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，涉及到知之甚少和非常复杂的膜相互作用。本提案的总体目标是阐明简单脂质系统中的膜融合，以便这些知识以及我们的新技术可以用来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，但它可以对生物膜融合的关键阶段——双层-双层融合进行异常详细的检查。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融合巨大的阴离子脂质囊泡。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泄漏，(b)双层囊泡可以以至少三种形态学上可区分的方式进行半融合（两个接触的单层融合），(c)正如快速显微荧光法所示，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）对接触后的结果有控制影响，即完全融合，半融合模式之一。或者是简单的粘附，(e)阳离子磷脂与阴离子磷脂混合时，其物理性质发生了根本性的变化，特别是，单层分子所占的面积减少，产生了非层状相（倒六边形或立方）。据推测，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，要么是由于接触区非层状脂质的积累，这两种过程都使接触的单层膜发生半融合，然后完全融合。具体目标是：1。鉴定在囊泡粘附条件下容易形成膜不稳定相的阳离子磷脂。2. 表征这种阳性磷脂囊泡与阴离子磷脂囊泡融合的过程。3. 将这些信息应用于：(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.