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Protein Dynamics in Living Cells

Protein Dynamics in Living Cells

Different fluorescence-based techniques are used to study the protein dynamics in living cells. These techniques include FRAP, FRET, and PET.
Fluorescent recovery after photobleaching (FRAP) is a fluorescent-protein-based detection technique used to quantify protein movement rates within the cell. This method exposes a small portion of the cell to an intense laser beam. The laser beam causes permanent photobleaching of the fluorophore-tagged proteins in the exposed region. As the bleached...

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Single-Cell Quantification of Protein Degradation Rates by Time-Lapse Fluorescence Microscopy in Adherent Cell Culture

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Published on: February 4, 2018

Quantifying E2F1 protein dynamics in single cells.

Bernard Mathey-Prevot^1,2, Bao-Tran Parker¹, Carolyn Im¹

¹Department of Pharmacology and Cancer Biology, Duke University School of Medicine, Durham, NC 27710, USA.

Quantitative Biology (Beijing, China)

|June 17, 2020

Summary

This summary is machine-generated.

Researchers developed fluorescent E2F1 protein reporters to track cell cycle dynamics. These tools accurately capture E2F1 protein levels during cell division and stress, aiding in understanding cell fate decisions.

Keywords:

E2F1 reporter cell cycle protein

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Last Updated: Dec 18, 2025

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Published on: February 4, 2018

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Monitoring Kinase and Phosphatase Activities Through the Cell Cycle by Ratiometric FRET

Monitoring Kinase and Phosphatase Activities Through the Cell Cycle by Ratiometric FRET

Published on: January 27, 2012

Area of Science:

Cell Biology
Molecular Biology
Genetics

Background:

E2F1 protein is crucial for cell cycle regulation, impacting proliferation, apoptosis, and differentiation.
E2F1 expression is tightly controlled by multiple mechanisms, but its precise role in different cellular outcomes requires further study.
Accurate monitoring of E2F1 protein dynamics in live cells is essential for understanding its functions.

Purpose of the Study:

To engineer and validate fluorescent E2F1 protein reporters for real-time monitoring of E2F1 dynamics.
To quantitatively capture E2F1 protein expression changes during the cell cycle and in response to genotoxic stress.
To investigate the influence of the E2F1 3' untranslated region (3'UTR) and Rb protein on E2F1 reporter dynamics.

Main Methods:

Developed reporter constructs expressing E2F1-Venus fusion proteins under the control of E2F1 promoters.
Constructs were designed with or without the E2F1 3'UTR to assess its regulatory role.
Validated and quantified reporter protein expression in single fibroblasts and epithelial cells using live imaging.

Main Results:

E2F1 reporters accurately reflected endogenous E2F1 behavior during cell cycle progression and genotoxic stress.
Reporter signal increased during the cell cycle, peaking before division and declining prior to mitosis.
Absence of the E2F1 3'UTR resulted in higher protein levels and a less complex dynamic profile; Rb presence/absence did not affect reporter levels.

Conclusions:

Validated E2F1 fluorescent reporters provide a novel tool for real-time, single-cell analysis of E2F1 dynamics.
These reporters complement existing tools for studying the Rb/E2F pathway.
The findings offer insights into the regulation of E2F1 expression and its role in cell fate determination.