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A Golgi-derived vesicle potentiates PtdIns4P to PtdIns3P conversion for endosome fission

Abstract

Endosome fission is essential for cargo sorting and targeting in the endosomal system. However, whether organelles other than the endoplasmic reticulum (ER) participate in endosome fission through membrane contacts is unknown. Here, we characterize a Golgi-derived vesicle, the SEC14L2 compartment, that plays a unique role in facilitating endosome fission through ternary contacts with endosomes and the ER. Localized to the ER-mediated endosome fission site, the phosphatidylinositol transfer protein SEC14L2 promotes phosphatidylinositol 4-phosphate (PtdIns4P) to phosphatidylinositol 3-phosphate (PtdIns3P) conversion before endosome fission. In the absence of SEC14L2, endosome fission is attenuated and more enlarged endosomes arise due to endosomal accumulation of PtdIns4P and reduction in PtdIns3P. Collectively, our data suggest roles of the Golgi network in ER-associated endosome fission and a mechanism involving ER–endosome contacts in the regulation of endosomal phosphoinositide conversion.

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Fig. 1: The Golgi-derived SEC14L2 compartment promotes ER-associated endosome fission.
Fig. 2: The SEC14L2 compartment contacts endosomes and the ER.
Fig. 3: The SEC14L2 compartment promotes endosomal PtdIns3P accumulation and PtdIns4P removal before fission in vivo.
Fig. 4: Endosomal PtdIns3P depletion or PtdIns4P accumulation attenuates ER-associated endosome fission.
Fig. 5: SEC14L2 depletion attenuates ER-associated endosome fission and PtdIns4P to PtdIns3P conversion.
Fig. 6: SEC14L2 promotes PtdIns4P removal of endosomes in parallel with the VAPA/B–OSBP machinery.
Fig. 7: Lipid-binding residues in the SEC14 domain are required for ER-associated endosome fission.
Fig. 8: Purified zSec14l3 binds and transports PtdIns3P/PtdIns4P in vitro.

Data availability

The authors declare that all data supporting the findings of this study are available within the article and its supporting information files (extended data, source data and supplementary videos) or from the corresponding author upon reasonable request. Source data are provided with this paper.

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Acknowledgements

We thank J. Lippincott-Schwartz (Janelia Research Campus, Howard Hughes Medical Institute) for essential suggestions to validate the feature and origin of Sec14l3 vesicles, H. Wang (J. Wu Lab, Tsinghua University) for discussion about the lipid transfer assay, W. Wang (Imaging Core Facility, Technology Center for Protein Sciences, Tsinghua University) for help with image processing using Imaris software, J. Wang and H. Cao (SLSTU-Nikon Biological Imaging Center) for assistance with NIS-Element software, X. Wang (Metabolomics Facility at Technology Center for Protein Sciences, Tsinghua University) for technical support with lipid extraction for LC–MS/MS analysis, Q. Feng (Core Facility of Center of Biomedical Analysis, Tsinghua University) for assistance with the fluorescence microplate reader (Thermo Scientific VARIOSKAN FLASH), W. Wang (State Key Laboratory of Membrane Biology, Tsinghua University) for assistance with the flow cytometry analysis, Y. Lu (Protein Preparation and Characterization Core Facility of Tsinghua University, Branch of National Protein Science Facility) for technical support with protein purification, and Y. Li (Cryo-EM Facility of Tsinghua University, Branch of National Protein Science Facility) for preparing ultrathin sections. Generous gifts of plasmids are acknowledged in the plasmid section of the Methods. We appreciate the help from members of the Meng Laboratory and especially for critical discussion with L. Yu (School of Life Sciences, Tsinghua University) and Y. Chen (School of Life Sciences, Tsinghua University) and their comments on this manuscript. This work was financially supported by grants from the National Natural Science Foundation of China, the Ministry of Science and Technology of China, the National Key Research and Development Program of China, and the China Postdoctoral Science Foundation (91754112, 91754202, 2019YFA0801403, 31801130, 32000480 and BX20190355).

