Electrostatically-mediated membrane fusion

描述（由申请人提供）：膜融合是一种基本的细胞功能，涉及知之甚少且非常复杂的膜相互作用。该提案的总体目标是阐明在一个简单的脂质系统中的膜融合，以便这种知识，沿着我们的新技术，可以用来帮助理解生物膜融合，并开发脂质体，有效地提供治疗细胞。虽然该系统没有精确的生物学对应物，但它允许对双层-双层合并进行异常详细的检查，这是生物膜融合的关键阶段。该建议是基于最近的发现，新的阳离子磷脂形成巨大的囊泡，与巨大的阴离子脂质囊泡融合。这些囊泡可以在荧光显微镜下通过电压梯度单独操作，这提供了一个独特的机会，可以比以前更详细地检查双层融合。现在已经发现，（a）这种双层囊泡可以融合而不渗漏，（B）双层可以进行半融合（两个接触单层的融合），（c）如快速显微荧光光谱法所示，完全融合通常在半融合之前，（d）囊泡组成（脂质种类和表面电荷密度）对接触后的结果具有控制性影响，即，完全融合（半融合模式之一）或简单粘附，以及（e）当阳离子磷脂与阴离子脂质混合时，阳离子磷脂的物理性质发生根本性变化，特别是单层中分子所占据的面积减少，并产生非层状相（倒六边形或立方体）。据推测，融合的结果从粘附膜之间的接触区域的不稳定，无论是因为电荷中和和随之而来的面积冷凝，或者可替代地，因为在接触区中的非层状脂质的积累，这两个过程处理的接触单层半融合，然后完全融合。具体目标是：1.鉴定在囊泡粘附条件下易于形成膜去稳定相的阳离子磷脂。2.表征这种正性磷脂囊泡与阴离子磷脂囊泡之间的融合过程。3.应用该信息开发：（a）与细胞融合的脂质体和（B）病毒膜组分的功能测试。

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.