Quantitative methods to analyze subnuclear protein organization in cell populations with varying degrees of protein expression

Ty C. Voss, Ignacio A. Demarco, Cynthia F. Booker, Richard N. Day

Research output: Contribution to journalArticle

3 Scopus citations

Abstract

The control of gene transcription is dependent on DNA-binding and coregulatory proteins that assemble in distinct regions of the cell nucleus. We use multispectral wide-field microscopy of cells expressing transcriptional coregulators labeled with fluorescent proteins (FP) to study the subnuclear localization and function of these factors in living cells. In coexpression studies, the glucocorticoid receptor interacting protein (GRIP) coactivator protein and the silencing mediator of retinoid and thyroid (SMRT) corepressor protein form spherical subnuclear focal bodies that are spatially distinct, suggesting that specific protein interactions concentrate these divergent proteins in separate subnuclear regions. However, the variability of these subnuclear bodies between cells within the population makes analysis based on "representative images" difficult, if not impossible. To address this issue, we develop a protocol for unbiased selection of cells from the population, followed by the automated quantification of the subnuclear organization of the labeled proteins. Statistical methods identify a significant linear correlation between the FP-coregulator expression level and subnuclear focal body formation for both FP-GRIP and FP-SMRT. Importantly, we confirm that these changes in subnuclear organization could be statistically normalized for differences in coregulator expression level. This integrated quantitative image analysis method will allow the rigorous comparison of different experimental cell populations that express variable levels of FP fusion proteins.

Original languageEnglish (US)
Article number024011
JournalJournal of Biomedical Optics
Volume10
Issue number2
DOIs
StatePublished - Mar 1 2005
Externally publishedYes

Keywords

  • Bioimaging
  • Fluorescence microscopy
  • Glucocorticoid receptor interacting protein
  • Green fluorescent protein
  • Nuclear coactivator
  • Nuclear structure

ASJC Scopus subject areas

  • Electronic, Optical and Magnetic Materials
  • Biomaterials
  • Atomic and Molecular Physics, and Optics
  • Biomedical Engineering

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