Lipid Rafts in Eye Lens: Discrimination by Pulse EPR

眼晶状体中的脂筏：通过脉冲 EPR 进行区分

• 批准号: 7171797
• 负责人: WITOLD K SUBCZYNSKI
• 金额: $ 29.51万
• 依托单位: MEDICAL COLLEGE OF WISCONSIN
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-12-02 至 2009-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7171797
• 关键词: Address; Affect; Age; Aging; Amino Acids; Biological; Biological Models; Blindness; Cataract; Cell membrane; Cells; Characteristics; Cholesterol; Choline; Condition; Consensus; Crystalline Lens; Data; Detergents; Developing Countries; Development; Diffusion; Discrimination; Disease; Elderly; Environment; Eye; Glycerophospholipids; Goals; Human; In Situ; Integral Membrane Protein; Interphase Cell; Lead; Lens Fiber; Lipid Bilayers; Lipids; Liquid substance; Literature; Lymphocyte-Specific p56LCK Tyrosine Protein Kinase; Measurement; Membrane; Membrane Lipids; Membrane Microdomains; Methods; Modeling; Molecular; N-terminal; NR4A1 gene; Nuclear; Optics; Oxygen; Peptides; Phase; Physiologic pulse; Physiological; Process; Proteins; Pulse taking; Rate; Relaxation; Resistance; Role; Sphingolipids; Sphingomyelins; Spin Labels; Staging; Structure; Surface; System; Techniques; Temperature; Testing; Time; Work; age related; aged; crosslink; fiber cell; influenzavirus; lens; lipid disorder; membrane model; oxygen transport; physical property; research study; size; two-dimensional

DESCRIPTION (provided by applicant): The cell membrane has a 2-dimensional liquid-like structure containing domains that form and disperse continuously on various time and space scales. Rafts are membrane domains that require lipid interactions for their formation. The long-term objective of this proposal is to better understand the molecular mechanisms by which rafts form, are maintained and disintegrate in biological membranes, in particular in the plasma membrane of fiber cells of the eye lens. Detergent insolubility, which has been used to define rafts biochemically, does not reflect pre-existing structures and organization of the membrane. Furthermore, such an approach is not useful for understanding the size, lifetime and dynamics of the raft-constituent molecules and the raft itself. To address these issues, it is proposed to apply the pulse EPR spin labeling technique "discrimination by oxygen transport (DOT)" for in situ studies of rafts in both model and cell membranes. Since the spin-lattice relaxation time of spin labels is sufficiently long, membrane dynamics can be observed on the time scale 0.1 - 100 mu s. The DOT method permits discrimination of different membrane domains because the collision rate between O2 and the nitroxide moiety of spin labels (oxygen diffusion-concentration product) can be quite different in these domains. Additionally, membrane domains can be characterized by profiles of the oxygen diffusion concentration product in situ without the need for separation. This method is especially suitable for obtaining time-space characteristics of small/transient domains. It is hypothesized that rafts form liquid-ordered domains in the plasma membrane liquid-disordered environment. Membrane lipid composition as well as protein content is expected to modulate raft size and dynamics. The DOT method will be used to test the hypothesis on well-defined model systems in which domain size and the lipid exchange rate will be controlled by membrane lipid composition, selected protein and peptide content, and temperature. Furthermore, it will be used to study domain structure in cell membranes. These studies will include mature and aged fiber cell membranes in which the increased cholesterol/lipid ratio and elevated level of sphingomyelin create conditions favoring the formation of rafts. It is proposed: 1) to detect coexisting liquid-ordered and liquid-disordered domains in membranes containing cholesterol; 2) to evaluate the size and stability of the raft domains in model membranes made from raft-forming mixtures; 3) to examine how membrane anchored proteins and transmembrane alpha-helical peptides affect the organization and dynamics of these lipid raft domains; and 4) to apply the DOT method to look for raft domains in fiber cell plasma membranes of the eye lens during maturation and aging, as well as membrane models of mature, aged and cataractous lenses. Age-related nuclear cataract is a primary cause of blindness in the elderly in third world countries.

描述（由申请人提供）：细胞膜具有二维液体样结构，该结构包含在各种时间和空间尺度上连续形成和分散的域。筏是需要脂质相互作用以形成的膜结构域。该建议的长期目标是更好地了解筏形成，维持并在生物膜中瓦解的分子机制，特别是在眼镜纤维细胞的质膜中。洗涤剂的不溶性已用于在生化上定义木筏，并不能反映膜的先前结构和组织。此外，这种方法对于理解筏子固定分子和木筏本身的大小，寿命和动力学并不有用。为了解决这些问题，建议将脉冲EPR自旋标记技术“氧气转运（DOT）歧视”用于模型和细胞膜中的筏的原位研究。由于自旋标记的自旋晶格松弛时间足够长，因此可以在时间尺度0.1-100 mu上观察到膜动力学。 DOT方法允许对不同的膜结构域进行区分，因为在这些域中，O2和自旋标记的氮氧化物部分之间的碰撞速率可能完全不同。另外，膜结构域的特征是原位氧扩散浓度产物的曲线而无需分离。该方法特别适合获得小/瞬态域的时空特性。假设木筏在质膜液体隔离环境中形成液体排序的结构域。膜脂质组成以及蛋白质含量有望调节木筏的大小和动力学。 DOT方法将用于测试定义明确的模型系统上的假设，在该模型系统中，域大小和脂质汇率将由膜脂质组成，选定的蛋白质和肽含量以及温度控制。此外，它将用于研究细胞膜中的域结构。这些研究将包括成熟的和老化的纤维细胞膜，其中胆固醇/脂质比增加和鞘磷脂水平升高创造有利于形成筏的疾病。提出：1）检测含有胆固醇的膜中的液体有序和液体溶质结构域的共存； 2）评估由筏形成混合物制成的模型膜中筏域的尺寸和稳定性； 3）检查膜如何锚定蛋白质和跨膜α-螺旋肽影响这些脂质筏结构域的组织和动力学； 4）应用DOT方法在成熟和衰老过程中查找眼镜纤维细胞质膜中的筏域，以及成熟，老化和白内障透镜的膜模型。与年龄相关的核白内障是第三世界国家老年人失明的主要原因。