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THE FAR C-TERMINUS OF TPX2 CONTRIBUTES TO SPINDLE MORPHOGENESISEstes, Brett 24 March 2017 (has links)
A cell must build a bipolar mitotic spindle in order to faithfully segregate replicated DNA. To do so, multiple microtubule nucleation pathways are utilized to generate the robust spindle apparatus. TPX2, a microtubule binding protein, holds crucial roles in both the Ran-dependent and Augmin-dependent pathways where microtubules are nucleated near the chromosomes and from pre-existing microtubules. However, the exact role TPX2 plays in branching microtubules is less understood. Here, we explored the effect of truncating the essential TPX2 C-terminal 37 amino acids on Augmin localization and branching microtubule activity. First, we depleted LLC-Pk1 cells of the Augmin subunit HAUS6 and show that microtubule nucleation around the chromosomes following a nocodazole washout is strongly reduced leading to exaggerated kinetochore microtubule growth. Next, we depleted endogenous TPX2 in LLC-Pk1 cells harboring full length or truncated TPX2 bacterial artificial chromosome (BAC) DNA. Results show that TPX2 710 LAP cells have reduced Augmin localization on the spindle fibers, which correlates with reduced microtubule regrowth in the chromosomal region. In TPX2 710 LAP cells, regrowth was like Augmin depleted cells. Therefore, we provide evidence that the far C-terminus of TPX2 is required for branching microtubule nucleation and that kinetochore microtubule growth is Augmin-independent. In addition, we investigated cell cycle regulation of TPX2 by mutating the S738 phosphosite in the C-terminal motor interacting region. We utilized BAC recombineering to create phospho-mimetic and phospho-null mutants. In combination with plasmid DNA knockdown/rescue, overexpression and spindle assembly assays, we show that the phosphorylation of the C-terminal domain contributes to early mitotic events. LLC-Pk1 cells showed a significant increase in aberrant spindle morphology and reduced spindle stability in the presence of 738A and absence of endogenous TPX2. While rescue with the alanine mutant caused in an increase in multipolar spindles, overexpression resulted in a strong dominant negative monopolar phenotype. Therefore, S738 appears to contribute to mitotic force regulation during mitosis. In conclusion, the far C-terminus of TPX2 and its regulation play a role in the formation of a proper mitotic spindle.
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Studies of Helicoidal wall formation and organization in Nitella : [a thesis] ...Billeter, Elaine Dallahite 01 January 1991 (has links)
Patterns of cellulose deposition within the cell wall directly affect the size and shape of plant cells, in turn affecting the overall structure of the plant. In the primary wall of Nitella, and in the walls of many other algae and higher plants, a correspondence has been observed between the organization of cellulose microfibrils and cortical microtubules, each lying on opposite sides of the plasma membrane. The present work examines the development of the secondary wall in maturing Nitella internodal cells in an attempt to determine whether a relationship exists between cellulose microfibril organization and the organization of cortical microtubules. Treatments that artificially rearrange or remove microtubules were used, and effects on cellulose organization in the cell wall were examined through the use of transmission electron microscopy in thin sections and by the replica technique. Removal of microtubules in very young cells had a randomizing effect on cellulose organization, but absence of microtubules or their rearrangement did not prevent the formation of secondary walls with characteristic helicoidal patterns. The findings on the timing of helicoidal development in Nitella and the appearance of the innermost surface of helicoidal layers are discussed in relation to models of helicoidal architecture.
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Engineering Nanotechnological Applications of Biomolecular Motors and MicrotubulesChaudhuri, Samata 12 January 2018 (has links)
Biomolecular motor based transport reconstituted in synthetic environment has been recently established as a promising component for the development of nanoscale devices. A minimal system consisting of microtubules propelled over a surface of immobilized kinesin motor proteins has been used to transport and manipulate cargo for molecular sorting, analyte detection, and other novel nanotechnological applications. Despite these achievements, further progress of the field and translation of the reported applications to a real-world setting require overcoming several key challenges, such as, development of effective cargo conjugation strategies and precise control of the transport directionality with the reconstituted biomolecular motor systems.
The challenge of cargo conjugation is addressed in this thesis through the development of a robust bioorthogonal strategy to functionalize microtubules. The versatility of the developed method is demonstrated by covalently conjugating various types of cargos to microtubules. Further, the effect of the linker length on cargo attachment to microtubules is investigated by attaching cargo to microtubules via linkers of different lengths. By using kinesin-driven transport of microtubules that are covalently conjugated to antibodies, detection of various clinically relevant analytes is demonstrated as proof-of-principle applications for biosensing. Finally, the challenge of gaining control over transport directionality is addressed through topographical guiding of microtubules in nanostructures, and optimization of assay parameters to achieve successful guiding of microtubules. Spatio-temporal analyte concentration, using transport in these nanostructues, is also explored to make the biomolecular-motor based applications more suitable for use real-world point-of-care setting.
Taken together, the experimental work in this thesis contributes to the field of nanotechnological applications of biomolecular motors. The developed microtubule functionalization method and understanding of the effect of cargo attachment via linkers provide useful design principles for efficient cargo loading to microtubules.
