Max Planck Institute of Molecular Cell Biology & Genetics, Dresden, Germany.
Biophys J. 2010 Oct 20;99(8):2443-52. doi: 10.1016/j.bpj.2010.08.033.
Obtaining quantitative kinetic parameters from fluorescence recovery after photobleaching (FRAP) experiments generally requires a theoretical analysis of protein mobility and appropriate solutions for FRAP recovery derived for a given geometry. Here we provide a treatment of FRAP recovery for a molecule undergoing a combined process of reversible membrane association and lateral diffusion on the plasma membrane for two commonly used bleach geometries: stripes, and boxes. Such analysis is complicated by the fact that diffusion of a molecule during photobleaching can lead to broadening of the bleach area, resulting in significant deviations of the actual bleach shape from the desired bleach geometry, which creates difficulty in accurately measuring kinetic parameters. Here we overcome the problem of deviations between actual and idealized bleach geometries by parameterizing, more accurately, the initial postbleach state. This allows for reconstruction of an accurate and analytically tractable approximation of the actual fluorescence distribution. Through simulated FRAP experiments, we demonstrate that this method can be used to accurately measure a broad range of combinations of diffusion constants and exchange rates. Use of this method to analyze the plextrin homology domain of PLC-δ1 in Caenorhabditis elegans results in quantitative agreement with prior analysis of this domain in other cells using other methods. Because of the flexibility, relative ease of implementation, and its use of standard, easily obtainable bleach geometries, this method should be broadly applicable to investigation of protein dynamics at the plasma membrane.
从荧光漂白后恢复（FRAP）实验中获取定量动力学参数通常需要对蛋白质的流动性进行理论分析，并为给定的几何形状提供适当的 FRAP 恢复解决方案。在这里，我们针对一种在质膜上经历可逆膜结合和横向扩散的组合过程的分子，为两种常用的漂白几何形状（条纹和方框）提供了 FRAP 恢复的处理方法。这种分析很复杂，因为在漂白过程中分子的扩散会导致漂白区域变宽，从而导致实际漂白形状与所需的漂白几何形状之间存在显著偏差，这给准确测量动力学参数带来了困难。在这里，我们通过更准确地参数化初始漂白后状态，克服了实际和理想化漂白几何形状之间的偏差问题。这允许对实际荧光分布进行准确且可分析处理的近似重建。通过模拟 FRAP 实验，我们证明该方法可用于准确测量扩散常数和交换率的广泛组合。使用这种方法分析秀丽隐杆线虫 PLC-δ1 的 pleckstrin 同源结构域的结果与使用其他方法在其他细胞中对该结构域进行的先前分析具有定量一致性。由于该方法具有灵活性、相对容易实现，并且使用了标准的、易于获得的漂白几何形状，因此该方法应该广泛适用于研究质膜上的蛋白质动力学。
