Dynamics of Raft Formation and Growth

筏形成和生长的动力学

基本信息

批准号：
8197570
负责人：
FREDRIC S COHEN
金额：
$ 28.52万
依托单位：
RUSH UNIVERSITY MEDICAL CENTER
依托单位国家：
美国
项目类别：
财政年份：
2003
资助国家：
美国
起止时间：
2003-01-01 至 2013-11-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8197570
关键词：
Address Affect Alanine Atherosclerosis Biological Biological Models Biological Process Body Temperature Cell membrane Cell physiology Cell surface Cells Centrifugation Cholesterol Collection Data Detergents Disease Ensure Gangliosides Grant Growth Health Height Ice Length Leucine Lipid Bilayers Lipids Liposomes Literature Location Malignant Neoplasms Measures Membrane Membrane Microdomains Membrane Proteins Methods Modeling Peptides Phase Phospholipids Physical Chemistry Physiological Play Positioning Attribute Procedures Property Protein Analysis Proteins Research Personnel Resistance Role Rupture Signal Transduction Sphingolipids Sphingomyelins Sterols Stroke Structure Sucrose System Temperature Testing Time Transmembrane Domain Tryptophan Ursidae Family Vesicle cell growth cold temperature density monolayer pressure protein distribution research study residence stoichiometry symposium theories

项目摘要

Summary Membrane proteins that are critical for cell signaling and other biological processes reside in rafts, but rafts have been difficult to study because they have submicroscopic sizes, dynamic structures, and components that do not have fixed stoichiometries. The compositions of rafts - proteins and lipids - must be determined in order to understand which proteins interacting in cellular cascades come into proximity with each other as a result of their residence in a raft. A new experimental procedure now allows cellular rafts to be isolated at the physiological temperature of 37¿C, rather than the previously necessary temperature of 4¿C, enabling basic question in raft studies to be addressed. The compositions of many domains that exist within a cell membrane at mammalian body temperature (37¿C) are likely to be different from those of cell membranes that are kept on ice (4¿C), so many domains cannot be reliably determined at low temperature. The composition of rafts at biological temperature can now be determined, and membrane anchors of proteins favored in rafts can be compared to data derived at 4¿C. The relationship between types of proteins (GPI-anchored, transmembrane domain, prenylated) and amounts of cholesterol in a raft will be classified. Comparison of cholesterol levels in different types of rafts will uncover mechanisms that cause cholesterol to move between rafts. The displacement of one protein by another inside a raft, and the resulting effect on cellular processes, if any, will be assessed. The physical chemistry that controls the relationship between cholesterol and sphingomyelin content will be studied in a model raft system. The model system also allows the experimentally elusive question of how proteins contribute to raft formation to be approached in a concrete manner. For cellular studies, a new method will be employed that exploits the fact that the pressure needed to rupture a vesicle depends directly on the lipid composition of its membrane. Combining this method with the traditional approach of separation by membrane density will allow collection of a large range of domains. This will provide, for the first time, analysis of protein and lipid raft content without alteration caused by low temperature, detergents, and/or alkaline pH required by all previous raft isolation procedures. Isolating model bilayer domains containing a peptide and measuring compositions will determine the physical mechanisms that create these domains. This will yield experimentally testable hypotheses of mechanisms of biological domain formation.

概括对于细胞信号传导和其他生物过程至关重要的膜蛋白位于筏中，但筏子很难研究，因为它们具有微观尺寸，动态结构和没有固定石化的组件。筏子的组成 - 蛋白质和脂质 - 必须确定以了解在细胞级联中相互作用的蛋白由于他们在木筏中的住所，彼此之间相互接近。现在是一个新的实验程序允许在37 c的物理温度下隔离细胞筏，而不是以前必要的温度为4？c，可以解决筏研究中的基本问题。这在哺乳动物体温下在细胞膜内存在的许多域的组成（37€）可能与保存在冰上的细胞膜的不同（4？c）有所不同，所以很多域不能在低温下确定可靠。筏子在生物学上的组成现在可以确定温度，并且可以在筏中偏爱的蛋白质的膜锚与在4¿C中得出的数据相比跨膜结构域，婚前化）和筏中的胆固醇量将被分类。在不同类型的筏子中比较胆固醇水平将发现引起的机制胆固醇在筏之间移动。一种蛋白质由另一种蛋白质的位移在筏中，而将评估对细胞过程的影响（如果有）。控制的物理化学胆固醇和鞘磷脂蛋白含量之间的关系将在模型筏中研究系统。模型系统还允许实验性的弹性问题，即蛋白质如何贡献以混凝土方式接近筏的形成。对于细胞研究，将是一种新方法采用利用这样一个事实，即破裂囊泡所需的压力直接取决于其膜的脂质组成。将此方法与传统的分离方法相结合按膜密度将允许收集各种域。这将提供第一个时间，分析蛋白质和脂质筏含量，而没有低温引起的改变，确定所有先前的筏隔离程序所需的pH值和/或酒精pH。隔离模型包含肽和测量成分的双层域将确定物理创建这些域的机制。这将产生实验可检验的假设生物领域形成的机制。