Vosolsobě Stanislav, Schwarzerová Kateřina, Petrášek Jan
Department of Experimental Plant Biology, University of Science, Charles University;
Department of Experimental Plant Biology, University of Science, Charles University.
J Vis Exp. 2018 Jun 15(136):57837. doi: 10.3791/57837.
This method provides a fast approach for the determination of plasma membrane partitioning of any fluorescently-tagged peripherally-associated protein using the profiles of fluorescence intensity across the plasma membrane. Measured fluorescence profiles are fitted by a model for membrane and cytoplasm fluorescence distribution along a line applied perpendicularly to the cell periphery. This model is constructed from the fluorescence intensity values in reference cells expressing a fluorescently-tagged marker for cytoplasm and with FM 4-64-labeled plasma membrane. The method can be applied to various cell types and organisms; however, only plasma membranes of non-neighboring cells can be evaluated. This fast microscopy-based method is suitable for experiments, where subtle and dynamic changes of plasma membrane-associated markers are expected and need to be quantified, e.g., in the analysis of mutant versions of proteins, inhibitor treatments, and signal transduction observations. The method is implemented in a multi-platform R package that is coupled with an ImageJ macro that serves as a user-friendly interface.
这种方法提供了一种快速途径，可利用跨质膜的荧光强度分布曲线来测定任何荧光标记的外周相关蛋白在质膜中的分配情况。通过一个沿垂直于细胞外周的直线应用的膜和细胞质荧光分布模型，对测量得到的荧光分布曲线进行拟合。该模型由表达荧光标记的细胞质标记物且质膜用FM 4-64标记的参考细胞中的荧光强度值构建而成。此方法可应用于各种细胞类型和生物体；然而，只能评估非相邻细胞的质膜。这种基于快速显微镜的方法适用于预期质膜相关标记物会发生细微动态变化且需要进行量化的实验，例如在蛋白质突变体版本分析、抑制剂处理以及信号转导观察中。该方法在一个多平台R包中实现，并与一个作为用户友好界面的ImageJ宏相结合。
