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Repeated ER–endosome contacts promote endosome translocation and neurite outgrowth

Abstract

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 mature 1, 2, 3 . One function of such sites is to enable dephosphorylation of the cytosolic tails of endosomal signalling receptors by an ER-associated phosphatase 4 , whereas others serve to negatively control the association of endosomes with the minus-end-directed microtubule motor dynein 5 or mediate endosome fission 6 . Cholesterol transfer and Ca 2+ exchange have been proposed as additional functions of such sites 2, 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 outgrowth 7 , 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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Figure 1: Protrudin forms RAB7- and PtdIns(3)P-dependent ER–LE contact sites.
Figure 2: Protrudin forms repeated contact sites with LEs and mediates their translocation to the cell periphery.
Figure 3: Protrudin loads kinesin 1 onto FYCO1-positive LEs.
Figure 4: Protrudin and FYCO1 cooperatively induce neurite outgrowth by facilitating LE fusion with the plasma membrane.

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Acknowledgements

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

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Authors

Contributions

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.

Corresponding authors

Correspondence to Camilla Raiborg or Harald Stenmark.

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Competing interests

The authors declare no competing financial interests.

Extended data figures and tables

Extended Data Figure 1 Protrudin forms RAB7- and PtdIns(3)P-dependent ER–LE contact sites.

a, RAB7(Q67L) causes excessive recruitment of GFP–protrudin to LAMP1-positive endosomes in HeLa cells. Confocal images are representative of at least 10 captures. b, GFP–protrudin is not recruited to RAB5(Q79L)-positive endosomes. Confocal images are representative of at least 10 captures. c, Quantification of GFP–protrudin recruitment to RAB7 endosomes in cells cotransfected with mCherry–RAB7(Q67L) or RAB7(T22N). Quantification of Olympus ScanR images. Error bars denote ± s.e.m. RAB7(Q67L): 4 experiments, 2,673 cells; RAB7(T22N): 3 experiments, 1,814 cells. ***P < 0.001 (unpaired t-test). d, Protrudin is pulled down with GFP–RAB7(Q67L), but not with GFP–RAB7(T22N) or GFP alone in a GFP-trap assay. e, The C-terminal part of Myc–protrudin, including LCR domains, is pulled down with GFP–RAB7(Q67L) in a GFP-trap assay. f, In vitro pulldown of a GST–protrudin–LCR construct (residues 208–274) shows direct binding to His–RAB7 loaded with GTP, but not to RAB7–GDP or RAB5–GTP. g, Protrudin-positive ER associates with RAB7/PtdIns(3)P-positive endosomes in HeLa cells transfected with low levels of Myc–protrudin, mCherry–RAB7 and GFP–2×FYVE, shown by confocal microscopy. Note that PtdIns(3)P-positive RAB7-negative endosomes are protrudin-negative (arrows). Confocal images are representative of at least 10 captures. h, Confocal micrographs showing that high levels of 2×FYVE compete with the recruitment of protrudin to RAB7(Q67L)-positive endosomes. Representative of 4 confocal captures and 240 wide-field images per condition. Quantification of Olympus ScanR images. Error bars denote ± s.e.m. from 3 experiments. GFP–2×FYVEC215S: 5,861 cells, GFP–2×FYVE: 4,641 cells. **P < 0.01 (one-sample t-test).

Extended Data Figure 2 LEs and ER form extensive contact sites upon expression of RAB7(Q67L).

a, Protrudin co-recruits the ER marker VAP-A to RAB7(Q67L) endosomes in a FYVE-dependent manner. HeLa cells were transfected with GFP–RAB7(Q67L) and Myc–protrudin or FYVE-deleted protrudin as shown by confocal microscopy. Images are representative of at least 10 captures. WT, wild type. b, c, Electron micrographs showing that coexpression of protrudin and RAB7(Q67L) leads to formation of endosomal clusters with strong recruitment of ER structures. The massive accumulation of ER–endosome contact sites on an endosome can be clearly seen (arrowheads in b). Continuity with the rough ER is indicated (arrows in b). Representative of 30 images.

Extended Data Figure 3 Redistribution of ER membranes in protrudin- and RAB7(Q67L)-transfected cells.

CLEM. HeLa cells were transfected with GFP–protrudin and mCherry–RAB7(Q67L), and four neighbouring cells with varying levels of overexpression were analysed by confocal microscopy before electron microscopy. A non-transfected cell (cell number 1, inset a) shows ER membranes distributed all over the cytosol. A cell expressing high levels of protrudin and RAB7(Q67L) (cell number 2, inset b) shows a complete redistribution of ER membranes to perinuclear structures. These structures are positive for protrudin and RAB7(Q67L) (arrows) as seen by the immunofluorescense-electron microscopy overlay and represent the structures seen at higher resolution in Fig. 1e and Extended Data Fig. 2b, c). Representative of 4 CLEM images.

