Letter | Published:

Repeated ER–endosome contacts promote endosome translocation and neurite outgrowth

Nature volume 520, pages 234238 (09 April 2015) | Download Citation


The main organelles of the secretory and endocytic pathways—the endoplasmic reticulum (ER) and endosomes, respectively—are connected through contact sites whose numbers increase as endosomes mature1,2,3. One function of such sites is to enable dephosphorylation of the cytosolic tails of endosomal signalling receptors by an ER-associated phosphatase4, whereas others serve to negatively control the association of endosomes with the minus-end-directed microtubule motor dynein5 or mediate endosome fission6. Cholesterol transfer and Ca2+ exchange have been proposed as additional functions of such sites2,3. However, the compositions, activities and regulations of ER–endosome contact sites remain incompletely understood. Here we show in human and rat cell lines that protrudin, an ER protein that promotes protrusion and neurite outgrowth7, forms contact sites with late endosomes (LEs) via coincident detection of the small GTPase RAB7 and phosphatidylinositol 3-phosphate (PtdIns(3)P). These contact sites mediate transfer of the microtubule motor kinesin 1 from protrudin to the motor adaptor FYCO1 on LEs. Repeated LE–ER contacts promote microtubule-dependent translocation of LEs to the cell periphery and subsequent synaptotagmin-VII-dependent fusion with the plasma membrane. Such fusion induces outgrowth of protrusions and neurites, which requires the abilities of protrudin and FYCO1 to interact with LEs and kinesin 1. Thus, protrudin-containing ER–LE contact sites are platforms for kinesin-1 loading onto LEs, and kinesin-1-mediated translocation of LEs to the plasma membrane, fuelled by repeated ER contacts, promotes protrusion and neurite outgrowth.

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We thank A. Sagona and K.-W. Tan for assistance with plasmid constructs, E. Rønning for yeast two-hybrid analyses and protein purifications, Y. Zhen for advice on RAB7 knockdowns, and A. Engen for expert help with cell cultures. We are grateful to W. Do Heo for providing mCitrine–protrudin and the protrudin(FYVE4A) mutant. The Core Facilities for Advanced Light Microscopy and Electron Microscopy at Oslo University Hospital are acknowledged for providing access to relevant microscopes. C.R. and E.M.W. are senior research fellows of the Norwegian Cancer Society and South-Eastern Norway Regional Health Authority, respectively. C.B. was supported by the Associazione Italiana per la Ricerca sul Cancro (Investigator Grant 14709), Telethon-Italy (grant GGP09145) and MIUR (PRIN2010-2011). T.J. was supported by grant 196898 from the Norwegian Research Council and grant 71043-PR-2006-0320 from the Norwegian Cancer Society. H.S. was supported by grants from the Norwegian Cancer Society and an Advanced Grant from the European Research Council. This work was partly supported by the Research Council of Norway through its Centres of Excellence funding scheme, project number 179571.

Author information


  1. Centre for Cancer Biomedicine, Faculty of Medicine, University of Oslo, Montebello, N-0379 Oslo, Norway

    • Camilla Raiborg
    • , Eva M. Wenzel
    • , Nina M. Pedersen
    • , Kay O. Schink
    • , Sebastian W. Schultz
    • , Marina Vietri
    • , Andreas Brech
    •  & Harald Stenmark
  2. Department of Molecular Cell Biology, Institute for Cancer Research, Oslo University Hospital, Montebello, N-0379 Oslo, Norway

    • Camilla Raiborg
    • , Eva M. Wenzel
    • , Nina M. Pedersen
    • , Kay O. Schink
    • , Sebastian W. Schultz
    • , Marina Vietri
    • , Andreas Brech
    •  & Harald Stenmark
  3. Institute of Medical Biology, University of Tromsø — The Arctic University of Norway, N-9037 Tromsø, Norway

    • Hallvard Olsvik
    •  & Terje Johansen
  4. Department of Biological and Environmental Sciences and Technologies (DiSTeBA), University of Salento, Via Provinciale Monteroni 165, 73100 Lecce, Italy

