• A high-throughput fluorescence lifetime-based assay to detect binding of myosin-binding protein C to F-actin

      Bunch, Thomas A.; Lepak, Victoria C.; Bortz, Kellan M.; Colson, Brett A.; Department of Cellular and Molecular Medicine, University of Arizona (Rockefeller University Press, 2021-02-18)
      Binding properties of actin-binding proteins are typically evaluated by cosedimentation assays. However, this method is time-consuming, involves multiple steps, and has a limited throughput. These shortcomings preclude its use in screening for drugs that modulate actin-binding proteins relevant to human disease. To develop a simple, quantitative, and scalable F-actin-binding assay, we attached fluorescent probes to actin's Cys-374 and assessed changes in fluorescence lifetime upon binding to the N-terminal region (domains C0-C2) of human cardiac myosin-binding protein C (cMyBP-C). The lifetime of all five probes tested decreased upon incubation with cMyBP-C C0-C2, as measured by time-resolved fluorescence (TR-F), with IAEDANS being the most sensitive probe that yielded the smallest errors. The TR-F assay was compared with cosedimentation to evaluate in vitro changes in binding to actin and actin-tropomyosin arising from cMyBP-C mutations associated with hypertrophic cardiomyopathy (HCM) and tropomyosin binding. Lifetime changes of labeled actin with added C0-C2 were consistent with cosedimentation results. The HCM mutation L352P was confirmed to enhance actin binding, whereas PKA phosphorylation reduced binding. The HCM mutation R282W, predicted to disrupt a PKA recognition sequence, led to deficits in C0-C2 phosphorylation and altered binding. Lastly, C0-C2 binding was found to be enhanced by tropomyosin and binding capacity to be altered by mutations in a tropomyosin-binding region. These findings suggest that the TR-F assay is suitable for rapidly and accurately determining quantitative binding and for screening physiological conditions and compounds that affect cMyBP-C binding to F-actin for therapeutic discovery. © 2021 Bunch et al.
    • The number of Z-repeats and super-repeats in nebulin greatly varies across vertebrates and scales with animal size

      Gohlke, J.; Tonino, P.; Lindqvist, J.; Smith, J.E.; Granzier, H.; Department of Cellular and Molecular Medicine, University of Arizona (Rockefeller University Press, 2021)
      Nebulin is a skeletal muscle protein that associates with the sarcomeric thin filaments and has functions in regulating the length of the thin filament and the structure of the Z-disk. Here we investigated the nebulin gene in 53 species of birds, fish, amphibians, reptiles, and mammals. In all species, nebulin has a similar domain composition that mostly consists of ∼30-residue modules (or simple repeats), each containing an actin-binding site. All species have a large region where simple repeats are organized into seven-module super-repeats, each containing a tropomyosin binding site. The number of super-repeats shows high interspecies variation, ranging from 21 (zebrafish, hummingbird) to 31 (camel, chimpanzee), and, importantly, scales with body size. The higher number of super-repeats in large animals was shown to increase thin filament length, which is expected to increase the sarcomere length for optimal force production, increase the energy efficiency of isometric force production, and lower the shortening velocity of muscle. It has been known since the work of A.V. Hill in 1950 that as species increase in size, the shortening velocity of their muscle is reduced, and the present work shows that nebulin contributes to the mechanistic basis. Finally, we analyzed the differentially spliced simple repeats in nebulin's C terminus, whose inclusion correlates with the width of the Z-disk. The number of Z-repeats greatly varies (from 5 to 18) and correlates with the number of super-repeats. We propose that the resulting increase in the width of the Z-disk in large animals increases the number of contacts between nebulin and structural Z-disk proteins when the Z-disk is stressed for long durations. © 2020 Gohlke et al.
    • Think globally, act locally: Centrosome-localized mRNAs ensure mitotic fidelity

      Zarnescu, Daniela C; Molecular and Cellular Biology, University of Arizona (Rockefeller University Press, 2020-11-20)
      The functional importance of mRNA localization to centrosomes is unclear. Ryder et al. (2020. J. Cell Biol. https://doi.org/10.1083/jcb.202004101) identify fragile-X mental retardation protein as a regulator of centrocortin (cen) mRNA dynamics in Drosophila. Mistargeting of cen impairs division and development, indicating that cen mRNA localization to centrosomes ensures mitotic fidelity.
    • Titin N2A: More than a signaling node?

      van der Pijl, R.J.; Ottenheijm, C.A.C.; Department of Cellular and Molecular Medicine, University of Arizona (Rockefeller University Press, 2021)
    • Toward an understanding of myofibrillar function in health and disease

      Moss, R.L.; Cremo, C.; Granzier, H.L.; Cellular and Molecular Medicine, University of Arizona (Rockefeller University Press, 2021)