RED CELL DEFORMABILITY IN VITRO AND SURVIVAL IN VIVO

描述：（改编自研究者摘要）长期 本建议的目的是详细了解 红细胞膜生理学的分子基础， 机械功能。本次更新申请的主要目的 是严格定义红细胞膜材料的来源 通过结合新的功能信息， 使用申请人开发的技术， 表征膜的生物物理行为。申请人 他建议通过三个目标来实现这一目标。 具体目标1是确定红细胞膜的分子基础 结构完整性，特别强调 骨架蛋白与膜结合力的侧向连接。他将 通过探索这种假设， 联系将导致膜骨架失败，建议重点 α和β血影蛋白和蛋白质4.1。努力将集中在一个 这些分子的功能结构域的数量由电流定义 结构/功能理解。掺入选定的重组体 肽、抗体和Fab片段将用于干扰 特定的功能结构域，使得机械稳定性的变化可以 可以通过Ektacytometry定量。此外，申请人将 探讨血影蛋白和蛋白4.1磷酸化的作用， 钙/钙调素在调节膜内聚性中的作用。这些 实验将采取的形式影响磷酸化的具体 蛋白质和对膜稳定性的监测作用。此外他 将继续表征现有材料的机械性能 具有确定突变的病理性红细胞，以获得进一步的见解 进入细胞骨架蛋白的功能区域。 具体目标2是确定膜失效的分子基础， 涉及脂质双层与下面的骨架的分离。 使用荧光Fab对某些蛋白质的特异性，他将首先 记录标记组分的荧光密度图，使用 他的荧光成像微量吸管的新技术 抽吸，以及使用FRAP来识别哪些蛋白质是真正的 参与连接双分子层和膜。这样做之后，他 将使用上述技术使完整的膜分离 从骨架中分离蛋白质，以辨别对膜内聚力的影响 （通过机械稳定性评估）。作为这些研究的一部分， 还将检测锚蛋白4.1和p55的贡献。同样地， 可用的病理性红细胞将用于检查 选择性缺陷的修复。从这个具体的数据 目的是确定膜失效的分子基础。 具体目标3将定义双层骨架蛋白的贡献 网络链接到膜变形性，跟进申请人的 关于血型糖蛋白A配体对 膜刚性特别是，抗体的作用， 血型糖蛋白A在带3上的迁移率将使用各种方法进行检查。 扰动和重构策略，选择性地影响 潜在的联系。第三个目标是提供一种分子 膜刚度调节基础的定义。

DESCRIPTION: (Adapted from investigator's abstract) The long-term objective of the present proposal is to develop a detailed understanding of the molecular basis for red cell membrane physiology as it relates to its mechanical function. The principal aim of this renewal application is to critically define the origins of red cell membrane material behavior at the molecular level by combining new functional information on individual proteins with techniques developed by the applicant for characterizing the biophysical behavior of the membrane. The applicant proposes to accomplish this by means of three aims. Specific Aim 1 is to define the molecular basis for red cell membrane structural integrity, with particular emphasis on the contributions of lateral linkages of skeletal proteins to membrane cohesion. He will approach this by exploring the hypothesis that weakening of such linkages will lead to membrane skeletal failure, the proposal focusing on alpha and beta spectrin and protein 4.1. Efforts will focus on a number of functional domains of these molecules defined by current structure/function understanding. Incorporation of selected recombinant peptides, antibodies and Fab fragments will be used to interfere with specific functional domains so that changes in mechanical stability can be quantitated by ektacytometry. In addition, the applicant will explore the role of phosphorylation of spectrin and protein 4.1 and the effects of calcium/calmodulin in regulating membrane cohesion. These experiments will take the form of affecting phosphorylation of specific proteins and monitoring effect on membrane stability. In addition, he will continue to characterize the mechanical properties of available pathologic red cells with defined mutations to gain further insights into the functional domains of cytoskeletal proteins. Specific Aim 2 is to define the molecular basis for membrane failure that involves separation of the lipid bilayer from the underlying skeleton. Using fluoresceinated Fab specific for certain proteins, he will first document the fluorescence density map of the labeled components using his new and novel technique of fluorescence-imaged micropipette aspiration, as well as use FRAP to identify which proteins are truly involved in linking the bilayer to the membrane. Having done so, he will use the above techniques to cause dissociation of integral membrane proteins from the skeleton to discern effect on membrane cohesion (assessed by mechanical stability). As part of these studies, contributions of ankyrin, 4.1, and p55 also will be examined. Likewise, available pathologic red cells will be used to examine effect of reconstitution on selective deficiencies. The data from this specific aim are expected to define the molecular basis for membrane failure. Specific Aim 3 will define a contribution of bilayer-skeletal protein network linkages to membrane deformability, following up the applicant's seminal observations regarding the effect of glycophorin A ligands on membrane rigidity. In particular, the effect that antibodies to glycophorin A have on band 3 mobility will be examined using various strategies of perturbation and reconstitution to selectively influence potential linkages. The third aim is expected to provide a molecular definition of the basis for regulation of membrane rigidity.