MEMBRANE REMODELING DURING VIRAL INFECTION, PARASITE INVASION, AND APOPTOSIS

病毒感染、寄生虫入侵和细胞凋亡期间的膜重塑

• 批准号: 6290226
• 负责人: JOSHUA ZIMMERBERG
• 金额: --
• 依托单位: EUNICE KENNEDY SHRIVER NATIONAL INSTITUTE OF CHILD HEALTH & HUMAN DEVELOPMENT
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/6290226
• 关键词: hemagglutinin; influenzavirus A; membrane fusion; microorganism hemagglutinin; protein folding; protein structure function; tissue /cell culture; video microscopy; virus cytopathogenic effect; virus protein

We continued our work on the membrane biology of pathogenic processes. Membrane fusion intermediates induced by the glycosylphosphatidylinositol-linked ectodomain of influenza hemagglutinin (GPI-HA) were investigated with simultaneous recordings of whole-cell admittance and fluorescence, in addition to rapidly frozen, freeze-substitution, thin section electron microscopy. Upon triggering with acid solution conformational changes in HA expressed by CHO cells and bound to red blood cells, the previously separated membranes developed a multiplicity of 10-50 nm points of membrane coalescence which did not enlarge further or combine. Physiological measurements showed fast lipid dye mixing between cells after acidification, and small fusion pores developed in a significant fraction of the cell pairs. Those GPI-HA induced pores behaved qualitatively similar to HA induced pores but never expanded. Lipid mixing was detected either prior to or during pore opening. These findings indicate that the transmembrane domain of HA, though not essential for the pore formation, is strictly required to compose the fusion complex in a way to ensure the pore opening and its subsequent expansion. Second, the energetics underlying the expansion of fusion pores, which determines pore growth, was studied. For two homogeneous fusing membranes under different tensions, pore growth can be quantitatively described by treating the pore as a quasi-particle that moves in a medium with a viscosity determined by that of the membranes. This treatment explains how increases in tension through osmotic swelling of vesicles cause enlargement of pores between the vesicles and planar bilayer membranes. The calculations also show that for biological fusion, pore expansion can be regulated by pore length: the membrane mechanics of pore lengthening is an energetically favored process.Third, membrane permeability changes in apoptosis were studied. Release of proteins through the outer mitochondrial membrane is a critical step in apoptosis, and the localization of apoptosis-regulating Bcl-2 family members there suggests they control this process. We used planar phospholipid membranes to test the effect of full-length Bax and Bcl-xL synthesized in vitro, and native Bax purified from bovine thymocytes. Instead of forming pores with reproducible conductance levels expected for ionic channels, Bax, but not Bcl-xL, created arbitrary and continuously variable changes in membrane permeability, and decreased the stability of the membrane, regardless of the origin of the protein. This breakdown of the membrane permeability barrier and destabilization of the bilayer was quantified using membrane lifetime measurements. Bax decreased membrane lifetime in a voltage and concentration-dependent manner. Bcl-xL did not protect against Bax induced membrane destabilization, supporting the idea that these two proteins function independently. Corresponding to a physical theory for lipidic pore formation, Bax potently diminished the linear tension of the membrane (i.e. the energy required to form the edge of a new pore). We suggest that Bax acts directly by destabilizing the lipid bilayer structure of the outer mitochondrial membrane, forming a pore -- the apoptotic pore -- large enough to allow mitochondrial proteins such as cytochrome c to be released into the cytosol. Bax could then enter and permeabilize the inner mitochondrial membrane through the same hole. - influenza, viral envelope, hemagglutinin, membrane tension, bax, bcl, mitochondria, phospholipid bilayer, fusion pore, hemifusion.

我们继续研究致病过程的膜生物学。通过同时记录全细胞入院和荧光，除了快速熟料，冷冻剂，薄片薄片，薄片，薄型，通过全细胞入院和荧光的同时记录，还研究了由流感血凝素（GPI-HA）诱导的膜融合中间体（GPI-HA）诱导的。在用酸溶液触发CHO细胞表达并与红细胞结合的HA的构象变化后，先前分离的膜形成了10-50 nm的膜聚合点的多重性，并未进一步扩大或结合。生理测量结果显示酸化后细胞之间的快速脂质染料混合，并且在很大一部分细胞对中形成的小融合孔。那些GPI-HA诱导的毛孔在质量上与HA诱发的毛孔相似，但从未扩展。在打开孔隙之前或期间检测到脂质混合。这些发现表明，虽然对于孔形成而言并不是必需的HA的跨膜结构域，但严格要求以确保孔口的打开及其后续扩展的方式组成融合络合物。其次，研究了融合孔扩展的基础的能量学，该融合孔的扩张确定了孔的生长。对于在不同紧张局部下的两个均匀融合膜的膜，可以通过将孔作为一个准颗粒来定量地描述孔，该准粒子在培养基中以粘度由膜的粘度移动。该治疗方法解释了囊泡渗透肿胀如何增加张力的增加，导致囊泡和平面双层膜之间的毛孔扩大。该计算还表明，对于生物融合，孔的膨胀可以通过孔长度调节：毛孔延长的膜力学是一个能量偏爱的过程。研究了凋亡的三分之二，膜通透性的变化。通过线粒体外膜释放蛋白质是细胞凋亡的关键步骤，在那里调节细胞凋亡的BCL-2家族成员的定位表明他们控制了这一过程。我们使用平面磷脂膜来测试体外合成的全长BAX和BCL-XL的作用，并从牛胸腺细胞中纯化的天然BAX。与其形成对离子通道预期可再现电导水平的孔，而不是bcl-xl，而是产生了膜通透性的任意和连续变化的变化，而不论蛋白质的起源如何，膜的渗透性变化并降低了膜的稳定性。使用膜寿命测量对双层的膜渗透性屏障和双层的不稳定分解。 Bax以电压和浓度依赖性方式降低膜寿命。 Bcl-XL不能预防BAX诱导的膜破坏稳定，这支持了这两种蛋白独立起作用的观念。与脂质孔形成的物理理论相对应，Bax有效地减少了膜的线性张力（即形成新孔的边缘所需的能量）。我们建议BAX直接通过破坏线粒体外膜的脂质双层结构的稳定，从而形成毛孔 - 凋亡孔 - 足够大，可以允许线粒体蛋白（例如细胞色素c）释放到细胞质中。然后，Bax可以进入并通过同一孔进入线粒体膜。 - 流感，病毒包膜，血凝素，膜张力，BAX，BCL，线粒体，磷脂双层，融合毛孔，半缩。