Measuring mobility in chromatin by intensity sorted FCS

Abstract

6The architectural organization of chromatin can play an important role in genome regulation 7 by affecting the mobility of molecules within its surroundings via binding interactions and 8 molecular crowding. The diffusion of molecules at specific locations in the nucleus can be 9 studied by Fluorescence Correlation Spectroscopy (FCS), a well-established technique based 10 on the analysis of fluorescence intensity fluctuations detected in a confocal observation 11 volume. However, detecting subtle variations of mobility between different chromatin regions 12 remains challenging with currently-available FCS methods. 13 Here we introduce a method that samples multiple positions by slowly scanning the FCS 14 observation volume across the nucleus. Analyzing the data in short time segments, we 15 preserve the high temporal resolution of single-point FCS while probing different nuclear 16 regions in the same cell. Using the intensity level of the probe (or a DNA marker) as a 17 reference, we efficiently sort the FCS segments into different populations and obtain average 18 correlation functions that are associated to different chromatin regions. This sorting and 19 averaging strategy renders the method statistically robust while preserving the observation of 20 intranuclear variations of mobility. 21Using this approach, we quantified diffusion of monomeric GFP in high versus low 22 chromatin density regions. We found that GFP mobility was reduced in heterochromatin, 23 especially within perinucleolar heterochromatin. Moreover, we found that modulation of 24 chromatin compaction by ATP depletion, or treatment with solution of different osmolarity, 25 differentially-affected the ratio of diffusion in both regions. Then, we used the approach to 26 probe the mobility of estrogen receptor-α (ER) in the vicinity of an integrated multicopy 27 prolactin gene array. Finally, we discussed the coupling of this method with stimulated 28 emission depletion (STED)-FCS, for performing FCS at sub-diffraction spatial scales. 29 30 31 32 33 34 74data at multiple observation volumes (25, 26). Another method that has been used to measure 75 fluctuations at, and between, different points in the nucleus is scanning FCS, which is easily 76 implemented on confocal laser scanning microscopes, but whose temporal resolution is 77 typically limited, by scanning, to the millisecond range (27, 28). Finally, diffusion maps have 78 been recently obtained from the analysis of raster image correlation spectroscopy (RICS) data 79 (29). However, these methods will only provide an accurate description of the mobility 80 properties in different nuclear regions if they are immobile during FCS data acquisition. 81A significant advantage can be gained if the different chromatin regions are identified with 82 the help of a reference marker (30). For instance, the intensity of a fluorescent protein can be 83 used to identify specific sub-nuclear regions or, simply, the intensity of a DNA marker (e.g. 84Hoechst) can be used to identify regions of different ch...

The interplay of chromatin phase separation and lamina interactions in nuclear organization

“…The dynamics of nuclear chromatin is governed by the Allen-Cahn evolution equations of the phase-field variables { φ, ψ }: where and are mobility coefficients which are proportional to the relaxation time of different phase-field variables. We have chosen the mobility coefficients to match the magnitudes of in vivo measurements of euchromatin and heterochromatin diffusion coefficients [43] The terms in Eq. (3) account for the fluctuations at the boundaries of EC/fHC and EC/cHC islands due to finite size nature of the droplets.…”

“…The diffusion coefficient of chromosomes D φ is fixed to 1 µm 2 /s . These values of diffusion coefficients are motivated by experimental measurements of euchromatin/heterochromatin mobility in live cells [43]. The interaction coefficient between chromosomal territories and nuclear envelope is chosen to be strong enough to maintain them inside the nucleus by setting β 0 = 66.7.…”

Chromatin nanoscale compaction in live cells visualized by acceptor‐to‐donor ratio corrected Förster resonance energy transfer between DNA dyes

“…For instance, techniques like fluorescence recovery after photobleaching and fluorescence correlation spectroscopy (FCS) have been widely used to measure molecular diffusion within the nucleus of live cells . Even if limited by diffraction, these methods detect differences in the diffusion of nanometer‐sized probes and can be used to indirectly infer properties of the nanoscale chromatin architecture . FCS can be eventually coupled with STED to probe diffusion at subdiffraction spatial scales .…”

High photon count rates improve the quality of super-resolution fluorescence fluctuation spectroscopy

“…Fluorescence correlation spectroscopy (FCS) has, since its introduction almost 50 years ago, become a widely applied technique to study diffusion dynamics in synthetic and biological applications [1][2][3] . It has greatly contributed to the understanding of molecular diffusion in model systems and living cells, both in 2D (in vitro models or cellular membranes) and in 3D (solution or cellular cytoplasm and nucleus) environments [4][5][6][7][8] . Notably, it has offered fundamental insights into the dynamic organisation of living systems at the molecular level, e.g.…”