Lipid Rafts in Eye Lens: Discrimination by Pulse EPR

描述（由申请人提供）：细胞膜具有二维液体样结构，含有在各种时间和空间尺度上连续形成和分散的结构域。筏是膜结构域，其形成需要脂质相互作用。该提议的长期目标是更好地理解筏在生物膜中，特别是在眼睛透镜的纤维细胞的质膜中形成、维持和分解的分子机制。洗涤剂不溶性，已被用于定义筏生物化学，不反映预先存在的结构和组织的膜。此外，这种方法对于理解筏组成分子和筏本身的大小、寿命和动力学是没有用的。为了解决这些问题，有人建议应用脉冲EPR自旋标记技术“歧视的氧运输（DOT）”的筏在模型和细胞膜的原位研究。由于自旋标记的自旋-晶格弛豫时间足够长，可以在0.1 - 100 μ s的时间尺度上观察到膜动力学。DOT方法允许不同的膜域的歧视，因为O2和自旋标签（氧扩散浓度的产品）的氮氧部分之间的碰撞率可以在这些域中是完全不同的。此外，膜域可以通过原位氧扩散浓度产物的分布来表征，而不需要分离。该方法特别适用于获得小/瞬态域的时空特性。据推测，筏形成液体有序领域的质膜液体无序的环境。膜脂组成以及蛋白质含量预计将调节筏的大小和动力学。DOT方法将用于检验定义明确的模型系统的假设，其中结构域大小和脂质交换速率将由膜脂质组成、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结构域结构。这些研究将包括成熟和老化的纤维细胞膜，其中胆固醇/脂质比增加和鞘磷脂水平升高创造了有利于形成筏的条件。建议：1）检测含有胆固醇的膜中共存的液体有序和液体无序结构域; 2）评估由筏形成混合物制成的模型膜中筏结构域的大小和稳定性; 3）检查膜锚定蛋白和跨膜α-螺旋肽如何影响这些脂筏结构域的组织和动力学; 4）应用DOT方法寻找成熟和老化过程中眼透镜纤维细胞质膜中的筏结构域，以及成熟、老化和白内障晶状体的膜模型。核性白内障是第三世界国家老年人失明的主要原因。

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.