Extended Data Figure 4 Protrudin mediates LE translocation to the cell periphery.

a, The positioning of early and recycling endosomes is not changed in GFP–protrudin-overexpressing HeLa cells (indicated by asterisks). Confocal images are representative of at least 10 captures. b, Endogenous FYCO1 localizes to protrudin and RAB7(Q67L)-positive endosomes. HeLa cells were transfected with GFP–protrudin and mCherry–RAB7(Q67L), stained with anti-FYCO1 antibody and imaged by confocal microscopy. Images are representative of at least 10 captures. c, Myc–protrudin is pulled down with GFP–FYCO1 but not with GFP in a GFP-trap assay. Input 1%. d, Still pictures from Supplementary Video 2, representative of 10 videos. A distal part of a GFP–protrudin- and mCherry–FYCO1-positive HeLa cell is shown. FYCO1 vesicles move towards the microtubule plus-end of the cell. The movement of each vesicle during 120 s is shown by colour-coded lines. The coloured dots indicate where the vesicles stop. Bottom panel: example for the calculation of the centre of mass of all tracked vesicles from Supplementary Video 2. The starting point of every track is set to x = 0 and y = 0. The endpoints of all tracks (blue dots) define the centre of mass (red cross) as their spatial averaged point.

Extended Data Figure 5 LAMP1 and FYCO1 cluster perinuclearly in cells depleted of protrudin.

a, Western blot showing the level of siRNA-mediated knockdown of protrudin and KLC1 as compared to control. b, c, HeLa (b) and RPE1 (c) cells depleted of protrudin by siRNA show perinuclear localization of LAMP1-positive LEs. Images are representative of at least 4 confocal and 290 wide-field captures. Cells were transfected with control siRNA, siRNA targeting protrudin (#1–3) or KLC1 (only RPE1) and analysed by Olympus ScanR high-throughput microscopy. Error bars denote ± s.e.m. HeLa, 3 experiments: control: 5,945 cells; siRNA protrudin #1: 2,965 cells; siRNA protrudin #2: 3,378 cells; siRNA protrudin #3: 3,986 cells. RPE1, 4 experiments: control: 3,655 cells; siRNA protrudin #1: 2,292 cells; siRNA protrudin #2: 1,961 cells; siRNA protrudin #3: 2,402 cells; siRNA KLC1 (3 experiments): 1,625 cells. *P < 0.05, **P < 0.01 (unpaired t-test). d, GFP–FYCO1 clusters perinuclearly in protrudin siRNA cells as compared to control cells, in which GFP–FYCO1 is mainly localized to the cell periphery as quantified by Olympus ScanR microscopy (representative of 240 images). Error bar denotes ± s.e.m. from 3 experiments. Control: 2,979 cells; siRNA protrudin: 2,011 cells. **P < 0.01 (one-sample t-test). e, In protrudin siRNA-treated cells, late endosomes cluster perinuclearly, but not in neighbouring cells expressing siRNA-resistant Myc–protrudin, as shown by confocal microscopy (representative of 2 confocal and 290 wide-field captures). The graph shows quantification of perinuclear localization of LAMP1 in siRNA-treated cells (siRNA protrudin #1) compared to neighbouring cells expressing siRNA-resistant Myc–protrudin by Olympus ScanR analysis. Error bars denote ± s.e.m. from 3 experiments. siRNA protrudin: 3,601 cells; siRNA protrudin/Myc–protrudin: 1,437 cells. ***P < 0.001 (unpaired t-test). Knockdown efficiency and level of overexpression of the siRNA-resistant Myc–protrudin construct is shown by western blotting. f, LAMP1-positive LEs cluster perinuclearly in cells depleted of FYCO1 as shown by Olympus ScanR quantitative microscopy (representative of 290 images). Error bars denote ± s.e.m. from 3 experiments. Control: 5,033 cells; siRNA FYCO1: 5,820 cells. **P < 0.01 (unpaired t-test). Knockdown efficiency of FYCO1 is shown by western blotting.

Extended Data Figure 6 Protrudin forms repeated contact sites with LEs and mediates their translocation to the cell periphery.

ad, Still images from videos showing how mCherry–FYCO1 vesicles move towards the plasma membrane while dynamically associating with GFP–protrudin (representative of 31 videos). The vesicles halt when close to protrudin, speed up when escaping protrudin and reassociate with a new pool of protrudin as they slow down. Images are captured every second. Owing to space limitations, selected frames are shown in a and c. The scatter plots show the inverse correlation of speed versus protrudin association of the FYCO1 vesicles, as indicated by a linear regression line.