    • Veronica Nisi
    •  & Cecilia Bucci


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C.R. designed the project and the experiments, performed most of the high-content image analyses and quantitations, mapped the interaction surfaces of protrudin with RAB7(Q67L), made most of the figures and participated in writing the manuscript. E.M.W. performed all live imaging, SIM and LE–PM fusion experiments, and participated in CLEM and immunofluorescense imaging. N.M.P. performed all GFP-trap experiments and messenger RNA analyses. H.O. and T.J. made the RAB7 and FYCO plasmids and performed the MBP pull-down assays. S.W.S. performed the CLEM. M.V. performed some of the quantitations of protrusions in RPE1 cells. K.O.S. wrote scripts for image quantitations using Fiji or ImageJ, made plasmid constructs and stable cell lines and participated in quantitations. V.N. and C.B. performed GST pull-down experiments with protrudin and RAB proteins. A.B. performed conventional electron microscopy and electron microscopy tomography. H.S. coordinated the project and wrote the manuscript. All co-authors gave comments on the manuscript and approved the final version.

Competing interests

The authors declare no competing financial interests.

Corresponding authors

Correspondence to Camilla Raiborg or Harald Stenmark.

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  1. 1.

    Electron tomography model of Protrudin mediated ER-LE contact sites.

    ER is displayed in blue, LE in red.

  2. 2.

    Plus end migration of mCherry-FYCO1 LEs in a GFP-Protrudin expressing cell.

    HeLa cells were transfected with GFP-Protrudin and mCherry-FYCO1 and imaged on a Delta Vision deconvolution microscope with a 60x objective. Images of a GFP-Protrudin expressing cell were acquired at 0.5 Hz for 2 minutes in the mCherry channel. FYCO1-LEs were tracked using the ImageJ plugin "Manual tracking" and showed an overall movement in the plus-end direction.

  3. 3.

    FYCO1 LEs show slow random movement while attached to the ER and to Protrudin.

    HeLa cells were transfected with the ER-marker mTq2-KDEL, mCitrine-Protrudin and mCherry-FYCO1 and imaged on an OMX V4 system (DeltaVision OMX) with a 60x objective. Triple-color live cell imaging was done at 0.33 Hz. Depicted is the overlay of all three colors or combinations of two colors. mCitrine-Protrudin is shown in green, mCherry-FYCO1 in red and mTq2-KDEL is shown in blue or white.

  4. 4.

    Repeated contacts of FYCO1 LEs with Protrudin promote plus-end translocation.

    HeLa cells were transfected with GFP-Protrudin and mCherry-FYCO1 and imaged on an OMX V4 system (DeltaVision OMX) with a 60x objective. Simultaneous dual-color live cell imaging was done at 1 Hz and 9 successive examples of FYCO1 LE movement are shown in total. GFP-Protrudin is displayed in green and mCherry-FYCO1 in red. Note that FYCO1 LEs seem to speed up in the plus-end direction (to the right) after having had contact (arrows) with Protrudin. Scale bar, 1 μm.

  5. 5.

    Protrudin dynamically associates with FYCO1 LEs that move along microtubules.

    HeLa cells were transfected with mTq2-α-Tubulin, mCitrine-Protrudin and mCherry-FYCO1 and imaged on an OMX V4 system (DeltaVision OMX) with a 60x objective. Triple-color live cell imaging was done at 0.33 Hz. Depicted is the overlay of all three colors or combinations of two colors. mCitrine-Protrudin is shown in green, mCherry-FYCO1 in red and mTq2-α-Tubulin is shown in white. Scale bar, 1 µm.

  6. 6.

    Protrudin-induced protrusion formation requires a functional FYVE domain.

    RPE1 cells were transfected with GFP-Protrudin wt or GFP-ProtrudinFYVE4A and imaged on a Delta Vision deconvolution microscope with a 40x objective. Images were taken every 10 minutes for 12 hours.

  7. 7.

    3D rendering of a PC12 cell positive for GFP-Protrudin and mCherry-FYCO1.

    PC12 cells were transfected with GFP-Protrudin (green) and mCherry-FYCO1 (red) and imaged on a Zeiss LSM780 confocal microscope with a 60x objective. Surface rendering was done in Imaris and the animated reconstruction is shown. Note the FYCO1 LEs in the periphery of the protrusions and how the Protrudin meshwork encloses FYCO1 vesicles.

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