Date of Award

12-1-2004

Degree Type

Restricted Access Dissertation

Degree Name

Doctor of Philosophy

Field of Study

Microbiology and Immunology

Department

Graduate School of Biomedical Sciences

First Advisor

Julian Borejdo

Second Advisor

Thomas Burghardt

Third Advisor

Ben Harris

Abstract

Shepard, Athena A., Anisotropy of Myosin and Actin in Contraction of Skeletal Muscle. Doctor of Philosophy (Molecular Biology and Immunology), December, 2004, 161 pp., 1 table, 42 illustrations, bibliography, 253 titles. Muscle contraction results from the interaction of myosin and actin proteins contained in the muscle sarcomere. During actomyosin interactions, myosin consumes ATP and imparts an impulsive force to actin resulting in sliding of myosin and actin filaments to produce work. These proteins constitute the elementary motor responsible for cellular motility. The overall goal of this research project was to elucidate the mechanism of the actomyosin interaction on a molecular level. Novel time-resolved optical microscopic techniques followed myosin and actin orientation changes during skeletal muscle contraction. Fluorescence anisotropy was used to study the real time orientation changes of myosin, actin, and nucleotide during a single cross bridge cycle beginning in a state of rigor. Rabbit psoas fibers were isolated on a microscopic slide and labeled with fluorescently labeled regulatory light chain to monitor orientation changes of the lever arm of myosin, with fluorescent phalloidin to monitor orientation changes of actin and/or with Alexa ADP to monitor ATP hydrolysis. Caged ATP was perfused into the fiber prior to analysis to allow a small population of cross-bridges to execute a single cross-bridge cycle. Flash photolysis with UV light during analysis converted caged ATP from an inactive from to an active from. Confocal and multi-photon imaging allowed illumination of a small population of fluorescently labeled cross-bridges to measure orientation changes over time. The conclusions of this dissertation are: 1) The regulatory light chain rotates during skeletal muscle contraction and the lever arm model is supported, 2) Release of ADP from S1 corresponds to a single rotation of the lever arm, 3) Actin rotates during skeletal muscle contraction, 4) The rotation of actin is passive, i.e. it rotates as a consequence of dissociation of S1 from actin. The results revealed orientation changes in key contractile proteins during muscle contraction in the non-disease state organism. By understanding the mechanism of muscle contraction in the healthy scenario, hopefully a better understanding of diseased states stemming from mutations in contractile proteins (Usher’s Syndrome, Snell’s Waltzer Disease, and certain familial hypertrophic cardiomyopathies) will be made available, leading to a better preventative measures or treatments to treat such diseases in the future.

Comments

Shepard, Athena A., Anisotropy of Myosin and Actin in Contraction of Skeletal Muscle. Doctor of Philosophy (Molecular Biology and Immunology), December, 2004, 161 pp., 1 table, 42 illustrations, bibliography, 253 titles. W 4 S547A 2004

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