Membrane Remodeling in Viral Infection, Parasite Replication, and Traumatic Brain Injury

1. 尽管流感每年造成约50万人死亡，但即使排除大流行，也只有一种抗病毒药物：神经氨酸酶抑制剂。通过使用一种新的方法，利用巨大的单层囊泡和传染性X-31流感病毒，并测试新确定的孔膜融合的中间体，我们观察到3087%的穿孔，这取决于脂质成分。测试的假设，即自发曲率（SC）的脂质单层控制膜穿孔，我们的泊松模型和玻尔兹曼充满活力的考虑建议从泄漏到非泄漏的融合途径的过渡取决于SC的目标膜。当靶膜SC低于约0.20 nm 1时，流感病毒和靶膜之间的融合主要是非渗漏的，而高于该值时，融合主要是渗漏的，这表明在融合期间流感血凝素（HA）催化的靶膜的拓扑转化与膜完整性的丧失相关。 2. 为了了解轻度冲击波诱导的TBI（bTBI）的病理生理学，并确定与原发性损伤阶段相关的物理力的目标，我们开发了一种系统，该系统将气动冲击波耦合到微流体通道，以精确和可重复地将剪切瞬变传递到解离的人中枢神经系统（CNS）细胞，其时间尺度与爆炸性冲击波相当，但具有最小的压力瞬变。使用荧光珠，我们已经表征了细胞所经历的剪切瞬变，并证明该系统能够准确地和可重复地提供均匀的剪切瞬变，并在整个细胞培养体积中具有最小的压力。该系统与高分辨率、延时光学显微镜兼容。使用这个系统，我们证明了爆炸样的剪切瞬变产生的最小的压力瞬变和亚毫秒的上升时间激活钙离子的反应，在解离的人中枢神经系统文化。细胞以增加的胞质游离钙响应于8-25 Pa之间的阈值剪切应力;这种钙响应的传播是嘌呤能信号传导的结果。我们建议该系统在体外模拟爆炸冲击波穿过人类中枢神经系统密度不均匀性后产生的剪切力产生的基本损伤波。 3. 细胞内疟原虫生长在由寄生虫空泡膜（PVM）界定的空泡中。这种膜对寄生虫在其细胞内生态位中的生存起着关键作用，例如蛋白质输出和营养获取。使用条件性敲除，我们在这里证明，丰富的积分PVM蛋白EXP 1是必不可少的寄生虫生存，但这是独立的，其先前假定的功能作为谷胱甘肽S-转移酶。膜片钳实验表明，EXP 1是关键的营养渗透通道活动在PVM。EXP 1的缺失废除了EXP 2的正确定位，EXP 2是PVM处营养渗透通道活性和蛋白质输出所需的成孔蛋白。然而，EXP 1的意外损失仅影响PVM的营养渗透通道活性，而不影响蛋白质输出。具有低水平的EXP 1的寄生虫变得对低营养条件过敏，表明EXP 1确实是营养吸收所需的，并且实验证实了长期存在的假设，即在PVM处测量的通道活性是寄生虫营养获取所需的。因此，EXP 1是EXP 2作为营养渗透通道的功能性表达所特别需要的，并且对于疟原虫的代谢物供应至关重要。 4. 疟原虫的存活。在宿主红细胞（RBC）内的免疫依赖于膜蛋白复合物的功能，该膜蛋白复合物被称为输出蛋白质的疟原虫易位子（PTEX），其将某些寄生虫蛋白质（统称为输出体）输出穿过寄生虫空泡膜（PVM），该空泡膜将寄生虫包裹在宿主RBC细胞质中。PTEX的核心由三种蛋白质组成：EXP 2，PTEX 150和HSP 101 ATP酶;在这三种蛋白质中，只有EXP 2是膜蛋白。研究出口组成员的PTEX依赖性转运，我们发现出口蛋白，如环感染的红细胞表面抗原（RESA），未能在寄生虫中转运，其中寄生虫棒状体蛋白RON 3被有条件地破坏。R 0 N 3缺陷型寄生虫也未能发展到年轮期之后，葡萄糖摄取显著降低。这些发现提供了证据表明，RON 3影响两个易位功能，即，通过PTEX运输寄生虫出口组和将葡萄糖从RBC细胞质运输到寄生虫空泡（PV）空间，在PV空间中，葡萄糖可以通过寄生虫质膜中的己糖转运蛋白（HT）进入寄生虫。

1. Although influenza kills about a half million people each year, even after excluding pandemics, there is only one set of antiviral drugs: neuraminidase inhibitors. By using a new approach utilizing giant unilamellar vesicles and infectious X-31 influenza virus, and testing for the newly identified pore intermediate of membrane fusion, we observed 3087% poration, depending upon lipid composition. Testing the hypothesis that spontaneous curvature (SC) of the lipid monolayer controls membrane poration, our Poisson model and Boltzmann energetic considerations suggest a transition from a leaky to a non-leaky fusion pathway depending on the SC of the target membrane. When the target membrane SC is below approximately 0.20 nm1 fusion between influenza virus and target membrane is predominantly non-leaky while above that fusion is predominantly leaky, suggesting that influenza hemagglutinin (HA)-catalyzed topological conversion of target membranes during fusion is associated with a loss of membrane integrity. 2. Towards the goal of understanding the pathophysiology of mild blast induced TBI (bTBI), and identifying the physical forces associated with the primary injury phase, we developed a system that couples a pneumatic blast to a microfluidic channel to precisely and reproducibly deliver shear transients to dissociated human central nervous system (CNS) cells, on a time scale comparable to an explosive blast but with minimal pressure transients. Using fluorescent beads, we have characterized the shear transients experienced by the cells, and demonstrate that the system is capable of accurately and reproducibly delivering uniform shear transients with minimal