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)
<p>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 an...
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| مؤلفون آخرون: | , , , , , |
| منشور في: |
2023
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| _version_ | 1864513511830323200 |
|---|---|
| author | Stanko N. Nikolić (14151438) |
| author2 | Sho Oasa (7244462) Aleksandar J. Krmpot (7244456) Lars Terenius (79411) Milivoj R. Belić (3958976) Rudolf Rigler (375415) Vladana Vukojević (165297) |
| author2_role | author author author author author author |
| author_facet | Stanko N. Nikolić (14151438) Sho Oasa (7244462) Aleksandar J. Krmpot (7244456) Lars Terenius (79411) Milivoj R. Belić (3958976) Rudolf Rigler (375415) Vladana Vukojević (165297) |
| author_role | author |
| dc.creator.none.fl_str_mv | Stanko N. Nikolić (14151438) Sho Oasa (7244462) Aleksandar J. Krmpot (7244456) Lars Terenius (79411) Milivoj R. Belić (3958976) Rudolf Rigler (375415) Vladana Vukojević (165297) |
| dc.date.none.fl_str_mv | 2023-10-02T03:00:00Z |
| dc.identifier.none.fl_str_mv | 10.1021/acs.analchem.3c01427 |
| dc.relation.none.fl_str_mv | https://figshare.com/articles/journal_contribution/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_/26021146 |
| dc.rights.none.fl_str_mv | CC BY 4.0 info:eu-repo/semantics/openAccess |
| dc.subject.none.fl_str_mv | Biological sciences Biochemistry and cell biology Genetics Biomedical and clinical sciences Neurosciences Chemical sciences Analytical chemistry Cell transport Fluorescence Fluorescence spectroscopy Nanoparticles Suspensions |
| dc.title.none.fl_str_mv | 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) |
| dc.type.none.fl_str_mv | Text Journal contribution info:eu-repo/semantics/publishedVersion text contribution to journal |
| description | <p>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.</p> <h2>Other Information</h2> <p>Published in: Analytical Chemistry<br> License: <a href="https://creativecommons.org/licenses/by/4.0/" target="_blank">https://creativecommons.org/licenses/by/4.0/</a><br> See article on publisher's website: <a href="https://dx.doi.org/10.1021/acs.analchem.3c01427" target="_blank">https://dx.doi.org/10.1021/acs.analchem.3c01427</a></p> <p>Additional institutions affiliated with: Arts and Sciences Program - TAMUQ</p> |
| eu_rights_str_mv | openAccess |
| id | Manara2_7fded8e14fccc1ce0ce9e0beafc9dcdc |
| identifier_str_mv | 10.1021/acs.analchem.3c01427 |
| network_acronym_str | Manara2 |
| network_name_str | Manara2 |
| oai_identifier_str | oai:figshare.com:article/26021146 |
| publishDate | 2023 |
| repository.mail.fl_str_mv | |
| repository.name.fl_str_mv | |
| repository_id_str | |
| rights_invalid_str_mv | CC BY 4.0 |
| spelling | 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)Stanko N. Nikolić (14151438)Sho Oasa (7244462)Aleksandar J. Krmpot (7244456)Lars Terenius (79411)Milivoj R. Belić (3958976)Rudolf Rigler (375415)Vladana Vukojević (165297)Biological sciencesBiochemistry and cell biologyGeneticsBiomedical and clinical sciencesNeurosciencesChemical sciencesAnalytical chemistryCell transportFluorescenceFluorescence spectroscopyNanoparticlesSuspensions<p>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.</p> <h2>Other Information</h2> <p>Published in: Analytical Chemistry<br> License: <a href="https://creativecommons.org/licenses/by/4.0/" target="_blank">https://creativecommons.org/licenses/by/4.0/</a><br> See article on publisher's website: <a href="https://dx.doi.org/10.1021/acs.analchem.3c01427" target="_blank">https://dx.doi.org/10.1021/acs.analchem.3c01427</a></p> <p>Additional institutions affiliated with: Arts and Sciences Program - TAMUQ</p>2023-10-02T03:00:00ZTextJournal contributioninfo:eu-repo/semantics/publishedVersiontextcontribution to journal10.1021/acs.analchem.3c01427https://figshare.com/articles/journal_contribution/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_/26021146CC BY 4.0info:eu-repo/semantics/openAccessoai:figshare.com:article/260211462023-10-02T03:00:00Z |
| spellingShingle | 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) Stanko N. Nikolić (14151438) Biological sciences Biochemistry and cell biology Genetics Biomedical and clinical sciences Neurosciences Chemical sciences Analytical chemistry Cell transport Fluorescence Fluorescence spectroscopy Nanoparticles Suspensions |
| status_str | publishedVersion |
| title | 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) |
| title_full | 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) |
| title_fullStr | 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) |
| title_full_unstemmed | 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) |
| title_short | 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) |
| title_sort | 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) |
| topic | Biological sciences Biochemistry and cell biology Genetics Biomedical and clinical sciences Neurosciences Chemical sciences Analytical chemistry Cell transport Fluorescence Fluorescence spectroscopy Nanoparticles Suspensions |