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1
Malkova, Barbora. "Structural studies of cytoplasmic dynein." Thesis, University of Leeds, 2010. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.540206.
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Liu, Siming. "TESTING THE MULTI-DYNEIN HYPOTHESIS BY MUTATING INNER ARM DYNEIN HEAVY CHAINS IN TETRAHYMENA THERMOPHILA." Miami University / OhioLINK, 2004. http://rave.ohiolink.edu/etdc/view?acc_num=miami1077152822.
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Villemant, Cecile Madeleine. "Investigating dynein light intermediate chains function." Thesis, University of Manchester, 2010. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.518455.
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Kardon, Julia R. "Regulation of the cytoplasmic dynein motor." Diss., Search in ProQuest Dissertations & Theses. UC Only, 2009. http://gateway.proquest.com/openurl?url_ver=Z39.88-2004&rft_val_fmt=info:ofi/fmt:kev:mtx:dissertation&res_dat=xri:pqdiss&rft_dat=xri:pqdiss:3359552.
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Gleave, Emma Sarah. "Structural and single-molecule studies of the cytoplasmic dynein motor domain." Thesis, University of Cambridge, 2014. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.708182.
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Diamant, Aristides G. "The structure of the cytoplasmic dynein tail." Thesis, University of Cambridge, 2015. https://www.repository.cam.ac.uk/handle/1810/249014.
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Cytoplasmic dynein is a molecular motor that moves cargos along microtubules. Dynein, together with its large co-factor dynactin, is responsible for the vast majority of traffic towards the centre of the cell. The largest subunit of the dynein complex is called the dynein heavy chain (DHC). The DHC includes a C-terminal motor domain, which converts ATP hydrolysis into mechanical force, an N-terminal tail domain, and a flexible linker domain to join the two together. An intermediate chain (DIC) and light intermediate chain (DLIC) bind directly to the DHC tail, while light chains (DLCs) bind to the DIC. This tail complex is important for both cargo binding as well as homodimerisation of the DHC, which is necessary for processive movement. Previous studies suggest that the DLCs play an important role in homodimerisation, but it remains unclear how else the DHCs are held together. Using S. cerevisiae as a model system, I co-expressed all four dynein subunits and purified functional dynein motors. In this background, I found that truncating the DHC to include only the first 1004 residues (out of the total 4092) eliminates the motor domain as well as the flexible linker domain, while preserving binding to the DIC, DLIC and DLC. However, truncating just another 50 residues off of the C-terminus led to a loss of all accessory subunits. I developed a protocol for expressing and purifying large quantities of the 1004 residue construct, thus I provide the first description of a recombinant dynein tail domain. Using negative stain electron microscopy (EM), I also present the first 3D structural information for the tail region of the cytoplasmic dynein motor. I then describe a construct including only the first 557 residues of the DHC, which dimerises despite not being able to bind any of the other subunits. I present a crystal structure of this smaller DHC fragment, which shows that the N-terminal 180 residues of the DHC constitute an intricate dimerisation domain made up of a β-sheet sandwiched between α-helices. Not only is this the first crystal structure of any part of the DHC N-terminus, but it reveals a previously undocumented dimerisation domain within the DHC itself. Furthermore, information garnered from this crystal structure allowed for interpretation of a recent cryo-EM structure of a triple complex containing the dynein tail, dynactin and the cargo adaptor BICD2 (TDB) that was solved by my colleagues in the Carter group. Only by docking the DHC N-terminus crystal structure within the TDB EM density did it become clear that the N-terminus of the DHC is responsible for the majority of the contacts the dynein tail makes with both dynactin and BICD2. Therefore the work that I present here sheds new light on the unexpected importance of the DHC N-terminus and allows two important conclusions to be made. First, the N-terminal 180 residues of the DHC constitute a dimerisation domain of its own. Second, the next ~400 residues of the DHC form a domain that plays a key role in the complex interface between dynein, dynactin and BICD2.
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Roberts, Anthony John. "Structural studies on the mechanism of dynein." Thesis, University of Leeds, 2009. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.511146.
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Lo, Wai Hong. "Biochemical, structural and functional characterization of the light chains of the microtubule-based motor dynein /." View Abstract or Full-Text, 2003. http://library.ust.hk/cgi/db/thesis.pl?BICH%202003%20LO.
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Thesis (Ph. D.)--Hong Kong University of Science and Technology, 2003.<br>Includes bibliographical references (leaves 133-154). Also available in electronic version. Access restricted to campus users.
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Nyarko, Afua A. "Structure and interactions of subunits of cytoplasmic dynein /." View abstract, 2005. http://gateway.proquest.com/openurl?url_ver=Z39.88-2004&res_dat=xri:pqdiss&rft_val_fmt=info:ofi/fmt:kev:mtx:dissertation&rft_dat=xri:pqdiss:3191709.
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Nettesheim, Guilherme, Rafael A. Longoria, Allyson M. Rice, and George T. Shubeita. "Kinesin and dynein respond differently to cytoplasmic drag." Universitätsbibliothek Leipzig, 2015. http://nbn-resolving.de/urn:nbn:de:bsz:15-qucosa-182714.
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Milisav-Ribaric, Irina. "Characterisation of human dynein-related genes from testis." Thesis, University of Cambridge, 1997. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.627168.
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Nettesheim, Guilherme, Rafael A. Longoria, Allyson M. Rice, and George T. Shubeita. "Kinesin and dynein respond differently to cytoplasmic drag." Diffusion fundamentals 20 (2013) 37, S. 1, 2013. https://ul.qucosa.de/id/qucosa%3A13605.
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Paschal, Bryce M. "Structure and Function of Cytoplasmic Dynein: a Thesis." eScholarship@UMMS, 1992. https://escholarship.umassmed.edu/gsbs_diss/82.
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In previous work I described the purification and properties of the microtubule-based mechanochemical ATPase cytoplasmic dynein. Cytoplasmic dynein was found to produce force along microtubules in the direction corresponding to retrograde axonal transport. Cytoplasmic dynein has been identified in a variety of eukaryotes including yeast and human, and there is a growing body of evidence suggesting that this "molecular motor" is responsible for the transport of membranous organelles and mitotic chromosomes.
The first part of this thesis investigates the molecular basis of microtubule-activation of the cytoplasmic dynein ATPase. By analogy with other mechanoenzymes, this appears to accelerate the rate-limiting step of the cross-bridge cycle, ADP release. Using limited proteolysis, site-directed antibodies, and N-terminal microsequencing, I identified the acidic C-termini of α and β-tubulin as the domains responsible for activation of the dynein ATPase.
The second part of this thesis investigates the structure of the 74 kDa subunit of cytoplasmic dynein. The amino acid sequence deduced from cDNA clones predicts a 72,753 dalton polypeptide which includes the amino acid sequences of nine peptides determined by microsequencing. Northern analysis of rat brain poly(A) revealed an abundant 2.9 kb mRNA. However, PCR performed on first strand cDNA, together with the sequence of a partially matching tryptic peptide, indicate the existence of three isoforms. The C-terminal half is 26.4% identical and 47.7% similar to the product of the Chlamydomonas ODA6 gene, a 70 kDa subunit of flagellar outer arm dynein. Based on what is known about the Chlamydomonas70 kDa subunit, I suggest that the 74 kDa subunit is responsible for targeting cytoplasmic dynein to membranous organelles and kinetochores of mitotic chromosomes.
The third part of this thesis investigates a 50 kDa polypeptide which co-purifies with cytoplasmic dynein on sucrose density gradients. Monoclonal antibodies were produced against the 50 kDa subunit and used to show that it is a component of a distinct 20S complex which contains additional subunits of 45 and 150 kDa. Moreover, like cytoplasmic dynein, the 50 kDa polypeptide localizes to kinetochores of metaphase chromosomes by light and electron microscopy. The 50 kDa-associated complex is reported to stimulate cytoplasmic dynein-mediated organelle motility in vitro. The complex is, therefore, a candidate for modulating cytoplasmic dynein activity during mitosis.
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Roger, Yvonne. "The cytoplasmic dynein motor complex at microtubule plus-ends and in long range motility of early endosomes, microtubule plus-end anchorage and processivity of cytoplasmic dynein." Thesis, University of Exeter, 2013. http://hdl.handle.net/10871/11022.
