Techniques

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The Greenberg Lab takes an interdisciplinary approach to understanding molecular motors over multiple levels of organization. Techniques used by the Greenberg Lab include:

Single Molecule Optical Trapping

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.

Stopped Flow Rapid Kinetics and Biochemical Assays

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.

In Vitro Reconstitution Assays

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.

Protein Expression and Purification

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).

Cell Culture and Stem Cell Engineering

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.

Traction Force Microscopy

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.

Immunofluorescence and Super Resolution Microscopy

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.

Modeling and Simulation

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