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Control of plasma membrane lipid homeostasis by the extended synaptotagmins

This article has been updated

Abstract

Acute metabolic changes in plasma membrane (PM) lipids, such as those mediating signalling reactions, are rapidly compensated by homeostatic responses whose molecular basis is poorly understood. Here we show that the extended synaptotagmins (E-Syts), endoplasmic reticulum (ER) proteins that function as PtdIns(4,5)P2- and Ca2+-regulated tethers to the PM, participate in these responses. E-Syts transfer glycerolipids between bilayers in vitro, and this transfer requires Ca2+ and their lipid-harbouring SMP domain. Genome-edited cells lacking E-Syts do not exhibit abnormalities in the major glycerolipids at rest, but exhibit enhanced and sustained accumulation of PM diacylglycerol following PtdIns(4,5)P2 hydrolysis by PLC activation, which can be rescued by expression of E-Syt1, but not by mutant E-Syt1 lacking the SMP domain. The formation of E-Syt-dependent ER–PM tethers in response to stimuli that cleave PtdIns(4,5)P2 and elevate Ca2+ may help reverse accumulation of diacylglycerol in the PM by transferring it to the ER for metabolic recycling.

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Figure 1: Localization of endogenous E-Syt1.
Figure 2: E-Syt1 is a Ca2+-dependent lipid transfer protein.
Figure 3: Generation of E-Syt1/2 DKO and E-Syt1/2/3 TKO HeLa cells using TALEN and CRISPR.
Figure 4: Rapid isolation and characterization of PM sheets.
Figure 5: Prolonged accumulation of DAG in E-Syt KO cells on phospholipase activation.
Figure 6: SMP-domain-dependent and Ca2+-regulated diacylglycerol transfer by E-Syt1.
Figure 7: PM DAG extraction mediated by E-Syts.

Change history

  • 14 April 2016

    In the version of this Article originally published online, '100 mM' in the x axes labels of Fig. 6c should have read '100 μM'. This has been corrected in all versions of the Article.

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Acknowledgements

We thank H. Shen, S. Ferguson, S. Tomita, R. Dong, W. Hancock-Cerutti, J. Lees and Y. Cai for discussion and/or sharing reagents. We thank F. Wilson, H. Czapla and L. Lucast for technical assistance. This work was supported in part by NIH grants R37NS036251, DK45735 and DA018343 to P.D.C. Y.Saheki was supported by fellowships from the Uehara Memorial Foundation and the Japan Society for the Promotion of Science, and X.B. by a Human Frontier Science Program long-term fellowship.

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Authors

Contributions

All authors participated in the design of experiments, data analysis and interpretation. Y.Saheki designed and performed all of the genetic manipulations, all of the imaging and biochemical studies and the isolation of plasma membrane sheets. X.B., F.P. and K.M.R. participated in the design of lipid transfer assays that were performed by X.B. C.M.S. and K.M.R. designed SMP domain mutations and performed lipid-binding assays. M.A.S and C.K. performed the lipidomic analysis. Y.Saheki and P.D.C. wrote the manuscript, which was then reviewed by all authors.

Corresponding author

Correspondence to Pietro De Camilli.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 4 Localization of E-Syt1.

(a) Lysates of control and E-Syt1/2 DKO HeLa cells were processed by SDS-PAGE and immunoblotting with the original YU1231 serum (left) or affinity-purified antibodies from YU1231 serum (right). The affinity-purified antibodies were further incubated with aldehyde fixed E-Syt1/2 DKO lysates and used for immunohistochemistry. (b) Confocal images of E-Syt1/2 DKO HeLa cells expressing mRFP-Sec61β. Endogenous localization of E-Syt1 was detected by the affinity-purified antibodies after incubation with aldehyde fixed E-Syt1/2 DKO lysates (see Methods). Note the absence of the E-Syt1 fluorescence signals. Scale bars, 10 μm. (c) TIRF microscopy images of a HeLa cell expressing transfected EGFP-E-Syt1 before and after stimulation with ionomycin (2 μM) in the presence of extracellular Ca2+ at the indicated time. (d) Time-course of ionomycin-induced recruitment of EGFP-E-Syt1 to the PM, as shown in c (mean ± s.e.m., n = 11 cells pooled from 2 independent experiments). Scale bars, 10 μm. Unprocessed original scans of blots are shown in Supplementary Fig. 7.

