Fluorescence fluctuation spectroscopy: an invaluable microscopy tool for uncovering the biophysical rules for navigating the nuclear landscape

Abstract

Nuclear architecture is fundamental to the manner by which molecules traverse the nucleus. The nucleoplasm is a crowded environment where dynamic rearrangements in local chromatin compaction locally redefine the space accessible toward nuclear protein diffusion. Here, we review a suite of methods based on fluorescence fluctuation spectroscopy (FFS) and how they have been employed to interrogate chromatin organization, as well as the impact this structural framework has on nuclear protein target search. From first focusing on a set of studies that apply FFS to an inert fluorescent tracer diffusing inside the nucleus of a living cell, we demonstrate the capacity of this technology to measure the accessibility of the nucleoplasm. Then with a baseline understanding of the exploration volume available to nuclear proteins during target search, we review direct applications of FFS to fluorescently labeled transcription factors (TFs). FFS can detect changes in TF mobility due to DNA binding, as well as the formation of TF complexes via changes in brightness due to oligomerization. Collectively, we find that FFS-based methods can uncover how nuclear proteins in general navigate the nuclear landscape.

Cohesin depleted cells rebuild functional nuclear compartments after endomitosis

“…In order to test this hypothesis, it is necessary to explore the space-time compaction and accessibility of CDs. Super-resolved fluorescence microscopy, including single molecule localization microscopy (SMLM) and stochastic optical reconstruction microscopy (STORM), may become the methods of choice to measure absolute differences of DNA/chromatin compaction with spatial resolution at the nanometer scale [79, 84, 85], whereas chromatin accessibility can be probed indirectly with methods that allow to measure molecular diffusion rates [86–89].…”

Spatiotemporal dynamics of 53BP1 dimer recruitment to a DNA double strand break

“…Microscopy data acquisition. NB FFS measurements of the different eGFP-tagged 53BP1 constructs involved selecting a DIvA cell exhibiting a sufficiently low expression level to observe fluctuations in eGFP fluorescence intensity 29,30 and then selecting a 10.6 μm region of interest (ROI) within that DIvA cell's nucleus (Figs. 1 and 4) (or an ROI that contained the entire cell nucleus (Fig.…”

“…Thus, here we set out to quantify the spatiotemporal dynamics of 53BP1 selfassociation during a DSB DDR and test whether the DSB histone code differentially regulates 53BP1 dimer recruitment, retention, and oligomer formation in the context of live-cell nuclear architecture. To do so we couple fluorescence fluctuation spectroscopy (FFS) 29,30 of fluorescently tagged constructs of 53BP1 with the DSB inducible via AsiSI cell system (DIvA) 31,32 .…”

“…FFS describes a family of methods that statistically analyse fluctuations in fluorescence intensity within live-cell microscopy data to extract single molecule dynamics 29,30 . DIvA cells are a stable cell line generated in the human U2OS background harbouring a 4-hydroxytamoxifen (4OHT)-inducible AsiSI restriction enzyme, which allows for induction of approximately 100 site-specific DSBs in the genome upon 4OHT treatment 31,32 .…”

A Comprehensive Review of Fluorescence Correlation Spectroscopy

“…The first important application of FCS is to measure the diffusion coefficient of biomolecules, which is directly related to the molecular size in a certain surrounding medium. For instance, Priest et al [109] used FCS to measure the size of an inert fluorescent tracer inside the nucleus of a living cell. Shang et al [110] utilized FCS to measure the hydrodynamic radius increase of the nanoparticles (NPs) due to protein adsorption.…”