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状结构，包含在不同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，需要脂质相互作用才能形成。这项建议的长期目标是更好地理解筏在生物膜中形成、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，这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体组成分子和筏体本身的大小、寿命和动力学是没有用的。为了解决这些问题，我们建议将脉冲EPR自旋标记技术“氧转运辨别（DOT）”应用于模型和细胞膜中筏的原位研究。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 - 100 μ s的时间尺度上观察到膜动力学。DOT方法允许区分不同的膜域，因为自旋标记的O2和氮氧化物部分（氧扩散浓度积）之间的碰撞率在这些域中可以有很大的不同。此外，膜结构域可以通过原位氧扩散浓度产物的轮廓来表征，而无需分离。该方法特别适用于小/瞬态域的时空特征的获取。假设筏在质膜液体无序环境中形成液体有序结构域。膜脂组成和蛋白质含量有望调节筏的大小和动态。DOT方法将用于在定义良好的模型系统上测试假设，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结构域的结构。这些研究将包括成熟和老化的纤维细胞膜，其中胆固醇/脂质比的增加和鞘磷脂水平的升高为筏的形成创造了条件。建议：1)检测含胆固醇膜中共存的液体有序结构域和液体无序结构域；2)评估由筏形混合物制成的模型膜中筏形结构域的大小和稳定性；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力学；4)应用DOT方法寻找成熟和衰老过程中晶状体纤维细胞膜中的筏结构域，以及成熟、衰老和白内障晶状体的膜模型。年龄相关性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.