Mapping the dynamical organization of the cell nucleus through fluorescence correlation spectroscopy

STORTZ, M; ANGIOLINI, JUAN F; MOCKSOS, E; WOLOSIUK, A; PECCI, A; LEVI, V

METHODS

ACADEMIC PRESS INC ELSEVIER SCIENCE

Lugar: Amsterdam; Año: 2018 vol. 140 p. 10 - 22

1046-2023

The hierarchical organization of the cell nucleus into specialized open reservoirs and the nucleoplasmovercrowding impose restrictions to the mobility of biomolecules and their interactions with nuclear targets.These properties determine that many nuclear functions such as transcription, replication, splicingor DNA repair are regulated by complex, dynamical processes that do not follow simple rules. Advancedfluorescence microscopy tools and, in particular, fluorescence correlation spectroscopy (FCS) providecomplementary and exquisite information on the dynamics of fluorescent labeled molecules movingthrough the nuclear space and are helping us to comprehend the complexity of the nuclear structure.Here, we describe how FCS methods can be applied to reveal the dynamical organization of the nucleusin live cells. Specifically, we provide instructions for the preparation of cellular samples with fluorescenttagged proteins and detail how FCS can be easily instrumented in commercial confocal microscopes. Inaddition, we describe general rules to set the parameters for one and two-color experiments and therequired controls for these experiments. Finally, we review the statistical analysis of the FCS data andsummarize the use of numerical simulations as a complementary approach that helps us to understandthe complex matrix of molecular interactions network within the nucleus