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Cytoplasmic dynein is a microtubule-dependent motor protein which participates in numerous cellular processes. The motor complex consists of two heavy chains, intermediate, light intermediate and 3 families of light chains. Dynein is able to bind to these accessory chains as well as to regulatory proteins which enables the motor protein to fulfil such a variety of cellular processes. The associated light chains participate in long-distance organelle and vesicle transport in interphase and in chromosome segregation during mitosis. However, how these light chains control the activity of the motor protein is still unknown. In this study, I combine molecular genetics and live cell imaging to elucidate the role of the associated dynein light intermediate and light chains in dynein behaviour and early endosome (EE) motility in hyphal interphase cells as well as the anchorage of dynein to the microtubule (MT) plus-end in interphase and mitotic cells. I show that the dynein light intermediate chain (DLIC) as well as the light chain 2 (DLC2, Roadblock) are involved in dynein processivity and EE movement in interphase. The downregulation of either protein results in short hyphal growth which could be caused by a decreased runlength of EE and dynein. In addition, both proteins participate in dynein anchorage to the microtubule plus-end in interphase and mitosis as well as in spindle elongation during mitosis. Each protein causes a decrease of the motor protein dynein at MT plus-ends. Surprisingly, I found only minor or no defects in LC8 or Tctex mutants in the observed functions of dynein. LC8 seems to affect the dynein but not the EE runlength. In this case, dynein is still able to move into the bipolar MT array from where kinesin3 is able to take over EEs and move them towards the cell center. In contrast, Tctex has no effect on dynein or EE runlength or any other observed dynein function in hyphal cells. However, it causes a reduction in spindle elongation. Taken together, DLIC and DLC2 are important for dynein behaviour in long distance transport as well as in spindle positioning and elongation during mitosis. Furthermore, I studied the involvement of the dynein regulators Lis1 and NudE as well as the plus-end binding protein Clip1 (Clip-170 homologue) in the anchorage of dynein to the astral microtubule plus-ends during mitosis. The disruption of the anchorage complex at the astral MT plus-end causes a decrease in dynein number at this site and therefore slower spindle elongation in Anaphase B. Taken together, all three proteins are involved in anchorage of dynein to the astral microtubule tip and the subsequent spindle elongation. Furthermore, these findings also show that Ustilago maydis evolved two different mechanisms to anchor the motor protein to MT plus-ends in hyphal and mitotic cells. The plus-end binding protein Peb1 (EB1 homologue) and the dynein regulator dynactin mediate the dynein anchorage in hyphal cells whereas in mitotic cells the plus-ends binding protein Clip1 and the dynein regulators Lis1 and NudE anchor dynein to astral MT plus-ends.
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Ananthanarayanan, Vaishnavi. "Dynein dynamics during meiotic nuclear oscillations of fission yeast." Doctoral thesis, Saechsische Landesbibliothek- Staats- und Universitaetsbibliothek Dresden, 2014. http://nbn-resolving.de/urn:nbn:de:bsz:14-qucosa-135620.
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Cytoplasmic dynein is a ubiquitous minus-end directed motor protein that is essential for a variety of cellular processes ranging from cargo transport to spindle and chromosome positioning. Specifically, in fission yeast during meiotic prophase, the fused nucleus follows the spindle pole body in oscillatory movements from one cell pole to the other. The three molecular players that are essential to this process are: (i) the motor protein dynein, which powers the movement of the nucleus, (ii) microtubules, which provide the tracts for the movement and (iii) Num1, the anchor protein of dynein at the cortex. Dyneins that are localized to the anchor protein at the cortex and simultaneously bound to the microtubule emanating from the spindle pole body, pull on that microtubule leading to the movement of the nucleus. The spindle pole body, by virtue of its movement establishes a leading and a trailing side.
Previous work by Vogel et al. has elucidated the mechanism of these oscillations as that of asymmetric distribution of dynein between the leading and trailing sides. This differential distribution is a result of the load-dependent detachment of dynein preferentially from the trailing microtubules. This self-organization model for dynein, however, requires a continuous redistribution of dynein from the trailing to the leading side. In addition, dyneins need to be bound to the anchor protein to be able to produce force on the microtubules. Anchored dyneins are responsible for many other important processes in the cell such as spindle alignment and orientation, spindle separation and rotation. So we set out to elucidate the mechanism of redistribution of dynein as well as the targeting mechanism of dynein from the cytoplasm to cortical anchoring sites where they can produce pulling force on microtubules.
By employing single-molecule observation using highly inclined laminated optical sheet (HILO) microscopy and tracking of fluorescently-tagged dyneins using a custom software, we were able to show that dyneins redistributed in the cytoplasm of fission yeast by simple diffusion. We also observed that dynein bound first to the microtubule and not directly to the anchor protein Num1. In addition, we were able to capture unbinding events of single dyneins from the microtubule to the cytoplasm. Surprisingly, dynein bound to the microtubule exhibited diffusive behaviour. The switch from diffusive to directed movement required to power nuclear oscillations occurred when dynein bound to its cortical anchor Num1. In summary, dynein employs a two-step targeting mechanism from the cytoplasm to the cortical anchoring sites, with the attachment to the microtubule acting as the intermediate step.
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Tolic-Nørrelykke, Iva M., Sven K. Vogel, Nenad Pavin, Nicola Maghelli, and Frank Jülicher. "Self-Organization of Dynein Motors Generates Meiotic Nuclear Oscillations." Saechsische Landesbibliothek- Staats- und Universitaetsbibliothek Dresden, 2015. http://nbn-resolving.de/urn:nbn:de:bsz:14-qucosa-180717.
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Meiotic nuclear oscillations in the fission yeast Schizosaccharomyces pombe are crucial for proper chromosome pairing and recombination. We report a mechanism of these oscillations on the basis of collective behavior of dynein motors linking the cell cortex and dynamic microtubules that extend from the spindle pole body in opposite directions. By combining quantitative live cell imaging and laser ablation with a theoretical description, we show that dynein dynamically redistributes in the cell in response to load forces, resulting in more dynein attached to the leading than to the trailing microtubules. The redistribution of motors introduces an asymmetry of motor forces pulling in opposite directions, leading to the generation of oscillations. Our work provides the first direct in vivo observation of self-organized dynamic dynein distributions, which, owing to the intrinsic motor properties, generate regular large-scale movements in the cell.
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Tolic-Nørrelykke, Iva M., Sven K. Vogel, Nenad Pavin, Nicola Maghelli, and Frank Jülicher. "Self-Organization of Dynein Motors Generates Meiotic Nuclear Oscillations." PLOS, 2009. https://tud.qucosa.de/id/qucosa%3A28924.
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Meiotic nuclear oscillations in the fission yeast Schizosaccharomyces pombe are crucial for proper chromosome pairing and recombination. We report a mechanism of these oscillations on the basis of collective behavior of dynein motors linking the cell cortex and dynamic microtubules that extend from the spindle pole body in opposite directions. By combining quantitative live cell imaging and laser ablation with a theoretical description, we show that dynein dynamically redistributes in the cell in response to load forces, resulting in more dynein attached to the leading than to the trailing microtubules. The redistribution of motors introduces an asymmetry of motor forces pulling in opposite directions, leading to the generation of oscillations. Our work provides the first direct in vivo observation of self-organized dynamic dynein distributions, which, owing to the intrinsic motor properties, generate regular large-scale movements in the cell.
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Kinzel, Kathryn Whitney. "Functional analysis of inner-arm dynein knockdowns in Trypanosoma brucei /." Connect to online version, 2008. http://ada.mtholyoke.edu/setr/websrc/pdfs/www/2008/268.pdf.
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Belanger-Nelson, Erika. "The regulation of orexin receptor function by dynein light chains." Thesis, McGill University, 2011. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=96954.
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Orexins (OX-A, OX-B) are involved in the regulation of sleep, feeding and reward. The action of these peptides is governed by Orexin Receptors 1 and 2 (OX1R, OX2R). In aim to understand the mechanisms involved upon activation of these receptors, we have identified the dynein light chains 1 and 3 (Dynlt1/3) as novel partners. We hypothesize that Dynlt1/3 are important for orexin receptor intracellular regulation. After identification of a strong interaction between OX1R and Dynlt1 and the importance of the OX1R C-terminal domain residues, the functional implication of this novel interaction was assessed. Ligand-induced internalization of OX1R was not altered by modification of Dynlt1/3 expression, yet its transit in early endosomes was accelerated by Dynlt1 over-expression. In conclusion, these data suggest that Dynlt1 promotes the exit of OX1R from early endosomes following ligand-induced internalization, in association with an accelerated signal termination as measured by the phosphorylation levels of ERK1/2.<br>Les orexines (OX-A, OX-B) sont impliquées dans le sommeil, l'alimentation et la récompense. Leur action est médiée par les récepteurs aux orexines 1 et 2 (OX1R, OX2R). Pour comprendre les mécanismes découlant de leur activation, nous avons identifié les chaînes légères de la dynéine 1 et 3 (Dynlt1/3) comme partenaires de ces récepteurs. Notre hypothèse est que les Dynlt1/3 sont importantes pour réguler les récepteurs. Après avoir identifié une forte interaction entre OX1R et Dynlt1 et l'importance du domaine C-terminal d'OX1R, l'importance fonctionnelle de cette nouvelle interaction a été caractérisée. Le départ d'OX1R de la membrane n'est pas affecté par une modification de l'expression des Dynlt1/3, mais sa transition dans les endosomes a été accélérée par la surexpression de Dynlt1. En conclusion, nos données suggèrent que Dynlt1 favorise la sortie d'OX1R des endosomes après l'internalisation, accélèrant la fin de signalisation du récepteur (mesurée par les niveaux de phosphorylation d'ERK1/2).
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Redwine, William Bret. "The Structural Basis for Microtubule Binding and Release by Dynein." Thesis, Harvard University, 2012. http://dissertations.umi.com/gsas.harvard:10667.
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Eukaryotic cells face a considerable challenge organizing a complicated interior with spatial and temporal precision. They do so, in part, through the deployment of the microtubule- based molecular motors kinesin and dynein, which translate chemo-mechanical force production into the movement of diverse cargo. Many aspects of kinesin’s motility mechanism are now known in detail, whereas fundamental aspects of dynein’s motility mechanism remain unclear. An important unresolved question is how dynein couples rounds of ATP binding and hydrolysis to changes in affinity for its track, a requisite for a protein that takes steps. Here we report a sub- nanometer cryo-EM reconstruction of the high affinity state of dynein’s microtubule binding domain in complex with the microtubule. Using molecular dynamics flexible fitting, we determined a pseudoatomic model of the high affinity state. When compared to previously reported crystal structure of the free microtubule binding domain, our model revealed the conformational changes underlying changes in affinity. Surprisingly, our simulations suggested that specific residues within the microtubule binding domain may tune dynein’s affinity for the microtubule. We confirmed this observation by directly measuring dynein’s motile properties using in vitro single molecule motility assays, which demonstrated that single point mutations of these residues dramatically enhance dynein’s processivity. We then sought to understand why dynein has been selected to be a restrained motor, and found that dynein-driven nuclear oscillations in budding yeast are defective in the context of highly processive mutants. Together, these results provide a mechanism for the coupling of ATPase activity to microtubule binding and release by dynein, and the degree to which evolution has fine-tuned this mechanism. I conclude with a roadmap of future approaches to gain further insight into dynein’s motility mechanism, and describe our work developing materials and methods towards this goal.
