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 sec) 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 Lim2). The formation and closure ofa 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.Toxoplasma gondii is an important pathogen of humans, causing congenital birth defects when acute infection occurs during pregnancy, and severe encephalitis in immunocompromised persons. Toxoplasmic encephalitis has recently emerged as one of the most serious opportunistic infections associated with AIDS (1, 2). Like other Apicomplexan parasites (including Plasmodium, the causative agent of malaria), T. gondii is an obligate intracellular parasite. During invasion, which is an active process distinctly different from phagocytosis (3), the parasite becomes surrounded by the PVM. The PVM pinches off from the host cell membrane at the end of invasion, to create a vacuole within which the parasite grows and replicates to continue the infection. As the interface between the parasite and the host cell, the PVM functions in metabolite uptake, nutrient transport, and protein trafficking (4-7).The origin of the PVM in cells infected with Toxoplasma and other Apicomplexan parasites is unknown, and has been the subject of considerable controversy (8-11). Two main models have been proposed to explain how the PVM is formed. In the bilayer insertion model, the PVM is thought to be formed from lipids that are secreted from apical organelles of the parasite and inserted into the host cell membrane during invasion. Evidence supporting this model includes the absence of host cell membrane proteins from the forming PVM (12-15), and the presence of membrane-like lamellar structures within and emanating from the parasite's apical secretory organelles (16)(17)(18)). An alternative model, induced invagination, proposes that the parasite induces the host cell membrane to invaginate to form the PVM. Evidence supporting this model includes the similar lipid compositions of the host cell membrane and the developing PVM (8). As discussed elsewhere (8, 9), neither model can be ...

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Protein kinase C (PKC) is a key enzyme for many cellular processes but its physiological roles are poorly understood. An excellent opportunity to investigate the function of PKC has been provided by the identification of an eye-specific PKC in Drosophila and a null PKC mutant, inaCP209 (refs 5,6). Bright conditioning lights delivered to inaC photoreceptors lead to an abnormal loss of sensitivity in whole cell recordings from dissociated ommatidia; this has been interpreted as 'hyper-adaptation' and PKC's role has been suggested to be distinct from light adaptation. A presumably related finding is that during intense light, the response of inaC declines to baseline. Invertebrate photoreceptors use the phosphoinositide signalling cascade, responding to single photons with so-called quantum bumps which sum to form the macroscopic response to light. Light adaptation allows photoreceptors to adjust their sensitivity over the enormous range of ambient intensities. Although the molecular mechanism of light adaptation remains obscure, it is a negative-feedback process mediated by a rise in cytosolic calcium and a decrease in bump size. We now show that under physiological conditions light adaptation is severely reduced in inaC, suggesting that eye-specific PKC, itself activated by a rise in cytosolic calcium and diacylglycerol, is required for adaptation. Furthermore, we show that in the absence of PKC individual bumps fail to terminate normally, an effect that can account for the pleiotropic manifestations of the inaC phenotype.

Heparanase is a heparan sulfate (HS) degrading endoglycosidase participating in extracellular matrix degradation and remodeling. Apart of its well characterized enzymatic activity, heparanase was noted to exert also enzymatic-independent functions. Non-enzymatic activities of heparanase include enhanced adhesion of tumor-derived cells and primary T-cells. Attempting to identify functional domains of heparanase that would serve as targets for drug development, we have identified heparin binding domains of heparanase. A corresponding peptide (residues Lys158-Asp171, termed KKDC) was demonstrated to physically associate with heparin and HS, and to inhibit heparanase enzymatic activity. We hypothesized that the pro-adhesive properties of heparanase are mediated by its interaction with cell surface HS proteoglycans, and utilized the KKDC peptide to examine this possibility. We provide evidence that the KKDC peptide interacts with cell membrane HS, resulting in clustering of syndecan-1 and syndecan-4. We applied classical analysis of cell morphology, fluorescent and time-lapse microscopy and demonstrated that the KKDC peptide efficiently stimulates the adhesion and spreading of various cell types, mediated by PKC, Src, and the small GTPase Rac1. These results support, and further substantiate the notion that heparanase function is not limited to its enzymatic activity.