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Related Concept Videos

Protein Dynamics in Living Cells01:19

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...
Protein Diffusion in the Membrane01:24

Protein Diffusion in the Membrane

Proteins show rotational as well as lateral diffusion across the membrane. The lateral diffusion of proteins was confirmed through the cell fusion experiment where mouse and human cells were fused, resulting in hybrid cells. When the human and mouse cells fused, the specific membrane proteins on human and mouse cells were marked with the red and green-fluorescent markers, respectively. Initially, the red and green fluorescence was located on the respective hemisphere of the cell. As time...

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Related Experiment Video

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Fluorescence Recovery after Merging a Droplet to Measure the Two-dimensional Diffusion of a Phospholipid Monolayer
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Published on: October 15, 2015

Measuring diffusion and binding kinetics by contact area FRAP.

Timothy P Tolentino1, Jianhua Wu, Veronika I Zarnitsyna

  • 1Wallace H. Coulter Department of Biomedical Engineering and George W. Woodruff School of Mechanical Engineering, Georgia Institute of Technology, Atlanta, Georgia 30332, USA.

Biophysical Journal
|April 9, 2008
PubMed
Summary
This summary is machine-generated.

Researchers developed a mathematical model and experimental method to study the immunological synapse. This approach measures kinetic rates and diffusion coefficients of interacting molecules within these crucial immune cell junctions.

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Fluorescence Biomembrane Force Probe: Concurrent Quantitation of Receptor-ligand Kinetics and Binding-induced Intracellular Signaling on a Single Cell
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Fluorescence Biomembrane Force Probe: Concurrent Quantitation of Receptor-ligand Kinetics and Binding-induced Intracellular Signaling on a Single Cell

Published on: August 4, 2015

Area of Science:

  • Immunology
  • Biophysics
  • Cell Biology

Background:

  • The immunological synapse is a specialized structure for immune cell communication.
  • Its formation involves the dynamic diffusion and binding of molecules within the contact area.
  • Synapse stability allows receptor-ligand interactions to reach equilibrium.

Purpose of the Study:

  • To extend a fluorescence recovery after photobleaching (FRAP) experiment to validate a mathematical model of the immunological synapse.
  • To investigate the reaction-diffusion dynamics of ligands within the established immunological synapse.
  • To develop a new method for in situ measurement of kinetic rates and diffusion coefficients.

Main Methods:

  • Developed a mathematical model for coupled reaction-diffusion processes in immunological synapses.
  • Extended contact area FRAP experiments to test the model's validity.
  • Analyzed fluorescence recovery time courses to extract kinetic and diffusion parameters.

Main Results:

  • Ligand accumulation within the synapse was observed due to binding activity and lateral mobility.
  • Reverse reaction rates in the 2D synapse were significantly slower (at least 100-fold) than in 3D solution.
  • A nonrecoverable fluorescence fraction was detected, indicating slow dissociation or diffusion of some ligands.

Conclusions:

  • The combined theoretical and experimental approach provides a novel method for in situ measurements.
  • This method quantifies kinetic rates, diffusion coefficients, and nonrecoverable fractions of interacting molecules.
  • The findings offer insights into the dynamics of immunological synapses and other cell-bilayer junctions.