Extended Data Figure 7 Protrudin loads kinesin 1 onto FYCO1 on LEs.

a, MBP–FYCO1585–1233 beads capture GFP–KLC2 or GFP-KIF5B from cell extracts. Input: 10%. b, Domain structure of FYCO1 plasmids used in this study. c, Confocal images showing the colocalization of KLC1 and LAMP1 with GFP–FYCO1, which is lost in the GFP–FYCO1(Δ735–773) mutant. Confocal images are representative of at least 10 captures. d, Endogenous kinesin 1 colocalizes with mCherry–FYCO1-positive vesicles and accumulates on such vesicles in cells coexpressing Myc–protrudin. HeLa cells were transfected with mCherry–FYCO1 and different Myc–protrudin constructs and analysed by confocal microscopy. Images are representative of at least 30 captures. Quantified in Extended Data Fig. 7g. e, GFP–FYCO1(Δ735–773)-positive LEs are unable to translocate to the cell periphery. Representative of 5 confocal and 440 wide-field images. Quantification was done using an Olympus ScanR microscope. Error bar denotes ± s.e.m. from 3 experiments. GFP–FYCO1: 1,150 cells; GFP–FYCO1(Δ735–773): 1,372 cells. **P < 0.01 (one-sample t-test). f, Western blot showing siRNA-mediated downregulation of RAB7 in HeLa cells stably expressing GFP–FYCO1. g, Quantification of the relative mean intensity of endogenous KIF5B on mCherry–FYCO1 vesicles from confocal images in Extended Data Fig. 7d. Error bars denote ± s.e.m. Mock: 5 experiments (133 cells, 353 vesicles) were set to 1; Myc–protrudin: 5 experiments (118 cells, 217 vesicles); Myc–protrudin(FYVE4A): 3 experiments (90 cells, 193 vesicles). **P < 0.01. Mock/protrudin, one-sample t-test; protudin/FYVE4A, unpaired t-test. h, Western blot showing 1% of the input used for the GFP-trap assay from HeLa cells stably expressing GFP–FYCO1 (Fig. 3e). i, Western blot showing siRNA-mediated downregulation of protrudin in RPE1 cells stably expressing GFP–FYCO1.

Extended Data Figure 8 Protrudin and FYCO1 cooperatively induce protrusion formation.

a, Overexpression of GFP–protrudin induces a protrusion phenotype in RPE1 cells (see also Supplementary Video 6) and co-expression of GFP–protrudin and mCherry–FYCO1 increases the protrusion phenotype. mCherry–FYCO1 vesicles localize to the very end of each protrusion. Arrows indicate the direction of two protrusions pointing at the FYCO1-positive ends. Cells were transfected with the indicated constructs for 48 h and analysed by confocal microscopy. Images are representative of at least 15 confocal captures. Quantified in Extended Data Fig. 8b. b, Quantification of the number (%) of cells with protrusions in RPE1 cells transfected with the indicated constructs for 48 h. Images were acquired with an Olympus ScanR microscope and cells with protrusions were quantified by visual inspection of GFP. Error bars denote ± s.e.m. from 4 experiments. GFP + mCherry: 1,131 cells; GFP + mCherrry–FYCO1: 1,203 cells; GFP–protrudin + mCherry: 1,226 cells; GFP–protrudin(ΔFYVE) + mCherry: 871 cells; GFP–protrudin(FYVE4A) + mCherry: 1,365 cells; GFP–protrudin + mCherry–FYCO1: 979 cells; GFP–protrudin(ΔFYVE) + mCherry–FYCO1: 766 cells; protrudin(FYVE4A) + mCherry–FYCO1: 1,365 cells. *P < 0.05, **P < 0.01, ***P < 0.001 (unpaired t-test). c, Quantification of the number of cells with protrusions in RPE1 cells transfected with the indicated constructs and measured by live imaging (example in Supplementary Video 6). Cells with protrusions were quantified by visual inspection of GFP in the videos. Error bars denote ± s.e.m. Tubulin: 3 experiments, 19 cells; GFP–protrudin: 4 experiments, 33 cells; GFP–protrudin(FYVE4A): 3 experiments, 24 cells; GFP–protrudin + mCherry–FYCO1: 2 experiments, 31 cells; *P < 0.05, **P < 0.01, ***P < 0.001 (unpaired t-test). d, Knockdown of FYCO1 reduces the number of cells forming protrusions upon overexpression of protrudin. FYCO1 knockdown efficiency is shown by western blotting. Error bars denote ± s.e.m. from 5 experiments. Control: 1,706 cells; siRNA FYCO1: 1,393 cells. ***P < 0.001 (unpaired t-test). e, Protrudin-mediated protrusion formation is dependent on RAB7. Quantification of the number (%) of cells with protrusions in RPE1 cells transfected with the indicated constructs. Images were acquired with an Olympus ScanR microscope, and cells with protrusions were quantified by visual inspection. RAB7 knockdown efficiency is shown by western blotting. Error bars denote ± s.e.m. from 3 experiments. Control GFP–protrudin + mCherry: 2,631 cells; siRNA RAB7 GFP–protrudin + mCherry: 2,428 cells; control GFP–protrudin + mCherry–FYCO1: 3,046 cells; siRNA RAB7 GFP–protrudin + mCherry–FYCO1: 3,207 cells. **P < 0.01 (unpaired t-test).