Author information

Affiliations

Authors

Contributions

B.G., A.M. and S.J. designed the experiments and conceived the project. B.G. did most of the experiments and data analyses. Y.G. performed the GI-SIM imaging and prepared the videos to observe dynamic contact and endosome fission. X.L. performed the LC–MS/MS analyses to quantify PIP levels from endosomes and the in vitro transfer assays. S.D. helped with the SEC14L2 KO mouse genotyping and propagation. D.L. helped with image analysis captured by GI-SIM. D.L., B.G., A.M. and S.J. prepared the manuscript.

Corresponding authors

Correspondence to Dong Li or Shunji Jia.

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Competing interests

The authors declare no competing interests.

Additional information

Peer review information Nature Cell Biology thanks Vytas Bankaitis, Aurélien Roux and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Peer reviewer reports are available.

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Extended data

Extended Data Fig. 1 SEC14L2/zSec14l3 colocalization analyses with secretory and endolysosomal system markers.

a, HomoloGene analysis of zebrafish sec14l3 genes among different species. Putative homologs of the zebrafish sec14l3 gene in human, mouse, xenopus, yeast and C. elegans are listed according to their protein and DNA identity. b, RT-PCR of SEC14L2 and SEC14L3 expression in COS-7 cells, monkey blood, and liver tissues. ACTIN served as a control. c, Colocalization (arrows) of endogenous SEC14L2 with EGFP-ARF1 in COS-7 cells. The region enclosed by the rectangle is shown at higher magnification in the inset. Scale bars, 10 μm (left), 5 μm (right), and 1 μm (inset). d-e, Colocalization of GM130 (cis-Golgi) with endogenous SEC14L2 (d) or zSec14l3-mCherry (e) in COS-7 cells. f-g, Colocalization of β-COP (COPI vesicles) with SEC14L2 (f) or zSec14l3-mCherry (g) in COS-7 cells. h-i, Colocalization of ERIGC53 (ER-Golgi intermediate compartment) with SEC14L2 (h) or zSec14l3-mCherry (i) in COS-7 cells. j-k, Colocalization of SEC23 (COPII vesicles) with SEC14L2 (j) or zSec14l3-mCherry (k) in COS-7 cells. l-m, Endogenous SEC14L2 immunostaining of COS-7 cells overexpressing EGFP-RAB4 (l) or EGFP-RAB7 (m). Recycling marker RAB4 and late endosome marker Rab7 are shown in green. n-o, Colocalization of SEC14L2 and endosome marker EEA1 (n) or lysosome marker LAMP1 (o) in COS-7 cells. Both markers are in green. Scale bars, 10 μm and 1 μm (inset). Golgi regions are indicated by dashed circles.

Source data

Extended Data Fig. 2 zSec14l3 resides on Golgi-derived vesicles depending on its SEC14 domain.

a, EM images of vesicles stained with APEX2 fused to zSec14l3 in COS-7 cells. Arrows highlight zSec14l3 compartments (L3+) based on electron density. The ER, mitochondrial (Mito), and multivesicular body (MVB) regions are indicated. b, Diagram of vesicle purification by immunoprecipitation. The Myc-zSec14l3-APEX2 plasmid was transfected into COS-7 cells. After 30 h, cells were lysed to determine affinity binding using an anti-Myc antibody to enrich the Myc-tagged zSec14l3 compartment for APEX staining. c, EM images of purified zSec14l3 compartments. The region in the dotted box is magnified below the panel. d, Size distribution of the zSec14l3 compartments after purification from COS-7 cells. A total of 163 vesicles from three batches were measured and plotted according to their diameters. e, Domain structures of full-length or truncated zSec14l3 proteins. Δ indicates deletion. N, CRAL-TRIO domain; SEC, SEC14 domain; G/GOLD, GOLD2 domain; NG, CRAL-TRIO and GOLD2 domains. f, Confocal image of digitonin-permeabilized COS-7 cells expressing full-length or truncated zSec14l3.

