MEMBRANE REMODELING DURING VIRAL INDUCED SYNCYTIA FORMATION & PARASITE INVASION

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We are continuing our work on fusion pores, the first detectable intermediate in biological fusion. We previously described pores induced by the influenza envelope protein hemagglutinin (HA). We now report features of fusion pores induced by another viral protein, baculovirus GP64. Unlike for HA, the GP64-induced fusion pore on Sf9 cells has a large initial conductance, does not flicker, and forms much faster with kinetics which fits best to a parallel model with many independent elements. To investigate if features of fusion pores are determined only by fusion proteins or they could be influenced by other viral envelope proteins or characteristics of target membranes, we express GP64 in a stably transfected cell line (Op-1D) and study fusion of Op-1D cells with erythrocytes. Red blood cells were previously used as target cells for research on HA-induced fusion. Our preliminary analysis showed that other baculovirus envelope proteins or features of target membrane unlikely interfere with initial pore conductance but rather have an impact on fusion pore kinetics. Another pore figures in membrane remodelling, the fission pore. Most intracellular pathogens avoid lysing their host cells during invasion by wrapping themselves in a vacuolar membrane. This parasitophorous vacuole membrane (PVM) is often retained, serving as a critical transport interface between the parasite and the host cell cytoplasm. To test whether the PVM formed by the parasite Toxoplasma gondii is derived from host cell membrane or from lipids secreted by the parasite, we used time-resolved capacitance measurements and video microscopy to assay host cell surface area during invasion. We observed no significant change in host cell surface area during PVM formation, demonstrating that the PVM consists primarily of invaginated host cell membrane. Pinching off of the PVM from the host cell membrane occurred after an unexpected delay (34-305 s) and was seen as a 0.219 +/- 0.006 pF drop in capacitance, which corresponds well to the predicted surface area of the entire PVM (30-33 m2). The formation and closure of a fission pore connecting the extracellular medium and the vacuolar space was detected as the PVM pinched off. This final stage of parasite entry was accomplished without any breach in cell membrane integrity.