Supplementary Figure 5 Characterization of the lipid transfer assay.

(a) The self-quenching property of fluorescent lipids was plotted as the ratio of the quenched fluorescence signals ‘F0’ over the fully dequenched fluorescence signal in the presence of detergent ‘F’ (mean ± s.e.m., n = 3 independent experiments) (b) Time-course of normalized fluorescence signals, as assessed by fluorometry, for the mixture of acceptor liposomes with or without PI(4,5)P2 and donor liposomes containing 1% NBD-PE during an incubation with E-Syt1cyto in the presence of 100 μM Ca2+. Note the much slower dequenching of NBD-PE in the absence of PI(4,5)P2 in the acceptor liposomes. (c) Time-course of normalized fluorescence signals, as assessed by fluorometry, for the mixture of liposomes containing 1% NBD-PE either in ER-like or in PM-like liposomes incubated with E-Syt1cyto in the presence of 100 μM Ca2+. Note the comparable dequenching of NBD-PE in the two conditions. (d) (left) Content mixing assay. Time-course of normalized fluorescence signals, as assessed by fluorometer, from the mixture of acceptor and donor liposomes containing self-quenched sulforhodamine B (see Methods), and either E-Syt1cyto or E-Syt1cytoΔSMP, incubated with indicated concentration of Ca2+ in the assay buffer. Note the minimal dequenching of sulforhodamine B that depend neither on the presence nor absence of Ca2+. (right) Lipid transfer assay. Time-course of normalized fluorescence signals, as assessed by fluorometer, from the mixture of acceptor liposomes and donor liposomes containing 1% NBD-PE, and either E-Syt1cyto or E-Syt1cytoΔSMP, incubated with indicated concentration of Ca2+ in the assay buffer. Note the Ca2+- and SMP domain-dependent dequenching of NBD-PE fluorescence signals overtime. (e) Time-course of normalized fluorescence signal, as assessed by fluorometry, for the mixture of acceptor PM-like liposomes and donor ER-like liposomes containing 1% NBD-PE at the indicated ratios and incubated with E-Syt1cyto in the presence of 100 μM Ca2+. (f) Time-course of normalized fluorescence signals, as assessed by fluorometry, from the mixture of acceptor PM-like liposomes and donor ER-like liposomes. Addition of non-labeled PE to acceptor liposomes did not affect the transfer of NBD-PE. For all the liposome-based assays except a, data are from one experiment; the experiments were repeated three times with similar results.

Supplementary Figure 6 Enrichment and characterization of genome-edited E-Syt knockout (KO) cells.

