Assaying Homodimers of NF-κB in Live Single Cells
NF-κB is a family of heterodimers and homodimers which are generated from subunits encoded by five genes. The predominant classical dimer RelA:p50 is presumed to operate as “NF-κB” in many contexts. However, there are several other dimer species which exist and may even be more functionally relevant...
| Main Authors: | , , , , , , , |
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| Format: | Article |
| Language: | English |
| Published: |
Frontiers Media S.A.
2019-11-01
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| Series: | Frontiers in Immunology |
| Subjects: | |
| Online Access: | https://www.frontiersin.org/article/10.3389/fimmu.2019.02609/full |
| _version_ | 1811207016018870272 |
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| author | Erik W. Martin Sayantan Chakraborty Diego M. Presman Francesco Tomassoni Ardori Kyu-Seon Oh Mary Kaileh Lino Tessarollo Myong-Hee Sung |
| author_facet | Erik W. Martin Sayantan Chakraborty Diego M. Presman Francesco Tomassoni Ardori Kyu-Seon Oh Mary Kaileh Lino Tessarollo Myong-Hee Sung |
| author_sort | Erik W. Martin |
| collection | DOAJ |
| description | NF-κB is a family of heterodimers and homodimers which are generated from subunits encoded by five genes. The predominant classical dimer RelA:p50 is presumed to operate as “NF-κB” in many contexts. However, there are several other dimer species which exist and may even be more functionally relevant in specific cell types. Accurate characterization of stimulus-specific and tissue-specific dimer repertoires is fundamentally important for understanding the downstream gene regulation by NF-κB proteins. In vitro assays such as immunoprecipitation have been widely used to analyze subunit composition, but these methods do not provide information about dimerization status within the natural intracellular environment of intact live cells. Here we apply a live single cell microscopy technique termed Number and Brightness to examine dimers translocating to the nucleus in fibroblasts after pro-inflammatory stimulation. This quantitative assay suggests that RelA:RelA homodimers are more prevalent than might be expected. We also found that the relative proportion of RelA:RelA homodimers can be perturbed by small molecule inhibitors known to disrupt the NF-κB pathway. Our findings show that Number and Brightness is a useful method for investigating NF-κB dimer species in live cells. This approach may help identify the relevant targets in pathophysiological contexts where the dimer specificity of NF-κB intervention is desired. |
| first_indexed | 2024-04-12T03:56:45Z |
| format | Article |
| id | doaj.art-fc8177a5b3dc4703a9e9ffb3a21f10b7 |
| institution | Directory Open Access Journal |
| issn | 1664-3224 |
| language | English |
| last_indexed | 2024-04-12T03:56:45Z |
| publishDate | 2019-11-01 |
| publisher | Frontiers Media S.A. |
| record_format | Article |
| series | Frontiers in Immunology |
| spelling | doaj.art-fc8177a5b3dc4703a9e9ffb3a21f10b72022-12-22T03:48:49ZengFrontiers Media S.A.Frontiers in Immunology1664-32242019-11-011010.3389/fimmu.2019.02609445991Assaying Homodimers of NF-κB in Live Single CellsErik W. Martin0Sayantan Chakraborty1Diego M. Presman2Francesco Tomassoni Ardori3Kyu-Seon Oh4Mary Kaileh5Lino Tessarollo6Myong-Hee Sung7Laboratory of Molecular Biology and Immunology, National Institute on Aging, National Institutes of Health, Baltimore, MD, United StatesLaboratory of Molecular Biology and Immunology, National Institute on Aging, National Institutes of Health, Baltimore, MD, United StatesLaboratory of Receptor Biology and Gene Expression, National Cancer Institute, National Institutes of Health, Bethesda, MD, United StatesMouse Cancer Genetics Program, National Cancer Institute, National Institutes of Health, Frederick, MD, United StatesLaboratory of Molecular Biology and Immunology, National Institute on Aging, National Institutes of Health, Baltimore, MD, United StatesLaboratory of Molecular Biology and Immunology, National Institute on Aging, National Institutes of Health, Baltimore, MD, United StatesMouse Cancer Genetics Program, National Cancer Institute, National Institutes of Health, Frederick, MD, United StatesLaboratory of Molecular Biology and Immunology, National Institute on Aging, National Institutes of Health, Baltimore, MD, United StatesNF-κB is a family of heterodimers and homodimers which are generated from subunits encoded by five genes. The predominant classical dimer RelA:p50 is presumed to operate as “NF-κB” in many contexts. However, there are several other dimer species which exist and may even be more functionally relevant in specific cell types. Accurate characterization of stimulus-specific and tissue-specific dimer repertoires is fundamentally important for understanding the downstream gene regulation by NF-κB proteins. In vitro assays such as immunoprecipitation have been widely used to analyze subunit composition, but these methods do not provide information about dimerization status within the natural intracellular environment of intact live cells. Here we apply a live single cell microscopy technique termed Number and Brightness to examine dimers translocating to the nucleus in fibroblasts after pro-inflammatory stimulation. This quantitative assay suggests that RelA:RelA homodimers are more prevalent than might be expected. We also found that the relative proportion of RelA:RelA homodimers can be perturbed by small molecule inhibitors known to disrupt the NF-κB pathway. Our findings show that Number and Brightness is a useful method for investigating NF-κB dimer species in live cells. This approach may help identify the relevant targets in pathophysiological contexts where the dimer specificity of NF-κB intervention is desired.https://www.frontiersin.org/article/10.3389/fimmu.2019.02609/fullRelANF-κBtranscription factornumber and brightnessmicroscopyoligomerization |
| spellingShingle | Erik W. Martin Sayantan Chakraborty Diego M. Presman Francesco Tomassoni Ardori Kyu-Seon Oh Mary Kaileh Lino Tessarollo Myong-Hee Sung Assaying Homodimers of NF-κB in Live Single Cells Frontiers in Immunology RelA NF-κB transcription factor number and brightness microscopy oligomerization |
| title | Assaying Homodimers of NF-κB in Live Single Cells |
| title_full | Assaying Homodimers of NF-κB in Live Single Cells |
| title_fullStr | Assaying Homodimers of NF-κB in Live Single Cells |
| title_full_unstemmed | Assaying Homodimers of NF-κB in Live Single Cells |
| title_short | Assaying Homodimers of NF-κB in Live Single Cells |
| title_sort | assaying homodimers of nf κb in live single cells |
| topic | RelA NF-κB transcription factor number and brightness microscopy oligomerization |
| url | https://www.frontiersin.org/article/10.3389/fimmu.2019.02609/full |
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