congresos y reuniones científicas
Exploring binding interactions in living cells through advanced fluorescence microscopy and numerical simulations
STORTZ MD; LEVI V; PECCI A
Congreso; Reunión Anual de Sociedades Biomedicas; 2017
Fluorescence microscopy is widely used to extract information on intracellular dynamics in living cells. Unfortunately, dynamical processes do not allow following simple rules. The combination of the experimental analysis with Monte Carlo simulations could help with the interpretation of the data and interrogate on the limitations and advantages of the used microscopy technique. We have previously studied the nuclear, dynamical distribution of the glucocorticoid receptor (GR) and one coactivator in living cells by fluorescence correlation methods. To get insight into these experimental observations, we used the Fluorescence Emission Recipes and Numerical Routines Toolkit to analyze whether fluorescence cross-correlation spectroscopy allows recovering information on the sequentiality of the interactions of GR and the coactivator with the same fixed targets. We also compared the performance of fluorescence correlation spectroscopy (FCS) and single molecule tracking (SMT) to explore GR-DNA binding interactions. The time-scale of these interactions obtained in SMT experiments are one order of magnitude higher than those obtained by FCS under similar conditions. Our simulations show that SMT may overestimate the residence time of GR on DNA when this is shorter than 1 s, even with a sampling time of 0.2 s. This is due to a preferential tracking of slow moving or fixed molecules in SMT. On the other hand, FCS may sample preferentially frequent, fast binding events and it is limited by the temporal window in which the autocorrelation functions are calculated. This work stresses the relevance of simulations to help to interpret experimental data from fluorescence microscopy.