Techniques

We use optical trapping to examine the contractile properties of single myosin molecules.  Our home-built optical trap enables us to measure nanometer movements and piconewton forces with high spatial and temporal resolution.  Moreover, our system enables us to simultaneously measure single molecule fluorescence using total internal reflectance microscopy.  We are using this system to reconstitute muscle contraction at the molecular level.

We use biochemical and biophysical techniques to examine the interactions between motor proteins and binding partners.  Our stopped-flow apparatus allows us to measure the rates of very fast biochemical reactions.  Using this technique, it is possible to dissect the individual steps of the myosin ATPase cycle.  We also use other biochemical and biophysical techniques including fluorimetry, sedimentation assays, and chromatography.

We use purified proteins to reconstitute contraction in vitro.  The video above shows fluorescently-labeled actin filaments, decorated with troponin and tropomyosin, moving over a bed of myosin.

For our molecular experiments, we use proteins that are either tissue purified or recombinantly expressed in E. coli, Sf9 cells, or mammalian cells.  Proteins are purified using several methods including fast protein liquid chromatography (FPLC).

Our lab uses stem cell technologies to model heart disease.  We use the CRISPR/Cas9 system to introduce disease-causing mutations into human stem cells and then we differentiate these cells to cardiomyocytes.  We also culture mammalian cells for use in engineered tissues.  Click on the image  above to see the beating cardiac tissue.

To examine the contractile properties of stem-cell derived cardiomyocytes, we use traction force microscopy.  We use microfabrication techniques to pattern extracellular matrix proteins on to a polyacrylamide hydrogel with defined mechanical properties.  As the cell beats, beads embedded in the gel are displaced, allowing for the calculation of the cellular force of contraction.  Click on the image above to see a beating cardiomyocyte on the hydrogel.

We examine the structural properties of cells using immunofluorescence.  We image these cells using confocal and structured illumination microscopy.  The cell structure can then be analyzed using custom written image processing software.

Our lab uses modeling and simulation to better understand contractility.  Our modeling scales from the level of molecules to models of whole tissues.