Biophysical Properties of Membranes: Lipid-Protein Bonds

描述（由申请方提供）：质膜的物理性质似乎直接调节几种膜机械化学功能的速率;内吞作用、运动性和膜重新密封。特别是，这些功能与将质膜区域移动到囊泡、板状伪足或伤口部位所需的能量成反比。使用激光镊子，我们可以测量将质膜移动到小的膜管或系绳中所需的力。事实上，我们已经表明，系绳力与内吞作用，肌动蛋白依赖的膜延伸和膜重新密封的速率呈负相关。两个因素有助于系绳力：张力在质膜和膜细胞骨架粘附。在这里，我们建议测量整个细胞的反应，以增加质膜张力和定量分析扰动膜细胞骨架粘附发展的分子模型的粘附。 为了测试单独的膜张力是否可以抑制内吞作用和运动性，我们将在系链上使用高静态力以在质膜中产生张力而不肿胀。人们相信，张力在整个细胞上是连续的，我们将测量整个细胞中张力振荡的耦合来测试这一点。耦合度将使我们能够从膜的已知弹性计算展开细胞的弹性参数。 膜-细胞骨架粘附的能量似乎主要由磷酸化肌醇脂质，主要是磷脂酰肌醇4，5二磷酸（PIP 2）的水平设定。为了测试PIP 2的游离浓度或质膜的表面电荷是否与粘附最密切相关，我们将测量粘附作为表达的EGFP-MARCKS效应物或几种不同EGFP-PH结构域的细胞质浓度的函数。显微注射相同的结构域将使我们能够确定粘附变化的时间过程是否与细胞骨架的改变或结合的直接抑制最相关。此外，如果粘附在显微注射后恢复，我们将专注于恢复的机制。这些研究将为粘附的分子模型提供基础，例如PIP 2-PH结构域键密度或肌动蛋白丝密度是粘附的主要决定因素。未来的研究将集中在完善粘附模型。 膜张力、膜-细胞骨架粘附和PIP 2水平涉及除了内吞作用、运动性和膜再密封之外的许多细胞功能，例如体积调节和胞吐作用。使用膜参数和细胞活性的定量物理和生物化学分析，将有可能联合收割机结合对物理和生物化学活性的理解，以开发给定细胞功能的集成模型。

DESCRIPTION (provided by applicant): Physical properties of the plasma membrane appear to directly regulate the rates of several membrane mechanochemical functions; endocytosis, motility, and membrane resealing. In particular, these functions are inversely dependent upon the energy required to move an area of plasma membrane into a vesicle, lamellipodium or wound site. Using the laser tweezers, we can measure the force required to move plasma membrane into small membrane tubes or tethers. Indeed, we have shown that tether force correlates inversely with the rates of endocytosis, actin-dependent membrane extension and membrane resealing. Two factors contribute to the tether force: tension in the plasma membrane and membrane-cytoskeleton adhesion. Here we propose to measure the response of the whole cell to an increase in plasma membrane tension and to quantitatively analyze perturbations of membrane-cytoskeleton adhesion to develop a molecular model of adhesion. To test if membrane tension alone can inhibit endocytosis and motility, we will use high static force on tethers to create tension in the plasma membrane without swelling. It is believed that tension is continuous over the whole cell, and we will measure the coupling of oscillations in tension across the cell to test this. The degree of coupling will enable us to calculate the elastic parameters of spread cells from the known elasticity of the membrane. The energy of membrane-cytoskeleton adhesion appears to be set primarily by the level of phosphorylated inositol lipids, primarily phosphatidylinositol 4,5 diphosphate (PIP2). To test whether the free concentration of PIP2 or the surface charge of the plasma membrane correlates most closely with adhesion, we will measure adhesion as a function of cytoplasmic concentration of expressed EGFP-MARCKS effector or several different EGFP-PH domains. Microinjection of the same domains will enable us to determine if the time course of change in adhesion correlates best with the alteration of the cytoskeleton or direct inhibition of binding. Further, if adhesion recovers after microinjection, we will focus on the mechanism of recovery. These studies will provide the basis for a molecular model of adhesion, e.g. PIP2-PH domain bond density or actin filament density is the primary determinant of adhesion. Future studies will be focussed on refining the model of adhesion. Membrane tension, membrane-cytoskeleton adhesion and PIP2 levels are implicated in many cellular functions in addition to endocytosis, motility, and membrane resealing, e.g. volume regulation and exocytosis. Using quantitative physical and biochemical analyses of membrane parameters and cell activities, it will be possible to combine an understanding of the physical and biochemical activities to develop an integrated model of a given cell function.