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Trott, Laurie Elizabeth. "A physical model describing the transport mechanisms of cytoplasmic dynein." Thesis, University of Sussex, 2017. http://sro.sussex.ac.uk/id/eprint/66461/.
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Cytoplasmic dynein 1 is crucial for many cellular processes including endocytosis and cell division. Dynein malfunction can lead to neurodevelopmental and neurodegenerative disease, such as intellectual disability, Charcot-Marie-Tooth disease and spinal muscular atrophy with lower extremity predominance. We formulate, based on physical principles, a mechanical model to describe the stepping behaviour of cytoplasmic dynein walking on microtubules. Unlike previous studies on physical models of this nature, we base our formulation on the whole structure of dynein to include the temporal dynamics of the individual components such as the cargo (for example an endosome or bead), two rings of six ATPase domains associated with diverse cellular activities and the microtubule binding domains. This mathematical framework allows us to examine experimental observations across different species of dynein as well as being able to make predictions (not currently experimentally measured) on the temporal behaviour of the individual components of dynein. Initially, we examine a continuous model using plausible force functions to model the ATP force and binding affinity to the microtubule. Our results show hand-over-hand and shuffling stepping patterns in agreement with experimental observations. We are able to move from a hand-overhand to a shuffling stepping pattern by changing a single parameter. We also explore the effects of multiple motors. Next, we explore stochasticity within the model, modelling the binding of ATP as a random event. Our results reflect experimental observations that dynein walks using a predominantly shuffling stepping pattern. Furthermore, we study the effects of mutated dynein and extend the model to include variable step sizes, backward stepping and dwelling. Independent stepping is studied and the results show that coordinated stepping is needed in order to obtain experimental run lengths.
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Hanson, Benjamin S. "Mesoscale modelling of cytoplasmic dynein using fluctuating finite element analysis." Thesis, University of Leeds, 2018. http://etheses.whiterose.ac.uk/19398/.
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At the forefront of biological experimentation and simulation technology is the attempt to understand the biological mesoscale, the regime in which thermal fluctuations are still vital for function but atomic resolution may no longer be required. There is a wealth of low-resolution biomolecular structural data of macromolecules available for study, and experimental developments are allowing these biomolecules to be visualised to near-atomic resolution without the need for crystallisation. It is clear that a new form of simulation is required to take advantage of this structural data in order to better understand the dynamics of proteins at the biological mesoscale, and their relationship to dynamics at both the microscale and the macroscale. The work presented in this thesis concerns the development of Fluctuating Finite Element Analysis (FFEA), a mesoscale simulation technique that treats globular macromolecules as visco-elastic continuum objects subject to an additional thermal stress, satisfying our definition of the mesoscale. I have further developed the constitutive continuum model to better represent biological macromolecules, and designed a new solution procedure in order to both increase the computational efficiency of the algorithm and to remove superfluous dynamical information. I also introduce a completely new kinetic framework that couples to the underlying simulation protocol, enabling us to simulate discrete biological events, such as conformational changes, within a continuous dynamical simulation. I apply FFEA to the molecular motor cytoplasmic dynein, a mesoscopic system exhibiting dynamical features that are beyond the scope of standard molecular dynamics simulations, but well within the mesoscopic regime FFEA was designed for. I determine the physical parameters that an FFEA model of dynein requires for consistency with both experimental and high-resolution molecular dynamics simulations. Finally, I consider the diffusional properties of dynein with respect to its microtubule track, with the aim of understanding the potential mechanisms that enable the motor to be processive.
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Yang, Yen Ching. "The regulation of dynein function in membrane movement by NudEL." Thesis, University of Manchester, 2014. https://www.research.manchester.ac.uk/portal/en/theses/the-regulation-of-dynein-function-in-membrane-movement-by-nudel(0156ad8a-546f-4ee1-8682-d68d8c782d56).html.
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The accurate regulation of cytoplasmic dynein-1 (dynein) is very important since dynein performs multiple functions in cells. In interphase, dynein is responsible for the correct positioning of membrane organelles, such as the Golgi complex and lysosomes. Previous work suggests that dynein's accessory proteins NudEL/Nde1/LIS1¬ may be involved in regulating dynein-dependent organelle movement. This study focuses on how NudEL regulates dynein-driven membrane movement. By using various NudEL fragments, this work presents the first evidence that NudEL is involved in the regulation of dynein-driven ER movement in vitro. Moreover, the in vivo organelle positioning assays also indicate additional regulatory function of NudEL.NudEL fragment (1-157 aa) which contains both the dynein and LIS1 binding domains is sufficient to activate dynein-driven membrane movement, since NudEL1-157 aa activates ER motility in vitro and enhances clustering of the Golgi complex and lysosomes in the peri-nuclear region in vivo. On the other hand, NudEL 96-206 aa containing the LIS1 binding domain alone inhibits ER motility in vitro and causes scattering of the Golgi complex and lysosomes in vivo, indicating an inhibition of dynein-dependent organelle movement. The activation of dynein activity requires the recruitment of LIS1 to the dynein complex by NudEL, since NudEL 1-157 aa has strong binding affinity to both LIS1 and dynein whereas NudEL 96-206 aa binds to LIS1 but not dynein which suggests the sequestering of LIS1 from the dynein complex. Interestingly, NudEL 1-206 aa, which also contains both the dynein and LIS1 binding domains, causes the dispersal of the Golgi complex and lysosomes in vivo, but to a lesser extent than NudEL 96-206 aa. The putative NudEL regulatory domain (157 -242 aa, which contains various phosphorylation sits and is less conserved between NudEL and Nde1) in NudEL 1-206 aa may regulate the interaction of LIS1 and the dynein complex, since NudEL 1-206 aa has strong binding affinity to LIS1 and weak binding affinity to dynein. However, further work is needed to understand the exact mechanism by which this putative NudEL domain regulates dynein activity.
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Subramanian, Aswati. "p28 DYNEIN LIGHT CHAINS AND CILIARY MOTILITY IN Tetrahymena thermophila." Miami University / OhioLINK, 2014. http://rave.ohiolink.edu/etdc/view?acc_num=miami1389719903.
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Granger, Elizabeth. "The interplay between dynein, accessory proteins and the endocytic pathway." Thesis, University of Manchester, 2014. https://www.research.manchester.ac.uk/portal/en/theses/the-interplay-between-dynein-accessory-proteins-and-the-endocytic-pathway(c456befe-2114-41a1-90cc-e5123624a2d3).html.
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Cytoplasmic dynein 1 (dynein) is a multi-subunit complex that transports cargo along microtubules towards their minus ends. These microtubule minus ends are normally located toward the centre of the cell. Dynein is involved in transport of endocytic and autophagic membranes and is tightly regulated by interactions between dynein subunits and by dynein-accessory proteins. Dynein accessory proteins that are involved in a wide range of dynein-driven transport events include dynactin, Lis1 and the paralogues Nde1 and Ndel1. Lis1 and Nde1/Ndel1 interact with each other and are involved in the recruitment of dynein to cargo and in regulating dynein activity. Although much is known about the specific interactions of dynein and accessory proteins, the interplay between dynein and its network of regulators in living cells is not well defined. This project used RNAi to investigate how the dynein subunits light intermediate chain (LIC) and intermediate chain (IC) as well as Lis1 and Nde1/Ndel1 influence the endocytic pathway, autophagy and cargo recruitment. Biochemical analysis of bulk membrane preparations showed that IC is important for dynein and dynactin association with intracellular membranes. In addition, dynein and dynactin recruitment to Rab interacting lysosomal protein (RILP)-positive membranes was shown to require LIC and there was redundancy between LIC1 and LIC2 in this role. Lis1 was also needed for dynactin-dynein recruitment to these membranes, in a context that was Nde1/Ndel1-independent. Loss of LIC, IC, Lis1 and Nde1 had differing effects on endocytic compartment size and distribution, but they all led to mislocalisation of early endosomes and lysosomes and caused lysosomes to become enlarged. Loss of LIC led to a specific phenotype whereby cells formed lamellipodia-like regions in which early endosomes and lysosomes accumulated. Loss of Lis1 prevented traffic from the early endosome to late endosomes and caused a striking enlargement of late endosomes and lysosomes. These enlarged lysosomes were LC3-positive, indicating that they were autophagic. In addition, loss of IC and LIC also led to an increase in LC3 puncta, but the LC3 did not colocalise specifically with lysosomes. In summary, the results from this project show that i) dynactin recruitment to intracellular membranes, including RILP-postivie membranes, requires dynein, ii) Lis1 and LIC1 or LIC2 are necessary but not sufficient, individually, to recruit dynein and dynactin to RILP-positive membranes iii) LIC, IC, Lis1 and Nde1/Ndel1 influence endocytic progression in specific ways, which may in turn affect autophagic flux.
