Open Access

ARTICLE

An Improved Mathematical Approach for Determination of Molecular Kinetics in Living Cells with FRAP

Tanmay Lele^1,1, Philmo Oh^1,1, Jeffrey A. Nickerson^1,1,2,2, Donald E. Ingber^1,1,3,3

1 Vascular Biology Program, Departments of Pathology and Surgery, Children’s Hospital, Harvard Medical School, Boston, MA 02115-5737
2 Department of Cell Biology and Cancer Center, University of Massachusetts Medical School, North Worcester, MA 01655
3 Donald Ingber, MD, PhDVascular Biology ProgramChildren’s Hospital / Harvard Medical SchoolKarp Family Research Laboratories, Room 11-127
4 00 Longwood Ave.Boston, MA 02115-5737 Ph: 617-919-2223 FAX: 617-730-0230 Email: donald.ingber@childrens.harvard.edu

Molecular & Cellular Biomechanics 2004, 1(3), 181-190. https://doi.org/10.3970/mcb.2004.001.181

Abstract

The estimation of binding constants and diffusion coefficients of molecules that associate with insoluble molecular scaffolds inside living cells and nuclei has been facilitated by the use of Fluorescence Recovery after Photobleaching (FRAP) in conjunction with mathematical modeling. A critical feature unique to FRAP experiments that has been overlooked by past mathematical treatments is the existence of an `equilibrium constraint': local dynamic equilibrium is not disturbed because photobleaching does not functionally destroy molecules, and hence binding-unbinding proceeds at equilibrium rates. Here we describe an improved mathematical formulation under the equilibrium constraint which provides a more accurate estimate of molecular reaction kinetics within FRAP studies carried out in living cells. Due to incorporation of the equilibrium constraint, the original non-linear kinetic terms become linear allowing for analytical solution of the transport equations and greatly simplifying the estimation process. Based on mathematical modeling and scaling analysis, two experimental measures are identified that can be used to delineate the rate-limiting step. A comprehensive analysis of the interplay between binding-unbinding and diffusion, and its effect on the recovery curve, are presented. This work may help to bring clarity to the study of molecular dynamics within the structural complexity of living cells.

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