1. Calcium in malaria parasite egress. A steady increase in cytoplasmic free Ca2+ is found to precede parasite egress. This increase is independent of extracellular Ca2+ for at least the last two hours of the cycle, but is dependent upon Ca2+ release from internal stores. Intracellular BAPTA chelation of Ca2+ within the last 45 minutes of the cycle inhibits egress prior to parasitophorous vacuole swelling and erythrocyte membrane poration, two characteristic morphological transformations preceding parasite egress. Inhibitors of the parasite endoplasmic reticulum (ER) Ca2+-ATPase accelerate parasite egress, indicating that Ca2+ stores within the ER are sufficient in supporting egress. Markedly accelerated egress of apparently viable parasites was achieved in mature schizonts using Ca2+ ionophore A23187. Ionophore treatment overcomes the BAPTA-induced block of parasite egress, confirming that free Ca2+ is essential in egress initiation. Ionophore treatment of immature schizonts had an adverse effect inducing parasitophorous vacuole swelling and killing the parasites within the host cell. In conclusion, the parasite egress programme requires intracellular free Ca2+ for egress initiation, vacuole swelling, and host cell cytoskeleton digestion. The evidence that parasitophorous vacuole swelling, a stage of unaffected egress, is dependent upon a rise in intracellular Ca2+ suggests a mechanism for ionophore-inducible egress and a new target for Ca2+ in the programme liberating parasites from the host cell. A regulatory pathway for egress that depends upon increases in intracellular free Ca2+ is proposed. 2.Chemical Imaging of Lipid Domains: To directly probe PM chemistry of HA domains, we have established a collaboration with Dr. Mary Kraft, UI, the pioneer of high-resolution imaging secondary ion mass spectrometry (SIMS) of lipid bilayers. We determined conditions for preserving PM molecular organization and measured the distributions of metabolically incorporated 15N-sphingolipids in the PM of mouse fibroblast cells stably expressing HA, detecting 100 nm 1μdiameter sphingolipid patches in the PM. Sphingolipid domains are strongly perturbed by disruption of the cytoskeleton, but not in response to cholesterol depletion, and exist independently of PM localized hemagglutinin (50% colocalization). These sphingolipid domains were temperature insensitive and hardly circular - their origin was not a tail-interaction dominated phase separation with typical line tensions. We were also successful in metabolically labeling fibroblasts expressing HA with 15N-sphingolipid such that 90% of the cellular sphingolipids contained one 15N isotope, and 60% of the cellular cholesterol contained one 18O isotope. After stabilizing the lipids using chemical fixation, as measured by direct real-time observation of fluorescent PM sphingomyelin during fixation of live cells, cells were imaged by scanning electron microscopy (SEM) followed by SIMS. Membrane domains with elevated 15N-enrichment, and thus high 15N-sphinoglipid abundance were visible, confirming our previous measurements. Surprisingly, the 18O-enrichment images of the same cell did not reveal cholesterol-enriched domains. No significant difference in the 18O-cholesterol abundance within the sphingolipid domain and non-domain regions was detected. Thus PM sphingolipid domains are not significantly enriched with cholesterol. The 18O-cholesterol instead appears to be evenly distributed within the plasma membrane. This work is highly significant because it reports for the first time that cholesterol and sphingolipids have been directly imaged in the plasma membrane of an intact cell without the use of potentially perturbing labels. 3. Interaction of the cytoskeleton with the clusters of influenza hemagglutinin. HA clusters co-localize with actin. Individual molecular trajectories in live cells show restricted HA mobility on actin. However, HA does not directly bind to actin, since it is mobile on timescales much shorter than those of actin remodeling. The actin binding protein cofilin was excluded from regions within several hundred nanometers of HA clusters, suggesting that HA controls a cytoskeletal agent. This suggests the concept of membrane protein-cytoskeletal crosstalk. While the idea of the cytoskeleton organizing membrane proteins is not new, here HA is organizing the cytoskeleton. The angular dependence is not random, consistent with the hypothesis that HA diffusion is constrained by boundary reflection due to diffusional barriers fences implying a relatively immobile set of pickets. Yet, even the restricted HA moves. Either there is another, as yet undetected, picket or this model is wrong. To test, we will carry out a pull-down of HA binding components in these cells, and perform proteomic mass spectrometry. We are aware that the cell lysis conditions (detergent, sonication and temperature) will be critical for these assays and may influence the proteins identified as binding partners. We will consider using osmotic stress to keep weak binding partners together throughout the pull-down procedure. differential imaging of cell vs. isolated membrane proteomic ultrastructure in situ.
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