ELECTROSTATICALLY MEDIATED MEMBRANE FUSION

描述(摘自摘要)：膜融合是一种重要的 所有动植物细胞特有的功能。它涉及到一个 一组复杂的膜相互作用，其中很少被理解为 细节。本提案的总体目标是了解膜 在简单的脂质系统中融合，从而获得的知识可以 应用于理解生物膜融合，回答了一些 膜静电学的基本问题及其发展 高效地将物质输送到细胞的脂质体。该提案是 促使人们发现一些O-烷基磷脂酰胆碱 化合物，一类新的具有净正电荷的磷脂 最近，通过另一个项目Form Giant可以使用 与阴离子类脂小泡融合的小泡。因为这些 在荧光作用下，可以对囊泡进行电泳性操作 显微镜下，一个独特的机会出现了研究双层融合 比目前为止可能的细节要多得多。既然是这样 发现了这一现象，发现a)双层囊泡 可以无泄漏地融合，b)双层可以进行半融合(融合 当小泡附着在一个小泡上时，接触到的两个单层 另一种)以几种形态不同的方式，c)囊泡 成分(随附的未带电类脂的种类和比例 荷电脂质)对可能的比例有控制的影响 结果，即融合是半融合的模式之一，还是稳定的 附着力占主导地位，d)阳离子的物理性质 当与磷脂混合时，磷脂发生了根本的变化 阴离子脂，尤指阴离子脂中分子所占据的面积。 单层被减少，并且这些混合物可以瞬时形成三个- 相互连接的双层的尺寸图案(立方相)。它是 假设，在相反电荷的囊泡融合中， 粘附膜之间的接触区不稳定是由于 为了中和电荷，导致接触的重组 曲面和融合。阳离子脂类已经被制备成形成相 这应该会促进融合，以及在荧光版本中将 便于监测膜融合的细节。具体目标是： 1.鉴定易形成的阳离子磷脂 囊泡粘连条件下的膜不稳定相。2. 为了刻画负泡和负泡之间的融合过程 含有脂类的阳性小泡被鉴定为易于融合。3.至 测量与这些工艺相关的膜静电特性。 4.将目标1-3中的信息应用于脂质体与细胞的融合 以及病毒膜组分的功能测试。

DESCRIPTION (Adapted from abstract): Membrane fusion is an essential and characteristic function of all plant and animal cells. It involves a complex set of membrane interactions, few of which are understood in detail. The overall goals of this proposal are to understand membrane fusion in a simple lipid system so that the knowledge acquired can be applied to understanding biological membrane fusion, answering some fundamental problems of membrane electrostatics, and developing liposomes that efficiently deliver materials to cells. The proposal is prompted by the discovery that some o-alkyl-phosphtidylcholinium compounds, a new class of phospholipids with a net positive charge that very recently became available through another project, form giant vesicles that fuse with vesicles of anionic lipids. Because these vesicles can be manipulated electrophoretically under the fluorescence microscope, a unique opportunity arises to examine bilayer fusion in considerably greater detail than possible heretofore. Since this phenomenon was discovered, it has been found that a) bilayer vesicles can fuse without leakage, b) bilayers can undergo hemifusion (fusion of the two monolayers that come into contact when vesicles adhere to one another) in several morphologically different ways, c) the vesicle composition (kind and proportion of uncharged lipid included with charged lipid) has a controlling influence on the proportion of possible outcomes, i.e. whether fusion, one of the modes of hemifusion, or stable adhesion predominates, and d) 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 these mixtures can transiently form a three- dimensional pattern of interconnected bilayers (cubic phase). It is hypothesized that, in fusion of oppositely-charged vesicles, destabilization of the contact region between adherent membranes is due to charge neutralization, leading to reorganization of the contacting surfaces and fusion. Cationic lipids have been prepared that form phases which should promote fusion as well as in fluorescent versions that will facilitate monitoring details of membrane 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 negative vesicles and positive vesicles containing lipids identified as fusion-prone. 3. To measure membrane electrostatic properties relevant to these processes. 4. To apply information from aims 1-3 to fusion of liposomes with cells and to functional tests of viral membrane components.