Swank Lab
Flight and Jump Testing
The first and most fun assay we use to characterize our mutants are ones that measure flight ability, wing beat frequency, wing stroke amplitude, and jump ability. This whole-organism data can be correlated with changes in muscle mechanical properties.
Techniques and Projects
Transgenic Fly Generation
Standard molecular biology techniques are employed to generate myosin transgenes that are injected into Drosophila embryos to create transgenic fly lines. Some of our transgenics are designed to explore the function of a specific region of a protein. Two specific domains being tested are the converter and the relay domain of muscle myosin. Other fly lines, generated by our collaborators, express myosins with mutations that cause muscle and heart diseases in humans.
Muscle Mechanics
We evaluate muscle mechanical properties using the skinned fiber technique. The mechanics apparatus includes a pair of hooks, one stationary and affixed to a force transducer, the other attached to a high-speed servomotor. The muscle fiber is mounted onto the hooks and lowered into a solution chamber. By varying the concentrations of the solutions and imposing various perturbations on the fiber, we measure muscle properties such as power, force, velocity, stiffness, and rate constants of the myosin cross-bridge cycle. We primarily test the mechanical properties of indirect flight and jump muscles from our transgenic Drosophila, and have begun evaluating cultured myotubes and fibroblasts.
In vitro Motility and Optical Trapping
We characterize actin and myosin interactions under unloaded conditions in the absence of any other proteins in the in vitro motility assay. Myosin is purified from our fruit flies and then adhered to a glass coverslip flow cell. Fluorescently-labeled actin filaments are moved by the myosin heads, powered by MgATP hydrolysis. By varying ATP, Pi, and ADP and measuring actin velocity under each condition, it is possible to determine rate constants of the myosin cross-bridge cycle of different myosin isoforms and mutants.
Protein structure determination
We use protein structure prediction software, molecular dynamics simulations, and NMR to help determine mechanisms by which mutations alter the biophysical and physiological function of myosin and muscle.
Collaborators:
Dr. Sanford Bernstein, San Diego State University, Biology Department
Dr. Belinda Bullard, University of York, Department of Biology
Dr. Kathleen Clark, University of Utah, Huntsman Cancer Institute
Dr. Roger Cooke, University of California San Francisco, Department of Biochemistry and Biophysics
Dr. David Corr, Rensselaer Polytechnic Institute, Department of Biomedical Engineering
Dr. David Maughan, University of Vermont Department of Physiology
Dr. Edward Pate, Washington State University, Department of Mathematics
Dr. Chunyu Wang, Rensselaer Polytechnic Institute, Department of Biology
Other Muscle Proteins
We can investigate other muscle proteins by many of the same methods employed to study myosin. Currently, isoforms of troponin C are being investigated for a potential link to stretch activation, an important mechanism in high speed or oscillatory muscle types. Mutations in the troponin C genes have been correlated to some forms of heritable human cardiomyopathy. Another protein we are investigating, muscle limb protein (MLP), is also implicated in heritable cardiomyopathies and in sensing stretch.
Dickinson et al, 1999
Grant Support:
Muscular Dystrophy Association
National Institutes of Health, NIAMS
American Heart Association
