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PIKfyve inhibition increases exosome release and induces secretory autophagy

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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.

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Abbreviations

CI-MPR:

Cation-independent mannose 6-phosphate receptor

EM:

Electron microscopy

ESCRTs:

Endosomal sorting complexes required for transport proteins

ILV:

Intraluminal vesicle

MS:

Mass spectrometry

MVB:

Multivesicular body

NTA:

Nanoparticle tracking analysis

PI(3)P:

Phosphatidylinositol-3-phosphate

PI(3,5)P2 :

Phosphatidylinositol-3,5-bisphosphate

PI(5)P:

Phosphatidylinositol-5-phosphate

TGN:

Trans-golgi network

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Acknowledgments

This work was funded by The South-Eastern Norwegian Regional Health Authority, The Norwegian Cancer Society and The Research Council of Norway, and supported by The Research Council of Norway through its Centers of Excellence funding scheme, project number 179571. We thank Anne Engen, Anne Grethe Myrann and Marianne Smestad for their excellent technical assistance and Tore Skotland for valuable and interesting discussions. We also thank the proteomics core facility, especially Bernd Thiede and Christian Koehler, at the University of Oslo.

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Authors and Affiliations

  1. Department of Molecular Cell Biology, The Norwegian Radium Hospital, Institute for Cancer Research, Oslo University Hospital, 0379, Oslo, Norway

    Nina Pettersen Hessvik, Anders Øverbye, Andreas Brech, Maria Lyngaas Torgersen, Ida Seim Jakobsen, Kirsten Sandvig & Alicia Llorente

  2. Centre for Cancer Biomedicine, University of Oslo, 0379, Oslo, Norway

    Nina Pettersen Hessvik, Anders Øverbye, Andreas Brech, Maria Lyngaas Torgersen, Ida Seim Jakobsen, Kirsten Sandvig & Alicia Llorente

  3. Department of Biosciences, University of Oslo, 0316, Oslo, Norway

    Andreas Brech & Kirsten Sandvig

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  1. Nina Pettersen Hessvik
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Hessvik, N.P., Øverbye, A., Brech, A. et al. PIKfyve inhibition increases exosome release and induces secretory autophagy. Cell. Mol. Life Sci. 73, 4717–4737 (2016). https://doi.org/10.1007/s00018-016-2309-8

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