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Zou, Sirui. "The Mechanistic Basis of Dynein Microtubule Binding and Its Regulation." Thesis, Harvard University, 2015. http://nrs.harvard.edu/urn-3:HUL.InstRepos:14226071.
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Eukaryotic cells use a diverse toolbox of cytoskeletal motors to transport and position cellular materials in space and time. Two microtubule-based motors—kinesin and dynein—transport organelles, RNA and protein cargos over long-distances. While multiple kinesin motors are used for long-distance plus- end-directed transport, a single type of dynein—cytoplasmic dynein 1— performs nearly all minus-end-directed tasks. Despite cytoplasmic dynein’s role in such diverse activities, many aspects of its molecular mechanism remain poorly understood. My thesis work uses a combination of cryo-electron microscope (EM) structural biology and single-molecule approaches to provide novel insights into the mechanistic basis of how dynein interacts with its microtubule track and how microtubule binding is regulated by the ubiquitous co-factor, Lis1.
First, we solved a 9.7A structure of dynein’s microtubule binding domain bound to microtubules. This structure allowed us to identify large conformational changes that occur in dynein’s microtubule-binding domain upon track binding. We hypothesize that these conformational changes allosterically regulate the ATP hydrolysis cycle in dynein’s motor domain, which is located over 25 nm from the site of microtubule binding. Molecular dynamics simulations, followed by single-molecule assays, allowed us to identify dynamic salt bridge switches in dynein, which can tune its affinity for the microtubule. The native dynein, which has been selected for submaximal processivity, might allow a broader dynamic range for regulation.
Second, we identified how dynein is regulated by its ubiquitous co-factor, Lis1. Our three-dimensional cryo-EM structures of the dynein-Lis1 complex showed dynein’s mechanical element, the linker, is in an altered position in the presence of Lis1. Fluorescence resonance energy transfer (FRET) and single- molecule studies indicated that Lis1 binding to dynein sterically blocks the dynein linker from reaching its normal docking site, which may interrupt dynein’s mechanochemical cycle and prevent its release from microtubules.
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Dunsch, Anja Katrin. "Control of the mitotic spindle by dynein light chain 1 complexes." Thesis, University of Oxford, 2013. http://ora.ox.ac.uk/objects/uuid:b2fd5670-a035-42ca-aaef-78a30aeaa084.
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Robust control mechanisms ensure faithful inheritance of an intact genome through the processes of mitosis and cytokinesis. Different populations of the cytoplasmic dynein motor defined by specific dynein adaptor complexes are required for the formation of a stable bipolar mitotic spindle. This study analysed how different dynein subcomplexes contribute to spindle formation and orientation. Various dynein subpopulations were identified by mass spectrometry. I have shown that the dynein light chain 1 (DYNLL1) directly interacts with the kinetochore localised Astrin-Kinastrin complex as well as the spindle microtubule associated complex formed by CHICA and HMMR. I have characterised both complexes and identified unique functions in chromosome alignment and mitotic spindle orientation, respectively. I have found that Kinastrin (C15orf23) is the major Astrin-interacting protein in mitotic cells and is required for Astrin targeting to microtubule plus ends proximal to the plus tip tracking protein EB1. Fixed cell microscopy revealed that cells over-expressing or depleted of Kinastrin mislocalise Astrin. Additionally, depletion of the Astrin-Kinastrin complex delays chromosome alignment and causes the loss of normal spindle architecture and sister chromatid cohesion before anaphase onset (Dunsch et al., 2011). Using immunoprecipitation and microtubule binding assays, I have shown that CHICA and HMMR interact with one another, and target to the spindle by a microtubule-binding site in the amino-terminal region of HMMR. CHICA interacts with DYNLL1 by a series of conserved TQT motifs in the carboxy-terminal region. Depletion of DYNLL1, CHICA or HMMR causes a slight increase in mitotic index but has little effect on spindle formation or checkpoint function. Fixed and live cell microscopy reveal, however, that the asymmetric distribution of cor tical dynein is lost and the spindle in these cells fails to orient correctly in relation to the culture surface (Dunsch et al., 2012). These findings presented here suggest that the Astrin-Kinastrin complex is required for normal spindle architecture and chromosome alignment, and that per turbations of this pathway result in delayed mitosis and non-physiological separase activation, whereas HMMR and CHICA act as par t of a dynein-DYNLL1 complex with a specific function defining or controlling spindle orientation.
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Hernandez-Lopez, Rogelio Antonio. "Mechanistic Studies of the Microtubule-Based Motors Dynein and Kinesin-8." Thesis, Harvard University, 2015. http://nrs.harvard.edu/urn-3:HUL.InstRepos:17467496.
Full textAbstract:
The precise delivery and organization of intracellular factors in space and time relies on a set of molecules that move along and regulate the dynamics of cytoskeletal filaments. The two families of microtubule-based motors-- dyneins and kinesins-- power vital biological processes such as intracellular transport, chromosome segregation and more broadly cell-cell communication and cell polarization. Despite their role in such diverse activities, their molecular mechanisms remain poorly understood. Combining biochemistry, cryo-electron microscopy, molecular dynamics simulations and single molecule biophysics, we provide novel insights into the mechanistic basis of how dynein and kinesin-8 interact with microtubules (MTs) to regulate their function.
Cytoplasmic dynein is a homodimer that moves for long distances along MTs without dissociating, a property known as processivity. Its movement requires coupling cycles of ATP binding and hydrolysis to changes in affinity for its track. Intriguingly, the main site of ATP hydrolysis in the motor is separated from the microtubule binding domain (MTBD) by 25 nm. How do these sites communicate with each other? What are the changes responsible for modulating the affinity between the motor and its track during dynein’s mechanochemical cycle? Furthermore, it has been shown that dynein’s stepping behavior is highly variable. Dynein walks by taking a broad distribution of step sizes; some of its steps are sideways and some are backwards. Is dynein’s stepping behavior dictated by the motor’s ATPase activity or dynein’s affinity for MTs? To address these important questions, first, we solved a cryo-EM reconstruction of dynein’s MTBD bound to the MT. We found that upon MT binding, dynein’s MTBD undergoes a large conformational change underlying changes in its affinity for MTs. Our structural model suggested specific negatively charged residues within the MTBD that tune dynein’s affinity for MTs. We mutated these residues to alanine and show a dramatic increase in dynein’s MT binding affinity resulting in enhanced (~5-6 fold) motor processivity. These mutants provide us with a tool to explore the role of MT-binding affinity in dynein’s stepping behavior. We characterized, using single molecule experiments, the stepping pattern of the high MT binding affinity dyneins. We found that an increased MT-binding affinity reduces dynein’s stepping rate and impairs the coupling between ATPase activity and stepping. Together, our results provide a model for how dynein has evolved a finely tuned mechanism that allows its MTBD to communicate MT-binding to its motor domain. This mechanism also regulates dyneins’s affinity for the MT and motor’s processivity.
We then sought to understand the unique functional properties of kinesin-8. Unlike other kinesins that have the ability to either move along microtubules or regulate the dynamics of MT-ends, kinesin-8s can do both. Kip3, the budding yeast kinesin-8, is a highly processive motor capable of dwelling at the MT plus-end and it is a MT depolymerase. Given the highly conserved sequence and structure of kinesin’s motor domain, how is that Kip3 can perform these two distinct functions? Does Kip3 interact with the MT-lattice in the same manner than that at the MT-end? We characterized, structurally, how Kip3 binds to microtubules that mimic the MT-lattice and the MT-end. We have identified and tested specific residues within Kip3 that are responsible for Kip3’s processivity, MT-end dwelling and depolymerization activity.<br>Chemical Physics
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Colantonio, Jessica Russell. "The role of GAS11 and the dynein regulatory complex in vertebrates." Diss., Restricted to subscribing institutions, 2007. http://proquest.umi.com/pqdweb?did=1495958921&sid=1&Fmt=2&clientId=1564&RQT=309&VName=PQD.
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Derr, Nathan Dickson. "Coordination of Individual and Ensemble Cytoskeletal Motors Studied Using Tools from DNA Nanotechnology." Thesis, Harvard University, 2013. http://dissertations.umi.com/gsas.harvard:10889.
Full textAbstract:
The cytoskeletal molecular motors kinesin-1 and cytoplasmic dynein drive many diverse functions within eukaryotic cells. They are responsible for numerous spatially and temporally dependent intracellular processes crucial for cellular activity, including cytokinesis, maintenance of sub-cellular organization and the transport of myriad cargos along microtubule tracks. Cytoplasmic dynein and kinesin-1 are processive, but opposite polarity, homodimeric motors; they each can take hundreds of thousands of consecutive steps, but do so in opposite directions along their microtubule tracks. These steps are fueled by the binding and hydrolysis of ATP within the homodimer's two identical protomers. Individual motors achieve their processivity by maintaining asynchrony between the stepping cycles of each protomer, insuring that at least one protomer always maintains contact with the track. How dynein coordinates the asynchronous stepping activity of its protomers is unknown. We developed a versatile method for assembling Saccharomyces cerevisiae dynein heterodimers, using complementary DNA oligonucleotides covalently linked to dynein monomers labeled with different organic fluorophores. Using two-color, single-molecule microscopy and high-precision, two-dimensional tracking, we found that dynein has a highly variable stepping pattern that is distinct from all other processive cytoskeletal motors, which use "hand-over-hand" mechanisms. Uniquely, dynein stepping is stochastic when its two motor domains are close together. However, coordination emerges as the distance between motor domains increases, implying that a tension-based mechanism governs these steps. Many cellular cargos demonstrate bidirectional movement due to the presence of ensembles of both cytoplasmic dynein and kinesin-1. To investigate the mechanisms that coordinate the interactions between motors within an ensemble, we constructed programmable synthetic cargos using three-dimensional DNA origami. This system enables varying numbers of DNA oligonucleotide-linked motors to be attached to the synthetic cargo, allowing for control of motor type, number, spacing, and orientation in vitro. In ensembles of one to seven identical- polarity motors, we found that motor number had minimal effect on directional velocity, whereas ensembles of opposite-polarity motors engaged in a tug-of-war resolvable by disengaging one motor species.
