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Related Concept Videos

Protein Dynamics in Living Cells01:19

Protein Dynamics in Living Cells

2.5K
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-Molecule Imaging of Nuclear Transport
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Combined Selective Plane Illumination Microscopy and FRAP Maps Intranuclear Diffusion of NLS-GFP.

Chad M Hobson1, E Timothy O'Brien1, Michael R Falvo1

  • 1Department of Physics and Astronomy, The University of North Carolina at Chapel Hill, Chapel Hill, North Carolina.

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|July 19, 2020
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Summary

Selective plane illumination microscopy combined with fluorescence recovery after photobleaching (SPIM-FRAP) offers faster, more detailed analysis of protein diffusion and dynamics in living cells, overcoming previous spatial limitations.

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Area of Science:

  • Cellular dynamics and biophysics
  • Microscopy and imaging techniques
  • Molecular and protein dynamics

Background:

  • Fluorescence recovery after photobleaching (FRAP) is a widely used technique for studying protein diffusion and dynamics in cells.
  • Traditional FRAP has limitations in resolving spatial heterogeneity within cellular structures.
  • Selective plane illumination microscopy (SPIM) offers advanced imaging capabilities for live samples.

Purpose of the Study:

  • To develop and validate a novel technique, SPIM-FRAP, for high-resolution, quantitative analysis of protein diffusion and recovery.
  • To overcome the spatial resolution limitations of conventional FRAP.
  • To investigate intranuclear diffusion and DNA damage response dynamics in live cells.

Main Methods:

  • Integration of selective plane illumination microscopy (SPIM) with fluorescence recovery after photobleaching (FRAP) to create SPIM-FRAP.
  • Utilizing a light sheet to bleach a 2D plane and subsequently imaging recovery within the same plane.
  • Mapping diffusion of NLS-GFP and recovery of 53BP1-mCherry in live MDA-MB-231 cells, with validation against simulated data and comparison to fluorescence-correlation spectroscopy.

Main Results:

  • SPIM-FRAP enables simultaneous quantification of diffusion or protein recovery for every pixel in a 2D slice, significantly enhancing spatial resolution.
  • The technique is an order of magnitude faster than fluorescence-correlation spectroscopy for similar measurements.
  • Observed large length-scale heterogeneity in NLS-GFP recovery times, with slower diffusion in nucleoli, and demonstrated that 53BP1-mCherry recovery is slowed at DNA damage sites.

Conclusions:

  • SPIM-FRAP provides a powerful and straightforward method for quantifying spatial distributions of protein recovery and diffusion in living cells.
  • The technique overcomes previous limitations of FRAP, offering improved spatial resolution and speed.
  • SPIM-FRAP validates quantitative accuracy and is poised to become a valuable tool with increasing SPIM system accessibility.