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）：细胞膜具有二维类液体结构，包含在不同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才能形成的膜结构域。该提案的长期目标是更好地了解生物膜中筏形成、维持和分解的分子机制，特别是在眼晶状体纤维细胞的质膜中。洗涤剂不溶性已用于在生物化学上定义筏，但并不反映膜的预先存在的结构和组织。此外，这种方法对于理解筏组成分子和筏本身的尺寸、寿命和动力学没有用。为了解决这些问题，建议应用脉冲 EPR 自旋标记技术“氧运输判别（DOT）”来对模型和细胞膜中的筏进行原位研究。由于自旋标记的自旋晶格弛豫时间足够长，因此可以在0.1-100μs的时间尺度上观察膜动力学。 DOT 方法允许区分不同的膜域，因为 O2 和自旋标记的硝基氧部分（氧扩散浓缩产物）之间的碰撞率在这些域中可能有很大不同。此外，膜域可以通过原位氧扩散浓度产物的分布来表征，而无需分离。该方法特别适合获取小/瞬态域的时空特征。据推测，筏在质膜液体无序环境中形成液体有序域。膜脂质组成和蛋白质含量预计会调节筏的大小和动力学。 DOT 方法将用于测试明确的模型系统的假设，其中结构域大小和脂质交换率将由膜脂组成、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和老化的纤维细胞膜，其中胆固醇/脂质比率的增加和鞘磷脂水平的升高创造了有利于筏形成的条件。建议：1）检测含有胆固醇的膜中共存的液体有序结构域和液体无序结构域； 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) 研究膜锚定蛋白和跨膜α螺旋肽如何影响这些脂筏结构域的组织和动态； 4）应用DOT方法寻找成熟和老化过程中眼睛晶状体纤维细胞质膜的筏域，以及成熟、老化和白内障晶状体的膜模型。与年龄相关的核性白内障是第三世界国家老年人失明的主要原因。