Non-vesicular lipid transport between bilayers at membrane contact sites plays important physiological roles. Mechanistic insight into the action of lipid-transport proteins localized at these sites requires determination of the distance between bilayers at which this transport can occur. Here we developed DNA-origami nanostructures to organize size-defined liposomes at precise distances and used them to study lipid transfer by the synaptotagmin-like mitochondrial lipid-binding protein (SMP) domain of extended synaptotagmin 1 (E-Syt1). Pairs of DNA-ring-templated donor and acceptor liposomes were docked through DNA pillars, which determined their distance. The SMP domain was anchored to donor liposomes via an unstructured linker, and lipid transfer was assessed via a Förster resonance energy transfer (FRET)-based assay. We show that lipid transfer can occur over distances that exceed the length of an SMP dimer, which is compatible with the shuttle model of lipid transport. The DNA nanostructures developed here can also be adapted to study other processes occurring where two membranes are closely apposed to each other.
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Saheki, Y. & De Camilli, P. Endoplasmic reticulum–plasma membrane contact sites. Annu. Rev. Biochem. 86, 659–684 (2017).
Wu, H., Carvalho, P. & Voeltz, G. K. Here, there, and everywhere: the importance of ER membrane contact sites. Science 361, eaan5835 (2018).
Wong, L. H., Gatta, A. T. & Levine, T. P. Lipid transfer proteins: the lipid commute via shuttles, bridges and tubes. Nat. Rev. Mol. Cell Biol. 20, 85–101 (2018).
Stefan, C. J., Manford, A. G. & Emr, S. D. ER–PM connections: sites of information transfer and inter-organelle communication. Curr. Opin. Cell. Biol. 25, 434–442 (2013).
AhYoung, A. P. et al. Conserved SMP domains of the ERMES complex bind phospholipids and mediate tether assembly. Proc. Natl Acad. Sci. USA 112, E3179–E3188 (2015).
Jeong, H., Park, J., Jun, Y. & Lee, C. Crystal structures of Mmm1 and Mdm12–Mmm1 reveal mechanistic insight into phospholipid trafficking at ER–mitochondria contact sites. Proc. Natl Acad. Sci. USA 114, E9502–E9511 (2017).
Kawano, S. et al. Structure–function insights into direct lipid transfer between membranes by Mmm1–Mdm12 of ERMES. J. Cell Biol. 217, 959–974 (2018).
Kopec, K. O., Alva, V. & Lupas, A. N. Homology of SMP domains to the TULIP superfamily of lipid-binding proteins provides a structural basis for lipid exchange between ER and mitochondria. Bioinformatics 26, 1927–1931 (2010).
Lee, I. & Hong, W. Diverse membrane-associated proteins contain a novel SMP domain. FASEB J. 20, 202–206 (2006).
Schauder, C. M. et al. Structure of a lipid-bound extended synaptotagmin indicates a role in lipid transfer. Nature 510, 552–555 (2014).
Wong, L. H. & Levine, T. P. Tubular lipid binding proteins (TULIPs) growing everywhere. Biochim. Biophys. Acta Mol. Cell Res. 1864, 1439–1449 (2017).
Lees, J. A. et al. Lipid transport by TMEM24 at ER–plasma membrane contacts regulates pulsatile insulin secretion. Science 355, eaah6171 (2017).
Alva, V. & Lupas, A. N. The TULIP superfamily of eukaryotic lipid-binding proteins as a mediator of lipid sensing and transport. Biochim. Biophys. Acta Mol. Cell Biol. Lipids 1861, 913–923 (2016).
Kornmann, B. et al. An ER–mitochondria tethering complex revealed by a synthetic biology screen. Science 325, 477–481 (2009).
Liu, L. K., Choudhary, V., Toulmay, A. & Prinz, W. A. An inducible ER–Golgi tether facilitates ceramide transport to alleviate lipotoxicity. J. Cell Biol. 216, 131–147 (2017).
Toulmay, A. & Prinz, W. A. A conserved membrane-binding domain targets proteins to organelle contact sites. J. Cell Sci. 125, 49–58 (2012).
Hirabayashi, Y. et al. ER–mitochondria tethering by PDZD8 regulates Ca2+ dynamics in mammalian neurons. Science 358, 623–630 (2017).
Min, S. W., Chang, W. P. & Sudhof, T. C. E-Syts, a family of membranous Ca2+-sensor proteins with multiple C2 domains. Proc. Natl Acad. Sci. USA 104, 3823–3828 (2007).
Manford, A. G., Stefan, C. J., Yuan, H. L., Macgurn, J. A. & Emr, S. D. ER-to-plasma membrane tethering proteins regulate cell signaling and ER morphology. Dev. Cell 23, 1129–1140 (2012).
Giordano, F. et al. PI(4,5)P2-dependent and Ca2+-regulated ER–PM interactions mediated by the extended synaptotagmins. Cell 153, 1494–1509 (2013).
Chang, C. L. et al. Feedback regulation of receptor-induced Ca2+ signaling mediated by E-Syt1 and Nir2 at endoplasmic reticulum–plasma membrane junctions. Cell Rep. 5, 813–825 (2013).
Fernandez-Busnadiego, R., Saheki, Y. & De Camilli, P. Three-dimensional architecture of extended synaptotagmin-mediated endoplasmic reticulum–plasma membrane contact sites. Proc. Natl Acad. Sci. USA 112, E2004–E2013 (2015).
Idevall-Hagren, O., Lu, A., Xie, B. & De Camilli, P. Triggered Ca2+ influx is required for extended synaptotagmin 1-induced ER–plasma membrane tethering. EMBO J. 34, 2291–2305 (2015).
