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

1.疟疾寄生虫排泄物中的钙。发现胞质游离Ca 2+的稳定增加先于寄生虫出口。这种增加至少在周期的最后两个小时内不依赖于细胞外Ca 2+，但依赖于内部储存的Ca 2+释放。在周期的最后45分钟内，细胞内BAPTA螯合Ca2+抑制寄生虫空泡肿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特征性形态学转变。寄生虫内质网（ER）Ca2 +-ATP酶的抑制剂加速寄生虫的出口，表明ER内的Ca2+储存足以支持出口。明显加速出口的明显可行的寄生虫实现了在成熟的cathionts使用Ca2+离子载体A23187。离子载体治疗克服了BAPTA诱导的寄生虫出口阻滞，证实游离Ca2+在出口启动中是必不可少的。离子载体处理未成熟的寄生虫有不良影响，诱导寄生空泡肿胀和杀死宿主细胞内的寄生虫。 总之，寄生虫出口程序需要细胞内游离Ca2+的出口启动，液泡肿胀，和宿主细胞骨架消化。寄生虫空泡肿胀，一个阶段的不受影响的出口，是依赖于细胞内Ca2+的上升的证据表明，离子载体诱导的出口机制和一个新的目标Ca2+在程序中解放寄生虫从宿主细胞。提出了一种依赖于细胞内游离Ca2+增加的出口调节途径。 2.脂质结构域的化学成像：为了直接探测HA结构域的PM化学，我们与UI的玛丽克拉夫特博士建立了合作关系，她是脂质双层的高分辨率成像二次离子质谱（SIMS）的先驱。我们确定的条件下，保留PM分子组织和测量的分布代谢纳入15 N-鞘脂在PM的小鼠成纤维细胞稳定表达HA，检测100 nm 1直径的鞘脂补丁在PM。μ鞘脂结构域被细胞骨架的破坏强烈干扰，但不响应胆固醇消耗，并且独立于PM定位的血凝素存在（50%共定位）。这些鞘脂结构域是温度不敏感的，几乎没有圆形-它们的起源不是具有典型线张力的尾部相互作用主导的相分离。我们还成功地用15 N-鞘脂代谢标记表达HA的成纤维细胞，使得90%的细胞鞘脂含有一种15 N同位素，60%的细胞胆固醇含有一种18 O同位素。在使用化学固定稳定脂质后，如通过在活细胞固定期间直接实时观察荧光PM鞘磷脂所测量的，通过扫描电子显微镜（SEM）随后通过SIMS对细胞成像。膜结构域与升高的15 N-富集，因此高15 N-鞘脂丰度是可见的，证实了我们以前的测量。令人惊讶的是，相同细胞的18O富集图像没有显示胆固醇富集结构域。鞘脂结构域和非结构域区域内的18 O-胆固醇丰度没有检测到显着差异。因此，PM鞘脂结构域不显著富含胆固醇。而18O-胆固醇似乎均匀分布在质膜内。这项工作是非常重要的，因为它首次报道了胆固醇和鞘脂在完整细胞的质膜中直接成像，而不使用潜在的干扰标记。 3.细胞骨架与流感病毒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显示肌动蛋白上的HA流动性受限。然而，HA不直接结合肌动蛋白，因为它是移动的时间尺度上远远短于肌动蛋白重塑。肌动蛋白结合蛋白cofilin被排除在几百纳米的HA簇内的区域，表明HA控制细胞骨架剂。这表明了膜蛋白-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HA正在组织细胞骨架。角度依赖性是不随机的，与假设HA扩散受到边界反射的约束，由于扩散障碍栅栏意味着一组相对固定的纠察。然而，即使是受限制的房委会也会采取行动。要么还有另一个尚未被发现的纠察队，要么这个模型是错误的。为了测试，我们将在这些细胞中进行HA结合组分的下拉，并进行蛋白质组学质谱分析。我们知道细胞裂解条件（去污剂、超声处理和温度）对于这些试验至关重要，可能会影响被鉴定为结合伴侣的蛋白质。我们将考虑在整个下拉过程中使用渗透压来保持弱结合配偶体在一起。细胞与分离的膜蛋白质组原位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.