Membrane Remodeling During Viral Infection, Parasite Invasion, And Apoptosis

本项目旨在了解生理和致病过程中膜重塑的物理化学机制。有两个组件： 1.细胞内的疟疾寄生虫在每个无性复制周期后离开宿主红细胞感染邻近细胞。目前还没有直接量化寄生虫释放的方法。为了量化寄生虫的释放过程，将感染恶性疟原虫的人红细胞以最佳密度注射到密封的小室中，在那里它们一直持续到红细胞周期的结束。每一次在红细胞破裂部位释放寄生虫的事件都会在腔壁上留下足迹，由1)分离的寄生虫、2)带有血球蛋白的消化液泡和3)破裂的膜碎片组成。这些足迹在数小时内都是稳定的，可以使用差分干涉对比(DIC)显微镜进行精确识别。寄生虫的相对释放率被定义为在37℃下注入并在室内孵化两小时的所有裂殖体中此类足迹的百分比。该方法重现性好，易于操作，不需要昂贵的设备。此外，这种方法允许分析细胞和释放部位的形态，这增加了有关释放过程和培养质量的信息。该方法用于证明无蛋白质介质引起的裂殖体膨胀抑制了寄生虫的释放。 总之，描述了一种利用显微镜计数寄生虫释放部位的新方法。除了直接估计受感染红细胞中寄生虫的释放外，该方法还提供了接近原虫生命周期末期的正常感染细胞的形态评估，或作为寄生虫释放过程中实验干预结果的病理形式的评估。现在，人们可以准确地估计在周期转换时的相对寄生虫释放速率，而不需要任何与寄生虫入侵的强制性耦合。 2.被包裹的病毒的形状严重依赖于内部蛋白质基质，但基质蛋白质如何控制被膜的几何形状尚不清楚。我们发现，从新城疫病毒中提纯的基质蛋白吸附在磷脂双层上，并凝聚成类流体结构域，引起膜变形和球状囊泡的萌发，如荧光和电子显微镜所见。膜导纳的测量解决了芽的逐渐生长和快速闭合，然后它的分离形成了一个游离囊。囊泡大小分布受萌发区域固有曲率的限制，但因它们的合并而变宽，与病毒大小分布相匹配。因此，基质蛋白实现了结构域驱动的出芽机制，这足以控制这些蛋白脂泡的形状。

This project is aimed at the understanding of the physico-chemical mechanisms of membrane remodeling during physiological and pathogenic events. There are two components: 1. Intracellular malaria parasites leave their host erythrocytes to infect neighbouring cells after each cycle of asexual replication. No method is currently available for the direct quantification of parasite release. To quantify parasite release process, human erythrocytes infected with Plasmodium falciparum were injected into sealed chambers at optimal density, where they progressed through the end of the erythrocyte cycle. Each event of parasite release inside the chamber at the site of erythrocyte rupture leaves on the chamber wall a footprint, composed of 1) separated parasites, 2) a digestive vacuole with haemozoin, and 3) fragments of the ruptured membranes. These footprints are stable for hours, allowing precise identification using differential interference contrast (DIC) microscopy. The relative rate of parasite release is defined as the percent of such footprints out of all schizonts injected and incubated into chamber at 37C for two hours. The method is highly reproducible, easy to perform, and does not require expensive equipment. Additionally, this method allows one to analyse cell and release site morphology, which adds information about the release process and the quality of the culture. The method is used here to show that swelling of schizonts caused by protein-free media inhibits parasite release. In conclusion, a novel method is described to count sites of parasite release by microscopy. Besides the direct estimation of parasite release from infected erythrocytes, this method provides a morphological evaluation of normal infected cells approaching the end of the plasmodial life cycle, or pathological forms accumulated as the result of experimental intervention in the parasite release process. One may now accurately estimate the relative parasite release rate at the time of cycle transition, without any obligatory coupling to parasite invasion. 2. The shape of enveloped viruses depends critically on an internal protein matrix, yet it remains unclear how the matrix proteins control the geometry of the envelope membrane. We found that matrix proteins, purified form Newcastle Disease Virus, adsorb on a phospholipid bilayer and condense into fluid-like domains that cause membrane deformation and budding of spherical vesicles, as seen by fluorescent and electron microscopy. Measurements of the electrical admittance of the membrane resolved the gradual growth and rapid closure of a bud, followed by its separation to form a free vesicle. The vesicle size distribution, confined by intrinsic curvature of budding domains, but broadened by their merger, matched the virus size distribution. Thus, matrix proteins implement domain-driven mechanism of budding, which suffices to control the shape of these proteolipid vesicles.