(a) Surveyor nuclease assay for TALEN- and Cas9-mediated cleavage at E-Syt2(left), E-Syt1(middle) and E-Syt3(right) loci in HeLa cells. (b) (top left) Schematics showing the design of the surrogate vector for E-Syt2 KO. (top right) Confocal images of cells expressing the indicated TALEN constructs together with the mCherry-E-Syt2_SMP-EGFP surrogate vector. Scale bars, 100 μm. (bottom left) Schematics showing the design of the surrogate vector for E-Syt1 KO. Protospacer-adjacent motif (PAM) sequences are indicated by yellow boxes; yellow allowheads indicate predicted cleavage sites. (bottom right) Confocal images of cells expressing hCas9 and the indicated guide RNA expression vectors together with the mCherry-E-Syt1(6,7)-EGFP surrogate vector. Scale bars, 10 μm. (c) Surveyor nuclease assay of Cas9-mediated cleavage of E-Syt1 locus. Note approximately 2-fold increase in the cleavage efficiency after FACS. (d) Detection of TALEN-mediated cleavage of E-Syt2 gene by PCR. Asterisks denote clonal cell lines with size changes of the PCR products. (e) Lysates of control HeLa cells (WT) and 8 candidate E-Syt2 KO cell lines were processed by SDS-PAGE and immunoblotting (IB) with indicated antibodies. Arrow indicates endogenous E-Syt2. (f) (top) Nucleotide sequence analysis of the E-Syt2 gene; TALEN-binding sites are highlighted in red, and bold and blue letters indicate TALEN-binding sites and additional nucleotide insertions, respectively. (bottom) Lysates of control HeLa cells (WT) and E-Syt2 KO cell lines transfected with EGFP-E-Syt1 were processed for immunoprecipitation with anti-GFP antibodies. Total lysates (Input) as well as anti-GFP immunoprecipitates were processed by SDS-PAGE and immunoblotted with indicated antibodies. Arrow indicates endogenous E-Syt2. (gh) KO of E-Syt1 and E-Syt3. (g, top) Sequencing analyses of the E-Syt1 gene of two E-Syt1/2 DKO cell lines show one nucleotide insertions. (g, bottom) Lysates of control HeLa cells (WT), E-Syt2 KO cell line and the same cell line treated with guide RNA expressing vectors were processed by SDS-PAGE and immunoblotted with indicated antibodies. (h) Sequencing analysis of the E-Syt3 gene of the E-Syt TKO cell line. Guide RNA-targeting sites and PAM sequences are highlighted in red and green. Unprocessed original scans of blots are shown in Supplementary Fig. 7.

Supplementary Figure 7 PI(4,5)P2 dynamics on muscarinic receptor stimulation is not affected in HeLa cells lacking E-Syts.

(a) (left) Time course of normalized mRFP fluorescence, as assessed by TIRF microscopy, in response to Oxo-M (10 μM) and atropine (50 μM), from WT control HeLa cells, E-Syt1/2 DKO HeLa cells and E-Syt TKO HeLa cells expressing mRFP-PH-PLCδ and M1R. (right) Values of ΔF/F0 corresponding to the indicated time point by arrow. [mean ± s.e.m., n = 11 cells (Ctrl), n = 13 cells (DKO#6-8), n = 12 cells (TKO#5); data are pooled from 2 independent experiments for each condition] Bonferroni’s multiple comparisons test, n.s. = not significant (P > 0.05). (b) Time-course of normalized EGFP, iRFP (left axis) and mCherry (right axis) fluorescence signals in response to Histamine (1 mM), as assessed by TIRF microscopy, from cells expressing endogenously-tagged E-Syt1 as well as iRFP-PH-PLCδ and C1-mCherry. (mean ± s.e.m., n = 13 cells from 3 independent dishes) (c) Time-course of normalized EGFP, iRFP (left axis) and mCherry (right axis) fluorescence signals in response to Oxo-M (10 μM) and atropine (50 μM), as assessed by TIRF microscopy, from cells expressing endogenously-tagged E-Syt1 as well as M1R, iRFP-PH-PLCδ and C1-mCherry. (mean ± s.e.m., n = 25 cells pooled from 4 independent experiments).

Supplementary Figure 8 Enhanced and prolonged increase of DAG in the PM of E-Syt KO cells in response to ionomycin.

(a) (left) Time-course of normalized YFP fluorescence, as assessed by TIRF microscopy, from WT control and E-Syt TKO cells expressing YFP-DBD (a DAG probe), in response to ionomycin (2 μM). (right) Values of ΔF/F0 corresponding to the end of the experiment as indicated by an arrow [mean ± s.e.m., n = 27 cells pooled from 6 independent experiments (Ctrl), n = 19 cells pooled from 3 independent experiments (TKO#5); two-tailed Student’s t-test with unequal variance, P < 0.0001].

Supplementary Figure 9 Dynamics of DAG in E-Syt KO cells.

