Computer-assisted image analysis protocol that quantitatively measures subnuclear protein organization in cell...

Area of Science:

• Cell Biology
• Molecular Biology
• Biophysics

Background:

• Nuclear proteins, including nuclear receptor co-repressor (NCoR), exhibit specific subnuclear localization.
• This localization pattern is maintained when NCoR is expressed as a fluorescent protein (FP) fusion.
• Studying factors influencing NCoR subnuclear organization necessitates unbiased cell selection for image analysis.

Purpose of the Study:

• To develop an unbiased method for selecting cells for image analysis to study NCoR subnuclear organization.
• To quantify the subnuclear organization of NCoR using automated image analysis.

Main Methods:

• Co-expression of monomeric red FP (mRFP) and yellow FP (YFP)-labeled NCoR in cells.
• Cell selection for imaging based on diffuse mRFP signal, independent of YFP-NCoR organization.
• Automated image analysis to identify regions of interest (ROIs), estimate FP expression, and quantify YFP-NCoR subnuclear organization (focal body size, relative intensity).

Main Results:

• An integrated image analysis protocol was established.
• The protocol allows for precise and consistent measurement of thousands of ROIs across numerous cells.
• The method provides an unbiased approach to assessing NCoR subnuclear positioning.

Conclusions:

• The developed protocol offers an unbiased and efficient method for quantifying NCoR subnuclear organization.
• This approach facilitates the study of factors influencing nuclear protein localization.
• Automated image analysis of fluorescently tagged proteins enables high-throughput investigation of subnuclear architecture.