We use a variety of techniques to study the biochemical and mechanical properties of molecular motors, the cytoskeleton and biopolymers.
Total internal reflectance fluorescence microscopy
TIRF microscopy is achieved by aiming an excitation laser onto a microscope glass slide beyond the critical angle of refraction. This can be done either through a microscope objective, as shown on the picture, or through a glass prism. In either case, the resulting evanescent wave excites fluorophores only very near the glass surface and greatly reduces fluorescent background from fluorophore in solution. This makes assays with large amounts of labeled protein in solution possible, such as assembly assays of labeled G-actin into F-actin filaments.
Actin filament mechanics
 
In this assay individual fluorescently labeled actin filaments are observed via fluorescence microscopy. Filament motion is constrained to two dimensions by constructing a narrow observation chamber and recorded with an ICCD camera. We use the thermal induced shape changes to quantify actin filament persistence length.
Unloaded in vitro motility assay
 
The unloaded in vitro motility assay has all the advantages of solution biochemistry (i.e., temp, ionic conditions, substrate concentrations etc. are easily manipulated); however, movement, one of the fundamental properties of molecular motor function, can also be quantified. In this assay myosin is adsorbed to a silanized coverslip surface. Fluorescently labeled actin filaments are then allowed to bind to the surface-bound myosin. Movement is initiated by the addition of ATP and recorded with an ICCD camera.
Frictional loading in vitro Motility assay
 The unloaded motility assay gives important information about the motion generating capacity of molecular motors. However, motor molecules also produce force. We assess the effects of myosin or thin filament regulatory protein mutations on relative (to wild-type) force generation. In this assay, load is applied to the myosin motor by applying it to the surface along with small amounts of non motile actin binding proteins (e.g., alpha-actinin). The alpha-actinin will introduce a frictional load that resists filament motion. The amount of alpha-actinin required to stop filament motion is an indicator of myosin force generating capability. Since the load is applied to all of the filaments in a field of view, this assay is useful for rapidly screening a variety of experimental conditions (e. g.: regulated actin, unregulated actin, submaximal calcium activation, etc.).
Optical trap
 Optical traps use photon momentum to capture and hold small transparent particles with a higher refractive index than the medium. As a trapped particle moves away form the laser focus there is a restoring force that is proportional to the distance the particle moves. Therefore, an optical trap can be used as a very sensitive force tranducer that can measure forces in the piconewton range.
Laser trap loading assay
 We use both optical trap and frictional loading based assays to measure the inherent force generating capacity of mutant myosin molecules. Frictional loading assays provide an indication of relative force while optical trap based assays measure the absolute isometric force generating capability of the myosin mutants one filament at a time.
|
