Dipartimento di Scienze Farmacologiche e Biomolecolari, Università degli Studi di Milano, via Trentacoste 2, 20134 Milano, Italy.
Microsc Microanal. 2013 Oct;19(5):1358-63. doi: 10.1017/S1431927613001682. Epub 2013 Jun 10.
Xenopus laevis oocytes are an interesting model for the study of many developmental mechanisms because of their dimensions and the ease with which they can be manipulated. In addition, they are widely employed systems for the expression and functional study of heterologous proteins, which can be expressed with high efficiency on their plasma membrane. Here we applied atomic force microscopy (AFM) to the study of the plasma membrane of X. laevis oocytes. In particular, we developed and optimized a new sample preparation protocol, based on the purification of plasma membranes by ultracentrifugation on a sucrose gradient, to perform a high-resolution AFM imaging of X. laevis oocyte plasma membrane in physiological-like conditions. Reproducible AFM topographs allowed visualization and dimensional characterization of membrane patches, whose height corresponds to a single lipid bilayer, as well as the presence of nanometer structures embedded in the plasma membrane and identified as native membrane proteins. The described method appears to be an applicable tool for performing high-resolution AFM imaging of X. laevis oocyte plasma membrane in a physiological-like environment, thus opening promising perspectives for studying in situ cloned membrane proteins of relevant biomedical/pharmacological interest expressed in this biological system.
非洲爪蟾卵母细胞是研究许多发育机制的有趣模型，因为它们的尺寸和易于操作。此外，它们还是广泛应用于异源蛋白表达和功能研究的系统，这些蛋白可以在其质膜上高效表达。在这里，我们应用原子力显微镜（AFM）研究非洲爪蟾卵母细胞的质膜。特别是，我们开发并优化了一种新的样品制备方案，基于蔗糖梯度超速离心纯化质膜，以在生理样条件下对非洲爪蟾卵母细胞质膜进行高分辨率 AFM 成像。可重复的 AFM 形貌图允许可视化和膜斑的尺寸特征化，其高度对应于单个脂质双层，以及存在嵌入质膜中的纳米结构，并鉴定为天然膜蛋白。所描述的方法似乎是在生理样环境下对非洲爪蟾卵母细胞质膜进行高分辨率 AFM 成像的一种可行工具，从而为在该生物系统中表达的相关生物医学/药理学感兴趣的原位克隆膜蛋白的原位研究开辟了有前景的前景。
