Development of a Single Myofibril Mechanochemical Assay and Fluorescence Detection System.

University Of Vermont & State Agricultural College, Burlington VT

Investigators

Abstract

This award is for developing a hybrid instrument designed to investigate the mechanochemistry of single myofibrils, the smallest subdivision of muscle that retains the organized myofilament lattice. The proposed instrument is an atomic force microscope oriented horizontally (so that longitudinal forces are measured along the long axis of the myofibril) and an optical system for measuring sarcomere and myofibril dimensions and for monitoring fluorescent probes. The optical system will be able to view the myofibril along its length, unlike commercial AFM-optical microscopes in which the fibril is perpendicular to the optical viewing plane. The proposed instrument is complementary to one funded previously by NSF MRI (the Single Molecule Laser Optical Trap and Fluorescence System) located in the Department of Molecular Physiology and Biophysics. Coordinated use of the two multi-user instruments will provide unique opportunities to probe the mechanisms of muscle force generation at both the molecular and sarcomeric level, thereby bridging the two levels of structural organization in ways that have not yet been possible. All the research and teaching projects will rely on the instrument's ability to measure force, velocity, and dynamic stiffness and/or to quantitatively measure fluorescence from calcium or other ion indicators in a single myofibril. The instrument is intended to serve both the research needs of investigators at the University of Vermont and the educational needs of students as a unique teaching tool for molecular biophysics. An important strategic goal is to lay the foundation for future work by characterizing the ensemble action of myosin using the same physical-chemical variables and underlying molecular models as those employed in single molecule research. The proposed instrument is designed to allow the users to investigate how muscle operates at the subcellular and molecular levels in ways that have not yet been possible. The proposed instrument combines an extremely sensitive device to measure the force developed in a myofibril, which is the smallest subdivision of muscle that retains the full complement of muscle proteins in an organized lattice. The force-sensing device - a customized, horizontally-oriented, atomic force microscope - works in tandem with another device - a servomotor - that allows the myofibril to be manipulated in ways that help elucidate the molecular mechanism of force production. Both devices are integrated into an optical system that allows the ordered pattern of the myofibril to be imaged and biochemical reactions to be monitored at high temporal resolution by fluorescent probes. The proposed projects will also help us understand how single motor molecules of the myofibril are summed and integrated into a highly organized, smooth-running biological machine. With this instrument, the investigators will be able to consider questions at the forefront of their respective fields. By stimulating other laboratories in the US to develop similar or improved versions, this equipment and the projects it supports will have a significant impact beyond the individual investigators and research community at the host institution.