Source data

Extended Data Fig. 3 SEC14L2 compartments are dynamically recruited into the ER-associated RAB5-labeled early endosome fission site.

a-b, STED imaging and line scan analysis of endogenous SEC14L2 in COS-7 cells overexpressing EGFP-RAB5 (a) or EGFP-RAB4 (b) after 16 h of transfection. Boxed regions are magnified in the insets. Arrows indicate contact sites between vesicles. Right, example intensity profiles of the lines. SEC14L2, magenta; EGFP-RAB5/RAB4, green. c, Predicted frequency of SEC14L2 compartment-endosome contacts due to chance. A still image was randomly extracted from a 2-min movie and the amount of the SEC14L2 compartment surface covered by endosomes determined. Statistical data are shown as the Mean ± SEM at the right from n = 7 cells over 2 independent experiments. d, Overlay of FIBSEM images with confocal pictures of the cell with zSec14l3-mCherry (purple) and iRFP-SEC61β (yellow) overexpression. e, Magnification of the region in the white box in d. Segmentation of the ER (yellow), SEC14L2 compartments (zSec14l3+ in purple), and endosomes (cyan). f, Contours and line scan analysis of the relative fluorescence intensity of zSec14l3, RAB5, and SEC61β at three time points as shown in Fig. 2b, t = 10 s (pre-fission), t = 12 s (fission), t = 18 s (post-fission). Arrows in the contours at the top right corner indicate plotted lines. Images in Fig. 2b and the line scan analysis reveal that a dynamic SEC14L2 compartment is recruited to the dividing surface between RAB5-positive endosomes before fission (t = 12 s).

Source data

Extended Data Fig. 4 Acute endosomal recruitment of MTM1 depletes endosomal PI3P level.

a, Confocal analysis of COS-7 cells after transfection with mCherry-FKBP or mCherry-FKBP-MTM1 in combination with iRFP-FRB-RAB5 and EGFP-FYVESARA plasmids with or without 1 μM rapamycin (Rap.) for 30 min. Boxed regions are shown at high magnification in the right corners. Scale bars, 20 µm. b, Contour diagram of the experimental design performed in (a). Upon rapamycin addition, mCherry-FKBP or mCherry-FKBP-MTM1 could be recruited to RAB5+ endosomes due to rapamycin-induced FKBP-FRB dimerization. The PI3P sensor plasmid, EGFP-FYVESARA, was also simultaneously transfected to reflect PI3P level before and after rapamycin treatment. c, Quantification of the relative fluorescence intensity of EGFP-FYVESARA in RAB5+ endosomes. n = 4888 endosomes from 14 cells without rapamycin treatment and n = 4831 endosomes from 22 cells with rapamycin treatment in the mCherry-FKBP transfected group; n = 8197 endosomes from 13 cells without rapamycin treatment and n = 5287 endosomes from 26 cells with rapamycin treatment in the mCherry-FKBP-MTM1 transfected group. Data are presented as the mean ± SD. Unpaired, Two-tailed student’s t-test was used. ***p = 1.82E-13.

Source data

Extended Data Fig. 5 SEC14L2 KO causes endosome defects and compromised Tf recycling.

a, Schematic of two Cas9 target sites in the SEC14L2 locus and postulated genomic organization after editing. Primers for mutant line identification are indicated. b, PCR of genomic fragments amplified by the indicated primers. ACTIN served as a control. c, Western blot of WT and three independent SEC14L2 KO COS-7 cell lines. d, EEA1 staining in WT and SEC14L2 KO COS-7 cells. e, Phalloidin staining in WT and SEC14L2 KO COS-7 cells. f, Fluorescence-activated cell sorting (FACS) analysis of cells with or without Alexa fluor 488-conjugated Tf incubation. Lines in red and cyan indicate cells without or with Tf incubation respectively. g, FACS analysis of Tf recycling over 60 min in WT and SEC14l2 KO COS-7 cells. Percentage of recycled Tf over time is the mean ± SD from three independent experiments. h, Schematic of the Cas9 target sites in the mouse Sec14l2 locus and postulated genomic organization after editing. gRNA targeting site and genotyping primers are indicated. i, Representative gel of PCR products from genomic DNA with different genotypes. WT, 329 bp; mutant band, 179 bp. j, Summary of ER-endosome contact duration before endosome fission. Mouse Sec14l3 siRNA was transfected into WT and Sec14l2 KO MEFs individually. n = 29 fission events from 10 WT MEF cells, n = 32 fission events from 11 WT MEF cells with mSec14l3 siRNA transfection, n = 34 fission events from 11 Sec14l2 KO MEF cells, and n = 37 fission events from 13 Sec14l2 KO MEF cells with mSec14l3 siRNA transfection. Data are presented as the mean ± SD. Unpaired, Two-tailed student’s t-test was used. *p = 0.0469, **p = 0.003. k, RT-PCR of mouse Sec14l1 and Sec14l3 expression in WT or mSec14l2 KO MEFs with or without mSec14l3 siRNA transfection. Gapdh served as a control. l, Western blot of total cell lysates (TCLs), plasma membrane and larger organelle fractions, endosome fractions, and cytosol fractions from WT and SEC14L2 KO COS-7 cells. All isolated fractions were lysed and examined by immunoblotting using anti-EEA1, anti-Na+/K+ ATPase, and anti-CALNEXIN antibodies.

Source data

Extended Data Fig. 6 SEC14L2 promotes endosomal PI4P removal in parallel with VAPA/B-OSBP machinery.

a-d, GI-SIM imaging of SEC14L2 KO COS-7 cells overexpressing iRFP-FRB-RAB5 (green and gray) and mEmerald-KDEL (magenta) at 1-s intervals after endosomal recruitment of VPS34 or SAC1ΔTMD. The main panel shows a partial cell periphery with endosome fission. The insets illustrate a single fission event. White arrows mark approximate sites of fission. Time 0 corresponds to the frame in which tubules first emerge. e, Summary of ER-endosome contact duration before endosome fission in (a-d). SEC14L2 KO COS-7 cells were transfected with mCherry-FKBP, mCherry-FKBP-VPS34, or mCherry-FKBP-SAC1ΔTMD in combination with iRFP-FRB-RAB5 and mEmerald-KDEL. After 1 μM rapamycin (Rap.) treatment for 30 min, cells were examined for endosome fission. n = 20 fission events from 8 WT cells with mCherry-FKBP transfection, n = 26 fission events from 13 SEC14L2 KO cells with mCherry-FKBP transfection, n = 22 fission events from 6 SEC14L2 KO cells with mCherry-FKBP-VPS34 transfection, and n = 48 fission events from 22 SEC14L2 KO mCherry-FKBP-SAC1ΔTMD transfection. Data are presented as the mean ± SD. Unpaired, Two-tailed student’s t-test was used. ***p = 6.47E-07. f, Confocal images of GFP-PHOSBP in WT and VAPA + VAPB siRNA or OSBP siRNA transfected COS-7 cells with or without zSec14l3-mCherry overexpression. Statistics are shown in Fig. 6c. g, Confocal images of iRFP-PHOSBP in WT and SEC14L2 KO COS-7 cells with OSBP-EGFP or VAPA-EGFP overexpression. Statistics are shown in Fig. 6d. h-k, GI-SIM imaging of endosome fission in NC siRNA transfected WT COS-7 cells (h), VAPs siRNA transfected WT (i), and SEC14L2 KO (j) COS-7 cells and OSBP siRNA transfected SEC14L2 KO cells (k) at 1-s intervals. mEmerald-RAB5 (green) and mCherry-KDEL (magenta) were co-expressed to mark endosomes and ER, respectively. The main panel shows a partial cell periphery with endosome fission. The insets illustrate a single fission event. White arrows mark approximate sites of fission. Time 0 corresponds to the frame in which tubules first emerge. Statistics are shown in Fig. 6e.

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Extended Data Fig. 7 Lipid binding activity of zSec14l3 or hSEC14L2.

a, Lipid strip binding assay for purified zSec14l3. Purified zSec14l3-His protein (1 μg) was incubated with lipid strips for 30 min at room temperature. The strips were then treated with a His antibody. b-c, BLI to test zSec14l3 (b) or hSEC14L2 protein (c) binding with PI3P at different concentrations. d-e, BLI to test zSec14l3 (d) or hSEC14L2 protein (e) binding with PtdIns at different concentrations. f, Summary of PI3P binding affinity of zSec14l3 protein and its variant mutant forms. Dissociation constant (Kd) of zSec14l3 variants are listed individually. N.D indicates not determined. g, Statistical data for ER-endosome contact duration before endosome fission in (h-k). SEC14L2 KO COS-7 cells were transfected with different zSec14l3 domain-depleted forms to examine endosome fission. n = 21 fission events from 13 SEC14L2 KO COS-7 cells, n = 21 fission events from 8 SEC14L2 KO COS-7 cells with zSec14l3-ΔN transfection, n = 21 fission events from 7 SEC14L2 KO COS-7 cells with zSec14l3-ΔS transfection, and n = 28 fission events from 10 SEC14L2 KO COS-7 cells with zSec14l3-ΔG transfection. Data are presented as the mean ± SD. Two-tailed t test was used. h-k, GI-SIM imaging of SEC14L2 KO COS-7 cells overexpressing mEmerald-RAB5 (green and gray) and mCherry-KDEL (magenta) at 1-s intervals in the presence of different zSec14l3 domain-depleted forms. The main panel shows a partial cell periphery with endosome fission. The insets illustrate a single fission event. White arrows mark approximate sites of fission. Time 0 corresponds to the frame in which tubules first emerge.

