and the Graduate School of Pharmaceutical Sciences; / Medicinal Chemistry / PhD; / Dissertation;

<p>Dendrite shape is a defining component of neuronal function. Yet, the mechanisms specifying diverse dendritic morphologies, and the extent to which their functioning depends on these morphologies, remain unclear. Here, we demonstrate a dendrite-specific requirement for the microtubule severing protein Katanin p60-like 1 (Kat-60L1) in regulating the elaborate branch morphology and nocifensive functions of Drosophila melanogaster larval class IV dendritic arborization (da) neuron dendrites. Through genetic loss of function analysis we show that loss of kat-60L1 reduced dendrite branching and process length, particularly during a period of normally extensive growth. This morphological defect was paralleled by a reduction in nocifensive responsiveness mediated by these neurons, indicating a tight correlation between neuronal function and the full extent of the dendritic arbor. To understand the mechanism underlying Kat-60L1's effects, we used in vivo imaging of the microtubule plus-end binding protein EB1, and found fewer polymerizing microtubules within mutant dendrites. Kat-60L1 thus promotes microtubule growth within class IV dendrites to establish the full arbor complexity and nocifensive functions of these neurons. </p><p>Although reduction of the related microtubule severing protein Spastin also compromised class IV dendrite arborization and nocifensive responses, microtubule polymerization in dendrites was unchanged in spastin mutants, and behavioral defects arose from generally compromised neuronal excitation. Kat-60L1 and Spastin thus function in distinct neuronal compartments to establish the complex dendritic morphology and sensory functions of class IV da neurons via distinct mechanisms of microtubule regulation. Whereas Spastin regulates stable microtubules affecting both pre- and post-synaptic compartments of these neurons, Kat-60L1 function is required specifically in dendrites to promote their complex arborization through the addition of growing microtubule numbers. Double mutant analysis demonstrated that Kat-60L1 and Spastin function antagonistically to promote dendritic aborization, likely involving other molecular players involved in regulating the microtubule cytoskeleton. Lastly, we identified Mi-2 as a transcriptional regulator of both kat-60L1 and spastin and show a genetic interaction between mi-2 and kat-60L1 in the class IV dendritic arbor, demonstrating that Mi-2 antagonizes Kat-60L1 function, possibly through the parallel upregulation of spastin. These data support a key role for the differential utilization of microtubule severing in generating distinct neuronal morphologies and subsequent function.</p> / Dissertation

Thesis (Ph. D.)--University of Washington, 2002. / Vita. Includes bibliographical references (leaves 90-103).

Normal brain function is dependent on the correct positioning and connectivity of neurons established during development. The Reelin signaling pathway plays a crucial role in cortical lamination. Reelin is a secreted glycoprotein that exerts its function by binding to lipoprotein receptors and inducing tyrosine phosphorylation of the intracellular adaptor protein Dab1. Mutations in genes of the Reelin signaling pathway lead to profound defects in neuronal positioning during brain development in both mice and humans. However, the molecular mechanisms by which Reelin controls neuronal morphology and migration are unknown. We have used a systems analysis approach to identify genes perturbed in the Reelin signaling pathway and identified microtubule stabilizing CLIP-associated protein 2 (CLASP2) as a key cytoskeletal modifier of Reelin mutant phenotypes. Currently, little is known about the role of CLASP2 in the developing brain. We propose that CLASP2 is a key effector in the Reelin signaling pathway controlling basic aspects of cortical layering, neuronal morphology, and function. CLASP2 is a plus-end tracking protein and this localization places CLASP2 in a strategic position to control neurite outgrowth, directionality, and responsiveness to extracellular cues. Our results demonstrate that CLASP2 expression correlates with neurite length and synaptic activity in primary neuron cultures; however, the role of CLASP2 during brain development was unknown. In this dissertation, we have characterized the role of CLASP2 during cortical development by in utero electroporation of shRNA plasmids and found that silencing CLASP2 in migrating neurons leads to mislocalized cells at deeper cortical layers, abnormal positioning of the centrosome-Golgi complex, and aberrant length/orientation of the leading process. In addition, we found that GSK3β-mediated phosphorylation of CLASP2 controls Dab1 binding and is required for regulating CLASP2 effects on neuron morphology and migration. This dissertation provides the first steps in gaining insight into how Reelin signaling affects cytoskeletal reorganization to regulate fundamental features of neuronal migration, positioning and morphogenesis.

The tubulin/FtsZ superfamily contains a large set of proteins that spans through all kingdoms of life, with αβ-tubulins being the eukaryotic representatives and FtsZ being the best studied prokaryotic homologue. It is believed that all tubulin/FtsZ-related proteins have evolved from a common ancestor, however, members from this superfamily have diverged in many aspects. αβ-tubulins polymerise into giant and hollow microtubules in the presence of GTP. Despite the size of around 25 nm wide, microtubules display sophisticated dynamics. In particular, dynamic instability, the stochastic change between fast growth and rapid shrinkage, is a hallmark of microtubules. In contrast to αβ-tubulins, FtsZ lacks the C-terminal domain of tubulins and it probably functions as single homopolymeric protofilaments, possibly through treadmilling dynamics. There is strong divergence of the biological functions in the tubulin/FtsZ superfamily. Microtubules are involved in fundamental processes such as motility, transport and chromosomal segregation, whereas FtsZ is involved in bacterial cytokinesis (bacterial cell division), and the equivalent role of FtsZ is carried out by actin-based and ESCRTIII-based systems in eukaryotes. It seems that there is a big evolutionary gap between αβ-tubulins and FtsZ, and the only properties that are conserved within the tubulin/FtsZ superfamily are fold, protofilament formation and GTPase activity. In 2002, a pair of tubulin-like genes, btuba and btubb were identified in Prosthecobacter bacteria, with higher sequence homology to eukaryotic tubulins than FtsZ or any other bacterial homologues. The crystal structures solved later revealed, again, a closer similarity to αβ-tubulins than to their prokaryotic equivalents. It has been known for a while that BtubAB form filaments in the presence of GTP, however, little knowledge has been available regarding the filament architecture. In this project, I determined the near atomic resolution structure of the in vitro BtubAB filament using cryoEM and cryoET, revealing a hollow tube that consists of four protofilaments. A closer look showed that BtubAB filaments have many conserved microtubule features including: an overall polarity, similar longitudinal contacts, M-loops in lateral interfaces, and the presence of the seam, a structural hallmark of microtubules. My study also shows that BtubC, a protein with a TPR fold, binds to the BtubAB filaments in a stoichiometric manner, similar to some MAPs on microtubules. Based on this work, I concluded that BtubAB from Prosthecobacter form bacterial ‘mini microtubules’, and my work provided interesting insight into the evolution of tubulin/FtsZ-related proteins.

