MEMBRANE TRANSPORT AND FUSION

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Membrane topology defines life: the inside of a cell is highly organized at the molecular level, at almost solid-state densities; the outside environment of cells is usually more chaotic. To understand the mechanisms by which cells control their membranes' topologies, we have continued to develop new methods and use new and old techniques to study two aspects of cell membrane remodeling. A new algorithm for cell capacitance measurement has been developed. This algorithm uses calibration jumps caused by a known change in the compensation circuit parameters of the patch clamp amplifier to determine the phase shift and artifactual attenuation, allowing automatic phase angle adjustments and reconstruction of the undistorted admittance values of circuits formed by fusing cells and calculation of the electrical parameters of a single cell. We used this software to measure the electrical properties of fusion induced by two similar but distinct proteins, GP64 for the Op and Ac strains of baculovirus. The conductance of the Op-mediated initial fusion pore varies within a range 0.3 - 1.9 nS with a mean of 0.7 plus minus 0.4 nS. This differs significantly from the Ac-induced pore conductance: 1.0 plus minus 0.3 nS. This lays the foundation for structure/function studies of fusion pore phenotype. In the second membrane event, endocytosis, we are exploring the electrical manifestations of parasite invasion, which culminates in the pinching-off of a parasitic vacuole (PVM). The application of simultaneous optical and capacitance measurements during parasite invasion enabled us for the first time to show that there is no significant change in host cell surface area during invasion of Toxoplasma gondii. Thus the parasite does not contribute a significant amount of lipid to the PVM formation. In addition, pinching off of the PVM from the host cell membrane is seen in each case as a sudden 0.2-0.3 pF drop in Cm. A fission pore forms during the pinching off process of the PVM. The parasitic fission pore is the endocytotic equivalent to the fusion pore. The parasitic fission pore exhibits flicker and semistable stages with an average conductance of 2 nS. The pore reaches a conductance of less than 1 nS just before closing. Correlated optical and electrical records of more than 100 invasions have shown that transient changes in membrane current (spike) occur at precisely the moment that the host cell is first contacted by the apical end of the parasite.