Fluorescence correlation spectroscopy experiments to quantify free diffusion coefficients in reaction-diffusion systems: The case of Ca 2 + and its dyes

SIGAUT, LORENA; VILLARRUEL, CECILIA; PONCE, MARÍA LAURA; PONCE DAWSON, SILVINA

PHYSICAL REVIEW E

American Physical Society

Año: 2017 vol. 95

2470-0045

Many cell signaling pathways involve the diffusion of messengersthat bind and unbind to and from intracellularcomponents. Quantifying their net transport rate under different conditions then requires having separate estimatesof their free diffusion coefficient and binding or unbinding rates. In this paper, we show how performing sets offluorescence correlation spectroscopy (FCS) experiments under different conditions, it is possible to quantify freediffusion coefficients and on and off rates of reaction-diffusion systems. We develop the theory and present a practicalimplementation for the case of the universal second messenger, calcium (Ca2+) and single-wavelength dyes thatincrease their fluorescence upon Ca2+ binding. We validate the approach with experiments performed in aqueoussolutions containing Ca2+ and Fluo4 dextran (both in its high and low affinity versions). Performing FCS experimentswith tetramethylrhodamine-dextran in Xenopus laevis oocytes, we infer the corresponding free diffusioncoefficients in the cytosol of these cells. Our approach can be extended to other physiologically relevant reactiondiffusionsystems to quantify biophysical parameters that determine the dynamics of various variables of interest.