Studies of Retinoschisin 1 on the membrane of retinal cell inner segments

AFM和荧光的结合对于这种类型的项目来说是一个强有力的工具，其中不仅是几何结构，而且是成像结构的识别。阻断抗体与样本的非特异性结合是很重要的，我们仍在微调这一过程，以优化荧光成像。为了获得尽可能纯净的质膜溶液，人们尝试了多种样品制备方法。我们已经成功地证明了内节段的质膜似乎具有与其相关的具有蜂窝状结构的结构元件。到目前为止，用荧光识别这些结构被证明是比较困难的，但正在进行的工作应该优化适当的条件。 我们最近一直使用人工模型支撑的双层膜来研究不同离子条件下RS1对双层膜的吸收的影响。我们观察到，对于由磷脂酰丝氨酸(一种阴离子脂质)组成的双层，蛋白质在钙存在下的吸附显著改变了双层的拓扑结构，似乎RS1形成了蛋白质丰富的结构域。在由磷脂酰丝氨酸和磷脂酰胆碱(1：3)组成的混合脂质中，蛋白质强烈结合到边界缺陷上，但也在随机位置形成小的富含蛋白质的结构域。因此，RS1似乎与阴离子脂类结合，结合荧光将有助于验证这一假说。所使用的蛋白质结构是RS1在e-Coli中表达的产物，因此，它似乎具有两种折叠构象，其对双层的影响略有不同。

The combination of AFM and fluorescence can be a powerful tool for this type of project, where not only geometries but also identification of the imaged structures is critical. It is important to block non-specific binding of antibodies to the samples, and we are still fine tuning that process to optimize fluorescence imaging. A number of sample preparation methods have been tried to get as pure plasma membrane solutions as possible. We have successfully shown that the plasma membranes of the inner segments appear to have structured elements associated with them which have honeycomb-like structure. The identification of these structures with fluorescence proved more difficult so far, but ongoing work should optimize the appropriate conditions. We have recently been using artificial model supported bilayers to investigate the effects of RS1 absorption to the bilayers under different ionic conditions. We observe that, for bilayers composed of phosphatidylserine, an anionic lipid, protein adsorption in the presence of calcium, dramatically alters the topology of the bilayer and it appears that RS1 forms protein rich domains. In mixed lipids made of phosphatidylserine and phosphatidylcholine (1:3), the protein strongly binds to boundary defects but also forms small protein-rich domains at random locations. It appears, therefore, that RS1 binds to anionic lipids and the combination with fluoresence will help validate this hypothesis. The protein construct used is a product of RS1 expression in e-Coli and, as such, it appears that it possesses two folding conformations whose effects on the bilayers are somewhat different.