Electrostatically-mediated membrane fusion

描述(申请人提供)：膜融合是一种基本的细胞功能，涉及到知之甚少且非常复杂的膜相互作用。这项建议的总体目标是阐明简单脂质系统中的膜融合，以便这一知识与我们的新技术一起，可以用于帮助理解生物膜融合，并开发有效地将治疗药物输送到细胞的脂质体。虽然该系统没有确切的生物对应物，但它允许对双层-双层融合进行异常详细的检查，这是生物膜融合的关键阶段。这一提议是基于最近的一项发现，即新型阳离子磷脂形成与巨大阴离子脂质囊泡融合的巨大囊泡。这些囊泡可以通过荧光显微镜下的电压梯度单独操作，这为研究双层融合提供了一个独特的机会，比以前可能的更详细。现在已经发现，(A)这种双层囊泡可以无泄漏地融合，(B)双层可以以至少三种形态上可区分的方式进行半融合(两个接触单分子层的融合)，(C)如快速显微荧光计量学所示，完全融合通常是在半融合之前，(D)囊泡的组成(脂的种类和表面电荷密度)对接触后的结果具有控制性的影响，即完全融合、半融合模式之一或简单的粘连，以及(E)当阳离子磷脂与阴离子脂混合时，尤其是阳离子磷脂的物理性质发生基本的变化分子在单层中占据的面积减少，并产生非片层相(倒六方或立方相)。假设融合是由于粘附膜之间接触区域的不稳定造成的，要么是由于电荷中和和随后的区域冷凝，要么是由于接触区域中非片层脂的积累，这两个过程都将接触单层处理为半融合然后完全融合。具体目的是：1.鉴定在囊泡附着条件下容易形成膜不稳定相的阳离子磷脂。2.研究阳离子磷脂小泡与阴离子磷脂小泡的融合过程。3.应用这些信息开发：(A)与细胞融合的脂质体和(B)病毒膜成分的功能测试。

DESCRIPTION (provided by applicant): Membrane fusion, an essential cellular function, involves poorly understood and very complex membrane interactions. The overall goals of this proposal are to elucidate membrane fusion in a simple lipid system so that this knowledge, along with our new techniques, can be used to help understand biological membrane fusion and to develop liposomes that efficiently deliver therapeutics to cells. While the system has no exact biological counterpart, it allows an unusually detailed examination of bilayer-bilayer merging, a critical stage in biological membrane fusion. The proposal is based on the recent discovery that novel cationic phospholipids form giant vesicles that fuse with giant anionic lipid vesicles. These vesicles can be individually manipulated by voltage gradients under the fluorescence microscope, presenting a unique opportunity to examine bilayer fusion in much greater detail than previously possible. It has now been found that, (a) such bilayer vesicles can fuse without leakage, (b) bilayers can undergo hemifusion (fusion of the two contacting monolayers) in at least three morphologically distinguishable ways, (c) as indicated by fast microspectrofluorometry, full fusion is generally preceded by hemifusion, (d) the vesicle composition (kind of lipid and surface charge density) has a controlling influence on outcomes after contact, i.e., full fusion, one of the hemifusion modes, or simple adhesion and, (e) that the physical properties of cationic phospholipids change in fundamental ways when they are mixed with anionic lipids, in particular, the area occupied by a molecule in a monolayer is reduced and non-lamellar phases (inverted hexagonal or cubic) are generated. It is hypothesized that fusion results from destabilization of the contact region between adherent membranes, either because of charge neutralization and consequent area condensation, or alternatively, because of accumulation of non-lamellar lipids in the contact zone, both of which processes dispose the contacting monolayers to hemifusion and then to full fusion. The specific aims are: 1. To identify cationic phospholipids that are prone to formation of membrane-destabilizing phases under conditions of vesicle adhesion. 2. To characterize the process of fusion between vesicles of such positive phospholipids with anionic phospholipid vesicles. 3. To apply this information to develop: (a) liposomes that fuse with cells and (b) functional tests of viral membrane components.