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

AFM和荧光的结合可以成为这类项目的强大工具，其中不仅几何形状而且成像结构的识别都是至关重要的。 阻断抗体与样品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。 已经尝试了许多样品制备方法以获得尽可能纯的质膜溶液。 我们已经成功地表明，内节的质膜似乎具有与它们相关的结构化元件，其具有蜂窝状结构。 到目前为止，用荧光鉴定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。 我们最近一直在使用人工模型支持的双层膜，研究在不同的离子条件下的RS1吸收的双层膜的影响。我们观察到，对于由磷脂酰丝氨酸，阴离子脂质，蛋白质吸附在钙的存在下，大大改变了双层的拓扑结构，似乎RS1形成富含蛋白质的结构域。 在由磷脂酰丝氨酸和磷脂酰胆碱（1：3）组成的混合脂质中，蛋白质强烈地结合到边界缺陷，但也在随机位置形成小的富含蛋白质的结构域。因此，似乎RS1与阴离子脂质结合，并且与荧光的结合将有助于验证这一假设。所用的蛋白质构建体是RS1在大肠杆菌中表达的产物，因此，它似乎具有两种折叠构象，其对双层的影响有些不同。

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.