Nuclear transport rates determination in single yeast cells

DURRIEU, LUCÍA; RIKARD JOHANSSON; ALAN BUSH; ULRIKE MÜNZNER; CEDERSUND, GUNNAR; COLMAN-LERNER, ALEJANDRO

Mannheim

Conferencia; International Conference on Systems Biology (ICSB); 2011

International Society for Systems Biology

Cell-to-cell variability plays an important role in cell fate decisions. Thus, to achieve a system-level understanding of cellular function it is essential to have quantitative measurements of dynamic processes in single cells. Nuclear transport is especially interesting since it is involved in regulation of gene expression, signal transduction and cell differentiation among other functions. Rates of protein movement between cellular compartments can be measured by FRAP. However, no standard and reliable methods to calculate transport rates exist. Usual analysis of FRAPs involves fitting an exponential function to obtain a time constant (tau) with limited biological meaning. Here we introduce a method that, instead of tau, extracts import and export rates, even from noisy single cell data. For this, we constructed a mass action kinetics to fit to the data. Analysis of simulated data revealed that rates obtained by FRAP are very sensitive to noise, but also suggested that introducing several perturbations to the system would help to overcome this problem. Thus, we set up a protocol involving sequential FRAPs on the same cell (?train FRAP? technique). Using this method, we successfully measured import and export rates in individual cells. For YFP, average transport rates are 0.17 s-1. We estimated confidence intervals for these parameters through likelihood profile analysis. We found large cell to cell variation (CV = 2.4), suggesting a hitherto unknown source of cellular heterogeneity. Given the passive nature of YFP diffusion, we attribute this variation to large difference among cells in the number of nuclear pores. For two yeast proteins (Ace2 and Fus3) transport rates were slower but comparable to free YFP.Through this technique we were able to estimate the ratio of the nuclear and cytosol volumes available for diffusion, an important parameter for modeling intracellular processes. This represents a novel approach, which is easier and more reliable than calculating volumes for all organelles individually. Our results indicate that this ratio is only 5.12 (CV=0.35). This is noteworthy, since the nucleus is only one fifteenth the size of the cell, as determined using geometrical considerations only. Estimation of nuclear transport rates, and their distribution in populations, is a useful tool to explore mechanistic hypothesis for regulation of nuclear localization and is a valuable resource for building dynamic models of cell function.