Analysis of diffusion and binding in cells using the RICS approach.
The movement of macromolecules in cells is assumed to occur either through active transport or by diffusion. However, the determination of the diffusion coefficients in cells using fluctuation methods or FRAP frequently give diffusion coefficient that are orders of magnitude smaller than the diffusion coefficients measured for the same macromolecule in solution. It is assumed that the cell internal viscosity is partially responsible for this decrease in the apparent diffusion. When the apparent diffusion is too slow to be due to cytoplasm viscosity, it is assumed that weak binding of the macromolecules to immobile or quasi immobile structures is taking place. In this article, we derive equations for fitting of the RICS (Raster-scan Image Correlations Spectroscopy) data in cells to a model that includes transient binding to immobile structures, and we show that under some conditions, the spatio-temporal correlation provided by the RICS approach can distinguish the process of diffusion and weak binding. We apply the method to determine the diffusion in the cytoplasm and binding of Focal Adhesion Kinase-EGFP to adhesions in MEF cells.
Digman
Michelle A
MA
Optical Biology Core, University of California, Irvine, California 92697, USA.
Gratton
Enrico
E
D000818:Animals; D002460:Cell Line; D004058:Diffusion; D005347:Fibroblasts / Q000166:cytology / Q000378:metabolism* / Q000502:physiology*; D005456:Fluorescent Dyes / Q000378:metabolism*; D051416:Focal Adhesion Protein-Tyrosine Kinases / Q000235:genetics / Q000378:metabolism*; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism*; D007091:Image Processing, Computer-Assisted / Q000379:methods*; D051379:Mice; D018613:Microscopy, Confocal / Q000379:methods; D013050:Spectrometry, Fluorescence / Q000379:methods*; D014162:Transfection
D005456:Fluorescent Dyes; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D051416:Focal Adhesion Protein-Tyrosine Kinases