L'objectif de cette thèse est de proposer des modèles mathématiques permettant de décrire l'instabilité dynamique d'une population de microtubules (MTs) et l'effet de médicaments sur cette instabilité. L'instabilité dynamique des MTs joue un rôle extrêmement important dans les processus de la mitose et de la migration cellulaire et donc dans la progression tumorale. L'instabilité dynamique est un processus complexe qui implique différents états de la tubuline (polymérisée ou non-polymérisée, tubuline-GTP ou tubuline-GDP qui correspondent à deux états énergétiques différents des dimères) et qui résulte de processus chimiques (polymérisation, dépolymérisation, hydrolyse, recyclage, nucléation) liant ces différents états de la tubuline. Décrire cette complexité par le biais de modèles mathématiques permet alors de tester des hypothèses biologiques quant à l'impact de chacun de ces processus et l'action de molécules anti-MTs. De récents travaux suggèrent que le "vieillissement" des MTs impacte leur dynamique. Nous avons testé dans ce travail l'hypothèse que ce "vieillissement" accélère l'hydrolyse du GTP au sein de la tubuline. Nous avons construit de nouveaux modèles couplant deux équations de transport multi-D avec deux équations différentielles ordinaires impliquant des termes intégraux. Nous avons calibrer notre nouveau modèle à partir des données expérimentales; tester l'hypothèse biologique sur le mécanisme du processus de vieillissement; analyser la sensibilité du modèle par rapport aux paramètres décrivant les processus; tester différentes hypothèses quant l'effet des médicaments anti-MTs. / The aim of this thesis is to design new mathematical models that are able to appropriately describe dynamic instability of a population of microtubules (MTs) and effect of drugs on MT dynamics. MT dynamic instability play an important role in the processes of mitosis and cell migration and, thus, in cancer progression. Dynamic instability is a complex process that involves different states of tubulin (polymerized or non-polymerized, GTP-tubulin or GDPtubulin that correspond to two different energetic states of tubulin dimers) that resulted from chemical processes (polymerization, depolymerization, hydrolysis, recycling, nucleation) linking these different states of tubulin. Description of this complexity by mathematical models enables one to test biological hypotheses concerning the impact of each process and action of drugs on microtubule dynamics. Recent observations show that MT dynamics depends on aging of MT. One of the aims of the work is to test the hypothesis that MT aging results from the acceleration of the GTP hydrolysis. We construct for that new models that couple two multidimensional transport equations with two ordinary differential equations involving integral terms. We have calibrated our models on the basis of experimental data; tested biological hypothesis on mechanism of aging process; performed a sensitivity analysis of the model with respect to parameters describing chemical processes; and tested hypotheses concerning actions of drugs.

In this work the functions of the microtubule motors kinesin-1, -2 and -3 have been analysed in various settings. The reconstitution of microtubule-dependent motor activity in vitro has been primarily used to dissect the contributions of individual motors to cargo motility in two specific scenarios. Initially the regulation of kinesin-1 in a cell cycle-dependent manner has been examined by studying the ability of rat liver endoplasmic reticulum (ER) tubules to move in cytosols prepared from Xenopus laevis egg extracts arrested in interphase, meiosis or mitosis. It was found that kinesin-1-driven ER motility is significantly disrupted during metaphase in vitro. This is likely due to the recruitment or loss of binding partners which has a concomitant influence upon kinesin-1 activity. This work presents the first evidence that kinesin-1-driven ER movement, and not simply network morphology, varies during cell division. Furthermore, it is postulated that the replication of such regulation of kinesin-1 activity in vivo may contribute to the well documented changes in organelle positioning and cargo transit through membrane trafficking pathways which occur during cell division.The fungal metabolite brefeldin A (BFA) induces tubulation of several compartments located within the secretory and endocytic pathways in a microtubule-dependent fashion. The identity of the motor(s) responsible for this motility remains unconfirmed and controversial since several reports with conflicting data have been published. The contributions of kinesin-1, -2 and -3 to these processes have been investigated using in vitro motility assays in which rat liver Golgi membranes were combined with Xenopus laevis egg extract cytosol in the presence of BFA. Function blocking antibodies and dominant negative proteins were used to perturb the activities of various kinesin motors. This data indicates a particular isoform of kinesin-3, KIF1C, is solely responsible for the movement of BFA-induced tubules in vitro. This work was complemented by in vivo immunofluorescence studies using the HeLaM cultured cell line. Transient transfections of dominant negative proteins, or siRNA-mediated depletion, were used to disrupt the activities of various kinesin motors, either in isolation or in combination with each other. This approach revealed a contribution of KIF1C and kinesin-1 to the movement of early endosomal BFA-induced tubules in vivo.