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.
Extended data figures
ER is displayed in blue, LE in red.
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.
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.
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.
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.
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.
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.