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Yang, J. Petruska, J. Breslin, J. Baird, B. Scharf, T. Tripodi, G. Ryan, J. Tivade, J. Du, D. Minn, C. Romfo, and C. Trotta for help and support with this project. This work was supported by grants from the SMA Foundation and the Harvard Stem Cell Institute. F. Hoffmann-La Roche and PTC Therapeutics have filed three patent applications entitled “Compounds for Treating Spinal Muscular Atrophy” (WO2013/101974 A1, WO2013/112788 A1, and WO2013/119916 A2). SUPPLEMENTARY MATERIALS www.sciencemag.org/content/345/6197/688/suppl/DC1 Materials and Methods Figs. S1 to S9 Tables S1 to S4 References (49–56) Movies S1 to S3 23 December 2013; accepted 24 June 2014 10.1126/science.1250127 LIPID CELL BIOLOGY Polyunsaturated phospholipids facilitate membrane deformation and fission by endocytic proteins Mathieu Pinot, 1,2 Stefano Vanni, 1 Sophie Pagnotta, 3 Sandra Lacas-Gervais, 3 Laurie-Anne Payet, 4 Thierry Ferreira, 4 Romain Gautier, 1 Bruno Goud, 2 Bruno Antonny, 1 * Hélène Barelli 1 Phospholipids (PLs) with polyunsaturated acyl chains are extremely abundant in a few specialized cellular organelles such as synaptic vesicles and photoreceptor discs, but their effect on membrane properties is poorly understood. Here, we found that polyunsaturated PLs increased the ability of dynamin and endophilin to deform and vesiculate synthetic membranes. When cells incorporated polyunsaturated fatty acids into PLs, the plasma membrane became more amenable to deformation by a pulling force and the rate of endocytosis was accelerated, in particular, under conditions in which cholesterol was limiting. Molecular dynamics simulations and biochemical measurements indicated that polyunsaturated PLs adapted their conformation to membrane curvature. Thus, by reducing the energetic cost of membrane bending and fission, polyunsaturated PLs may help to support rapid endocytosis. M ost cellular membranes contain phospho- lipids (PLs) with saturated and mono- unsaturated acyl chains. However, in a few specialized organelles, such as syn- aptic vesicles, up to 80% of PLs contain at least one polyunsaturated acyl chain (1, 2). Such high levels suggest that polyunsaturated lipids might endow membranes with specific physicochemical properties. In photoreceptor discs, which are perfectly flat, polyunsaturated PLs facilitate the conforma- tional change of rhodopsin (3). The influence of polyunsaturated PLs on protein machineries that act on curved membranes is unclear. Nevertheless, exogenous treatment of neurons with polyun- saturated fatty acids facilitates SNARE (soluble N-ethylmaleimide–sensitive factor attachment protein receptor) assembly (4) and the recycling of synaptic vesicles (5, 6). Furthermore, poly- unsaturated PLs make pure lipid bilayers more flexible (7). We studied the effect of polyunsaturated PLs on the activity of the guanosine triphosphatase (GTPase) dynamin and the banana-shaped pro- tein endophilin, which cooperate in membrane fission by assembling into spirals around the neck of membrane buds (8, 9). We used liposomes or giant unilamellar vesicles (GUVs) with a fixed composition in terms of PL polar head groups and varied the ratio between mono and polyun- saturated PLs (tables S1 and S2; also see sup- plementary materials and methods). We chose C16:0-C18:1 and C18:0-C22:6 PLs (Fig. 1A) because they are the most abundant of each PL class. Dynamin hydrolyzed guanosine triphosphate (GTP) 7.5 times faster on large liposomes con- taining polyunsaturated PLs compared with mono- unsaturated PLs (Fig. 1B and fig. S1A). Moreover, polyunsaturated PLs eliminated the sharp re- sponse of dynamin to membrane curvature (Fig. 1C and fig. S1B) (10). Because GTP hydrolysis oc- curs through contacts between dynamin molecules within the assembled spiral (11), these results suggest that polyunsaturated PLs facilitate dynamin self-assembly on flat membranes. Using electron microscopy, we analyzed our liposomes incubated with dynamin, endophilin, and GTP, a mixture optimal for membrane fission (12). Before incubation, the liposomes displayed a similar size distribution (radius R = 30 to 200 nm) (Fig. 1, D and E). After incubation, polyunsatu- rated liposomes were consumed into small vesi- cles (R ≈ 20 nm), whereas monounsaturated liposomes appeared unchanged (Fig. 1, D and E, and fig. S2, A and D). When GTP hydrolysis was blocked, characteristic endophilin-dynamin spi- rals (8, 9) formed on polyunsaturated liposomes, but not on monounsaturated liposomes (Fig. 1D and fig. S2, B, C, and E). Thus, polyunsaturated membranes are sensitized to the mechanical ac- tivities of the endophilin-dynamin complex. Adhesion of liposomes to the electron micros- copy grid can lead to an overestimate of au- thentic fission owing to membrane breakage on the stiff support (13). To overcome this caveat, we used fission assays based on visualization of mod- el membranes by fluorescence microscopy. First, we incubated GUVs with dynamin, endophilin, and GTP and monitored the GUV diameter over time (12). GUVs containing polyunsaturated PLs showed a 14 to 30% decrease in size within 1 hour, suggesting consumption by membrane fission, whereas GUVs made of monounsaturated SCIENCE sciencemag.org 8 AUGUST 2014 • VOL 345 ISSUE 6197 693 1 Institut de Pharmacologie Moléculaire et Cellulaire, Université Nice Sophia Antipolis and CNRS, 06560 Valbonne, France. 2 Unité Mixte de Recherche 144, Institut Curie and CNRS, F-75248 Paris, France. 3 Centre Commun de Microscopie Appliquée, Université Nice Sophia Antipolis, Parc Valrose, 06000 Nice, France. 4 Signalisation et Transports Ioniques Membranaires, Université de Poitiers and CNRS, Poitiers, France. *Corresponding author. E-mail: antonny@ipmc.cnrs.fr RESEARCH | REPORTS