Analysis of Active Transport by Fluorescence Recovery after Photobleaching

Abstract

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.