Extended Data Figure 9 Protrudin and FYCO1 cooperatively induce neurite outgrowth by facilitating LE fusion with the plasma membrane.

a, Protrudin overexpression induces neurite outgrowth in PC12 cells. NGF treatment and overexpression of FYCO1 increase protrudin-mediated neurite outgrowth. WT, wild type. Error bars denote ± s.e.m. from 3 experiments. Without NGF: GFP + mCherry: 336 cells; GFP–protrudin + mCherry: 326 cells; GFP–protrudin + mCherry–FYCO1: 215 cells. With NGF: GFP + mCherry: 333 cells; GFP–protrudin + mCherry: 224 cells; GFP–Protrudin + mCherry–FYCO1: 173 cells. **P < 0.01, ***P < 0.001 (unpaired t-test). b, Deconvolved projections of z-stacks recorded by a DeltaVision microscope showing PC12 cells with protrusions (mediated by protrudin and FYCO1, left panel representative of 80 images) and without protrusions (inhibited by mutant FYCO1(Δ735–773), right panel representative of 30 images). Whereas mCherry–FYCO1 is transported into forming protrusions, mCherry–FYCO1(Δ735–773) (unable to engage kinesin 1) is not. The circles have a diameter of 30 µm. Cells with protrusions reaching out of the circle are regarded as protrusion-positive. Quantified in Fig. 4c. c, Western blots showing levels of knockdown in PC12 cells. d, Knockdown of SYT7 reduces the number of RPE1 cells with protrusions upon overexpression of protrudin or coexpression of protrudin and FYCO1. Images were acquired with an Olympus ScanR microscope, and cells with protrusions were quantified by visual inspection. Error bars denote ± s.e.m. from 3 experiments. Control GFP–protrudin + mCherry: 4,278 cells; siRNA SYT7 GFP–protrudin + mCherry: 901 cells; control GFP–protrudin + mCherry–FYCO1: 3,045 cells; siRNA SYT7 GFP–protrudin + mCherry–FYCO1: 1,731 cells. ***P < 0.001 (unpaired t-test). Confocal images are shown (representative of at least 10 confocal and 240 wide-field captures). Knockdown efficiency of SYT7 is shown by quantitative realtime PCR. Error bar denotes ± s.e.m. from 3 experiments. **P < 0.01 (one-sample t-test). e, Surface-accumulated LAMP1 is detected with a reporter construct consisting of a double-tagged LAMP1 construct. The surface-exposed GFP variant pHluorin is recognized with an anti-GFP-antibody on non-permeabilized cells. Indirect immunofluorescence amplifies the surface signal. f, Confocal images (representative of at least 30 captures) of cells expressing Cerulean–FYCO1 and the reporter construct pHluorin–LAMP1–mCherry show accumulation of surface LAMP1 in the presence of mCitrine–protrudin wild type, but not mCitrine–protrudin(FYVE4A). For quantification, the total fluorescence in the Alexa647 channel was normalized to the total fluorescence in the mCherry channel to take expression levels of the reporter construct into account. Quantified in Fig. 4e, f.

Extended Data Figure 10 Model for the functions of protrudin and FYCO1 in LE translocation and neurite outgrowth.

The kinesin-1-binding ER protein protrudin contacts LEs via coincident detection of RAB7–GTP and PtdIns(3)P on LEs. This allows kinesin 1 to be handed over to the RAB7–GTP- and PtdIns(3)P-binding LE protein FYCO1, which in turn mediates the transport of LEs towards the plasma membrane along microtubules. LEs are reloaded with kinesin 1 at ER–LE contact sites as the vesicles move into the growing neurite and ultimately fuse with the plasma membrane by a SYT7-dependent mechanism.

Supplementary information

Supplementary Tables

This file contains Supplementary Table 1. (PDF 48 kb)

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

ER is displayed in blue, LE in red. (MP4 19979 kb)

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. (MOV 1784 kb)

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. (MOV 702 kb)

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. (MOV 2530 kb)

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. (MOV 316 kb)

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. (MOV 3165 kb)

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. (MOV 2053 kb)

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Raiborg, C., Wenzel, E., Pedersen, N. et al. Repeated ER–endosome contacts promote endosome translocation and neurite outgrowth. Nature 520, 234–238 (2015). https://doi.org/10.1038/nature14359

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