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Extended Data Fig. 8 zSec14l3 and hSEC14L2 promote lipid transfer and VPS34 kinase activity in vitro.

a, Cartoon schematic of PI3P/PI4P extraction assays. NBD-PHFAPP protein was mixed with PI3P- or PI4P-containing liposomes with or without DGS-NiNTA in the presence or absence of 3 µM His-zSec14l3 protein. Fluorescence spectra of NBD-PHFAPP were obtained after the addition of proteins at 5 min, 15 min, and 30 min. The percentage of PI3P/PI4P extraction was calculated according to the fluorescence intensity at 540 nm. b, Quantification of PI3P/PI4P extraction by zSec14l3. The lipid extraction assay was performed as in (a) and the amount of extracted PI3P/PI4P was calculated. Data are presented as the mean ± SD from 3 independent experiments. Unpaired, Two-tailed student’s t-test. ***p = 0.0008 and *p < 0.05. c, LC-MS/MS-based PI3P or PI4P transfer assay in vitro. 200 nM hSEC14L2-His protein was incubated for 15 min at 25 °C. d, Quantification of in vitro PI3P or PI4P transfer efficiency by hSEC14L2 protein. The in vitro transfer assay was performed as shown in (c) and acceptor liposomes recovered for LC-MS/MS analysis to quantify acquired PI3P or PI4P content. Data are presented as the mean ± SD of three independent experiments. Unpaired, Two-tailed student’s t-test was used. **p = 0.003 (PI3P) and **p = 0.002 (PI4P). e, Schematic of the liposome-based fusion assay. The change in fluorescence intensity (ΔF = Ft-F0) could reflect the liposome fusion percentage. f, Quantification of fluorescence changes when excited at 460 nm in the liposome-based fusion assay with addition of zSec14l3 or hSEC14L2 protein. Data are presented as the mean ± SD of three independent experiments. Unpaired, Two-tailed student’s t-test was used. ***p = 3.02E-05 and *p = 0.029. g, VPS34 interacts with zSec14l3 and its lipid binding deficient form zSec14l3-M5. h, Quantification of VPS34 catalyzed PI3P content in the presence of zSec14l3 or zSec14l3-M5. SEC14L2 KO COS-7 cells were transfected as indicated and then cells were lysed for immunoprecipitation using anti-EGFP antibody. The immunoprecipitated pellets were used for in vitro kinase assays and lipid extracted for LC-MS/MS. Data are presented as the mean ± SD of three independent experiments. Unpaired, Two-tailed student’s t-test was used. *p = 0.031 (M5), **p = 0.005 (WT).

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Supplementary Video 1

ER-associated endosome fission in COS-7 cells after DMSO or BFA treatment. COS-7 cells were transfected with mEmerald–RAB5 (green) and mCherry–KDEL (magenta) for live-cell imaging by GI-SIM at 1-s intervals for 2 min. Before imaging, DMSO or 10 μg ml−1 BFA was added to the cells for 10 min. ER-associated endosome fission events are indicated by arrows. Scale bars, 2 μm or 1 μm (insets).

Supplementary Video 2

The SEC14L2 compartment is dynamically associated with RAB5-positive endosomes. COS-7 cells were transfected with zSec14l3–mCherry (magenta) and EGFP–RAB5 (cyan) and imaged using an ANDOR Dragonfly spinning disc confocal microscope at 5-s intervals for 2 min. Three examples showing persistent (Ex. 1), temporary (Ex. 2) and no (Ex. 3) contact are arranged in the right panel at high resolution. Magenta arrows indicate SEC14L2 compartments and cyan indicate RAB5-positive endosomes. Scale bars, 2 μm or 0.5 μm (insets).

