Dr. Bernstein's research laboratory is located on the beautiful campus of San Diego State University within the Biology Department. Our research program is directed toward the molecular analysis of Drosophila muscle gene and protein structure and function.
Let us introduce ourselves to you.
• Jared Camillo
Introduction to Dr. Bernstein's Research
The molecular motor of muscle is myosin heavy chain, a protein that interacts with actin to cause muscle contraction. We determined that Drosophila possesses a single muscle myosin heavy chain gene, and that multiple forms of the myosin protein are produced by the process of alternative RNA splicing. Specific regions of the myosin RNA are included in specific muscle types to yield myosin protein isoforms. The regions that differ among the isoforms must be critical for the myosin-mediated ultrastructural and physiological differences among muscle types. In collaboration with Dr. Ron Milligan (the Scripps Research Institute), we determined the location of the isoform differences on the three dimensional map of the myosin molecule. In collaboration with Dr. Tom Huxford (SDSU), we determined the structures of two myosin isoforms via X-ray crystallography. Using these data, we developed hypotheses regarding how these differences can affect myosin function.
To experimentally test our hypotheses on myosin isoform function, we developed transgenic strains of Drosophila that express the wrong isoforms in particular muscle types. As an initial approach, we showed that the normal myosin gene can rescue muscle defects associated with myosin null mutations. Interestingly, additional copies of the myosin gene result in defective muscles as well, due to an overabundance of myosin-containing thick filaments. We next succeeded in expressing the embryonic form of myosin in adult flight muscle. To our surprise, the adult muscle assembles normally using the embryonic protein, indicating that structural properties of the muscle are not affected by the isoform of myosin present. However, the flight muscles do not function when the embryonic myosin substitutes for the normal form, indicating that myosin isoforms are functionally different.
We have produced additional transgenic fly lines that express other myosin isoforms, particular mutant myosins and putative suppressor mutations. Currently, we are isolating single isoforms of the myosin protein from these lines to determine the biochemical and biophysical properties that are encoded by particular variable regions of the protein, e.g. actin binding ability, ATPase kinetics, in vitro thin filament motility. Transient kinetic studies are performed in collaboration with Dr. Michael Geeves (University of Kent at Canterbury). Muscles with altered myosin isoforms are studied in collaboration with Dr. Douglas Swank (Rensselaer Polytechnique Institute) to assess how mechanical properties are affected by the alterations. Overall our approach should lead to a more complete understanding of the in vitro and in vivo properties imparted by particular regions of the myosin molecule.
We are dissecting the role of myosin domains and residues in Drosophila cardiac muscle in collaboration with Dr. Anthony Cammarato (Johns Hopkins University) as well as with Dr. Rolf Bodmer and Dr. Karen Ocorr (Sanford-Burnham-Prebys Medical Discovery Institute). Using their quantitative videomicroscopy set-up, we have documented myosin-based defects that mimic human dilated cardiomyopathy and restrictive cardiomyopathy. We are also examining the effects of myosin mutations known to cause cardiomyopathy in humans, with the goal of developing approaches to suppress these defects by genetic or pharmacological treatments.
We are producing and analyzing Drosophila models of several myosin-based human diseases: inclusion body myopathy type 3, myosin storage myopathy and Freeman-Sheldon syndrome. The goal is to determine how the mutations affect muscle development and aging using an organism with a defined genetic background. Studies on muscle ultrastructure, myosin biochemistry and muscle physiology will aid in understanding the cause and effects of each disease.
We are also studying Drosophila UNC-45, a molecular chaperone/co-chaperone that aids in folding of muscle myosin heavy chain and possibly other muscle or non-muscle proteins. We solved the structure of the Drosophila UNC-45 protein in collaboration with Dr. Tom Huxford (SDSU). This will aid us in pursuing a detailed structure/function analysis of this protein in vitro and in vivo. Our research on UNC-45 and myosin mutations will lead to insights as to how mutations that result in production of abnormally folded contractile proteins cause phenotypic defects and how these may be ameliorated. Since several neuromuscular diseases arise from aberrant protein folding and accumulation of misfolded protein aggregates, our work will contribute to understanding the disease process and may yield insight into genetic and pharmacological approaches that prove to be therapeutic in humans.