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Mapping the Direction of Nucleocytoplasmic Transport of Glucocorticoid Receptor (GR) in Live Cells Using Two-Foci Cross-Correlation in Massively Parallel Fluorescence Correlation Spectroscopy (mpFCS)

journal contribution
submitted on 2024-07-04, 06:05 and posted on 2024-07-04, 06:09 authored by Stanko N. Nikolić, Sho Oasa, Aleksandar J. Krmpot, Lars Terenius, Milivoj R. Belić, Rudolf Rigler, Vladana Vukojević

Nucleocytoplasmic transport of transcription factors is vital for normal cellular function, and its breakdown is a major contributing factor in many diseases. The glucocorticoid receptor (GR) is an evolutionarily conserved, ligand-dependent transcription factor that regulates homeostasis and response to stress and is an important target for therapeutics in inflammation and cancer. In unstimulated cells, the GR resides in the cytoplasm bound to other molecules in a large multiprotein complex. Upon stimulation with endogenous or synthetic ligands, GR translocation to the cell nucleus occurs, where the GR regulates the transcription of numerous genes by direct binding to glucocorticoid response elements or by physically associating with other transcription factors. While much is known about molecular mechanisms underlying GR function, the spatial organization of directionality of GR nucleocytoplasmic transport remains less well characterized, and it is not well understood how the bidirectional nucleocytoplasmic flow of GR is coordinated in stimulated cells. Here, we use two-foci cross-correlation in a massively parallel fluorescence correlation spectroscopy (mpFCS) system to map in live cells the directionality of GR translocation at different positions along the nuclear envelope. We show theoretically and experimentally that cross-correlation of signals from two nearby observation volume elements (OVEs) in an mpFCS setup presents a sharp peak when the OVEs are positioned along the trajectory of molecular motion and that the time position of the peak corresponds to the average time of flight of the molecule between the two OVEs. Hence, the direction and velocity of nucleocytoplasmic transport can be determined simultaneously at several locations along the nuclear envelope. We reveal that under ligand-induced GR translocation, nucleocytoplasmic import/export of GR proceeds simultaneously but at different locations in the cell nucleus. Our data show that mpFCS can characterize in detail the heterogeneity of directional nucleocytoplasmic transport in a live cell and may be invaluable for studies aiming to understand how the bidirectional flow of macromolecules through the nuclear pore complex (NPC) is coordinated to avoid intranuclear transcription factor accretion/abatement.

Other Information

Published in: Analytical Chemistry
License: https://creativecommons.org/licenses/by/4.0/
See article on publisher's website: https://dx.doi.org/10.1021/acs.analchem.3c01427

Additional institutions affiliated with: Arts and Sciences Program - TAMUQ

Funding

Qatar National Research Fund (PPM 04-0131-200019), Early diagnosis of proteinopathies using massively parallel nano-spectroscopy with single-molecule sensitivity. Advanced clinical diagnostics for the development of personalized treatments.

Swedish Research Council (2018-05337, 2022-03402), Quantitative spatio-temporally resolved fluorescence microscopy imaging of fast dynamic processes via massively parallel Fluorescence Correlation Spectroscopy (mpFCS).

Olle Engkvists Foundation (199-0480).

Magnus Bergvall Stiftelse Foundation (2020-04043).

Karolinska Institutet’s Research Foundation Grants (2020-02325).

Swedish Foundation for Strategic Research (SBE13-0115), Molecular imaging in neuropathology.

National Institute on Alcohol Abuse and Alcoholism (R01AA028549), Drug targeting the dynamics of opioid systems in alcohol dependence.

Strategic Research Program in Neuroscience - StratNeuro, (N/A).

ERASMUS+: European Union Programme for Education, Training, Youth and Sport, (N/A).

Science Fund of the Republic of Serbia (6066079).

Ministarstvo Prosvete, Nauke i Tehnoloskog Razvoja (N/A).

Strategic Research Program in Neuroscience (N/A).

History

Language

  • English

Publisher

American Chemical Society

Publication Year

  • 2023

License statement

This Item is licensed under the Creative Commons Attribution 4.0 International License.

Institution affiliated with

  • Texas A&M University at Qatar