Cooperative & competitive binding of anti-myosin tail antibodies revealed by super-resolution microscopy.

Autor: Quedan D; Department of Biological Sciences, Division of Biochemistry and Molecular Biology, University of North Texas, Denton, TX, 76203, USA., Singh R; Department of Biological Sciences, Division of Biochemistry and Molecular Biology, University of North Texas, Denton, TX, 76203, USA., Akel A; Department of Biological Sciences, Division of Biochemistry and Molecular Biology, University of North Texas, Denton, TX, 76203, USA., Bernardino AL; Department of Biological Sciences, Division of Biochemistry and Molecular Biology, University of North Texas, Denton, TX, 76203, USA., Thang C; Department of Biological Sciences, Division of Biochemistry and Molecular Biology, University of North Texas, Denton, TX, 76203, USA., Bhaskaruni M; Department of Biological Sciences, Division of Biochemistry and Molecular Biology, University of North Texas, Denton, TX, 76203, USA., Haldankar A; Department of Biological Sciences, Division of Biochemistry and Molecular Biology, University of North Texas, Denton, TX, 76203, USA., Tanner BCW; Department of Integrative Physiology & Neuroscience, Washington State University, Pullman, WA, 99164, USA., Root DD; Department of Biological Sciences, Division of Biochemistry and Molecular Biology, University of North Texas, Denton, TX, 76203, USA. Electronic address: droot@unt.edu.
Jazyk: angličtina
Zdroj: Archives of biochemistry and biophysics [Arch Biochem Biophys] 2023 Oct 01; Vol. 747, pp. 109753. Date of Electronic Publication: 2023 Sep 14.
DOI: 10.1016/j.abb.2023.109753
Abstrakt: The MF30 monoclonal antibody, which binds to the myosin subfragment-2 (S2), was found to increase the extent of myofibril shortening. Yet, previous observations found no effect of this antibody on actin sliding over myosin during in vitro motility assays with purified proteins in which myosin binding protein C (MyBPC) was absent. MF30 is hypothesized to enhance the availability of myosin heads (subfragment-1 or S1) to bind actin by destabilizing the myosin S2 coiled-coil and sterically blocking S2 from binding S1. The mechanism of action likely includes MF30's substantial size, thereby inhibiting S1 heads and MyBPC from binding S2. Hypothetically, MF30 should enhance the ON state of myosin, thereby increasing muscle contraction. Our findings indicate that MF30 binds preferentially to the unfolded heavy chains of S2, displaying positive cooperativity. However, the dose-response curve of MF30's enhancement of myofibril shortening did not suggest complex interactions with S2. Single, double, and triple-stained myofibrils with increasing amounts of antibodies against myosin rods indicate a possible competition with MyBPC. Additional assays revealed decreased fluorescence intensity at the C-zone (central zone in the sarcomere, where MyBPC is located), where MyBPC may inhibit MF30 binding. Another monoclonal antibody named MF20, which binds to the light meromyosin (LMM) without affecting myofibril contraction, showed less reduction in fluorescence intensity at the C-zone in expansion microscopy than MF30. Expansion microscopy images of myofibrils labeled with MF20 revealed labeling of the A-band (anisotropic band) and a slight reduction in the labeling at the C-zone. The staining pattern obtained from the expansion microscopy image was consistent with images from photolocalization microscopy which required the synthesis of unique photoactivatable quantum dots, and Zeiss Airyscan imaging as well as alternative expansion microscopy digestion methods. Consistent with the hypothesis that MF30 competes with MyBPC binding to S2, cardiac tissue from MyBPC knockout mice was stained more intensely, especially in the C-zone, by MF30 compared to the wild type.
Competing Interests: Declaration of competing interest None.
(Copyright © 2023 Elsevier Inc. All rights reserved.)
Databáze: MEDLINE
Externí odkaz: