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FERARI is required for Rab11-dependent endocytic recycling

Nature Cell Biology volume 22, pages 213–224 (2020)Cite this article

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Abstract

Endosomal transport is essential for cellular organization and compartmentalization and cell–cell communication. Sorting endosomes provide a crossroads for various trafficking pathways and determine recycling, secretion or degradation of proteins. The organization of these processes requires membrane-tethering factors to coordinate Rab GTPase function with membrane fusion. Here, we report a conserved tethering platform that acts in the Rab11 recycling pathways at sorting endosomes, which we name factors for endosome recycling and Rab interactions (FERARI). The Rab-binding module of FERARI consists of Rab11FIP5 and rabenosyn-5/RABS-5, while the SNARE-interacting module comprises VPS45 and VIPAS39. Unexpectedly, the membrane fission protein EHD1 is also a FERARI component. Thus, FERARI appears to combine fusion activity through the SM protein VPS45 with pinching activity through EHD1 on SNX-1-positive endosomal membranes. We propose that coordination of fusion and pinching through a kiss-and-run mechanism drives cargo at endosomes into recycling pathways.

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Fig. 1: Conserved interactions between FERARI components.
Fig. 2: Loss of FERARI affects endosomal recycling.
Fig. 3: Rab11FIP5 is part of FERARI and provides the link to Rab11.
Fig. 4: FERARI may act on sorting endosomes but is not essential for transport to lysosomes.
Fig. 5: FERARI is present on and required for the maintenance of SNX-1 active recycling compartments in C. elegans.
Fig. 6: The SNAREs SYX-6 and SYX-7 act in the FERARI-dependent RAB-11 recycling pathway.
Fig. 7: FERARI promotes kiss-and-run vesicle dynamics and quantal cargo uptake.

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Data availability

Source Data for Figs. 1–3 (unprocessed blots), Figs. 1–7 (statistical source data), Extended Data Figs. 1–5, 7 and 8 and Extended Data Figs. 1–7 and 9 (statistical source data) are provided with the paper. All other data supporting the findings of this study are available from the corresponding author upon reasonable request.

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Acknowledgements

We thank S. Begum and J. Fürst for excellent technical assistance with some of the experiments. The proteomics analysis was performed in the Proteomics Core Facility of the Biozentrum with the help of S. Moes and P. Jenö. The Imaging Core Facility of the Biozentrum facilitated the generation of videos and, in particular, L. Guérard provided support for image analysis. Cells were sorted in the FACS Core Facility of the Biozentrum. We are grateful to I. G. Macara for critical comments on the manuscript. B. Grant and P. Liu are acknowledged for sharing strains. Some strains were provided by the CGC, which is funded by the NIH Office for Research Infrastructure Programs (P40 OG010440). This work was supported by the Swiss National Science Foundation (CRSII3_141956, 31003A_141207) and the University of Basel.

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  1. These authors contributed equally: Jachen A. Solinger, Harun-Or Rashid.

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  1. Biozentrum, University of Basel, Basel, Switzerland

    Jachen A. Solinger, Harun-Or Rashid, Cristina Prescianotto-Baschong & Anne Spang

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Contributions

J.A.S., H.-O.R. and A.S. conceived and designed the study. J.A.S., H.-O.R. and C.P.-B. performed the experiments. J.A.S., H.-O.R., C.P.-B. and A.S. analysed and discussed the data. J.A.S. and A.S. wrote the manuscript with input from all authors.

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Correspondence to Anne Spang.

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Extended data

Extended Data Fig. 1 Pull-downs of FERARI subunits show multiple interactions.

a, Schematic drawing of the putative FERARI tether. b, Pull-downs were performed with extracts from yeasts expressing SPE-39-FLAG and the HA-tagged proteins indicated at the top. Control pull-downs with beads coupled to IgG were also carried out. Arrows on the left indicate the sizes of expressed proteins. n=3 independent experiments. Quantification is shown in (b’); mean ± s. d. is given. c, Pull-downs showing interactions between SPE-39-FLAG and HA-RME-1 fragments. n=3 independent experiments. Quantification is shown in (c’); mean ± s. d. is given. d, Pull-downs experiments using SPE-39-FLAG and fragments of UNC-44 (with HA tag). UNC-44D fragment expression is low and can be seen on longer exposure as indicated. n=3 independent experiments. Quantification is shown in (d’); mean ± s. d. is given. See Unprocessed Blots Extended Data 1 and Statistical Source Data Extended Data 1.

Source data

Extended Data Fig. 2 Determination of FERARI subunits interactions.

a, Pull-downs showing interaction between HA-RABS-5 and fragments of UNC-44 (coupled to FLAG). UNC-44Z was not expressed, anti-FLAG antibody cross-reacted with RABS-5 (indicated by asterisk). n=3 independent experiments. Quantification is shown in (a’); mean ± s. d. is given. b, Western blot of the interactions between transiently over-expressed human FERARI components rabenosyn-5, VIPAS39 and VPS45. GFP-Rabenosyn-5 was precipitated with GFP-trap beads, and myc-VIPAS39 and myc-VPS45 co-precipitated. n= 3 independent experiments. Quantification is shown in (b’); mean ± s. d. is given. c, Pull-down experiment showing the interaction of SNX-1 with SPE-39 and RABS-5. n=3 independent experiments. Quantification is shown in (c’); mean ± s. d. is given. d, Interactions between SPE-39 and VPS-45 as well as VPS-45 and RABS-5 in pull-down experiments. n=3 independent experiments. Quantification is shown in (d’); mean ± s. d. is given. See Unprocessed Blots Extended Data 2 and Statistical Source Data Extended Data 2.

Source data

Extended Data Fig. 3 Interactions of C. elegans and mammalian FERARI subunits.

a, Yeast two hybrid growth assay showing interactions between SPE-39 (bait) and RABS-5, SNX-1 and RME-1 (preys). n=3 biologically independent experiments. b, Western blot depicting the interactions between human protein Rabenosyn-5 with EHD1 and Rab11FIP5, co-expressed in HEK-293 cells. GFP-Rabenosyn-5 was used as bait to co-immunoprecipitate myc-tagged EHD1 and Rab11FIP5. n= 3 independent experiments. Quantification is shown in (b’); mean ± s. d. is given. c, Interaction between RBSN and EHD1 is reduced in rab11fip5 KO cells. ctr KO and rab11fip5 KO HEK293 cells were co-transfected with GFP-RBSN and myc-EHD1 as indicated and myc-trap beads were used as bait. Immunoprecipitated proteins and inputs were then probed with antibodies against myc, GFP and actin. n=3 independent experiments Quantification is shown in (c’); mean ± s. d. is given (P=0.0232). d, Quantification of interactions shown in Fig. 1a, e, Fig. 3a, f, Fig. 3b and g, Fig. 3i. The mean values ± standard deviations are shown; n=3 independent experiments; *P < 0.05, **P < 0.01, ***P < 0.00, ****P < 0.0001, and n.s. P > 0.05. Two-tailed Student’s t-tests were used for all analyses. See Unprocessed Blots Extended Data 3 and Statistical Source Data Extended Data 3.

Source data

Extended Data Fig. 4 Human FERARI components interact and co-localize in HeLa cells.

a, Co-localization of rabenosyn-5 with human FERARI components VPS45, EHD1, and Rab11FIP5. HeLa cells co-transfected with GFP-Rabenosyn 5 and myc-VPS45, myc-EHD1, myc-Rab11FIP5 were subjected to immunofluorescence staining with GFP and myc antibodies. b, Quantification of co-localization shown in (a). n=95 cells were analyzed from n=3 independent biological replicates. Data show the mean ± s.d. c, Western blot depicting the efficiencies of CRISPR-Cas9 KO of indicated proteins in HeLa cells used for analyses in Fig. 2a and 3c. Corresponding quantifications are shown below each western blot. n=3 independent biological replicates (Data show the mean ± s.d). d, RT-qPCR data represents levels of ank1, ank2 and ank3 mRNA in HeLa cells at endogenous level and in the ank3 KO. n= 3 independent experiments Data show the mean ± s.d. See Unprocessed Blots Extended Data 4 and Statistical Source Data Extended Data 4.

Source data

Extended Data Fig. 5 Analysis of FERARI platform stability and phenotype.

a & b, Protein stability of FERARI components in the KO cells. Endogenous levels of Rab11FIP5 and VIPAS39 were assessed in different FERARI KO HeLa (a) and HEK-293 (b) cell lines as indicated. n=3 independent biological replicates. Data show the mean ± s.d. c, Quantification of RAB-11 and RAB-10 phenotypes shown in Fig. 2e. RAB-11 compartments and RAB-10 tubules were measured in n=6 worms (n=10 structures each), RAB-10 globular compartments were quantified semi-automatically and comprise n=50-120 structures (per worm) in n=6 different worms. Mean ± s.d. is given. For the box plot, the box represents 25-75 percentile box with median and the whiskers 1-99 percentile. See Unprocessed Blots Extended Data 5 and Statistical Source Data Extended Data 5.

Source data

Extended Data Fig. 6 Morphology of Rab11 compartments is affected by FERARI knock-downs in HeLa cells.

a, Electron micrographs showing the structure of immunogold labeled Rab11 compartments. HeLa cells with the indicated CRISPR knock-outs were processed for immuno-electron microscopy. Globular sorting endosomes are marked with “SE”. Tubular recycling endosomes with Rab11 signals are marked with “RE”. Arrows point to densely labeled Rab11 structures containing tubular membranes. Scale bars: 500 nm. b, Quantification of recycling endosome sizes in different KO HeLa cells shown in (a). n=23 for mock, n=8 for vipas39 KO, n=6 for rab11fip5 KO and n=4 for ehd1 KO cells (mean ± s.d.). n: number of cells. A schematic representation of sorting endosomes (SE) and recycling endosomes (RE) is shown. For size measurements the recycling endosomes were encircled as shown in the example pictures below the graph. Scale bars: 500 nm. See Statistical Source Data Extended Data 6.

Source data

Extended Data Fig. 7 Function of spe-39 and vps-45 in endosomal recycling.

a, Knock-down of vps-33.2 in worms has no effect on RAB-11 compartments. n=3 independent RNAi experiments with n=20 animals. Scale bar: 10 µm. b, Recycling of hTfR-GFP is abolished in spe-39(RNAi) and vps-45(RNAi) worms but not in vps-33.2(RNAi). Most hTfR-GFP signal is lost in spe-39(RNAi) and vps-45(RNAi) worms. n=3 independent RNAi experiments with n=20 animals. Scale bars: 10 µm. c, Schematic representation of the last 3 oocytes prior to the spermatheca in the worm gonad. Yolk uptake defect of spe-39(RNAi) and vps-45(RNAi) worms. n=3 independent RNAi experiments with n=20 animals. Wild-type uptake of VIT-2-GFP was quantified in the first 3 oocytes before the spermatheca. The measurements were normalized to the total fluorescent signal of all 3 wild-type cells. Knock-down of spe-39 and vps-45 show a reduction of yolk uptake. n=6 gonads were quantified for each RNAi experiment. Scale bars: 10 µm. d, The yolk receptor RME-2-GFP is mis-localized in spe-39(RNAi) and vps-45(RNAi) worms. n=3 independent RNAi experiments (n=20 animals). Scale bars: 10 µm. e, Transferrin recycling is reduced in rab11fip5KO Hela cells. After 1 h starvation ctrl KO and rab11fip5KO cells were treated with Transferrin Biotin. Cells were then chased with holo transferrin up to 30 min, and cell lysates were subjected to immunoblot with Streptavidin HRP. Quantification of the immunoblot: The mean ± s.d. values are shown; n=3 independent experiments. (P= 0.5249 at 2.5 min; P=0.2764 at 5 min; P=0.2169 at 10 min; P=0.4093 at 15 min; P=0.1949 at 20 min; P=0.01901 at 30 min). *P < 0.05, **P < 0.01, ***P < 0.00, ****P < 0.0001 and n.s., P > 0.05. Two-tailed Student’s t-tests were used for all analyses. See Unprocessed Blots Extended Data 7 and Statistical Source Data Extended Data 7.

Source data

Extended Data Fig. 8 Loss of recycling by spe-39(RNAi) or vps-45(RNAi) partially rescues a sand-1 mutant and endogenous VIPAS39 interacts with SNX1.

a, Knock-down of spe-39 or vps-45 induces the degradation pathway in coelomocytes. In sand-1(ok1963) coelomocytes BSA-TR is not transported to the lysosomes (LMP-1 compartment). spe-39(RNAi) and vps-45(RNAi) will favor the transport of BSA-TR to coelomocytes, probably by abolishing recycling. Arrows indicate LMP-1-positive compartments containing BSA-TR. n=3 independent RNAi experiments (n=20 animals). Scale bars:5 µm. b, GFP-RME-1 levels were unchanged upon FERARI (RNAi). Western blot analysis of GFP-RME-1 levels in FERARI(RNAi) worm lysates. n=3 independent experiments. c, Endogenous VIPAS39 does not interact with Retromer subunit VPS35 in HEK-293 cells transfected with GFP-VPS35. n=3 independent experiments. d, GFP-SNX1 transfected HEK-293 cells show slight interaction between SNX1 and endogenous VIPAS39. n=3 independent experiments. Very weak interaction (<1%) could not be measured due to overexposed bands in the input. See Unprocessed Blots Extended Data 8.

Source data

Extended Data Fig. 9 Kiss-and-run at sorting endosomes is quantal.

a, Schematic drawing of the C. elegans FERARI tether. The Rab interaction modules RFIP-2 and RABS-5 are highlighted. b, Quantification of GFP-RAB-11 vesicle residence times. Residence times occur in groups with an approximate 7 s -interval. The longer residence times are abolished in FERARI knock-downs. c, Quantification of residence times of cargo vesicles containing hTfR-GFP and Glut1-GFP. The quantal distribution of residence times is similar to RAB-11 vesicles and longer residence times are abolished in FERARI RNAi conditions. Numbers of vesicles analyzed are indicated. See Statistical Source Data Extended Data 9.

Source data

Extended Data Fig. 10 Glut1-GFP vesicle dynamics.

Movie stills showing Glut1-GFP vesicles docking to and pinching off from mCherry-SNX-1 compartments in mock-treated and rme-1(RNAi) worms. (see also movies S20 and S21). n=56 for GLUT1_wt, n=29 for GLUT1_rme-1. n represents number of vesicles. Scale bars: 2 µm.

Supplementary information

Reporting Summary

Supplementary Tables

Supplementary Tables 1–3.

Supplementary Video 1

RAB-5 compartments docked to SNX-1 tubules. A rotating 3D projection of a z stack recorded with a Zeiss LSM880 microscope in living worms. See Fig. 5b.

Supplementary Video 2

RAB-11 compartments docked to SNX-1 tubules. A rotating 3D projection of a z stack recorded with a Zeiss LSM880 microscope in living worms. See Fig. 5b.

Supplementary Video 3

RAB-11 compartments docked to SNX-1 tubules. A rotating 3D projection of a z stack recorded with a Zeiss LSM880 microscope in living worms (second video). See Fig. 5b.

Supplementary Video 4

RAB-7 compartments docked to SNX-1 tubules. A rotating 3D projection of a z stack recorded with a Zeiss LSM880 microscope in living worms. See Fig. 5b.

Supplementary Video 5

RME-1 patches on SNX-1 tubules. A rotating 3D projection of a z stack recorded with a Zeiss LSM880 microscope in living worms. See Fig. 5b.

Supplementary Video 6

DHS-3 lipid droplets near but not overlapping with SNX-1 tubules. A rotating 3D projection of a z stack recorded with a Zeiss LSM880 microscope in living worms. First negative control for SNX-1 interaction. See Fig. 5b.

Supplementary Video 7

MANS Golgi marker near but not overlapping with SNX-1 tubules. A rotating 3D projection of a z stack recorded with a Zeiss LSM880 microscope in living worms. Second negative control for SNX-1 interaction. See Fig. 5b.

Supplementary Video 8

Docking of a RAB-11 vesicle with an SNX-1 compartment. A single-plane video recorded with an Olympus FV3000 microscope in a living worm. First example. See Supplementary Fig. 6c.

Supplementary Video 9

Docking of a RAB-11 vesicle with an SNX-1 compartment. A single-plane video recorded with an Olympus FV3000 microscope in a living worm. Second example. See Supplementary Fig. 6c.

Supplementary Video 10

Docking of a RAB-11 vesicle with an SNX-1 compartment. A single-plane video recorded with an Olympus FV3000 microscope in a living worm. Third example. See Supplementary Fig. 6c.

Supplementary Video 11

Fission of a RAB-11 vesicle from an SNX-1 compartment. A single-plane video recorded with an Olympus FV3000 microscope in a living worm. First example. See Supplementary Fig. 6d.

Supplementary Video 12

Fission of a RAB-11 vesicle from an SNX-1 compartment. A single-plane video recorded with an Olympus FV3000 microscope in a living worm. Second example. See Supplementary Fig. 6d.

Supplementary Video 13

Fission of a RAB-11 vesicle from an SNX-1 compartment. A single-plane video recorded with an Olympus FV3000 microscope in a living worm. Third example. See Supplementary Fig. 6d.

Supplementary Video 14

‘Kiss-and-run’ interaction of a RAB-11 vesicle with an SNX-1 compartment in a WT worm. A single-plane video recorded with a Zeiss LSM880 microscope in a living worm. First example. See Fig. 7a.

Supplementary Video 15

‘Kiss-and-run’ interaction of a RAB-11 vesicle with an SNX-1 compartment in a WT worm. A single-plane video recorded with a Zeiss LSM880 microscope in a living worm. Second example. See Fig. 7a.

Supplementary Video 16

‘Kiss-and-run’ interaction of a RAB-11 vesicle with an SNX-1 compartment in an rme-1(RNAi) worm. A single-plane video recorded with a Zeiss LSM880 microscope in a living worm. See Fig. 7a.

Supplementary Video 17

‘Kiss-and-run’ interaction of an hTfR vesicle with an SNX-1 compartment in a WT worm. A single-plane video recorded with a Zeiss LSM880 microscope in a living worm. First example. See Fig. 7b.

Supplementary Video 18

‘Kiss-and-run’ interaction of an hTfR vesicle with an SNX-1 compartment in a WT worm. A single-plane video recorded with a Zeiss LSM880 microscope in a living worm. Second example. See Fig. 7b.

Supplementary Video 19

‘Kiss-and-run’ interaction of an hTfR vesicle with an SNX-1 compartment in an rme-1(RNAi) worm. A single-plane video recorded with an LSM880 microscope in a living worm. See Fig. 7b.

Supplementary Video 20

‘Kiss-and-run’ interaction of a Glut1 vesicle with an SNX-1 compartment in a WT worm. A single-plane video recorded with an LSM880 microscope in a living worm. See Supplementary Fig. 8.

Supplementary Video 21

‘Kiss-and-run’ interaction of a Glut1 vesicle with an SNX-1 compartment in an rme-1(RNAi) worm. A single-plane video recorded with an LSM880 microscope in a living worm. See Supplementary Fig. 8.

Supplementary Video 22

A mechanistic model for FERARI function. Protein labels appear in the video, processes are described on the right and a small timer indicates the ‘loading time’ during quantal cargo uptake. The video corresponds to a vesicle with a residence time of approximately 18 s in Fig. 7 and Supplementary Fig. 7d,e (4 s for docking and membrane fusion followed by two cycles of cargo uptake of 7 s each). See also Supplementary Fig. 8b for a static model.

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Solinger, J.A., Rashid, HO., Prescianotto-Baschong, C. et al. FERARI is required for Rab11-dependent endocytic recycling. Nat Cell Biol 22, 213–224 (2020). https://doi.org/10.1038/s41556-019-0456-5

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