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Tharia, Hazel Ann. "Biochemical characterization of 14S dynein isolated from the cilia of Tetrahymena thermophila." Thesis, University of Leicester, 1995. http://hdl.handle.net/2381/35168.
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Dynein is the ATPase responsible for cilia and flagella motility in eukaryotic organisms. At least two ATPases have been identified in Tetrahymena thermophila and are termed 22S and 14S on account of their differing sedimentation coefficients. 14S dynein was fractionated, using anion-exchange Fast Protein Liquid Chromatography, into four fractions (designated 1-4). Electron microscopy analysis revealed that the four fractions were structurally distinct. Fraction 1 comprised two globular heads interconnected via two stems; fraction 2 consisted of at least two clearly different globular structures; fraction 3 was a single globular head; and fraction 4 comprised three globular heads interconnected via three stems to a base. Further structural characterisation involved the use of hydrodynamic techniques in which the mass and sedimentation coefficient were determined for each fraction. In the presence of 40mM NaCl, fraction 1 had a mass of 654kDa and a sedimentation coefficient of 20.IS. Fraction 2 had a variable mass due to aggregation (616-966kDa), and a sedimentation coefficient of 16.6S, whereas fractions 3 and 4 had variable sedimentation coefficients but were of mass 701kDa and 527kDa respectively. These parameters were then utilised, in conjunction with electron microscopy data, in the construction of low-resolution bead models to represent the fractions. The four fractions had a unique polypeptide composition as shown by SDS polyacrylamide gel electrophoresis. At least four unique heavy chains were identified immunologically; two associated with fraction 1, one associated with fraction 2, and one associated with fractions 3 and 4. Fractions 1 and 2 were readily distinguished from fractions 3 and 4 using the technique of vanadate-dependent photolysis. The requirement for ATP in the photolysis reaction indicated differences in the structure of the heavy chain. The fractions were also distinguished with respect to the number and location of V2 sites, and the specific ATPase activity. These latter studies also showed that, for all four fractions, an increase in ionic strength resulted in a decrease in ATPase activity. This was coincident with an observed change in the structure and/or conformation of the fractions as determined by hydrodynamic analysis under equivalent conditions.
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Kuta, A. "Investigation of subunits of the cytoplasmic dynein complex using novel mouse models." Thesis, University College London (University of London), 2011. http://discovery.ucl.ac.uk/1324544/.
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Cytoplasmic dynein is a multisubunit complex responsible for the transport of cellular components from the cell periphery towards the nucleus. The role of the dynein complex in vesicle trafficking, organelle positioning and chromosome segregation during mitosis has been extensively studied but still little is known of specific roles of distinct subunits of the complex. Cytoplasmic dynein is a dimeric complex consisting of heavy chains, intermediate chains, light intermediate chains and three light chains. In order to investigate the roles of the cytoplasmic dynein subunits, two mouse lines with chemically generated single point mutations in the intermediate chain 1 and 2 genes (Dync1i1, Dync1i2) were subjected to a behavioural analysis. The mouse line carrying a mutation in the intermediate chain 2 showed working memory deficits which suggested impairment in hippocampal functions. In order to examine the effects of mutation at the cellular level primary mouse embryonic fibroblasts (MEFs) lines were derived from embryos carrying mutations in the intermediate chains and used as a model system. Cell functions, such as trafficking of epidermal growth factor (EGF) positive endosomes, Golgi assembly were examined. Furthermore, biochemical analyses were performed focused on the expression of dynein subunits and their assembly in the functional complex. Alternative splicing is known to produce multiple isoforms of the intermediate chains. The analysis of various splice variants of these genes in a panel of mouse tissues resulted in detecting new isoforms which were compared with bioinformatics data available for human and rat thus establishing the splicing pattern of the mouse intermediate chains. Legs at odd angles (Dync1h1Loa) is another mutant mouse line carrying a point mutation in the dynein heavy chain which results in neurological defects. Here the effects of the Loa mutation in the trafficking of membranous organelles were investigated by an infection of cultured MEFs with Salmonella enterica serovar Typhimurium. Furthermore, upon the induction of a cellular stress the wildtype and the Loa homozygous cells showed significant differences in stress granule assembly suggesting the impairment in the stress signaling.
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Tai, Chin-Yin. "Roles of Lissencephaly Gene, LIS1, in Regulating Cytoplasmic Dynein Functions: a Dissertation." eScholarship@UMMS, 2002. https://escholarship.umassmed.edu/gsbs_diss/31.
Full textAbstract:
Spontaneous mutations in the human LIS1 gene are responsible for Type I lissencephaly ("smooth brain"). The distribution of neurons within the cerebral cortex of lissencephalic children appears randomized, probably owing to a defect in neuronal migration during early development.
LIS1 has been implicated in the dynein pathway by genetic analyses in fungi. We previously reported that the vertebrate LIS1 co-localized with dynein at prometaphase kinetochores, and interference with LIS1 function at kinetochore caused misalignment of chromosomes onto the metaphase plate. This leads to a hypothesis that LIS1 might regulate kinetochore protein targeting. In order to test this hypothesis, I created dominant inhibitory constructs of LIS1. After removal of the endogenous LIS1 from the kinetochore by overexpression of the N-terminal self-association domain of LIS1, dynein and dynactin remained at the kinetochores. This result indicated that LIS1 is not required for dynein to localize at the kinetochore. Next, CLIP-170 was displaced from the kinetochores in the LIS1 full-length and the C-terminal WD-repeat overexpressers, suggesting a role for LIS1 in targeting CLIP-170 onto kinetochores.
LIS1 was co-immunoprecipitated with dynein and dynactin. Its association with kinetochores was mediated by dynein and dynactin, suggesting LIS1 might interact directly with subunits of dynein and/or dynactin complexes. I found that LIS1 interacted with the heavy and intermediate chains (HC and IC) of dynein complex, and the dynamitin subunit of dynactin complex. In addition to kinetochore targeting, the LIS1 C-terminal WD-repeat domain was responsible for interactions with dynein and dynactin. Interestingly, LIS 1 interacted with two distinct sites on HC: one in the stem region containing the subunit-binding domain, and the other in the first AAA motif of the motor domain, which is indispensable for the ATPase function of the motor protein. This LIS1-dynein motor domain interaction suggests a role for LIS1 in regulating dynein motor activity. To test this hypothesis, changes of dynein ATPase activity was measured in the presence of LIS1 protein. The ATPase activity of dynein was stimulated by the addition of a recombinant LIS1 protein.
Besides kinetochores, others and we have found LIS1 also localized at microtubule plus ends. LIS1 may mediate dynein and dynactin mitotic functions at these ends by interacting with astral microtubules at cortex, and associating with the spindle microtubules at kinetochores. Overexpression of LIS1 displaced dynein and dynactin from the microtubule plus ends, and mitotic progression was severely perturbed in LIS1 overexpressers. These results suggested that the role for LIS1 at microtubule plus ends is to regulate dynein and dynactin interactions with various subcellular structures.
Results from my thesis research clearly favored the conclusion that LIS1 activates dynein ATPase activity through its interaction with the motor domain, and this activation is important to establish an interaction between dynein and microtubule plus ends during mitosis. I believe that my thesis work not only has provided ample implications regarding dynein dysfunction in disease formation, but also has laid a significant groundwork for more future studies in regulations of the increasing array of dynein functions.
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Faulkner, Nicole E. "Mitotic Roles for Cytoplasmic Dynein and Implications for Brain Developmental Disease: a Dissertation." eScholarship@UMMS, 2003. http://escholarship.umassmed.edu/gsbs_diss/182.
Full textAbstract:
Cytoplasmic dynein has been implicated in a wide range of functions. Originally characterized as being responsible for retrograde axonal transport, its has also been shown to traffic vesicular organelles (Golgi, endosome and lysosome distribution), transport viral particles to the nucleus, and participate in microtubule organization. During mitosis, cytoplasmic dynein is thought to function in spindle pole focusing and prometaphase kinetochore capture. This thesis explores the mitotic roles of cytoplasmic dynein.
The first chapter addresses the role of cytoplasmic dynein in kinetochore activity. Using immunofluoresence microscopy, a number of motors and related proteins were observed at the primary, but not secondary, constrictions of prometaphase multicentric chromosomes. The proteins assessed included the cytoplasmic dynein intermediate chains, three components of the dynactin complex (dynamitin, Arp1, and p150Glued), the kinesin related proteins CENP-E and MCAK, and the proposed structural and checkpoint proteins CENP-F, HZW10, and MAD2. The differential localization of these proteins offered new insight into the assembly and composition of both active and inactive centromeres, and provided a molecular basis for the apparent inactivity of the latter during chromosome segregation.
