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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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Deep-Learning-Enhanced Diffusion Imaging Assay for Resolving Local-Density Effects on Membrane Receptors.

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We developed a deep-learning diffusion imaging assay to measure local G-protein-coupled receptor (GPCR) density effects on live cells. This method reveals how receptor density influences ligand interactions, offering new insights into receptor regulation.

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Area of Science:

  • Cellular Biology
  • Biophysics
  • Pharmacology

Background:

  • G-protein-coupled receptor (GPCR) function is regulated by cell surface density.
  • Measuring local density effects on GPCRs is challenging due to receptor mobility and density heterogeneity.
  • Understanding these local effects is crucial for comprehending receptor-ligand interactions.

Purpose of the Study:

  • To develop a novel assay for measuring local-density effects on G-protein-coupled receptor (GPCR) interactions in live cells.
  • To investigate the relationship between receptor density and ligand binding affinity.
  • To provide a new tool for studying receptor regulation in real-time.

Main Methods:

  • Development of a deep-learning (DL) enhanced diffusion imaging assay.
  • Utilizing DL algorithms to generate accurate receptor density maps from image data.
  • Establishing a spatially resolved diffusion assay to measure receptor diffusion coefficients and their relationship with local density.
  • Application of the assay to study chemokine receptor CXCR4 and its interactions with various ligands.

Main Results:

  • The DL-enhanced assay accurately quantifies receptor numbers and density.
  • Spatially resolved measurements revealed density-dependent effects on ligand-receptor interactions.
  • Chemokine receptor CXCR4 showed distinct preferences for agonists at low densities and inverse agonists at high densities.
  • Demonstrated a novel relationship between receptor diffusivity and local density.

Conclusions:

  • Local receptor density significantly influences G-protein-coupled receptor (GPCR) function and ligand interactions.
  • The developed DL-enhanced diffusion imaging assay provides unprecedented capabilities for studying receptor dynamics in live cells.
  • This assay is broadly applicable to various receptors and ligand types, advancing the understanding of receptor pharmacology and regulation.