Albert Kalganov1, Nabil Shalabi2, Nedjma Zitouni3, Linda Hussein Kachmar3, Anne-Marie Lauzon4, Dilson E Rassier5. 1. Department of Kinesiology and Physical Education, Faculty of Education, McGill University, Canada. 2. Department of Mechanical Engineering, Faculty of Engineering McGill University, Canada. 3. Meakins-Christie Laboratories, McGill University, Canada; Department of Experimental Medicine, Faculty of Medicine McGill University, Canada. 4. Meakins-Christie Laboratories, McGill University, Canada; Department of Experimental Medicine, Faculty of Medicine McGill University, Canada; Departments of Physics, Faculty of Science, McGill University, Canada; Department of Physiology, Faculty of Medicine, McGill University, Canada. 5. Department of Kinesiology and Physical Education, Faculty of Education, McGill University, Canada; Meakins-Christie Laboratories, McGill University, Canada; Department of Experimental Medicine, Faculty of Medicine McGill University, Canada; Departments of Physics, Faculty of Science, McGill University, Canada; Department of Physiology, Faculty of Medicine, McGill University, Canada. Electronic address: dilson.rassier@mcgill.ca.
Abstract
BACKGROUND: There is evidence that the actin-activated ATP kinetics and the mechanical work produced by muscle myosin molecules are regulated by two surface loops, located near the ATP binding pocket (loop 1), and in a region that interfaces with actin (loop 2). These loops regulate force and velocity of contraction, and have been investigated mostly in single molecules. There is a lack of information of the work produced by myosin molecules ordered in filaments and working cooperatively, which is the actual muscle environment. METHODS: We use micro-fabricated cantilevers to measure forces produced by myosin filaments isolated from mollusk muscles, skeletal muscles, and smooth muscles containing variations in the structure of loop 1 (tonic and phasic myosins). We complemented the experiments with in-vitro assays to measure the velocity of actin motility. RESULTS: Smooth muscle myosin filaments produced more force than skeletal and mollusk myosin filaments when normalized per filament overlap. Skeletal muscle myosin propelled actin filaments in a higher sliding velocity than smooth muscle myosin. The values for force and velocity were consistent with previous studies using myosin molecules, and suggest a close correlation with the myosin isoform and structure of surface loop 1. GENERAL SIGNIFICANCE: The technique using micro-fabricated cantilevers to measure force of filaments allows for the investigation of the relation between myosin structure and contractility, allowing experiments to be conducted with an array of different myosin isoforms. Using the technique we observed that the work produced by myosin molecules is regulated by amino-acid sequences aligned in specific loops.
BACKGROUND: There is evidence that the actin-activated ATP kinetics and the mechanical work produced by muscle myosin molecules are regulated by two surface loops, located near the ATP binding pocket (loop 1), and in a region that interfaces with actin (loop 2). These loops regulate force and velocity of contraction, and have been investigated mostly in single molecules. There is a lack of information of the work produced by myosin molecules ordered in filaments and working cooperatively, which is the actual muscle environment. METHODS: We use micro-fabricated cantilevers to measure forces produced by myosin filaments isolated from mollusk muscles, skeletal muscles, and smooth muscles containing variations in the structure of loop 1 (tonic and phasic myosins). We complemented the experiments with in-vitro assays to measure the velocity of actin motility. RESULTS: Smooth muscle myosin filaments produced more force than skeletal and mollusk myosin filaments when normalized per filament overlap. Skeletal muscle myosin propelled actin filaments in a higher sliding velocity than smooth muscle myosin. The values for force and velocity were consistent with previous studies using myosin molecules, and suggest a close correlation with the myosin isoform and structure of surface loop 1. GENERAL SIGNIFICANCE: The technique using micro-fabricated cantilevers to measure force of filaments allows for the investigation of the relation between myosin structure and contractility, allowing experiments to be conducted with an array of different myosin isoforms. Using the technique we observed that the work produced by myosin molecules is regulated by amino-acid sequences aligned in specific loops.
Authors: Michael Heymann; Sven K Vogel; Haiyang Jia; Johannes Flommersfeld; Henri G Franquelim; David B Brückner; Hiromune Eto; Chase P Broedersz; Petra Schwille Journal: Nat Mater Date: 2022-05-26 Impact factor: 47.656
Authors: Anabelle S Cornachione; Felipe S Leite; Junling Wang; Nicolae A Leu; Albert Kalganov; Denys Volgin; Xuemei Han; Tao Xu; Yu-Shu Cheng; John R R Yates; Dilson E Rassier; Anna Kashina Journal: Cell Rep Date: 2014-07-10 Impact factor: 9.423