Intensity Sorted Fluorescence Correlation Spectroscopy: A Novel Method to Probe Nuclear Dynamics and Chromatin Organization in Living Cells

The architectural organization of chromatin can play an important role in gene expression by regulating the diffusion of molecules via binding interactions and molecular crowding [1]. In this respect, understanding how variations of chromatin architecture affect nuclear dynamics is of fundamental importance. Fluorescence Correlation Spectroscopy (FCS) is able to probe chromatin accessibility and molecular crowding in live cells by measuring fast diffusion of molecules in the range between microseconds and milliseconds [2]. In particular, single point FCS (spFCS) has a high temporal resolution but lacks spatial information. Conversely, spatially-resolved methods, like scanning FCS, have in general limited temporal resolution. Here, we introduce a novel technique that samples several nuclear positions by slowly scanning the FCS observation spot in a continuous line: by analyzing the data in short time sequences, we preserve the high temporal resolution of spFCS while probing different nuclear compartments in the same cell. We use the intensity as a reference to sort the short sequences into Auto-Correlation Functions associated to different nuclear regions. Interestingly, with respect to different chromatin states, we show that the mobility of a small inert probe like monomeric GFP is reduced in heterochromatin vs euchromatin, especially in the perinucleolar heterochromatin; moreover, we show that compaction due to ATP depletion or hyperosmolar treatment affects this ratio in a different way. More mechanistically, we also use this method to characterize hormone-induced changes in the mobility and binding of estrogen receptor-a to an engineered prolactin gene array. Finally, we discuss coupling with STimulated Emission Depletion (STED), for performing spot-variation FCS in different nuclear regions at sub-diffraction spatial scales[3]. [1] Misteli T., Science 2001 [2] Elson E., Biophys J, 2011 [3] Lanzanò et al, Nat Commun 2017.