Supplementary Video 3

The SEC14L2 compartment is at the ER-associated dividing site of endosomes. COS-7 cells were transfected with EGFP–RAB5 (cyan), zSec14l3–mCherry (magenta) and iRFP–SEC61β (yellow) and captured by GI-SIM at 2-s intervals for 2 min. An endosome fission event with 14-s duration is shown. Fission occurs in frame 5. Scale bars, 2 μm or 0.5 μm (insets).

Supplementary Video 4

The SEC14L2 compartment replenishes PtdIns3P to RAB5-positive endosomes before division. COS-7 cells were co-transfected with iRFP–FYVESARA (magenta), EGFP–RAB5 (yellow) and zSec14l3–mCherry (cyan) for live-cell imaging using an ANDOR Dragonfly spinning disc confocal microscope at 6-s intervals for 2 min. The PtdIns3P signal (top left, magenta) is gradually replenished to RAB5-positive endosomes (yellow) from 12 s, and an endosome fission event occurs at 24 s, with newborn endosomes indicated by two yellow arrows. The upper left panel shows the iRFP–FYVESARA signal, upper right the iRFP–FYVESARA signal in RAB5-positive endosomes, lower left the iRFP–FYVESARA signal in the SEC14L2 compartment, and lower right is all merged channels. Scale bars, 1 μm.

Supplementary Video 5

The SEC14L2 compartment removes PtdIns4P from RAB5-positive endosomes before division. SEC14L2 KO COS-7 cells were co-transfected with SAC1 siRNA and EGFP–PHOSBP (cyan), iRFP–FRB–RAB5 (yellow) and mCherry–FKBP (magenta, left panel) or mCherry–FKBP–ΔNG–zSec14l3–KDEL (magenta, right panel) plasmids for live-cell imaging by GI-SIM at 2-s intervals for 30 min after the addition of 1 μM rapamycin. A PtdIns4P transport event from RAB5-positive endosomes is indicated by cyan arrows. After removal of PtdIns4P, the endosome undergoes fission at 18 s (yellow arrows). The upper panel shows the PtdIns4P signal (cyan), the middle panel the PtdIns4P signal (cyan) in RAB5-positive endosomes (yellow) and the lower panel all merged channels. Scale bars, 1 μm.

Supplementary Video 6

Endosomal recruitment of MTM1 mitigates ER-associated endosome fission. COS-7 cells were co-transfected with iRFP–FRB–RAB5 (green and grey), mCherry–FKBP (yellow) or mCherry–FKBP–MTM1 (yellow), and mEmerald–KDEL (magenta) for live-cell imaging by GI-SIM at 1-s intervals for 30 min after 1 μM rapamycin treatment. Scale bars, 2 μm.

Supplementary Video 7

Endosomal recruitment of PtdIns4KAC1001 mitigates ER-associated endosome fission. COS-7 cells were co-transfected with iRFP–FRB–RAB5 (green and grey), mCherry–FKBP–PtdIns4K(D1957A) (yellow) or mCherry–FKBP–PtdIns4K (yellow), and mEmerald–KDEL (magenta) for live-cell imaging by GI-SIM at 1-s intervals for 30 min after 1 μM rapamycin treatment. Scale bars, 2 μm.

Supplementary Video 8

SEC14L2 depletion attenuates ER-associated endosome fission. WT (left panel) and SEC14L2 KO (right panel) COS-7 cells were transfected with mEmerald–RAB5 (green) and mCherry–KDEL (magenta) for live-cell imaging by GI-SIM at 1-s intervals for 2 min. ER-associated endosome fission events are indicated by arrows. Top, mEmerald–RAB5 signal; bottom, merged channels. Scale bars, 1 μm.

Supplementary Video 9

Simultaneous depletion of mSec14l2 and mSec14l3 attenuates ER-associated endosome fission in MEFs. WT and mSec14l2 KO MEF cells were transfected with or without mSec14l3 siRNA to observe endosome fission by GI-SIM at 1-s intervals for 2 min. ER-associated endosome fission events are indicated by arrows. mEmerald–RAB5 (green) and mCherry–KDEL (magenta) were co-transfected to label the endosome and the ER, respectively. WT MEFs (part I), mSec14l2 KO MEFs (part II), mSec14l3 siRNA transfected WT MEFs (part III) and mSec14l3 siRNA transfected mSec14l2 KO MEFs (part IV) are sequentially shown. Scale bars, 1 μm.

