PIKfyve Inhibition Increases Exosome Release and Induces Secretory Autophagy

Cell Mol Life Sci. 2016 Dec;73(24):4717-4737. doi: 10.1007/s00018-016-2309-8. Epub 2016 Jul 20.

Abstract

Exosomes are vesicles released from cells by fusion of multivesicular bodies (MVBs) with the plasma membrane. This study aimed to investigate whether the phosphoinositide kinase PIKfyve affects this process. Our results show that in PC-3 cells inhibition of PIKfyve by apilimod or depletion by siRNA increased the secretion of the exosomal fraction. Moreover, quantitative electron microscopy analysis showed that cells treated with apilimod contained more MVBs per cell and more intraluminal vesicles per MVB. Interestingly, mass spectrometry analysis revealed a considerable enrichment of autophagy-related proteins (NBR1, p62, LC3, WIPI2) in exosomal fractions released by apilimod-treated cells, a result that was confirmed by immunoblotting. When the exosome preparations were investigated by electron microscopy a small population of p62-labelled electron dense structures was observed together with CD63-containing exosomes. The p62-positive structures were found in less dense fractions than exosomes in density gradients. Inside the cells, p62 and CD63 were found in the same MVB-like organelles. Finally, both the degradation of EGF and long-lived proteins were shown to be reduced by apilimod. In conclusion, inhibition of PIKfyve increases secretion of exosomes and induces secretory autophagy, showing that these pathways are closely linked. We suggest this is due to impaired fusion of lysosomes with both MVBs and autophagosomes, and possibly increased fusion of MVBs with autophagosomes, and that the cells respond by secreting the content of these organelles to maintain cellular homeostasis.

Keywords: Exophagy; Exosomes; Extracellular vesicles; PIKfyve; Phosphoinositides; Secretory autophagy.

MeSH terms

  • Autophagosomes / drug effects
  • Autophagosomes / metabolism
  • Autophagy* / drug effects
  • Autophagy-Related Proteins / metabolism
  • Cell Line, Tumor
  • Epidermal Growth Factor / metabolism
  • Exosomes / drug effects
  • Exosomes / metabolism*
  • Exosomes / ultrastructure
  • Gene Knockdown Techniques
  • Humans
  • Lysosomes / drug effects
  • Lysosomes / metabolism
  • Morpholines / pharmacology
  • Multivesicular Bodies / drug effects
  • Multivesicular Bodies / metabolism
  • Multivesicular Bodies / ultrastructure
  • Phosphatidylinositol 3-Kinases / metabolism
  • Phosphoinositide-3 Kinase Inhibitors*
  • Proteolysis / drug effects
  • Proteomics
  • RNA-Binding Proteins / metabolism
  • Secretory Pathway* / drug effects
  • Tetraspanin 30 / metabolism
  • Triazines / pharmacology
  • Ubiquitin / metabolism
  • Ubiquitination / drug effects
  • Up-Regulation / drug effects

Substances

  • Autophagy-Related Proteins
  • Morpholines
  • P62 protein, human
  • Phosphoinositide-3 Kinase Inhibitors
  • RNA-Binding Proteins
  • Tetraspanin 30
  • Triazines
  • Ubiquitin
  • apilimod mesylate
  • Epidermal Growth Factor
  • PIKFYVE protein, human