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The SNARE Sec22b has a non-fusogenic function in plasma membrane expansion

Nature Cell Biology volume 16, pages 434–444 (2014)Cite this article

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Abstract

Development of the nervous system requires extensive axonal and dendritic growth during which neurons massively increase their surface area. Here we report that the endoplasmic reticulum (ER)-resident SNARE Sec22b has a conserved non-fusogenic function in plasma membrane expansion. Sec22b is closely apposed to the plasma membrane SNARE syntaxin1. Sec22b forms a trans-SNARE complex with syntaxin1 that does not include SNAP23/25/29, and does not mediate fusion. Insertion of a long rigid linker between the SNARE and transmembrane domains of Sec22b extends the distance between the ER and plasma membrane, and impairs neurite growth but not the secretion of VSV-G. In yeast, Sec22 interacts with lipid transfer proteins, and inhibition of Sec22 leads to defects in lipid metabolism at contact sites between the ER and plasma membrane. These results suggest that close apposition of the ER and plasma membrane mediated by Sec22 and plasma membrane syntaxins generates a non-fusogenic SNARE bridge contributing to plasma membrane expansion, probably through non-vesicular lipid transfer.

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Figure 1: The R-SNARE Sec22b is present in neurites and growth cones where it localizes to the ER.
Figure 2: Impairment of Sec22b inhibits neurite growth.
Figure 3: Sec22b interacts with PM Q-SNARE Stx1, but does not mediate exocytosis.
Figure 4: Sec22b comes in close proximity to PM Stx1, and elongating Sec22b increases the distance between the ER and PM, and impairs neurite growth.
Figure 5: Sec22b is not required for SOCE, but overexpression of elongated Sec22b mutant impairs SOCE.
Figure 6: Sec22 interacts with lipid transfer proteins, and inhibition of Sec22 leads to defects in lipid metabolism at contact sites between ER and PM in yeast.

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Change history

  • 11 July 2014

    In the version of this Article originally published, the first sentence of the Immunoprecipitation section in Methods was incomplete. The correct wording reads: "Whole mouse brain or transfected COS7 cells were homogenized in homogenization buffer (50 mM Hepes pH 7.2, 100 mM NaCl, 4 mM EGTA, 2 mM MgCl2 and protein inhibitors (Roche, Complete without EDTA)) at 4 °C. Proteins were then extracted by addition of 1% final concentration of Triton X-100 and lysis at 4 °C for 2 h or 30 min respectively. The insoluble material was removed by centrifugation at 13,000 r.p.m. for 30 min with a Beckman tabletop centrifuge. Total protein in the extracts was quantified with Bradford assays." This has been corrected in the online versions of the Article.

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Acknowledgements

We are grateful to F. Polleux, V. Medvedeva and A. Assali for advice on in utero electroporation, N. Rouach for calcium imaging and F. Perez for the gift of RUSH reagents. We are grateful to Y. N. Jan for support to M.P. in revising the manuscript, and L. Bosanac for support with software. We are grateful to A. Pierani and members of the Galli laboratory for discussions. We acknowledge France-BioImaging infrastructure supported by the French National Research Agency (ANR-10-INSB-04, ‘Investments for the future’). Work in our group was financially supported in part by grants from INSERM, CNRS, the Association Française contre les Myopathies (AFM), the Association pour la Recherche sur le Cancer (ARC), the Mairie de Paris Medical Research and Health Program, the Fondation pour la Recherche Médicale (FRM), the Ecole des Neurosciences de Paris (ENP), and awards of the Association Robert Debré pour la Recherche Médicale to T.G., ANR JC grant to D.T. and the Ecole des Neurosciences de Paris (ENP) and award of the Fondation des Treilles to M.P. F.D. is supported by a PhD fellowship from Paris Descartes University and funds by the PhD Program Ecole Doctorale Frontières du Vivant (FdV)—Programme Bettencourt.

Author information

Author notes

  1. Maja Petkovic

    Present address: Present address: Howard Hughes Medical Institute, Departments of Physiology, Biochemistry, and Biophysics, University of California, San Francisco, San Francisco, California 94158, USA.,

  2. Aymen Jemaiel and Frédéric Daste: These authors contributed equally to this work.

Authors and Affiliations

  1. INSERM, U950, F-75013 Paris, France

    Maja Petkovic, Frédéric Daste, David Tareste & Thierry Galli

  2. Université Paris Diderot, Sorbonne Paris Cité, ERL U950, F-75013 Paris, France

    Maja Petkovic, Frédéric Daste, David Tareste & Thierry Galli

  3. CNRS, UMR 7592, Institut Jacques Monod, F-75013 Paris, France

    Maja Petkovic, Aymen Jemaiel, Frédéric Daste, Jean-Marc Verbavatz, David Tareste, Catherine L. Jackson & Thierry Galli

  4. Ecole des Neurosciences de Paris (ENP), F-75006 Paris, France

    Maja Petkovic

  5. Membrane Dynamics and Intracellular Trafficking, Institute Jacques Monod, F-75013 Paris, France

    Aymen Jemaiel & Catherine L. Jackson

  6. Ecole Doctorale Frontières du Vivant (FdV) – Programme Bettencourt, Université Paris Descartes, Sorbonne Paris Cité, F-75004 Paris, France