The second chapter characterizes LIS1, a protein that is defective in the developmental brain disease type1 lissencephaly. Mutations in the LIS1 gene cause gross histological disorganization of the developing cerebral cortex resulting in a nearly smooth brain surface. Because genetic evidence from lower eukaryotes suggested that LIS1 acted within the cytoplasmic dynein pathway, it was of interest to analyze LIS1 in terms of cytoplasmic dynein function. LIS1 was found to coimmunoprecipitate with cytoplasmic dynein and its companion complex dynactin. During mitosis LIS1 localized to the prometaphase kinetochore, spindle microtubules and the cell cortex, known sites for cytoplasmic dynein binding. Interference with endogenous LIS1 in cultured mammalian cells displaced dynein localization and disrupted mitotic progression. LIS1 was concluded to participate in cytoplasmic dynein functions, but only during mitosis.
Data presented in the final chapter further characterizes LIS1's interactions with microtubules, cytoplasmic dynein and the mammalian homologue of NUDC. LIS1 was not found to co-fractionate with microtubules, nor did overexpression of LIS1 cause visible effects on microtubule organization or dynamics. GFP-LIS1 was shown to ride along the plus ends of growing microtubules. Though LIS1 was not found to have a direct effect on microtubules, it may regulate dynein's microtubule binding activity. LIS1 was found to co-immunoprecipitate with a co-overexpressed cytoplasmic dynein subunit substantiating the existence of a dynein LIS1 supercomplex. Furthermore, association of these proteins increased markedly in mitotically blocked samples. LIS1's regulation of cytoplasmic dynein may change the capacity of the motor to efficiently manipulate its mitotic cargoes, dramatically effecting the timing of cell division. This disruption has implications for the fundamental role of cytoplasmic dynein during early embryonic development.
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Salter, Hannah Kate. "Molecular mechanisms underpinning Bicaudal-D's role in linking cargoes to the dynein motor." Thesis, University of Cambridge, 2013. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.648370.
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Faulkner, Nicole E. "Mitotic Roles for Cytoplasmic Dynein and Implications for Brain Developmental Disease: a Dissertation." eScholarship@UMMS, 2001. https://escholarship.umassmed.edu/gsbs_diss/182.
Full textAbstract:
Cytoplasmic dynein has been implicated in a wide range of functions. Originally characterized as being responsible for retrograde axonal transport, its has also been shown to traffic vesicular organelles (Golgi, endosome and lysosome distribution), transport viral particles to the nucleus, and participate in microtubule organization. During mitosis, cytoplasmic dynein is thought to function in spindle pole focusing and prometaphase kinetochore capture. This thesis explores the mitotic roles of cytoplasmic dynein.
The first chapter addresses the role of cytoplasmic dynein in kinetochore activity. Using immunofluoresence microscopy, a number of motors and related proteins were observed at the primary, but not secondary, constrictions of prometaphase multicentric chromosomes. The proteins assessed included the cytoplasmic dynein intermediate chains, three components of the dynactin complex (dynamitin, Arp1, and p150Glued), the kinesin related proteins CENP-E and MCAK, and the proposed structural and checkpoint proteins CENP-F, HZW10, and MAD2. The differential localization of these proteins offered new insight into the assembly and composition of both active and inactive centromeres, and provided a molecular basis for the apparent inactivity of the latter during chromosome segregation.
The second chapter characterizes LIS1, a protein that is defective in the developmental brain disease type1 lissencephaly. Mutations in the LIS1 gene cause gross histological disorganization of the developing cerebral cortex resulting in a nearly smooth brain surface. Because genetic evidence from lower eukaryotes suggested that LIS1 acted within the cytoplasmic dynein pathway, it was of interest to analyze LIS1 in terms of cytoplasmic dynein function. LIS1 was found to coimmunoprecipitate with cytoplasmic dynein and its companion complex dynactin. During mitosis LIS1 localized to the prometaphase kinetochore, spindle microtubules and the cell cortex, known sites for cytoplasmic dynein binding. Interference with endogenous LIS1 in cultured mammalian cells displaced dynein localization and disrupted mitotic progression. LIS1 was concluded to participate in cytoplasmic dynein functions, but only during mitosis.
Data presented in the final chapter further characterizes LIS1's interactions with microtubules, cytoplasmic dynein and the mammalian homologue of NUDC. LIS1 was not found to co-fractionate with microtubules, nor did overexpression of LIS1 cause visible effects on microtubule organization or dynamics. GFP-LIS1 was shown to ride along the plus ends of growing microtubules. Though LIS1 was not found to have a direct effect on microtubules, it may regulate dynein's microtubule binding activity. LIS1 was found to co-immunoprecipitate with a co-overexpressed cytoplasmic dynein subunit substantiating the existence of a dynein LIS1 supercomplex. Furthermore, association of these proteins increased markedly in mitotically blocked samples. LIS1's regulation of cytoplasmic dynein may change the capacity of the motor to efficiently manipulate its mitotic cargoes, dramatically effecting the timing of cell division. This disruption has implications for the fundamental role of cytoplasmic dynein during early embryonic development.
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Sladewski, Thomas Edward. "Molecular Mechanisms Of Mrna Transport By A Class V Myosin And Cytoplasmic Dynein." ScholarWorks @ UVM, 2017. http://scholarworks.uvm.edu/graddis/689.
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mRNA localization ensures correct spatial and temporal control of protein synthesis in the cell. Using a single molecule in vitro approach, we provide insight into the mechanisms by which localizing mRNAs are carried by molecular motors on cytoskeletal tracks to their destination.
Budding yeast serves as a model system for studying the mechanisms of mRNA transport because localizing mRNAs are moved on actin tracks in the cell by a single class V myosin motor, Myo4p. Molecular motors that specialize in cargo transport are generally double-headed so that they can "walk" for many microns without dissociating, a feature known as processivity. Thus, is was surprising when Myo4p purified from yeast was shown by in vitro assays to be non-processive. The reason for its inability to move processively is that the Myo4p heavy chain does not dimerize with itself, but instead binds tightly to the adapter protein She3p to form a single-headed motor complex. The mRNA-binding adapter protein She2p links Myo4p to mRNA cargo by binding She3p.
To understand the molecular mechanisms of mRNA transport in budding yeast, we fully reconstituted a messenger ribonucleoprotein (mRNP) complex from purified proteins and a localizing mRNA (ASH1) found in budding yeast. Using single molecule in vitro assays, we find that She2p recruits two Myo4p-She3p complexes, forming a processive double-headed motor complex that is stabilized by mRNA at physiological ionic strength. Thus, only in the presence of mRNA is Myo4p capable of continuous mRNA transport, an elegant mechanism that ensures that only cargo bound motors are motile.
We next wished to understand if the principles of mRNA transport in budding yeast are conserved in higher eukaryotes. In Drosophila, mRNA is transported on microtubule tracks by cytoplasmic dynein, and the adapters that link the motor to localizing transcripts are well-defined. The adapter protein bicaudal D (BicD) coordinates dynein motor activity with mRNA cargo binding. The N-terminus of BicD binds dynein, and the C-terminus interacts with the mRNA-binding protein Egalitarian. Unlike mammalian dynein alone, it was recently shown that an N-terminal fragment of BicD (BicD2CC1), in combination with a large 1.2MDa multi-subunit accessory complex called dynactin, forms a complex (DDBCC1) that is activated for long processive runs. But unlike the constitutively activated BicD2CC1 fragment, the full-length BicD molecule fails to recruit dynein-dynactin because it is auto-inhibited by interactions between the N-terminal dynein binding domain and the C-terminal cargo binding domain. To understand how dynein is activated by native cargo and full-length adapters, we fully reconstituted a mRNP complex in vitro from tissue-purified dynein and dynactin, expressed full-length adapters BicD and Egalitarian, and a synthesized localizing mRNA found in Drosophila. We find that only mRNA-bound Egalitarian is capable of relieving BicD auto-inhibition for the recruitment of dynein-dynactin, and activation of mRNA transport in vitro. Thus, the presence of an mRNA cargo for activation of motor complexes is a conserved mechanism in both budding yeast and higher eukaryotes to ensure that motor activity is tightly coupled to cargo selection.
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Tynan, Sharon H. "The Role of the Light Intermediate Chains in Cytoplasmic Dynein Function: a Dissertation." eScholarship@UMMS, 2000. https://escholarship.umassmed.edu/gsbs_diss/85.
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Cytoplasmic dynein is a multisubunit complex involved in retrograde transport of cellular components along microtubules. The heavy chains (HC) are very large catalytic subunits which possess microtubule binding ability. The intermediate chains (IC) are responsible for targeting dynein to its appropriate cargo by interacting with the dynactin complex. The light intermediate chains (LIC) are previously unexplored subunits that have been proposed to modulate dynein activity by regulating the motor or the IC-dynactin interaction. The light chains (LC) are a newly identified class of subunit which are also thought to have regulatory functions.
In the first part of this work, I analyzed the relationship between the four SDS-PAGE gel bands that comprise the light intermediate chains. 1- and 2-D electrophoresis before and after alkaline phosphatase treatment revealed that the four bands are derived from two different polypeptides, each of which is phosphorylated. Peptide microsequencing of these subunits yielded sequences that indicated similarity between them. cDNA cloning of the rat LICs revealed the presence of a conserved P-loop sequence and a very high degree of homology between the two different rat LICs and among LICs from different species.
