Membrane Remodeling in Viral Infection, Parasite Invasion, Apoptosis, and Cancer

1.疟疾寄生虫出口中的钙。在寄生虫排出之前，细胞质内游离钙离子的稳定增加被发现。这种增加至少在细胞周期的最后两个小时是独立于细胞外钙离子的，但依赖于细胞内钙离子的释放。在周期的最后45分钟内，细胞内BAPTA与钙离子的络合抑制了寄生虫空泡膨胀和红细胞膜穿孔之前的出口，这是寄生虫出口之前的两个典型的形态变化。寄生虫内质网(ER)Ca~(2+)-ATPase的抑制剂加速寄生虫的外流，表明内质网内的Ca~(2+)储存足以支持寄生虫的外流。在使用钙离子载体A23187的成熟裂殖体中，明显加速了明显存活的寄生虫的排出。离子载体处理克服了BAPTA诱导的寄生虫出口的阻断，证实了游离钙离子在出口启动中是必不可少的。离子载体处理未成熟裂殖体会导致寄生空泡肿胀，杀死寄主细胞内的寄生虫。 总之，寄生虫的出口程序需要细胞内的游离钙离子来启动出口、液泡膨胀和宿主细胞的细胞骨架消化。寄生性液泡肿胀是一种不受影响的出口阶段，它依赖于细胞内钙离子的增加，这一证据表明了离子载体诱导出口的机制，以及将寄生虫从宿主细胞中释放出来的程序中钙离子的新靶点。提出了一种依赖于细胞内游离钙离子增加的外流调节途径。 2.脂类结构域的化学成像：为了直接探测HA结构域的PM化学，我们与高分辨率成像二次离子质谱仪(SIMS)的先驱Mary Kraft，UI博士建立了合作关系。我们确定了保存PM分子组织的条件，并测量了稳定表达HA的小鼠成纤维细胞PM中代谢结合的15N-鞘磷脂的分布，检测了PM中直径100 nm的鞘磷脂斑块鞘磷脂结构域被细胞骨架的破坏强烈干扰，但不对胆固醇耗竭做出反应，并且独立于PM定位的血凝素(50%共定位)而存在。这些鞘脂结构域是温度不敏感的，几乎不是圆形的-它们的起源不是尾部相互作用主导的具有典型线张力的相分离。我们还成功地用15N-鞘磷脂对表达HA的成纤维细胞进行了代谢标记，使90%的细胞鞘脂含有一个15N同位素，60%的细胞胆固醇含有一个18O同位素。在用化学固定法稳定脂质后，通过直接实时观察活细胞固定过程中荧光PM鞘磷脂的变化，用扫描电子显微镜(SEM)和SIMS对细胞进行成像。可以看到15N-富集度升高的膜域，因此可以看到高15N-磷脂丰度，这证实了我们之前的测量结果。令人惊讶的是，同一细胞的18O富集区并没有显示出胆固醇富集区。鞘磷脂结构域和非结构域中的18O-胆固醇丰度没有显著差异。因此，PM鞘脂结构域不会因胆固醇而显着丰富。相反，18O-胆固醇似乎均匀地分布在质膜中。这项工作具有非常重要的意义，因为它首次报道了胆固醇和鞘脂已经在完整细胞的质膜上直接成像，而不使用潜在的扰动标记。 3.细胞骨架与流感血凝素簇的相互作用。HA簇与肌动蛋白共定位。活细胞中的单个分子轨迹表明，HA在肌动蛋白上的迁移率受到限制。然而，HA并不直接与肌动蛋白结合，因为它在比肌动蛋白重塑时间短得多的时间内是可移动的。肌动蛋白结合蛋白cofilin被排除在HA簇几百纳米范围内的区域，这表明HA控制着一种细胞骨架试剂。这就提出了膜蛋白-细胞骨架串扰的概念。虽然细胞骨架组织膜蛋白的想法并不新鲜，但在这里，HA正在组织细胞骨架。这种角度依赖关系不是随机的，这与HA扩散受到边界反射的假设一致，这是由于扩散障碍栅栏意味着一组相对固定的纠察队。然而，即使是受到限制的房委会也在行动。要么是有另一个尚未被发现的纠察队，要么是这个模式错了。为了进行测试，我们将对这些细胞中的HA结合成分进行下拉，并进行蛋白质组质谱分析。我们知道，细胞裂解条件(洗涤剂、超声波和温度)对这些分析至关重要，并可能影响被确定为结合伙伴的蛋白质。我们将考虑使用渗透胁迫在整个下拉过程中将弱结合伙伴保持在一起。原位细胞与离体膜蛋白质组超微结构的差异成像。

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 1μ diameter 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.