Bian, X., Saheki, Y. & De Camilli, P. Ca2+ releases E-Syt1 autoinhibition to couple ER–plasma membrane tethering with lipid transport. EMBO J. 37, 219–234 (2018).
Saheki, Y. et al. Control of plasma membrane lipid homeostasis by the extended synaptotagmins. Nat. Cell Biol. 18, 504–515 (2016).
Yu, H. et al. Extended synaptotagmins are Ca2+-dependent lipid transfer proteins at membrane contact sites. Proc. Natl Acad. Sci. USA 113, 4362–4367 (2016).
Orci, L. et al. From the cover: STIM1-induced precortical and cortical subdomains of the endoplasmic reticulum. Proc. Natl Acad. Sci. USA 106, 19358–19362 (2009).
Douglas, S. M. et al. Self-assembly of DNA into nanoscale three-dimensional shapes. Nature 459, 414–418 (2009).
Rothemund, P. W. Folding DNA to create nanoscale shapes and patterns. Nature 440, 297–302 (2006).
Dietz, H., Douglas, S. M. & Shih, W. M. Folding DNA into twisted and curved nanoscale shapes. Science 325, 725–730 (2009).
Grome, M. W., Zhang, Z., Pincet, F. & Lin, C. Vesicle tubulation with self-assembling DNA nanosprings. Angew. Chem. Int. Ed. Engl. 57, 5330–5334 (2018).
Xu, W. et al. A programmable DNA origami platform to organize SNAREs for membrane fusion. J. Am. Chem. Soc. 138, 4439–4447 (2016).
Yang, Y. et al. Self-assembly of size-controlled liposomes on DNA nanotemplates. Nat. Chem. 8, 476–483 (2016).
Zhang, Z., Yang, Y., Pincet, F., Llaguno, M. C. & Lin, C. Placing and shaping liposomes with reconfigurable DNA nanocages. Nat. Chem. 9, 653–659 (2017).
Chan, Y. H., van Lengerich, B. & Boxer, S. G. Effects of linker sequences on vesicle fusion mediated by lipid-anchored DNA oligonucleotides. Proc. Natl Acad. Sci. USA 106, 979–984 (2009).
Franquelim, H. G., Khmelinskaia, A., Sobczak, J. P., Dietz, H. & Schwille, P. Membrane sculpting by curved DNA origami scaffolds. Nat. Commun. 9, 811 (2018).
Perrault, S. D. & Shih, W. M. Virus-inspired membrane encapsulation of DNA nanostructures to achieve in vivo stability. ACS Nano 8, 5132–5140 (2014).
Beales, P. A. & Vanderlick, T. K. Specific binding of different vesicle populations by the hybridization of membrane-anchored DNA. J. Phys. Chem. A 111, 12372–12380 (2007).
Kumar, N. et al. VPS13A and VPS13C are lipid transport proteins differentially localized at ER contact sites. J. Cell Biol. 217, 3625–3639 (2018).
Valverde, D. P. et al. ATG2 transports lipids to promote autophagosome biogenesis. J. Cell Biol. 218, 1787–1798 (2019).
Lin, C., Perrault, S. D., Kwak, M., Graf, F. & Shih, W. M. Purification of DNA-origami nanostructures by rate-zonal centrifugation. Nucleic Acids Res. 41, e40 (2013).
Sun, E. W. et al. Lipid transporter TMEM24/C2CD2L is a Ca2+-regulated component of ER–plasma membrane contacts in mammalian neurons. Proc. Natl Acad. Sci. USA 116, 5775–5784 (2019).
Xu, W., Wang, J., Rothman, J. E. & Pincet, F. Accelerating SNARE-mediated membrane fusion by DNA–lipid tethers. Angew. Chem. Int. Ed. Engl. 54, 14388–14392 (2015).
Ma, L. et al. Single-molecule force spectroscopy of protein–membrane interactions. eLife 6, e30493 (2017).
Xu, J. et al. Structure and Ca2+-binding properties of the tandem C2 domains of E-Syt2. Structure 22, 269–280 (2014).
Stefan, C. J. et al. Osh proteins regulate phosphoinositide metabolism at ER–plasma membrane contact sites. Cell 144, 389–401 (2011).
Eden, E. R., White, I. J., Tsapara, A. & Futter, C. E. Membrane contacts between endosomes and ER provide sites for PTP1B–epidermal growth factor receptor interaction. Nat. Cell Biol. 12, 267–272 (2010).
Kauert, D. J., Kurth, T., Liedl, T. & Seidel, R. Direct mechanical measurements reveal the material properties of three-dimensional DNA origami. Nano Lett. 11, 5558–5563 (2011).
Liedl, T., Hogberg, B., Tytell, J., Ingber, D. E. & Shih, W. M. Self-assembly of three-dimensional prestressed tensegrity structures from DNA. Nat. Nanotechnol. 5, 520–524 (2010).
Castro, C. E., Su, H. J., Marras, A. E., Zhou, L. & Johnson, J. Mechanical design of DNA nanostructures. Nanoscale 7, 5913–5921 (2015).
We thank Y. Cai for discussion. This work was supported by NIH grants NS036251 and DA018343, the HHMI and the Kavli Foundation to P.D.C.; an NIH Director’s New Innovator Award (GM114830) and a Yale University faculty startup fund to C.L.; a Human Frontier Science Program Long-term Fellowship to X.B.; and an Agency for Science, Technology and Research Graduate Scholarship (Singapore) to Q.X.
The authors declare no competing interests.
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Bian, X., Zhang, Z., Xiong, Q. et al. A programmable DNA-origami platform for studying lipid transfer between bilayers. Nat Chem Biol 15, 830–837 (2019). https://doi.org/10.1038/s41589-019-0325-3
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