Browsing by Subject "Microtubules"

Now showing 1 - 3 of 3
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    A biochemical and molecular analysis of functional differences between dystrophin and utrophin
    (2013-11) Belanto, Joseph John
    The DMD gene encodes the protein dystrophin, a 427kD cytoplasmic protein responsible for linking the actin cytoskeleton to the extracellular matrix via the dystrophin-glycoprotein complex. Mutations in dystrophin that abolish its expression lead to Duchenne muscular dystrophy (DMD). Patients with DMD become wheelchair bound in their early teens and succumb to fatal cardiac and/or respiratory failure in their mid-twenties to early thirties. There is currently no effective or widely available treatment for DMD beyond ventilatory support and the use of corticosteroids. Many therapies for treating dystrophin deficiency aim at upregulating its autosomal homolog utrophin due to its structural similarity and ability to bind an almost identical repertoire of proteins that dystrophin binds. It was previously shown that utrophin cannot bind neuronal nitric oxide synthase (nNOS) even though dystrophin binds nNOS, establishing for the first time a functional difference between dystrophin and utrophin. Here, we show that transgenic overexpression of utrophin on the mdx mouse background (Fiona-mdx) is not sufficient to rescue the disorganized microtubule network of the mdx mouse. Thus, we have elucidated a second functional difference between dystrophin and utrophin. Additionally, Fiona-mdx mice lack full recovery of cage activity after mild exercise. Our results suggest that any deficiency in nNOS binding or microtubule lattice function caused by loss of dystrophin may not be restored by upregulation of utrophin. Previously, our lab reported that dystrophin directly binds to microtubules and organizes them beneath the sarcolemma. Using in vitro microtubule cosedimentation assays, we show that dystrophin binds to microtubules with strong affinity (KD=0.33µM). Through the use of various recombinant constructs tested via in vitro microtubule cosedimentation we show that spectrin-like repeats 20-22 of the dystrophin central rod are responsible for microtubule binding activity. However, we show that these repeats require flanking regions of dystrophin for proper binding activity, making microtubule binding context-dependent. Additionally, we show that recombinant utrophin does not bind microtubules in vitro, corroborating our in vivo findings of the disorganized subsarcolemmal microtubule lattice of the Fiona-mdx mouse. We also provide evidence showing that dystrophin functions as a molecular guidepost to organize microtubules into a rectilinear lattice.
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    The Role of Oxidative Stress in Remodeling the Cardiac Microtubule Cytoskeleton
    (2021-05) Goldblum, Rebecca
    Microtubules are cylindrical cytoskeletal polymers composed of α/β-tubulin heterodimers that make up an ordered tubulin lattice. In cells, microtubules form a network that is a key component of the cellular cytoskeleton. Under pathological conditions of oxidative stress, we and others have found that cardiomyocytes, the contractile cells in the heart, display a denser microtubule cytoskeleton, which may lead to the progressive structural and functional cellular changes associated with myocardial ischemia and systolic dysfunction. This reorganization of the microtubule network occurs despite only small increases in tubulin expression, suggesting that alterations to microtubule length regulation and stability are involved. Using biophysical reconstitution experiments and live-cell imaging, we found that oxidative stress may synergistically increase the density of microtubules inside of cells by simultaneously increasing the length of dynamic, short-lived microtubules, while fostering the longevity of stable, long-lived microtubules. We found that microtubules subjected to oxidative stress undergo cysteine oxidation, and our electron and fluorescence microscopy experiments revealed that the locations of oxidized tubulin subunits within the microtubule had structural damage within the cylindrical tubulin lattice, consisting of holes and lattice openings. For dynamic microtubules, incorporation of stabilizing GTP-tubulin into these damaged lattice regions led to an increased frequency of rescue events (the transition from shortening to growth), and thus longer microtubules. For long-lived microtubules, these same structural defects facilitate entry of the enzyme αTAT1 into the microtubule lumen, where it catalyzes the acetylation of α-tubulin. This intraluminal acetylation has been shown to increase the lifetime of stable microtubules by conferring mechanical stability to the microtubule lattice. In this way, oxidative stress triggers a dramatic, pathogenic shift from a sparse microtubule network into a dense, longitudinally aligned microtubule network inside of cardiac myocytes, likely contributing to increased cellular stiffness and contractile dysfunction. Our results provide insight into myocardial changes in ischemic heart disease by describing a mechanism for the dramatic remodeling of the microtubule cytoskeletal network within cardiac myocytes subjected to oxidative stress.
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    Skeletal Muscle Microtubule Organization and Stability is Regulated by the Dystrophin-Glycoprotein Complex and Cortical Actin
    (2020-08) Nelson, D'anna
    Duchenne muscular dystrophy (DMD) is a fatal X-linked myopathy caused by the loss of dystrophin in striated muscle. DMD is frequently studied in the mdx mouse which also lacks dystrophin. It has been observed by multiple independent research groups that mdx skeletal muscle presents with a disorganized cortical microtubule lattice that primarily lacks transverse microtubules as compared to the orthogonal microtubule lattice of wildtype mouse skeletal muscle. While transgenic expression of dystrophin in mdx skeletal muscle does restore microtubule organization, it is not understood how dystrophin regulates microtubule organization in vivo. This thesis implicates two regions of the dystrophin rod domain as regulators of microtubule organization and stability. Singular absence of dystrophin spectrin like repeats R4-15 or R20-24 does not impact basal microtubule organization. However, removal of both R4-15 and R20-23 from micro-dystrophin constructs results in a miniaturized dystrophin that is incapable of fully restoring microtubule organization when transgenically expressed in mdx muscle. In addition to the intermediate microtubule organization by micro-dystrophins lacking R4-15 and R20-23, we have characterized a novel microtubule pathology where transverse microtubules are lost upon eccentric contraction in the absence of either R4-15 or R20-24. Transverse microtubule loss is specific to eccentric contractions and occurs rapidly via a ROS mediated mechanism. Multiple sources of ROS appear to be involved including NOX2 but not nNOS. Moreover, loss of γ-cytoplasmic actin, β-cytoplasmic actin, or the dystrophin-glycoprotein complex (DGC) member α-dystrobrevin all cause a highly similar microtubule phenotype where transverse microtubules are lost post eccentric contraction. While both the intermediate microtubule organization and microtubule susceptibility to eccentric contraction exhibited by micro-dystrophin rescued muscle may have implications for micro-dystrophin gene therapy, the work presented in this thesis has also widened our understanding of skeletal muscle microtubule regulation to include cytoplasmic actins and DGC stability.