    Frédéric Daste

  7. Institut de Biologie de l’Ecole Normale Supérieure (IBENS), Biologie Cellulaire de la Synapse, INSERM U1024, CNRS UMR8197, F-75005 Paris, France

    Christian G. Specht & Antoine Triller

  8. Institut de Biologie de l’Ecole Normale Supérieure (IBENS), Functional Imaging of Transcription, INSERM U1024, CNRS UMR8197, F-75005 Paris, France

    Ignacio Izeddin & Xavier Darzacq

  9. Max Planck Institute of Molecular Cell Biology and Genetics, 01307 Dresden, Germany

    Daniela Vorkel & Jean-Marc Verbavatz

  10. Linda Crnic Institute for Down Syndrome and Department of Pediatrics, University Colorado School of Medicine, Aurora, Colorado 80045, USA

    Karl H. Pfenninger

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Contributions

M.P., C.L.J. and T.G. conceived the project. M.P. generated and analysed data in mammalian cells and neurons, and A.J. in budding yeast. F.D. and D.T. performed and analysed liposome fusion assays. M.P., C.G.S. and I.I. performed and C.G.S and I.I analysed results from super-resolution microscopy; X.D. and A.T. set-up the super-resolution microscopes. M.P. and K.H.P. performed and analysed subcellular fractionation experiments. D.V. and J-M.V. generated and analysed electron microscopy data. M.P., C.L.J. and T.G. wrote the manuscript.

Corresponding authors

Correspondence to Catherine L. Jackson or Thierry Galli.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1

(a) Immunochemistry of VAMP4 and Ykt6 in mouse cortical neurons, further showing observed strong growth cone enrichment is not a general characteristic of R-SNAREs, but specific to a subset of them (Sec22b, VAMP2 and TI-VAMP). VAMP4 is abundant in the perikaryon, but sparsely present in the neurites, while Ykt6 is clearly detectable in neurites as well as perikaryon, but does not have strong localization in the growth cones. Scale bar 10 μm. (b) Impact of Sec22b knockdown in vivo on the distribution of neurons between subdivisions of cortex. In utero electroporation in wild type mouse brains was performed with shRNA1, shRNA2 or scrambled construct as control (scrambled: n = 13 brain sections from 3 embryos; shRNA1: n = 3 brain sections from 2 embryos; shRNA2: n = 9 brain sections from 3 embryos), co-electoporated with GFP under CAG promoter. Lower panel: Brain sections with similar targeting of the electroporation in the same coronal plane were subdivided into regions based on DAPI staining: cortical plate (CP), IZ (intermediate zone) and SVZ/VZ (subventricular/ventricular zone) (first row). Selected regions were transferred to threshold images of the GFP signal (second row), where GFP-positive neurons were outlined (third to fifth row) and measured with Analyze particle tool in ImageJ, using the same parameters for all images. Upper panel: GFP+ positive cells in each subdivision were expressed as percentage of total number of GFP+ cells ± s.e.m. One way Anova showed no significant difference between conditions for all three regions. P ≥ 0.5 n.s. non-significant. Scale bar 100μm.

Supplementary Figure 2

(a) SDS-PAGE gel of R-SNARE (Sec22b) and Q-SNARE (co-expressed Stx1/SNAP25, separately expressed Stx1+SNAP25 or Stx1 alone) liposomes used in the lipid mixing assay (Fig. 3g, h). Separately expressed Q-SNARE was formed by incubating Stx1 liposomes overnight on ice with SNAP25 in a 1:3 (Stx1:SNAP25) molar ratio followed by isolation on a Nycodenz flotation gradient. The smaller size of Stx1 in the case of co-expressed Q-SNAREs is due to the absence of His6-tag. Lipids can be seen at the bottom of the gel. (b) Upper row: Immunolocalization of GFP-Sec22b with a 33 proline linker and calreticulin antibody in cortical neurons. Scale bar: 10 μm, inset: 2 μm. Lower row: COS7 cells co-transfected with GFP-Sec22b containing 33 proline linker and RFP-Sec22b. (c) Immunoprecipitation of GFP-Sec22b with 33 proline linker. COS7 cells were transfected with mCherry-Stx1 and GFP-Sec22b or the GFP-Sec22b with 33 proline linker. Immunoprecipitation was performed with mouse Stx1 antibody, mouse GFP antibody or mouse immunoglobulins as negative control of specificity of immunoprecipitation. Western blot was revealed with rabbit GFP and syntaxin 1 antibodies. mCherry-Stx1 co-immunoprecipitated both GFP-Sec22b variants and vice versa. (d, e) RUSH secretory assay. HeLa cells were co-transfected with GFP-Sec22b or GFP-Sec22bP33 and mCherry tagged VSV-G RUSH construct blocked in the ER. Secretion is released with addition of biotin, and cells are fixed after 30min (n = 76 cells for WT; n = 96 cells for P33), 60min (n = 92 for WT; n = 99 for P33) and 6h (n = 72 for WT; n = 71 for P33). Amount of surface VSV-G was evaluated by surface staining and expressed as ratio to total VSV-G, and normalized to the negative control (n = 77 for WT; n = 72 for P33), which are cells never exposed to biotin (t = 0 min). (d) Representative images are presented, with scale bar 10 μm. (e) Mean value per condition and time point are shown, where error bars represent the s.e.m. Student t-test did not reveal any significant difference between the WT and P33 Sec22b constructs at any of the time points measured. P ≥ 0,05 n.s.