(a) Time course of normalized EGFP fluorescence, as assessed by TIRF microscopy, in response to Oxo-M (10 μM) and atropine (50 μM), from WT control HeLa cells, E-Syt1/2 DKO HeLa cells and E-Syt TKO HeLa cells expressing C1-EGFP and M1R. [mean ± s.e.m., n = 11 cells (Ctrl), n = 13 cells (DKO#6-8), n = 12 cells (TKO#5); data are pooled from 2 independent experiments for each condition] (b) (left) Time-course of normalized EGFP, iRFP (left axis) and mCherry (right axis) fluorescence signals in response to Oxo-M (10 μM), atropine plus DGKi (50 μM each) and ionomycin (2 μM) in the absence of extracellular Ca2+, as assessed by TIRF microscopy, from cells expressing endogenously-tagged E-Syt1 as well as M1R, iRFP-PH-PLCδ and C1-mCherry. (right) Values of ΔF/F0 corresponding to the end of the experiment as shown here and in Fig. 7d with red arrows [mean ± s.e.m., n = 20 cells (ionomycin with Ca2+), n = 20 cells (ionomycin without Ca2+); data are pooled from 3 independent experiments for each condition; two-tailed Student’s t-test with unequal variance, denotes P = 0.0001]. (c) (left) Time-course of normalized mCherry signal, in response to Oxo-M (10 μM), atropine plus DGKi (50 μM each) and ionomycin (2 μM), as assessed by TIRF microscopy, from control (Ctrl) and E-Syt TKO cells expressing C1-mCherry. Re-expression of EGFP-E-Syt1 in E-Syt TKO cells rescued the accumulation of DAG, as assessed by C1-mCherry, while EGFP-E-Syt1 Cyto that lacks the ER anchor did not. (right) Values of F/F0 corresponding to the end of the experiment as shown with an arrow [mean ± s.e.m., n = 5 cells (Ctrl), n = 7 cells (TKO#5), n = 11 cells (TKO#5 + EGFP-E-Syt1), n = 13 cells (TKO#5 + EGFP-E-Syt1 Cyto)]; data are pooled from 2 independent experiments for each condition; Bonferroni’s multiple comparisons test, denotes P < 0.0001]. NS, not significant (P > 0.05).

Supplementary Figure 10 Unprocessed original scans of blots used for the figures and Supplementary Figures.

Supplementary Table 1 Primary and secondary antibodies used in this study.
Supplementary Table 2 Sequences of primers and oligos used in this study.
Supplementary Table 3 Moles percent of lipids used for the acceptor and donor liposomes in FRET-based lipid transfer experiments.

Supplementary information

Supplementary Information

Supplementary Information (PDF 640 kb)

41556_2016_BFncb3339_MOESM17_ESM.avi

Rapid translocation of endogenously tagged E-Syt1 to the cortical regions of a cell, as monitored by spinning disc confocal microscopy, in response to stimulation with ionomycin (2 μM). (AVI 1043 kb)

41556_2016_BFncb3339_MOESM18_ESM.avi

Rapid translocation of endogenously tagged E-Syt1 to the cortical regions of a cell, as monitored by spinning disc confocal microscopy, in response to thapsigargin (2 μM). (AVI 1340 kb)

Simultaneous TIRF imaging of Nir2-mCherry and endogenously-tagged E-Syt1 (endoEGFP-E-Syt1) in response to the sequential application of Oxo-M (10 μM), atropine plus DGKi (50 μM each) (ATR&DGKi) and ionomycin (2 μM) (Ion.) as indicated.

The increase of fluorescence in the TIRF field is both diffuse and spotted for Nir2 (as this protein is both soluble and partially anchored in the ER via its interaction with VAP36), while it is only spotted for E-Syt1, which is an intrinsic protein of the ER. Dark spots represent ER-PM contacts. (AVI 2390 kb)

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Saheki, Y., Bian, X., Schauder, C. et al. Control of plasma membrane lipid homeostasis by the extended synaptotagmins. Nat Cell Biol 18, 504–515 (2016). https://doi.org/10.1038/ncb3339

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