pressure across the cell culture volume. This system is compatible with high resolution, time-lapse optical microscopy. Using this system, we demonstrate that blast-like shear transients produced with minimal pressure transients and sub-millisecond rise times activate calcium responses in dissociated human CNS cultures. Cells respond with increased cytosolic free calcium to a threshold shear stress between 8-25 Pa; the propagation of this calcium response is a result of purinergic signaling. We propose that this system models, in vitro, the fundamental injury wave produced by shear forces consequent to blast shock waves passing through density inhomogeneity in human CNS. 3. Intracellular malaria parasites grow in a vacuole delimited by the parasitophorous vacuolar membrane (PVM). This membrane fulfills critical roles for survival of the parasite in its intracellular niche such as in protein export and nutrient acquisition. Using a conditional knockout, we here demonstrate that the abundant integral PVM protein EXP1 is essential for parasite survival but that this is independent of its previously postulated function as a glutathione S-transferase. Patch-clamp experiments indicated that EXP1 is critical for the nutrient-permeable channel activity at the PVM. Loss of EXP1 abolished the correct localization of EXP2, a pore-forming protein required for the nutrient-permeable channel activity and protein export at the PVM. Unexpectedly loss of EXP1 however affected only the nutrient-permeable channel activity of the PVM but not protein export. Parasites with low levels of EXP1 became hypersensitive to low nutrient conditions, indicating that EXP1 indeed is needed for nutrient uptake and experimentally confirming the long standing hypothesis that the channel activity measured at the PVM is required for parasite nutrient acquisition. Hence, EXP1 is specifically required for the functional expression of EXP2 as the nutrient-permeable channel and is critical for the metabolite supply of malaria parasites. 4. The survival of Plasmodium spp. within the host red blood cell (RBC) depends on the function of a membrane protein complex, termed the Plasmodium translocon of exported proteins (PTEX), that exports certain parasite proteins, collectively referred to as the exportome, across the parasitophorous vacuolar membrane (PVM) that encases the parasite in the host RBC cytoplasm. The core of PTEX consists of three proteins: EXP2, PTEX150, and the HSP101 ATPase; of these three proteins, only EXP2 is a membrane protein. Studying the PTEX-dependent transport of members of the exportome, we discovered that exported proteins, such as ring-infected erythrocyte surface antigen (RESA), failed to be transported in parasites in which the parasite rhoptry protein RON3 was conditionally disrupted. RON3-deficient parasites also failed to develop beyond the ring stage, and glucose uptake was significantly decreased. These findings provide evidence that RON3 influences two translocation functions, namely, transport of the parasite exportome through PTEX and the transport of glucose from the RBC cytoplasm to the parasitophorous vacuolar (PV) space where it can enter the parasite via the hexose transporter (HT) in the parasite plasma membrane.