A live cell reporter of exosome secretion and uptake reveals pathfinding behavior of migrating cells

Nat Commun. 2020 Apr 29;11(1):2092. doi: 10.1038/s41467-020-15747-2.

Abstract

Small extracellular vesicles called exosomes affect multiple autocrine and paracrine cellular phenotypes. Understanding the function of exosomes requires a variety of tools, including live imaging. Our previous live-cell reporter, pHluorin-CD63, allows dynamic subcellular monitoring of exosome secretion in migrating and spreading cells. However, dim fluorescence and the inability to make stably-expressing cell lines limit its use. We incorporated a stabilizing mutation in the pHluorin moiety, M153R, which now exhibits higher, stable expression in cells and superior monitoring of exosome secretion. Using this improved construct, we visualize secreted exosomes in 3D culture and in vivo and identify a role for exosomes in promoting leader-follower behavior in 2D and 3D migration. Incorporating an additional non-pH-sensitive red fluorescent tag allows visualization of the exosome lifecycle, including multivesicular body (MVB) trafficking, MVB fusion, exosome uptake and endosome acidification. This reporter will be a useful tool for understanding both autocrine and paracrine roles of exosomes.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Amino Acid Sequence
  • Cell Line, Tumor
  • Cell Movement*
  • Cell Survival
  • Exosomes / metabolism*
  • Exosomes / ultrastructure
  • Extracellular Space / metabolism
  • Green Fluorescent Proteins / metabolism
  • Humans
  • Multivesicular Bodies / metabolism
  • Multivesicular Bodies / ultrastructure
  • Nanoparticles / chemistry
  • Nanoparticles / ultrastructure
  • Tetraspanin 30 / chemistry
  • Tetraspanin 30 / metabolism
  • Time Factors

Substances

  • PHluorin
  • Tetraspanin 30
  • Green Fluorescent Proteins