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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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Updated: Jul 2, 2025

Immunofluorescence Analysis of Endogenous and Exogenous Centromere-kinetochore Proteins

Immunofluorescence Analysis of Endogenous and Exogenous Centromere-kinetochore Proteins

Published on: March 3, 2016

Fluorescence complementation-based FRET imaging reveals centromere assembly dynamics.

Zhen Dou¹, Ran Liu¹, Ping Gui^1,2,3

¹MOE Key Laboratory for Membraneless Organelles and Cellular Dynamics, Center for Advanced Interdisciplinary Science and Biomedicine of IHM, Hefei National Center for Cross-disciplinary Sciences, University of Science and Technology of China, Hefei 230027, China.

Molecular Biology of the Cell

|February 21, 2024

Summary

This summary is machine-generated.

We developed a novel fluorescence complementation-based Förster resonance energy transfer (FC-FRET) method to visualize protein assembly dynamics in live cells. This technique reveals how CDK1 kinase activity regulates protein interactions during cell division.

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Last Updated: Jul 2, 2025

Immunofluorescence Analysis of Endogenous and Exogenous Centromere-kinetochore Proteins

Immunofluorescence Analysis of Endogenous and Exogenous Centromere-kinetochore Proteins

Published on: March 3, 2016

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Quantitative Immunofluorescence Assay to Measure the Variation in Protein Levels at Centrosomes

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Published on: December 20, 2014

Area of Science:

Cell Biology
Molecular Biology
Biophysics

Background:

Understanding cellular dynamics requires real-time visualization of molecular assembly.
The centromere-kinetochore core complex (CCAN) is crucial for cell division.
Previous methods lacked the resolution to observe dynamic protein interactions in live cells.

Purpose of the Study:

To introduce and validate a new optical imaging method for observing multimeric protein interactions.
To investigate the spatiotemporal dynamics of protein complexes involved in cell division.
To elucidate the role of CDK1 kinase in protein recruitment during cell division.

Main Methods:

Developed fluorescence complementation-based Förster resonance energy transfer (FC-FRET) for live-cell imaging.
Utilized complementary fluorescent proteins to visualize protein dimerization.
Applied FRET measurements to quantify protein interactions at nanometer resolution.
Observed the assembly dynamics of centromere CENP-SXTW tetramers and interactions involving CENP-T and kinetochore proteins Spc24/25.

Main Results:

Successfully visualized centromere CENP-SXTW tetramer assembly dynamics in live cells.
Delineated dimeric interactions between CENP-TW and Spc24/25 dimers, and monomeric CENP-T with Spc24/25 dimers.
Discovered that CDK1 kinase activity is critical for the initial recruitment of Spc24/25 by CENP-T.
Found that CENP-T and Spc24/25 interactions during chromosome segregation are independent of CDK1 activity.

Conclusions:

FC-FRET is a powerful technique for studying spatiotemporal dynamics of trimeric and tetrameric proteins at the nanometer scale.
Established a platform for precise reporting of multimeric protein interactions in live cells.
Revealed a novel regulatory mechanism involving CDK1 in protein recruitment during cell division.