RED CELL DEFORMABILITY IN VITRO AND SURVIVAL IN VIVO

描述：(改编自调查人员摘要)长期 本提案的目标是制定一项详细的理解 红细胞膜生理学的分子基础 它的机械功能。本次续签申请的主要目的是 是批判性地定义红细胞膜材料的起源 结合新的功能信息在分子水平上的行为 用申请人开发的技术对单个蛋白质进行研究 表征膜的生物物理行为。申请人 建议通过三个目标来实现这一目标。 具体目的1是确定红细胞膜的分子基础 结构完整性，特别强调 骨骼蛋白与膜凝聚力的横向连接。他会的 通过探索这样一种假设来探讨这一点 连接将导致膜骨骼衰竭，该提案重点 关于阿尔法和贝塔血影蛋白和蛋白质4.1。努力的重点将是 由电流定义的这些分子的功能结构域的数量 了解结构/功能。经筛选的重组体的掺入 多肽、抗体和Fab片段将被用来干扰 特定的功能域，因此机械稳定性的变化可以 用细胞计数法进行定量。此外，申请者将 探讨血影蛋白和蛋白4.1的磷酸化作用及其机制 钙/钙调蛋白在调节膜凝聚力中的作用这些 实验将采取影响特定蛋白磷酸化的形式 蛋白质和监测对膜稳定性的影响。此外，他还 将继续表征可用的机械性能 具有明确突变的病理红细胞以获得进一步的洞察 进入细胞骨架蛋白的功能域。 具体目标2是定义膜失效的分子基础， 包括从底层骨架中分离脂类双层。 使用针对某些蛋白质的荧光Fab，他将首先 使用以下工具记录已标记组件的荧光密度图 他的荧光成像微吸管新技术 抽吸，以及使用FRAP来鉴定哪些蛋白质是真正的 参与将双层膜连接到膜上。在这样做之后，他 将使用上述技术导致整体膜的解离 从骨骼中识别蛋白质对膜凝聚力的影响 (通过机械稳定性进行评估)。作为这些研究的一部分， 此外，还将研究Ankyrin、4.1和P55的贡献。同样， 可获得的病理红细胞将被用来检验 对选择性缺陷的重构。来自该特定项目的数据 AIM有望确定膜失效的分子基础。 特定目标3将定义双层骨架蛋白的贡献 与膜变形性有关的网络连接，跟进申请人的 血糖素A配体对血管紧张素转换酶影响的精液观察 膜的刚性。尤其是，抗体对 血糖素A在带3上的流动性将用不同的方法检测 有选择地施加影响的扰动和重构策略 潜在的联系。第三个目标预计将提供一种分子 膜刚性调节依据的定义。

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.