使用融合到与激活过程的组成部分结合的蛋白质的GFP,通过生命细胞成像研究了质膜的激活过程。这样,用CRAC-GFP监测PIP3的形成,带有RBD-Raf-GFP的Ras-GTP,和Rap-GTP与Ral-GDS-GFP。荧光传感器在过程激活后从细胞质转移到质膜。虽然这种易位分析可以提供非常令人印象深刻的图像和电影,方法不太敏感,并且质膜处的GFP传感器的量与活化剂的量不是线性的。细胞边界处的像素中的荧光部分地来自与活化膜结合的GFP传感器,并且部分地来自该边界像素的胞浆体积中的未结合的GFP传感器。边界像素中胞质溶胶的可变和未知量导致GFP易位测定的低灵敏度和非线性。在这里,我们描述了一种方法,其中GFP传感器与胞质-RFP共表达。对于每个边界像素,例如,RFP荧光用于确定该像素的胞质溶胶的量,并从该像素的GFP荧光中减去,产生与该像素中的质膜特异性相关的GFP传感器的量。这种使用GFP传感器/RFP的GRminusRD方法至少敏感十倍,更具可重复性,与单独的GFP传感器相比,与活化剂呈线性。
Activation processes at the plasma membrane have been studied with life-cell imaging using GFP fused to a protein that binds to a component of the activation process. In this way, PIP3 formation has been monitored with CRAC-GFP, Ras-GTP with RBD-Raf-GFP, and Rap-GTP with Ral-GDS-GFP. The fluorescent sensors translocate from the cytoplasm to the plasma membrane upon activation of the process. Although this translocation assay can provide very impressive images and movies, the method is not very sensitive, and amount of GFP-sensor at the plasma membrane is not linear with the amount of activator. The fluorescence in pixels at the cell boundary is partly coming from the GFP-sensor that is bound to the activated membrane and partly from unbound GFP-sensor in the cytosolic volume of that boundary pixel. The variable and unknown amount of cytosol in boundary pixels causes the low sensitivity and nonlinearity of the GFP-translocation assay. Here we describe a method in which the GFP-sensor is co-expressed with cytosolic-RFP. For each boundary pixels, the RFP fluorescence is used to determine the amount of cytosol of that pixel and is subtracted from the GFP fluorescence of that pixel yielding the amount of GFP-sensor that is specifically associated with the plasma membrane in that pixel. This GRminusRD method using GFP-sensor/RFP is at least tenfold more sensitive, more reproducible, and linear with activator compared to GFP-sensor alone.