Dynamic and unique nucleolar microenvironment revealed by fluorescence correlation spectroscopy

Abstract

Organization and functions of the nucleolus is maintained by mobilities and interactions of nucleolar factors. Because the nucleolus is a densely packed structure, molecular crowding effects determined by the molecular concentrations and mobilities in the nucleolus should also be important for regulating nucleolar organization and functions. However, such molecular property of nucleolar organization is not fully understood. To understand the biophysical property of nucleolar organization, the diffusional behaviors of inert green fluorescent protein (GFP) oligomers with or without nuclear localization signals (NLSs) were analyzed under various conditions by fluorescence correlation spectroscopy. Our result demonstrates that the mobility of GFPs inside the nucleolus and the nucleoplasm can be represented by single free diffusion under normal conditions, even though the mobility in the nucleolus is considerably slower than that in the chromatin region. Moreover, the free diffusion of GFPs is found to be significantly size-and NLS-dependent only in the nucleolus. Interestingly, the mobility in the nucleolus is highly sensitive to ATP depletion, as well as actinomycin D (ActD) treatment. In contrast, the ultra-structure of the nucleolus was not significantly changed by ATP depletion but was changed by ActD treatment. These results suggest that the nucleolus behaves similarly to an open aqueous-phase medium with an increased molecular crowding effect that depends on both energy and transcription.-Park, H., Han, S.-S., Sako, Y., Pack, C.-G. Dynamic and unique nucleolar microenvironment revealed by fluorescence correlation spectroscopy. FASEB J. 29, 837-848 (2015). www.fasebj.org

Local raster image correlation spectroscopy generates high-resolution intracellular diffusion maps

“…More specifically, the study of protein dynamics in the nucleus can be relevant for understanding many nuclear processes and for characterizing the complex nuclear architecture 1 – 3 . For instance, by using biologically inert macromolecules and measuring their mobility, it is possible to gather insights about the chromatin architecture at a scale comparable to the size of the macromolecules 3 – 5 . For these reasons many techniques have been applied over the years for studying protein diffusion in the nuclear environment 6 – 11 .…”

Comprehensive correlation analysis for super-resolution dynamic fingerprinting of cellular compartments using the Zeiss Airyscan detector

“…The study of protein dynamics and the use of EGFP as a probe for investigating the structure of cellular compartments have been used for decades 1,2,4,6,11,13,33,34 while, on a parallel effort, many microscopy techniques have been developed from the super-resolution imaging field (STED, ISM…) and from the fluorescence fluctuations field (pCF, iMSD, spot-variation FCS, N&B…) in order to provide increased spatiotemporal resolution to address dynamic as well as structural information of the cellular environment. Although many studies have focused on the coupling of STED with fluorescence fluctuation techniques, our work is, at the best of our knowledge, the first coupling ISM concepts to such techniques enabling, on a different spatial scale, the super-resolution investigation of many dynamic properties in a single analysis in a few seconds.…”

Physicochemical Properties of Chromosomes in Live Cells Characterized by Label-Free Imaging and Fluorescence Correlation Spectroscopy

“…Interestingly, the D value in DM cell cultured by hypertonic media showed much smaller than the theoretical value. Since hypertonic stress enforces molecular crowding in the cellular components due to the osmotic extraction of water from the cells (Richter et al, 2007), these results suggest that molecular diffusion in the cytosol and chromosome at high osmolality is largely affected by molecular crowding induced by the hypertonic conditions (Golkaram et al, 2016;Park et al, 2015).…”

“…While it remains unclear how protein factors find and interact with their targets, the structure and function of cellular bodies are likely maintained by mobility and interaction with related factors during the cell cycle. Previous studies using biophysical methods such as fluorescence correlation spectroscopy (FCS) demonstrated that various probe molecules freely diffuse in the nucleus and various subnuclear bodies, such as chromatin, chromosomes, and nucleoli (Hihara et al, 2012;Pack et al, 2006;Park et al, 2015;Wachsmuth et al, 2008). FCS based on confocal laser scanning microscopy (CLSM) was applied as a useful and highly sensitive technique for quantitatively assessing molecular concentration and diffusion of fluorescent probe in aqueous solutions and living cells (Eigen and Rigler, 1994;Erdel et al, 2011;Magde et al, 1974;Pack et al, 2006).…”

“…The cellular microenvironment and molecular crowding are key physicochemical characteristics of cellular bodies with a high macromolecular content (Gnutt et al, 2015;Hancock, 2004;Matera et al, 2009;Richter et al, 2007;Richter et al, 2008), and diffusion coefficients (Ds) of fluorescent probe molecules such as green fluorescent protein (GFP) in live cells have been systematically characterized to investigate these traits (Finan et al, 2011;Hihara et al, 2012;Konopka et al, 2009;Mika et al, 2014;Park et al, 2015;Schavemaker et al, 2018;Verkman, 2002). In particular, observations using FCS suggested that heterochromatin and mitotic chromosomes are accessible to protein factors with a molecular size up to 150 kDa, despite their densely packed structure (Hihara et al, 2012).…”