Supplementary Figure 3

(a) Growth defect of the temperature sensitive sec22-3 yeast strain is rescued by expression of GFP-Sec22p. RSY279 sec22-3 cells expressing GFP-Sec22 from a low-copy centromeric plasmid (pCM188-GFP-Sec22) were serially diluted onto medium selective for the plasmid and grown at 24 °C (permissive) and at 30 °C and 37 °C (restrictive temperatures) for 2 days. (b) Correct PM localization of GFP-Sso1 in wild type yeast cells. BY4742 GFP-Sso1 expressing cells in exponential growth were imaged by confocal microscopy at 24 °C; similar results were obtained for wild type cells (SEY6210). Scale bar 3 μm. (c) Sac1 (the PI4P phosphatase acting on the PM PI4P pool) is correctly localized in sec22-3. RSY279 sec22-3 cells expressing GFP-Sac1 were incubated for 0, 30 or 60 minutes at 37 °C, then imaged; representative images show normal ER localization of Sac1 at both time points after shift to restrictive temperature.

Supplementary Figure 4

Uncropped Western blots referring to main figures 1.a, 1.b, 3.b-f. Regions shown in main figures are marked with red squares. All Western blots were named according to the main figure to which they relate to. (Fig. 1a) Distribution analysis of growth cone fractionation showing specific enrichment of GAP43, axonal growth cone marker. (Fig. 1b) Enrichment of R-SNAREs in growth cone membrane fraction compared to total brain homogenate. GAP43 and nucleoporins were used as positive and negative control, respectively. (Fig. 3b) Western blot showing Stx1 immunoprecipitation from embryonic and adult brain, blotted for Stx1 on the left, and reblotted on the same membrane for Sec22b on the right. (Fig. 3c) Enrichment of SNAPs in growth cone membrane fraction compared to total brain homogenate. (Fig. 3d) Western blot showing SNAP25 immunoprecipitation from embryonic and adult brain, blotted for SNAP25 and VAMP2, and Sec22b. Membrane on the left shows SNAP25 and VAMP2, and membrane on the right from a separate gel done in the same way shows SNAP-25 and Sec22b. (Fig. 3e) Western blot showing SNAP23 immunoprecipitation from embryonic and adult brain, blotted for SNAP23 and VAMP2 on the left, and reblotted on the same membrane for Sec22b on the right. (Fig. 3f) Western blot showing SNAP29 immunoprecipitation from embryonic and adult brain, blotted for Stx1, Sec 22b and VAMP2 on the left, and membrane on the right from a separate gel done in the same way shows SNAP-29.

Supplementary Figure 5

Uncropped Western blots referring to main figures 6.a-c, 6.e. Regions shown in main figures are marked with red squares. All Western blots were named according to the main figure to which they relate for simplicity. (Fig. 6a) Western blot showing GFP-Sso1 and GFP-Sec22p immunoprecipitation from yeast, blotted for GFP, Sso1 and Sec22p. (Fig. 6b) Western blot showing GFP-Sso1 expression over time from plasmid under tetracycline-repressible promoter (pAP87-GFP-Sso1) stimulated with doxycycline. Membrane is blotted for Sec22. (Fig. 6c) Western blot showing GFP immunoprecipitation from GFP-Sso1 and GFP-Vam7 expressing yeast cells, blotted for Sec22. (Fig. 6e) Western blot showing GFP immunoprecipitation from GFP-Osh2 and GFP-Osh3 expressing yeast cells, blotted for GFP, Sso1 and Sec22p.

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Sec22b colocalizes with ER marker in COS7 cells.

Videomicroscopy of COS7 cells co-transfected with mCherry-Sec22b and GFP-Sec61 reveals colocalization of the two constructs and clear ER morphology of the Sec22b compartment. Addition to (Fig. 1g). (AVI 127 kb)

Sec22b compartment has tubule-reticular morphology in neurites.

Videomicroscopy of DIV2 mouse cortical neurons transfected with EGFP-Sec22b reveals clear tubulo-reticular morphology of the Sec22b compartment. Addition to (Fig. 1g). (AVI 986 kb)

Sec22b tubules enter growth cone filopodia.

Confocal microscopy of DIV2 mouse cortical neurons transfected with EGFP-Sec22b reveals active dynamic of Sec22b tubules. Addition to (Fig. 1g). (AVI 560 kb)

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Petkovic, M., Jemaiel, A., Daste, F. et al. The SNARE Sec22b has a non-fusogenic function in plasma membrane expansion. Nat Cell Biol 16, 434–444 (2014). https://doi.org/10.1038/ncb2937

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