Punctatools

Punctatools quantifies features of fluorescently-labeled biomolecular condensates from live-cell fluorescence microscopy images to measure puncta properties and thermodynamic partitioning associated with liquid-liquid phase separation (LLPS).

Key Features:

• Live-cell image processing: Processes live-cell fluorescence microscopy images to identify and analyze puncta.
• Puncta quantification: Detects and quantifies puncta number, volume, shape, and location.
• Fluorescence intensity measurement: Measures fluorescence intensities of labeled biomolecules inside and outside puncta.
• Concentration determination: Converts fluorescence intensity to apparent molar concentration using a standard curve correlating fluorescence intensity with protein concentration.
• Thermodynamic metrics: Calculates partition coefficient (Kp) and Gibbs free energy of transfer (ΔGTr) to quantify partitioning favorability.
• Reporting: Generates comprehensive reports of puncta features across hundreds of cells.
• Statistical analysis: Provides a library of R functions for statistical analysis tailored to experimental designs.

Scientific Applications:

• LLPS characterization: Quantitative analysis of biomolecular condensates formed by liquid-liquid phase separation (LLPS) in cells.
• NUP98 fusion analysis: Analysis of nucleoporin 98 (NUP98) fusion oncoproteins, including NUP98-HOXA9 transcriptional condensates relevant to leukemogenesis.
• Comparative studies: Comparing puncta properties formed under different cellular conditions or by distinct biomolecules.
• Thermodynamic investigation: Quantifying thermodynamic drivers of partitioning to investigate misregulation implicated in neurodegenerative disorders and cancer.

Methodology:

Live-cell fluorescence microscopy image processing based on existing methodologies and open-source tools; quantification of puncta numbers, volumes, shapes, locations, and fluorescence intensities; conversion of intensities to apparent molar concentration via a standard curve; calculation of partition coefficient (Kp) and Gibbs free energy of transfer (ΔGTr); and statistical analysis using an R function library with report generation.