Albert H. Gough

Individual Leaflets of a Membrane Bilayer Can Independently Regulate Permeability

Water rapidly crosses most membranes, but only slowly crosses apical membranes of barrier epithelia such as bladder and kidney collecting duct, a feature essential to barrier function. How apical membrane structure reduces permeabilities remains unclear. Cell plasma membranes contain two leaflets of distinct lipid composition; the role of this bilayer asymmetry in membrane permeability is unclear. To determine how asymmetry of leaflet composition affects membrane permeability, effects on bilayer permeation of reducing single leaflet permeability were determined using two approaches: formation of asymmetric bilayers in an Ussing chamber, with only one of two leaflets containing cholesterol sulfate, and stabilization of the external leaflet of unilamellar vesicles with praeseodymium (Pr). In both systems, permeability measurements showed that each leaflet acts as an independent resistor of water permeation. These results show that a single bilayer leaflet can act as the barrier to permeation and provide direct evidence that segregation of lipids to create a low permeability exofacial leaflet may act to reduce the permeability of barrier epithelial apical membranes.

Automated interactive microscopy: measuring and manipulating the chemical and molecular dynamics of cells and tissues

The Automated Interactive Microscope is a robotic light microscope workstation that combines high performance light microscopy and computing to explore the chemical and molecular dynamics of cells and tissues.

Mapping intracellular biochemistry with fluorescence anisotropy imaging microscopy

Fluorescence polarization anisotropy can be used to determine the rotational mobility of a fluorescent analog, detect anisotropic orientation distributions, or measure the fluorescence lifetime of a fluorophore. Steady state fluorescence anisotropy can be simply measured in a standard fluorescence microscope equipped with excitation and emission polarizers and therefore, two dimensional maps of fluorescence anisotropy can be easily acquired. We are using steady state fluorescence anisotropy imaging microscopy to study the biochemistry of cell motility. The optimum fluorophore for fluorescence polarization measurements has a fluorescence lifetime that is comparable to the rotational correlation time of the molecule of interest. In order to make imaging measurements with high sensitivity and reasonable time resolution, however, this general rule has to be adjusted, and we have found that FITC-calmodulin has a useful combination of the above features. Calmodulin is a key regulatory protein, that is proposed to be involved in the regulation of the actin-myosin II based force generation in non-muscle cells. The rotational mobility of macromolecules is very sensitive to molecular interactions, yet is relatively insensitive to any surrounding gel matrix. We have taken advantage of this feature to map FITC-calmodulin interactions in the complex cytomatrix in living cells by steady state Fluorescence Anisotropy Imaging Microscopy (FAIM). In addition, we are investigating the use of FAIM for mapping variations in molecular orientation distributions, and fluorescence lifetime distributions.