Using Fluorescence Recovery After Photobleaching data to uncover filament dynamics

International audienceFluorescence Recovery After Photobleaching (FRAP) has been extensively used to understand molecular dynamics in cells. This technique when applied to soluble, globular molecules driven by diffusion is easily interpreted and well understood. However, the classical methods of analysis cannot be applied to anisotropic structures subjected to directed transport, such as cytoskeletal filaments or elongated organelles transported along microtubule tracks. A new mathematical approach is needed to analyze FRAP data in this context and determine what information can be obtain from such experiments. To address these questions, we analyze fluorescence intensity profile curves after photobleaching of fluorescently labelled intermediate filaments anterogradely transported along microtubules. We apply the analysis to intermediate filament data to determine information about the filament motion. Our analysis consists of deriving equations for fluorescence intensity profiles and developing a mathematical model for the motion of filaments and simulating the model. Two closed forms for profile curves were derived, one for filaments of constant length and one for filaments with constant velocity, and three types of simulation were carried out. In the first type of simulation, the filaments have random velocities which are constant for the duration of the simulation. In the second type, filaments have random velocities which instantaneously change at random times. In the third type, filaments have random velocities and exhibit pausing between velocity changes. Our analysis shows: the most important distribution governing the shape of the intensity profile curves obtained from filaments is the distribution of the filament velocity. Furthermore, filament length which is constant during the experiment, had little impact on intensity profile curves. Finally, gamma distributions for the filament velocity with pauses give the best fit to asymmetric fluorescence intensity profiles of intermediate filaments observed in FRAP experiments performed in polarized migrating astrocytes reported in (1). Our analysis also shows that the majority of filaments are stationary. Overall, our data give new insight into the regulation of intermediate filament dynamics during cell migration. AUTHOR SUMMARY Fluorescence Recovery After Photobleaching (FRAP) is a commonly-used technique to analyze the dynamics of fluorescently-tagged proteins or structures in biology. After photochemical altering the fluorophor in a specific region, fluorescent material from the surrounding region moves into the photobleached region. Usually applied to the diffusion of soluble or membrane associate proteins, the existing models of analysis are not suitable for the elucidation of directional transport of elongated structures. Different modes of motions for the elongated structures with distributed lengths and velocities in cells are considered. First, we observe that filament lengths can be inferred from the level of noisiness. We further show the characteristics of fluorescence profile curves mainly depend on the occurrence of changes in velocities and distributions of velocities; whereas length distributions have negligible impact. Analysis of experimental data using this new framework indicates intermediate filaments transported by kinesins along microtubules in polarized migrating cells have gamma distributed velocities changing over time between pausing. Most filaments are found to be very slow or stationary with a few moving fast. This new computational approach should permit the interpretation of FRAP experimental data obtained with any directionally moving elongated structures of various lengths