The second series of experiments was designed to analyze the association of pericentrin with cytoplasmic dynein. First, various dynein and dynactin subunits were co-associate with pericentrin in these experiments. Co-precipitation from 35S labeled cell extracts revealed a direct interaction between LIC and pericentrin. Comparison of pericentrin binding by LICl and LIC2 showed that only LICl was able to bind. Further investigation of the relationship between LICl and LIC2 demonstrated that each LIC will self-associate, but they will not form heterooligomers. Additionally, using co-overexpression and immunoprecipitation of LICl, LIC2, and HC, I have shown that binding of the two LICs to HC is mutually exclusive.
Finally, I investigated the relationships between dynein HC, IC, and LIC by examining the interactions among the subunits. IC and LIC were both found to bind to the HC, but not to each other. Despite the lack of interaction between IC and LIC, they are, in fact, present in the same dynein complexes and they have partially overlapping binding sites within the N-terminal sequence of the HC. The HC dimerization site was determined to extend through a large portion of the N-terminus, and it includes both the IC and LIC binding sites, although these subunits are not required for dimerization.
Together these studies implicate the light intermediate chains in dynein targeting. Targeting of dynein to its cargo has been thought to be performed by the dynactin complex, and for one particular cargo, the kinetochore, there is considerable evidence to support this model. The results presented here suggest that the light intermediate chains appear to function in a separate, non-dynactin-based targeting mechanism.
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Vernon, Geraint Grrffydd. "Mechanical activity and its propagation along the flagellar axoneme : studies using caged ATP." Thesis, University of Bristol, 1996. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.319140.
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Mali, Girish Ram. "Multisystem functional characterisation of motile ciliopathy genes HEATR2 and ZMYND10." Thesis, University of Edinburgh, 2015. http://hdl.handle.net/1842/21683.
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Cilia are polarized extensions of the cells microtubule-based cytoskeleton dedicated to sensory, signaling and motility-related functions. In mammals, there are two main types of cilia, immotile and motile, where motile cilia generate/modulate fluid flow at the embryonic node, in respiratory airways, cerebral ventricles and the oviduct in addition to sperm propulsion via the flagellum. Defects in cilia motility cause a rare genetic disorder called Primary Ciliary Dyskinesia (PCD). In this thesis, I present functional and molecular characterisation of two PCD causing genes HEATR2 and ZMYND10. Core cilia genes are transcriptionally activated by members of the winged-helix transcription factors of the RFX family. The forkhead transcription factor FOXJ1, additionally activates motility genes such as the ones encoding components of axonemal dynein motors which transfer the chemical energy released from ATP hydrolysis to kinetic motion necessary for ciliary motility. I present data in this thesis which show that Heatr2 and Zmynd10 are both targets of the RFX3-FOXJ1 transcriptional module which co-operatively switches on genes required to make motile cilia Mutations in both HEATR2 and ZMYND10 cause the same subtype of PCD (loss of inner and outer arm dyneins in cilia). I characterise a human PCD causing mutation in HEATR2 in this thesis. Additionally, using genetic null mouse models generated using the CRISPR technology, I describe the phenotypic effects of complete loss of Zmynd10 in mice. Zmynd10 mutant mice display characteristic PCD-like features. Adding to my functional studies, I present proteomic data to propose mechanisms by which HEATR2 and ZMYND10 proteins control cilia motility. Mass spectrometry and protein interaction studies support distinct roles for HEATR2 and ZMYND10 in intracellular transport and pre-assembly of axonemal dynein motors. The multisystem approaches described in this thesis to characterise the roles of HEATR2 and ZMYND10 highlight the molecular complexity underlying the assembly and delivery of axonemal dyneins to motile cilia and provide novel functional and molecular insights into the pathophysiology of PCD.
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Meaders, Johnathan Lee. "Growth, Morphology, and Positioning of Microtubule Asters in Large Zygotes:." Thesis, Boston College, 2020. http://hdl.handle.net/2345/bc-ir:109018.
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Thesis advisor: David R. Burgess<br>Microtubule (MT) asters are radial arrays of MTs nucleated from a microtubule organizingcenter (MTOC) such as the centrosome. Within many cell types, which display highly diverse size and shape, MT asters orchestrate spatial positioning of organelles to ensure proper cellular function throughout the cell cycle and development. Therefore, asters have adopted a wide variety of sizes and morphologies, which are directly affects how they migrate and position within the cell. In large cells, for example during embryonic development, asters growth to sizes on the scales of hundreds of microns to millimeters. Due to this relatively enormous size scale, it is widely accepted that MT asters migrate primarily through pulling mechanisms driven by dynein located in the cytoplasm and/or the cell cortex. Moreover, prior to this dissertation, significant contributions from pushing forces as a result of aster growth and expansion against the cell cortex have not been detected in large cells. Here we have reinvestigated sperm aster growth, morphology, and positioning of MT asters using the large interphase sperm aster of the sea urchin zygote, which is historically a powerful system due to long range migration of the sperm aster to the geometric cell center following fertilization. First, through live-cell quantification of sperm aster growth and geometry, chemical manipulation of aster geometry, inhibition of dynein, and targeted chemical ablation, we show that the sperm aster migrates to the zygote center predominantly through a pushing-based mechanism that appears to largely independent of proposed pulling models. Second, we investigate the fundamental principles for how sperm aster size is determined during growth and centration. By physically manipulating egg size, we obtain samples of eggs displaying a wide range of diameters, all of which are at identical developmental stages. Using live-cell and fluorescence microscopy, we find strong preliminary evidence that aster diameter and migration rates show a direct, linear scaling to cell diameter. Finally, we hypothesize that a collective growth model for aster growth, or centrosome independent MT nucleation, may explain how the sperm aster of large sea urchin zygotes overcomes the proposed physical limitations of a pushing mechanism during large aster positioning. By applying two methods of super resolution microscopy, we find support for this collective growth model in the form of MT branching. Together, we present a model in which growth of astral MTs, potentially through a collective growth model, pushes the sperm aster to the zygote center<br>Thesis (PhD) — Boston College, 2020<br>Submitted to: Boston College. Graduate School of Arts and Sciences<br>Discipline: Biology
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Ahrens, Nikolai. "Evidence for and characterization of cytoplasmic dynein and kinesin in renal medulla and cortex." Thesis, University of Oxford, 1994. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.386898.
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Nandini, Swaran. "Characterization of Motility Alterations Caused By the Impairment of Dynein/Dynactin Motor Protein Complex." Master's thesis, University of Central Florida, 2013. http://digital.library.ucf.edu/cdm/ref/collection/ETD/id/5824.
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Transport of intracellular cargo is an important and dynamic process required for cell maintenance and survival. Dynein is the motor protein that carries organelles and vesicles from the cell periphery to the cell center along the microtubule network. Dynactin is a protein that activates dynein for this transport process. Together, dynein and dynactin forms a motor protein complex that is essential for transport processes in all the vertebrate cells. Using fluorescent microscope based live cell imaging techniques and kymograph analyses, I studied dynein/dynactin disruptions on the intracellular transport in two different cell systems. In one set of experiments, effects of dynein heavy chain (DHC) mutations on the vesicular motility were characterized in the fungus model system Neurospora crassa. I found that many DHC mutations had a severe transport defect, while one mutation linked to neurodegeneration in mice had a subtle effect on intracellular transport of vesicles. In a different set of experiments in mammalian tissue culture CAD cells, I studied the effects of dynactin knockdown and dynein inhibition on mitochondrial motility. My results indicated that reductions in dynactin levels decrease the average number of mitochondrial movements and surprisingly, increase the mitochondrial run lengths. Also, I determined that the dynein inhibitory drug Ciliobrevin causes changes in mitochondrial morphology and decreases the number of mitochondrial movements inside cells. Overall, my research shows that distinct disruptions in the dynein and dynactin motor complex alters intracellular motility, but in different ways. So far, my studies have set the ground work for future experiments to analyze the motility mechanism of motor proteins having mutations that lead to neurodegenerative disorders.<br>M.S.<br>Masters<br>Molecular Biology and Microbiology<br>Medicine<br>Biotechnology
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Alami, Nael H. "The Role of Myosin Va and the Dynein/Dynactin Complex in Neurofilament Axonal Transport." The Ohio State University, 2009. http://rave.ohiolink.edu/etdc/view?acc_num=osu1259091406.
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Wadzinski, Thomas. "Light Intermediate Chain 1: a Multifunctional Cargo Binder for Cytoplasmic Dynein 1: a Dissertation." eScholarship@UMMS, 2006. https://escholarship.umassmed.edu/gsbs_diss/167.
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Cells as dynamic, interactive, and self contained units of life have a need for molecular motors that can create physical forces to move cargoes within the cell. Cytoplasmic dynein 1 is one such molecular motor that has many functions in the cell. The number and variety of functions that involve cytoplasmic dynein 1 suggest that there are a number of different binding sites on dynein for different proteins. Cytoplasmic dynein 1 is a multiprotein complex made up of six different subunit families. The many different combinations of subunits that could be used to make up a cytoplasmic dynein 1 holocomplex provides the variety of different binding sites for cargoes that can be individually regulated.
The following chapters flush out how light intermediate chain 1 (LIC1), a subunit of cytoplasmic dynein 1, is involved with multiple dynein functions involving the binding of different cargoes to the cytoplasmic dynein 1 holocomplex, and how the binding of these cargoes can be regulated. First, LIC1 is found to be involved in the spindle assembly checkpoint. LIC1 appears to facilitate the removal of Mad1-Mad2, a complex important in producing a wait anaphase signal, from kinetochores. Second, the involvement of LIC1 in the spindle assembly checkpoint requires the phosphorylation of LIC1 at a putative Cdk1 phosphorylation site. This site is located in a domain of LIC1 that binds various proteins suggesting that this phosphorylation could also regulate these interactions. Third, LIC1 is involved in the centrosomal assembly of pericentrin, an important centrosomal protein. From the data presented herein, LIC1 is shaping up as a multifunctional cargo binder for cytoplasmic dynein 1 that requires regulation of its various cargoes.
