Browsing by Author "Lindsay, Angus"

Now showing 1 - 3 of 3
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    Essential nucleotide- and protein-dependent functions of Actb/β-actin
    (National Academy of Sciences, 2018-07-31) Patrinostro, Xiaobai; Roy, Pallabi; Lindsay, Angus; Chamberlain, Christopher M.; Sundby, Lauren J.; Starker, Colby G.; Voytas, Daniel F.; Ervasti, James M.; Perrin, Benjamin J.; Biochemistry and Molecular Biology, School of Medicine
    The highly similar cytoplasmic β- and γ-actins differ by only four functionally similar amino acids, yet previous in vitro and in vivo data suggest that they support unique functions due to striking phenotypic differences between Actb and Actg1 null mouse and cell models. To determine whether the four amino acid variances were responsible for the functional differences between cytoplasmic actins, we gene edited the endogenous mouse Actb locus to translate γ-actin protein. The resulting mice and primary embryonic fibroblasts completely lacked β-actin protein, but were viable and did not present with the most overt and severe cell and organismal phenotypes observed with gene knockout. Nonetheless, the edited mice exhibited progressive high-frequency hearing loss and degeneration of actin-based stereocilia as previously reported for hair cell-specific Actb knockout mice. Thus, β-actin protein is not required for general cellular functions, but is necessary to maintain auditory stereocilia.
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    Impaired muscle relaxation and mitochondrial fission associated with genetic ablation of cytoplasmic actin isoforms
    (Wiley, 2018) O'Rourke, Allison R.; Lindsay, Angus; Tarpey, Michael D.; Yuen, Samantha; McCourt, Preston; Nelson, D'anna M.; Perrin, Benjamin J.; Thomas, David D.; Spangenburg, Espen E.; Lowe, Dawn A.; Ervasti, James M.; Biology, School of Science
    While α-actin isoforms predominate in adult striated muscle, skeletal muscle-specific knockouts (KOs) of nonmuscle cytoplasmic βcyto- or γcyto-actin each cause a mild, but progressive myopathy effected by an unknown mechanism. Using transmission electron microscopy, we identified morphological abnormalities in both the mitochondria and the sarcoplasmic reticulum (SR) in aged muscle-specific βcyto- and γcyto-actin KO mice. We found βcyto- and γcyto-actin proteins to be enriched in isolated mitochondrial-associated membrane preparations, which represent the interface between mitochondria and sarco-endoplasmic reticulum important in signaling and mitochondrial dynamics. We also measured significantly elongated and interconnected mitochondrial morphologies associated with a significant decrease in mitochondrial fission events in primary mouse embryonic fibroblasts lacking βcyto- and/or γcyto-actin. Interestingly, mitochondrial respiration in muscle was not measurably affected as oxygen consumption was similar in skeletal muscle fibers from 12 month-old muscle-specific βcyto- and γcyto-actin KO mice. Instead, we found that the maximal rate of relaxation after isometric contraction was significantly slowed in muscles of 12-month-old βcyto- and γcyto-actin muscle-specific KO mice. Our data suggest that impaired Ca2+ re-uptake may presage development of the observed SR morphological changes in aged mice while providing a potential pathological mechanism for the observed myopathy.
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    Loss of peroxiredoxin-2 exacerbates eccentric contraction-induced force loss in dystrophin-deficient muscle
    (Springer Nature, 2018-11-30) Olthoff, John T.; Lindsay, Angus; Abo-Zahrah, Reem; Baltgalvis, Kristen A.; Patrinostro, Xiaobai; Belanto, Joseph J.; Yu, Dae-Yeul; Perrin, Benjamin J.; Garry, Daniel J.; Rodney, George G.; Lowe, Dawn A.; Ervasti, James M.; Biology, School of Science
    Force loss in skeletal muscle exposed to eccentric contraction is often attributed to injury. We show that EDL muscles from dystrophin-deficient mdx mice recover 65% of lost force within 120 min of eccentric contraction and exhibit minimal force loss when the interval between contractions is increased from 3 to 30 min. A proteomic screen of mdx muscle identified an 80% reduction in the antioxidant peroxiredoxin-2, likely due to proteolytic degradation following hyperoxidation by NADPH Oxidase 2. Eccentric contraction-induced force loss in mdx muscle was exacerbated by peroxiredoxin-2 ablation, and improved by peroxiredoxin-2 overexpression or myoglobin knockout. Finally, overexpression of γcyto- or βcyto-actin protects mdx muscle from eccentric contraction-induced force loss by blocking NADPH Oxidase 2 through a mechanism dependent on cysteine 272 unique to cytoplasmic actins. Our data suggest that eccentric contraction-induced force loss may function as an adaptive circuit breaker that protects mdx muscle from injurious contractions.