An Image Analysis Pipeline for Quantifying the Features of Fluorescently-Labeled Biomolecular Condensates in Cells

Area of Science:

• Cellular Biology
• Biophysics
• Computational Biology

Background:

• Biomolecular condensates, formed by liquid-liquid phase separation (LLPS), regulate crucial cellular functions.
• Misregulation of these condensates is linked to diseases like cancer and neurodegeneration.
• Analyzing condensate features provides insights into LLPS thermodynamics and disease mechanisms.

Purpose of the Study:

• To develop an objective computational pipeline for quantifying features of biomolecular condensate puncta.
• To derive thermodynamic parameters associated with LLPS from microscopy data.
• To provide a tool for analyzing NUP98 fusion oncoprotein condensates in pediatric leukemia.

Main Methods:

• Developed a live-cell fluorescence microscopy image processing pipeline (Punctatools).
• Quantified puncta number, volume, and fluorescence intensity.
• Calculated apparent molar concentration, partition coefficient (Kp), and Gibbs free energy of transfer (ΔGTr).
• Provided R functions for statistical analysis and open-source code on GitHub.

Main Results:

• The Punctatools pipeline objectively quantifies puncta features from microscopy images.
• It enables calculation of thermodynamic parameters governing LLPS.
• Demonstrated application in analyzing NUP98-HOXA9 fusion oncoprotein condensates relevant to leukemia.

Conclusions:

• Punctatools offers a reliable method for analyzing biomolecular condensate puncta and their thermodynamics.
• This tool facilitates quantitative comparisons across different cellular conditions and biomolecules.
• The pipeline aids research into condensate-related diseases and provides open-source resources for the scientific community.