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Babic, Milos. "Molecular Mechanisms of Mitochondrial Transport in Neurons." Diss., The University of Arizona, 2015. http://hdl.handle.net/10150/556433.
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Dynamic mitochondrial transport into axons and dendrites of neuronal cells is critical for sustaining neuronal excitability, synaptic transmission, and cell survival. Failure of mitochondrial transport is the direct cause of some neurodegenerative diseases, and an aggravating factor for many others. Mitochondrial transport regulation involves many proteins; factoring prominently among them are the atypical mitochondrial GTPase Miro and the Milton/TRAK adaptor proteins, which link microtubule (MT) motors to mitochondria. Motors of the kinesin family mediate mitochondrial transport towards the plus ends of microtubules, while motors of the dynein family mediate mitochondrial transport towards the minus ends. Selective use of these motors determines the ultimate subcellular distribution of mitochondria, but the underlying control mechanisms remain poorly understood. Drosophila Miro (dMiro) is required for kinesin-driven transport of mitochondria, but its role in dynein-driven transport remains controversial. In Chapter 2 of this study, I show that dMiro is also required for the dynein-driven transport of mitochondria. In addition, we used the loss-of-function mutations dMiroT25N and dMiroT460N to analyze the function of dMiro's N- and C-terminal GTPase domains, respectively. We show that dMiroT25N causes lethality and impairs mitochondrial distribution and transport in a manner indistinguishable from dmiro null mutants. Our analysis suggests that both kinesin- and dynein-driven mitochondrial transport require the activity of Miro's N-terminal GTPase domain, which likely controls the transition from a stationary to a motile state irrespective of the transport direction. dMiroT460N reduced only dynein motility during retrograde axonal transport but had no effect on distribution of mitochondria in neurons, indicating that the C-terminal GTPase domain of Miro most likely has only a small modulatory role on transport. Furthermore, we show that commonly used substitutions in Miro's GTPase domains, based on the constitutively active Ras-G12V mutation, appear to cause neomorphic phenotypic effects which are probably unrelated to the normal function of the protein. In mammalian neurons, kinesin and dynein motors are linked to mitochondria via a Miro complex with the adapter proteins TRAK1 and TRAK2, respectively. Differential linkage of TRAK-motor complexes provides a mechanism for determining the direction of transport and controlling mitochondrial distributions within the cell. Drosophila has only one TRAK gene homolog, Milton, which expresses several protein isoform. Milton has been previously been shown to facilitate mitochondrial transport by binding to kinesin and dMiro, a role analogous to TRAK1. However, the question whether Milton might be able mediate dynein-based transport in a manner similar to TRAK2 has remained unknown. In Chapter 3 of this study, I show that protein isoforms A and B of Milton, generated through alternative mRNA splicing, facilitate differential motor activities analogous to mammalian TRAKs. Specifically, overexpression (OE) of Milton-A caused a mitochondrial redistribution and accumulation at axon terminals, which requires kinesin-driven MT plus end directed transport; while OE of Milton-B caused a redistribution of axonal mitochondria into the soma, which requires dynein-driven MT minus end directed transport. I further show that Milton-motor complex binding to mitochondria requires Miro exclusively, and that transport with either of the motor complexes absolutely requires the activity of Miro's N-terminal GTPase domain. Together, these results suggest that Miro controls the transition of mitochondria from a stationary to a motile phase. Thereafter the direction of transport is likely determined by an alternative binding of opposing Milton/TRAK-motor complexes to Miro, a process which appears to be regulated by a Miro-independent mechanism.
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Straube, Anne. "Struktur und zelluläre Funktionen von cytoplasmatischem Dynein und Organisation des Mikrotubuli-Cytoskeletts in Ustilago maydis." [S.l.] : [s.n.], 2003. http://archiv.ub.uni-marburg.de/diss/z2003/0175.
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Bähr, Jennifer. "Einfluss der Dynein-Mutation auf zelluläre Mediatoren der neuronalen Signaltransduktion im Cra1-Mausmodell der Motoneurondegeneration." Diss., lmu, 2009. http://nbn-resolving.de/urn:nbn:de:bvb:19-106332.
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Ban, Kenneth. "Localized regulation of the anaphase-promoting complex by the Emi1/NuMA/Dynein-dynactin (END) network /." May be available electronically:, 2007. http://proquest.umi.com/login?COPT=REJTPTU1MTUmSU5UPTAmVkVSPTI=&clientId=12498.
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Osseman, Quentin. "Analyse du transport intracytoplasmique de la capside du virus de l’hépatite B : analyse des interactions entre les capsides du VHB et les chaînes du complexe de la dynéine." Thesis, Bordeaux, 2014. http://www.theses.fr/2014BORD0304/document.
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Le virus de l’hépatite B (VHB) utilise la machinerie transcriptionnelle nucléaire pour sa réplication. Le génome viral est transporté de la périphérie cellulaire à l’enveloppe nucléaire. Généralement, ce transport intracytoplasmique rétrograde est facilité par le réseau de Mt via l’utilisation du complexe moteur de la dynéine. Nous avons montré que le transport des capsides du VHB dépend des Mt, ce qui permet l’adressage des capsides aux complexes du pore nucléaire (NPC) ; lequel est requis pour l’étape de libération du génome de la capside dans le noyau.Dans cette étude, nous avons utilisé des capsides provenant de virus récupérés dans du surnageant de HepG2.2.15, qui contiennent le génome mature partiellement double brin (capsides matures), et des capsides exprimées chez E.coli. Ces dernières sont utilisées telles quelles, capsides E.coli contenant de l’ARN, ou bien sont utilisées pour préparer des capsides vides. Après microinjection dans des ovocytes de Xenopus laevis, nous avons observé que les capsides vides et les capsides matures sont transloquées aux NPC avec une cinétique similaire. Les capsides contenant de l’ARN ne sont pas identifiées aux NPCs ce qui implique que le transport des deux autres types de capsides est actif. Cela a été confirmé par la pré-injection d’anticorps anti tubuline qui neutralisent le transport assuré par les Mt.L’attachement spécifique des capsides matures et vides aux Mt a été confirmé en utilisant des Mt polymérisés in vitro, nous avons montré que cette interaction nécessitait des protéines cytosoliques. En utilisant des expériences de coïmmunoprécipitation et de cosédimentation nous avons identifié une chaîne légère de la dynéine (DynLL1 membre de la famille Lc8) comme partenaire des capsides. Dans les expériences de microinjection, la comicroinjection d’un excès de DynLL1 avec les capsides inhibe leur transport vers les NPCs, indiquant que DynLL1 est impliquée dans le transport actif des capsides.DynLL2 qui n’interagit pas avec les capsides diffère de DynLL1 de seulement six acides aminés. Par mutagénèse dirigée de DynLL1, nous avons montré l’implication de deux acides aminés dans l’interaction directe avec les capsides. Ces deux acides aminés sont présents à la surface du dimère de DynLL1 et absents dans le sillon résultant de la dimérisation de DynLL1, sillon impliqué dans l’interaction avec la DynIC. Nous avons partiellement reconstitué le complexe DynIC, DynLL1 et capsides vides qui doit en partie refléter la situation in vivo<br>Hepatitis B virus (HBV) needs the nuclear transcription machinery for replication. The virus thus depends on the transport of its genome from the cell periphery to the nuclear envelope. In general this retrograde intracytoplasmic trafficking is facilitated along Mt (MT) using motor protein complexes of the dynein family. As we showed earlier HBV capsid transport also depends upon intact MT in order to allow their arrival at the nuclear pores, which in turn is required for genome liberation from the capsid.In the analysis we used virus-derived HBV capsids obtained from the supernatant of HepG2.2.15, which contain the mature partially double-stranded DNA genome (mature capsids) and capsids expressed in E. coli. The latter were applied in two forms: as unspecific E. coli RNA- containing capsids and as empty capsids. Upon microinjection into Xenopus laevis oocytes we observed that mature and empty capsids were translocated to the nuclear pores with a similar kinetic. RNA-containing capsids failed to arrive at the pores implying that transport of the two other capsid types was active. Active translocation was confirmed by pre-injecting anti tubulin antibodies which interfere with MT-mediated translocation.In vitro reconstitution assays confirmed the specific attachment of mature and empty capsids to MTs and showed the need of further cytosolic proteins. Using pull-down and co-sedimentation experiments we identified one dynein light chain (DYNLL1, member of the Lc8 family) as interaction partner of the capsids. Injecting an excess of recombinant DYNLL1 with empty capsids into Xenopus laevis oocytes inhibited capsid transport to the nuclear pores indicating that DYNLL1 was only functional interaction partner implied in active transport.DNYLL2 did not interact with the capsids although differing from DYNLL1 by just six amino acids. Site directed mutagenesis of DYNLL1 revealed that two amino acids were critical for a direct interaction with the capsids. Both localized at the exterior of the DYNLL1 dimer and not in the groove of DYNLL1, which interacts with the dynein intermediate chain. Accordingly we could reconstitute a complex consisting of empty capsids, DYNLL1 and dynein intermediate chain as it should be in the in vivo situation