Quantitative Cell Cycle Analysis Based on an Endogenous All-in-One Reporter for Cell Tracking and Classification

Area of Science:

• Cell Biology
• Molecular Biology
• Biophysics

Background:

• Cell cycle kinetics significantly influence cell fate decisions.
• Live imaging offers single-cell insights but is limited by the number of fluorescent markers for simultaneous protein and cell cycle analysis.
• Linking protein dynamics to cell cycle progression is essential for understanding cell fate determination.

Purpose of the Study:

• To develop an all-in-one cell cycle reporter for simultaneous analysis of cell cycle progression and protein dynamics.
• To establish an automated image analysis pipeline for cell cycle phase classification.
• To investigate G1 phase regulation and cellular responses to perturbations by combining the reporter with specific protein dynamics.

Main Methods:

• Fluorescently tagging endogenous proliferating cell nuclear antigen (PCNA) as an all-in-one cell cycle reporter.
• Developing an automated image analysis pipeline for cell segmentation, tracking, and classification.
• Simultaneously analyzing cell cycle progression and the dynamics of labeled endogenous cyclin D1 and p21.

Main Results:

• Demonstrated the utility of fluorescently tagged PCNA for tracking cell cycle progression, including quiescence entry.
• Enabled simultaneous quantitative analysis of cell cycle phase and protein dynamics.
• Provided insights into G1 phase regulation and cellular responses to perturbations.

Conclusions:

• The all-in-one PCNA reporter facilitates integrated analysis of cell cycle and protein dynamics.
• The developed image analysis pipeline automates cell cycle phase classification.
• This approach enhances understanding of cell fate decisions and responses to external stimuli.