Department of Physics, University of Surrey, Guildford, Surrey GU2 7XH, United Kingdom.
Integr Biol (Camb). 2011 Mar;3(3):197-207. doi: 10.1039/c0ib00098a. Epub 2011 Jan 14.
We combine Fluorescence Recovery After Photobleaching (FRAP) experiments with mathematical modelling to study the dynamics inside the nucleus of both the TGF-β-sensitive transcriptional regulator Smad2, and Green-Fluorescent Protein (GFP). We show how combining modelling with bleaching strips of different areas allows a rigorous test of whether or not a protein is moving via diffusion as a single species. As noted recently by others, it is important to consider diffusion during the bleaching process. Neglecting it can cause serious error. Also, it is possible to use the bleaching process itself to provide an extra consistency test to the models predicting the recovery. With our method we show that the dynamics of GFP are consistent with it diffusing as a single species in a uniform environment in which flow is negligible. In contrast, the dynamics of the intracellular signal transducer Smad2 are never consistent with it moving as a single species via simple diffusion in a homogeneous environment without flow. Adding TGF-β slows down the dynamics of Smad2 but even without TGF-β, the Smad2 dynamics are influenced by one or more of: association, flow, and inhomogeneity in space of the dynamics. We suggest that the dynamics inside cells of many proteins may be poorly described by simple diffusion of a single species, and that our methodology provides a general and powerful way to test this hypothesis.
我们将荧光恢复后光漂白（FRAP）实验与数学建模相结合，研究 TGF-β 敏感转录调节剂 Smad2 和绿色荧光蛋白（GFP）在核内的动力学。我们展示了如何将建模与不同区域的漂白条带相结合，可以严格测试蛋白质是否作为单个物种通过扩散移动。正如最近其他人所指出的，在漂白过程中考虑扩散很重要。忽略它可能会导致严重的错误。此外，还可以使用漂白过程本身为预测恢复的模型提供额外的一致性测试。通过我们的方法，我们表明 GFP 的动力学与其在无流动的均匀环境中作为单个物种扩散一致。相比之下，细胞内信号转导 Smad2 的动力学与其在无流动的均匀环境中作为单个物种通过简单扩散移动的动力学不一致。添加 TGF-β 会降低 Smad2 的动力学，但即使没有 TGF-β，Smad2 的动力学也会受到：缔合、流动和动力学空间的非均一性的影响。我们认为，许多蛋白质在细胞内的动力学可能无法用单个物种的简单扩散来很好地描述，我们的方法为检验这一假设提供了一种通用而强大的方法。
