Ciocanel Maria-Veronica, Kreiling Jill A, Gagnon James A, Mowry Kimberly L, Sandstede Björn
Division of Applied Mathematics, Brown University, Providence, Rhode Island.
Department of Molecular Biology, Cell Biology, and Biochemistry, Brown University, Providence, Rhode Island.
Biophys J. 2017 Apr 25;112(8):1714-1725. doi: 10.1016/j.bpj.2017.02.042.
Fluorescence recovery after photobleaching (FRAP) is a well-established experimental technique to study binding and diffusion of molecules in cells. Although a large number of analytical and numerical models have been developed to extract binding and diffusion rates from FRAP recovery curves, active transport of molecules is typically not included in the existing models that are used to estimate these rates. Here we present a validated numerical method for estimating diffusion, binding/unbinding rates, and active transport velocities using FRAP data that captures intracellular dynamics through partial differential equation models. We apply these methods to transport and localization of mRNA molecules in Xenopus laevis oocytes, where active transport processes are essential to generate developmental polarity. By providing estimates of the effective velocities and diffusion, as well as expected run times and lengths, this approach can help quantify dynamical properties of localizing and nonlocalizing RNA. Our results confirm the distinct transport dynamics in different regions of the cytoplasm, and suggest that RNA movement in both the animal and vegetal directions may influence the timescale of RNA localization in Xenopus oocytes. We also show that model initial conditions extracted from FRAP postbleach intensities prevent underestimation of diffusion, which can arise from the instantaneous bleaching assumption. The numerical and modeling approach presented here to estimate parameters using FRAP recovery data is a broadly applicable tool for systems where intracellular transport is a key molecular mechanism.
光漂白后荧光恢复（FRAP）是一种成熟的实验技术，用于研究细胞中分子的结合和扩散。尽管已经开发了大量的分析和数值模型来从FRAP恢复曲线中提取结合和扩散速率，但分子的主动运输通常不包括在用于估计这些速率的现有模型中。在这里，我们提出了一种经过验证的数值方法，使用FRAP数据估计扩散、结合/解离速率和主动运输速度，该数据通过偏微分方程模型捕捉细胞内动力学。我们将这些方法应用于非洲爪蟾卵母细胞中mRNA分子的运输和定位，其中主动运输过程对于产生发育极性至关重要。通过提供有效速度和扩散的估计值，以及预期的运行时间和长度，这种方法可以帮助量化定位和非定位RNA的动力学特性。我们的结果证实了细胞质不同区域中不同的运输动力学，并表明RNA在动物和植物方向上的移动可能会影响非洲爪蟾卵母细胞中RNA定位的时间尺度。我们还表明，从FRAP漂白后强度提取的模型初始条件可防止因瞬时漂白假设而导致的扩散低估。这里提出的使用FRAP恢复数据估计参数的数值和建模方法是一种广泛适用于细胞内运输是关键分子机制的系统的工具。
