MEMBRANE REMODELING DURING VIRAL INFECTION, PARASITE INVASION, AND APOPTOSIS

我们继续研究致病过程的膜生物学。通过同时记录全细胞导纳和荧光，以及快速冷冻、冷冻取代和薄切片电镜，研究了由流感血凝素（GPI-HA）的糖基磷脂酰肌醇连接外结构域诱导的膜融合中间体。在酸溶液触发CHO细胞表达的HA构象变化并与红细胞结合后，先前分离的膜产生了多个10-50 nm的膜聚结点，这些聚结点不会进一步扩大或合并。生理测量显示，酸化后细胞间的脂质染料快速混合，并且在相当一部分细胞对中形成了小的融合孔。GPI-HA诱导的孔隙在性质上与HA诱导的孔隙相似，但没有扩张。在开孔之前或开孔过程中检测脂质混合。这些发现表明，透明质酸的跨膜结构域虽然不是孔形成所必需的，但在形成融合复合物的过程中是严格需要的，以确保孔的打开和随后的扩张。其次，研究了决定孔隙生长的熔合孔膨胀背后的能量学。对于处于不同张力下的两种均匀融合膜，孔隙的生长可以通过将孔隙视为准颗粒来定量描述，准颗粒在由膜的粘度决定的介质中运动。这种治疗解释了通过囊泡渗透性膨胀而增加的张力是如何导致囊泡和平面双层膜之间的毛孔扩大的。计算还表明，对于生物融合，孔的扩展可以由孔的长度来调节：孔的延长的膜力学是一个能量有利的过程。第三，研究细胞凋亡过程中细胞膜通透性的变化。通过线粒体外膜释放蛋白质是细胞凋亡的关键步骤，凋亡调节Bcl-2家族成员在线粒体外膜的定位表明它们控制着这一过程。我们用平面磷脂膜来测试体外合成的全长Bax和Bcl-xL，以及从牛胸腺细胞中纯化的天然Bax的效果。与离子通道所期望的具有可重复电导水平的孔隙形成不同，Bax（而不是Bcl-xL）产生了任意和连续可变的膜通透性变化，并降低了膜的稳定性，而与蛋白质的来源无关。使用膜寿命测量来量化膜渗透性屏障的破坏和双层的不稳定。Bax以电压和浓度依赖的方式降低膜寿命。Bcl-xL对Bax诱导的膜失稳没有保护作用，这支持了这两种蛋白独立起作用的观点。与脂质孔隙形成的物理理论相对应，Bax有效地降低了膜的线性张力（即形成新孔隙边缘所需的能量）。我们认为Bax的直接作用是破坏线粒体外膜的脂质双层结构，形成一个孔——凋亡孔——大到足以允许线粒体蛋白（如细胞色素c）释放到细胞质溶胶中。然后Bax可以通过相同的孔进入并渗透线粒体内膜。-流感、病毒包膜、血凝素、膜张力、bax、bcl、线粒体、磷脂双分子层、融合孔、半融合。

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.