Walter D. Niles

Reconstituting channels into planar membranes : a conceptual framework and methods for fusing vesicles to planar bilayer phospholipid membranes

Protocols to reconstitute channels into planar bilayers via fusion methods have now been developed. The greater the intravesicular pressures generated, the greater is the fusion. These pressures can be calculated exactly for any experimental configuration. For some of the configurations, adding nystatin channels to the vesicle membrane will greatly aid fusion. The configurations of the 1990 Method (Figs. 4 and 5) are optimal for fusing vesicles that are reconstituted with ion-selective channels to planar membranes. Greater binding, and ultimately greater fusion, is achieved by ejecting vesicles directly at the membrane rather than by simply adding material to the cis compartment.

Planar bilayer membranes made from phospholipid monolayers form by a thinning process.

We investigated the manner in which planar phospholipid membranes form when monolayers are sequentially raised. Simultaneous electrical and optical recordings showed that initially a thick film forms, and the capacitance of the film increases with the same time course as the observed thinning. The diameter of fully thinned membranes varies from membrane to membrane and a torus is readily observed. The frequency-dependent admittance of the membrane was measured using a wide-bandwidth voltage clamp whose frequency response is essentially independent of capacitative load. The membrane capacitance dominates the total admittance and the membrane dielectric is not lossy. The specific capacitance of membranes of several mixtures was measured. A schematic diagram of the formation of these membranes is presented.

Single event recording shows that docking onto receptor alters the kinetics of membrane fusion mediated by influenza hemagglutinin

The initial steps of membrane fusion, receptor binding and membrane destabilization, are mediated by the envelope glycoprotein hemagglutinin of influenza virus. Interaction between these functions was determined from the time course of individual virion fusions to a planar membrane with and without receptor. With receptor, fusion was described by a Poisson process. In the absence of receptor, the time course was more complicated and could not be described with exponential rate constants. The conversion of a non-Markovian process into a simple Markov chain is direct evidence that receptor binding fundamentally alters the route of fusion.

Radiometric calibration of a video fluorescence microscope for the quantitative imaging of resonance energy transfer

A video microscope for radiometric imaging of resonance energy transfer (RET) between donor and acceptor fluorophores is described and calibrated. The donor is excited by epi‐illumination with monochromatic light. Light emitted by donor and acceptor fluorophores in the specimen is filtered into separate emission images, which are viewed with a pair of multichannel plate image intensifiers in tandem with charge‐coupled device (CCD) cameras. The RET rate constant is calculated at each location in the specimen from the ratio of the donor and acceptor emission intensities. These intensities are computed from the video brightnesses after transformation to irradiances using an experimentally obtained calibration, which accounts for the spectral transmission of the optical pathways and the spectral response of the detectors to determine the energies emitted by the probes in the specimen. We describe procedures for measuring the input irradiance–output brightness relations of the intensifier‐CCD cameras. With the...