Moreover, establishment of assay components for successful guiding of microtubules in nanostructures is a vital step forward for practical translation of future nanoscale devices.
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Characterization of pathogenic BicD2 mutations in vitro and in vivo.Yi, Julie Young Joo January 2022 (has links)
Microtubule motor proteins play fundamental roles in transporting a broad range of cellular cargoes in most eukaryotic cells. While there are over 40 kinesins helping to accommodate these diverse cellular demands, there is only one major form of cytoplasmic dynein (dynein, hereafter) carrying out almost all aspects of microtubule (MT) minus end-directed cargo transport. Dynein achieves this versatility by utilizing a wide range of adaptor proteins. Bicaudal D2 (BicD2) is a dynein adaptor protein responsible for linking cytoplasmic dynein to multiple forms of subcellular cargo. These include Rab6A, which contributes to Golgi function (Grigoriev et al., 2007; Matanis et al., 2002); the nucleoporin RanBP2 (Splinter et al., 2010); and the nucleus-cytoplasmic linker LINC complex protein Nesprin-2 (Goncalves et al., 2020). The latter two proteins were found to play important specific roles in the developing brain, respectively in the oscillatory interkinetic nuclear migration (INM) behavior characteristic of Radial Glial Progenitor (RGP) cells and in the directed nuclear migration in postmitotic neurons traveling to the expanding cortical plate.
The BICD2 gene was implicated in the autosomal dominant forms of neuromuscular diseases such as Spinal Muscular Atrophy with Lower Extremity Dominance 2 (SMA-LED2) (Neveling et al., 2013; Oates et al., 2013; Peeters et al., 2013; Synofzik et al., 2014) and, more recently in at least three developmental brain pathologies: polymicrogyria (Ravenscroft et al., 2016), cerebellar hypoplasia (Fiorillo et al., 2016), and Lissencephaly (Tsai et al., 2020) expanding the clinical spectrum from neuromuscular only to potentially the entire central nervous system. It is largely unknown how these diverse clinical presentations have any relations to the BicD2 mutational sites. To investigate the genotype-phenotype relationship, it is inevitable to study each point mutation molecularly and characterize the mutational effects in in vivo setting.
To investigate the mutational effects of BicD2, I used five different BicD2 fragments and nine missense mutants and characterized their behavior using biochemical and cellular approaches. Four of the missense mutations were further tested in the rat embryonic brain system, in which our lab has previously elucidated BicD2's roles during neurogenesis and post-mitotic neuronal migration. The chapters are organized by mutational effects on the BicD2-cargo interactions (Chapter 2) and the BicD2-dynein interactions (Chapter 3). In Chapter 4, I include supplemental materials to Chapters 2 and 3. In Chapter 5, I summarize, discuss, and provide my perspectives on all the mutational phenotypes found in the previous chapters. Lastly, all experimental procedures and reagents are described in Chapter 6.
Here, I describe the first identification of novel gain-of-function (GoF) defects in BicD2- nuclear cargo interaction associated with two pathogenic mutations, R690C and E770G (Chapter 2). Furthermore, I characterize the GoF defects in the embryonic rat brain by in utero electroporation. The cell-type specific expression of R690C or E770G constructs revealed mutation-specific impairment of nuclear migrations in the developing cerebral cortex. In addition, I found GoF defects in BicD2-dynein interaction associated with three pathogenic mutations, L679R, R690C, and T699, surprisingly, in the C-terminal cargo binding domain of BicD2 (Chapter 3). I demonstrate that these missense mutations cause a defect in BicD2 autoinhibition control, which in turn results in abnormally enhanced dynein association. I provide evidence for hyper-activation of BicD2 for dynein binding contributes to Golgi fragmentation, which has been associated with many neuromuscular diseases (Martinez-Menarguez et al., 2019), including SMA-LED2 (Martinez-Carrera and Wirth, 2015).
The entirety of this work describes molecular defects in 9 representative BicD2 mutations in vitro and demonstrates the mutational effects in vivo. I propose that the differential mutational effects associated with the BicD2 mutations might contribute to the broad spectrum of clinical phenotypes seen in patients with BicD2 diseases.
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The role of CCDC103 in the cytoskeletal dynamics, metabolic regulation, and functional maturation of zebrafish and human neutrophilsFalkenberg, Lauren 23 August 2022 (has links)
No description available.
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Characterization of the Parkinson's Disease Associated Protein, Leucine-Rich Repeat Kinase 2 (LRRK2), as a Ras-Related GTPaseGandhi, Payal January 2008 (has links)
No description available.
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FoxO limits microtubule stability and is itself negatively regulated by microtubule disruptionNechipurenko, Inna 26 June 2012 (has links)
No description available.
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Exploring Microtubule Structural Mechanics through Molecular Dynamics SimulationsJiang, Nan 30 October 2017 (has links)
No description available.
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Characterization of Microtubule Depolymerization by the HIV Protein RevBedi, Shimpi 02 February 2009 (has links)
No description available.
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The Role of Apoptosis in HeLa Cells Expressing HIV-1 RevPage, Elizabeth 16 April 2010 (has links)
No description available.
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