Supplementary Video 10

Endosomal recruitment of VPS34 or SAC1 cannot restore ER-associated endosome fission defects in SEC14L2 KO COS-7 cells. SEC14L2 KO COS-7 cells were co-transfected with iRFP–FRB–RAB5 (green and grey), mCherry–FKBP–VPS34 (yellow and part III) or mCherry–FKBP–SAC1ΔTMD (yellow and part IV), and mEmerald–KDEL (magenta) for live-cell imaging by GI-SIM at 1-s intervals for 30 min after 1 μM rapamycin treatment. WT and SEC14L2 KO COS-7 cells were co-transfected with mCherry–FKBP as controls in parts I and II. Scale bars, 2 μm.

Supplementary Video 11

Knocking down OSBP or VAPA/B in SEC14L2 KO COS-7 cells cannot aggravate ER-associated endosome fission defects, and zSec14l3 partially ameliorates ER-associated endosome fission defects in OSBP knockdown COS-7 cells. WT and SEC14L2 KO COS-7 cells were co-transfected with VAPA/B or OSBP siRNA to observe endosome fission by GI-SIM at 1-s intervals for 2 min. ER-associated endosome fission events are indicated by arrows. mEmerald–RAB5 (green) and mCherry–KDEL (magenta) were co-transfected to label endosomes and the ER, respectively. NC siRNA transfected WT and SEC14L2 KO COS-7 cells are shown in parts V and III, respectively; VAPA/B siRNA transfected WT and SEC14L2 KO COS-7 cells in parts VI and VII, respectively; and OSBP siRNA transfected WT and SEC14L2 KO COS-7 cells in parts I and VIII, respectively. zSec14l3–mCherry (part II) or OSBP–EGFP plasmid (part IV) was transfected into OSBP siRNA-transfected or SEC14L2 KO COS-7 cells to observe endosome fission by GI-SIM at 1-s intervals for 2 min. For the zSec14l3 overexpression group, Calnexin–Halo (magenta) and mEmerald–RAB5 (green) were co-transfected to label the ER and endosomes, respectively. For the OSBP overexpression group, mCherry–KDEL (magenta) and Halo–RAB5 (green) labelled the ER and endosomes, respectively. Scale bars, 1 μm.

Supplementary Video 12

zSec14l3 and hSEC14L2 restore ER-associated endosome fission defects in SEC14L2 KO COS-7 cells. hSEC14L2–mCherry (part III), zSec14l3–mCherry (part V), zSec14l3-M5–mCherry (part VI) or hSEC14L2-M5–mCherry plasmid (part IV) was individually transfected into SEC14L2 KO COS-7 cells to observe endosome fission by GI-SIM at 1-s intervals for 2 min. ER-associated endosome fission events are indicated by arrows. mEmerald–RAB5 (green) and Calnexin–Halo (magenta) were co-transfected to label endosomes and the ER, respectively. mCherry transfected WT and SEC14L2 KO COS-7 cells are shown in parts I and II, respectively. Scale bars, 1 μm.

Supplementary Video 13

Domain-depleted forms of zSec14l3 fail to rescue ER-associated endosome fission defects in SEC14L2 KO COS-7 cells. mCherry (part I), zSec14l3–ΔN–mCherry (part II), zSec14l3–ΔS–mCherry (part III) or zSec14l3–ΔG–mCherry (part IV) plasmids were individually transfected into SEC14L2 KO COS-7 cells to observe endosome fission by GI-SIM at 1-s intervals for 2 min. ER-associated endosome fission events are indicated by arrows. mEmerald–RAB5 (green) and Calnexin–Halo (magenta) were co-transfected to label endosomes and the ER, respectively. Scale bar, 1 μm.

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Gong, B., Guo, Y., Ding, S. et al. A Golgi-derived vesicle potentiates PtdIns4P to PtdIns3P conversion for endosome fission. Nat Cell Biol 23, 782–795 (2021). https://doi.org/10.1038/s41556-021-00704-y

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