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。用全细胞导纳和荧光同时记录法，以及快速冷冻、冷冻置换、薄层切片电镜技术，研究了流感血凝素（GPI-HA）糖基磷脂酰肌醇连接胞外域诱导的膜融合中间体。在用酸溶液触发由CHO细胞表达并与红细胞结合的HA的构象变化后，先前分离的膜产生了许多10-50 nm的膜聚结点，其没有进一步扩大或联合收割机结合。生理测量表明，快速脂质染料混合后酸化细胞之间，和小融合孔开发的一个显着的分数的细胞对。这些GPI-HA诱导的孔在性质上与HA诱导的孔相似，但从不扩张。在孔打开之前或期间检测脂质混合。这些发现表明，HA的跨膜结构域，虽然不是孔形成所必需的，但严格要求以确保孔打开及其随后的扩展的方式组成融合复合物。其次，研究了决定孔生长的熔合孔膨胀的能量学。对于两种不同张力下的均质融合膜，可以通过将孔处理为在介质中移动的准颗粒来定量描述孔生长，所述介质具有由膜的粘度确定的粘度。这种治疗解释了如何通过囊泡的渗透溶胀增加张力，导致囊泡和平面双层膜之间的孔扩大。计算还表明，对于生物融合，孔的扩张可以通过孔的长度来调节：孔延长的膜力学是一个能量上有利的过程。第三，研究了细胞凋亡中膜通透性的变化。通过线粒体外膜释放蛋白质是细胞凋亡的关键步骤，并且细胞凋亡调节Bcl-2家族成员在那里的定位表明它们控制这一过程。我们使用平面磷脂膜来测试体外合成的全长Bax和Bcl-xL以及从牛胸腺细胞纯化的天然Bax的效果。而不是形成孔与可重现的电导水平预期的离子通道，Bax，但不是Bcl-xL，创造任意和连续可变的变化，膜的渗透性，并降低了膜的稳定性，无论起源的蛋白质。使用膜寿命测量来量化膜渗透性屏障的这种破坏和双层的不稳定。Bax以电压和浓度依赖性方式降低膜寿命。Bcl-xL不能保护Bax诱导的膜不稳定，支持这两种蛋白独立发挥功能的观点。与多孔形成的物理理论相对应，Bax有效地减少了膜的线性张力（即形成新孔边缘所需的能量）。我们认为，Bax的作用直接通过破坏外线粒体膜的脂质双层结构，形成一个孔-凋亡孔-足够大，允许线粒体蛋白质，如细胞色素c释放到胞质溶胶。然后Bax可以通过同一个孔进入线粒体内膜并渗透。- 流感，病毒包膜，血凝素，膜张力，bax，bcl，线粒体，磷脂双层，融合孔，半融合。