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Analysis of Active Transport by Fluorescence Recovery after Photobleaching.
Ciocanel MV, Kreiling JA, Gagnon JA, Mowry KL, Sandstede B.
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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.
Axelrod,
Mobility measurement by analysis of fluorescence photobleaching recovery kinetics.
1976, Pubmed
Axelrod,
Mobility measurement by analysis of fluorescence photobleaching recovery kinetics.
1976,
Pubmed Beaudouin,
Dissecting the contribution of diffusion and interactions to the mobility of nuclear proteins.
2006,
Pubmed Bertrand,
Localization of ASH1 mRNA particles in living yeast.
1998,
Pubmed Birsoy,
Vg 1 is an essential signaling molecule in Xenopus development.
2006,
Pubmed
,
Xenbase Braeckmans,
Three-dimensional fluorescence recovery after photobleaching with the confocal scanning laser microscope.
2003,
Pubmed Braga,
Intracellular macromolecular mobility measured by fluorescence recovery after photobleaching with confocal laser scanning microscopes.
2004,
Pubmed Braga,
A reaction-diffusion model to study RNA motion by quantitative fluorescence recovery after photobleaching.
2007,
Pubmed Chen,
Intracellular spatial localization regulated by the microtubule network.
2012,
Pubmed Coller,
Tethered function assays: an adaptable approach to study RNA regulatory proteins.
2007,
Pubmed Dale,
Secretion and mesoderm-inducing activity of the TGF-beta-related domain of Xenopus Vg1.
1993,
Pubmed
,
Xenbase Gagnon,
Directional transport is mediated by a Dynein-dependent step in an RNA localization pathway.
2013,
Pubmed
,
Xenbase Hancock,
Bidirectional cargo transport: moving beyond tug of war.
2014,
Pubmed Holt,
Subcellular mRNA localization in animal cells and why it matters.
2009,
Pubmed Hughes,
Kinesins with extended neck linkers: a chemomechanical model for variable-length stepping.
2012,
Pubmed Jonasson,
A pixel-based likelihood framework for analysis of fluorescence recovery after photobleaching data.
2008,
Pubmed Jung,
Modeling the slowing of neurofilament transport along the mouse sciatic nerve.
2009,
Pubmed Kang,
Validation of Normalizations, Scaling, and Photofading Corrections for FRAP Data Analysis.
2015,
Pubmed Kang,
A quantitative approach to analyze binding diffusion kinetics by confocal FRAP.
2010,
Pubmed Kang,
A generalization of theory for two-dimensional fluorescence recovery after photobleaching applicable to confocal laser scanning microscopes.
2009,
Pubmed Khuc Trong,
Cortical microtubule nucleation can organise the cytoskeleton of Drosophila oocytes to define the anteroposterior axis.
2015,
Pubmed Klumpp,
Cooperative cargo transport by several molecular motors.
2005,
Pubmed Li,
Deciphering the axonal transport kinetics of neurofilaments using the fluorescence photoactivation pulse-escape method.
2014,
Pubmed Lorén,
Fluorescence recovery after photobleaching in material and life sciences: putting theory into practice.
2015,
Pubmed McNally,
Quantitative FRAP in analysis of molecular binding dynamics in vivo.
2008,
Pubmed Medioni,
Principles and roles of mRNA localization in animal development.
2012,
Pubmed Messitt,
Multiple kinesin motors coordinate cytoplasmic RNA transport on a subpopulation of microtubules in Xenopus oocytes.
2008,
Pubmed
,
Xenbase Mowry,
Vegetal messenger RNA localization directed by a 340-nt RNA sequence element in Xenopus oocytes.
1992,
Pubmed
,
Xenbase Mueller,
Evidence for a common mode of transcription factor interaction with chromatin as revealed by improved quantitative fluorescence recovery after photobleaching.
2008,
Pubmed Müller,
Tug-of-war as a cooperative mechanism for bidirectional cargo transport by molecular motors.
2008,
Pubmed Newby,
Quasi-steady state reduction of molecular motor-based models of directed intermittent search.
2010,
Pubmed Powrie,
Using in vivo imaging to measure RNA mobility in Xenopus laevis oocytes.
2016,
Pubmed
,
Xenbase Sadegh Zadeh,
A class of exact solutions for biomacromolecule diffusion-reaction in live cells.
2010,
Pubmed Shaner,
Improved monomeric red, orange and yellow fluorescent proteins derived from Discosoma sp. red fluorescent protein.
2004,
Pubmed Sirajuddin,
Regulation of microtubule motors by tubulin isotypes and post-translational modifications.
2014,
Pubmed Sprague,
Analysis of binding reactions by fluorescence recovery after photobleaching.
2004,
Pubmed Thomsen,
Processed Vg1 protein is an axial mesoderm inducer in Xenopus.
1993,
Pubmed
,
Xenbase Thorn,
Engineering the processive run length of the kinesin motor.
2000,
Pubmed Weiss,
Challenges and artifacts in quantitative photobleaching experiments.
2004,
Pubmed Zimyanin,
In vivo imaging of oskar mRNA transport reveals the mechanism of posterior localization.
2008,
Pubmed
