COPI Buds 60-Nm Lipid Droplets from Reconstituted Water–Phospholipid–Triacylglyceride Interfaces, Suggesting a Tension Clamp Function

COPI buds 60-nm lipid droplets from reconstituted water–phospholipid–triacylglyceride interfaces, suggesting a tension clamp function

Abdou Rachid Thiama,b, Bruno Antonnya,c,1, Jing Wanga, Jérôme Delacotteb, Florian Wilﬂinga, Tobias C. Walthera, Rainer Becka,d, James E. Rothmana, and Frédéric Pinceta,b,1

aDepartment of Cell Biology, School of Medicine, Yale University, New Haven, CT 06520; bLaboratoire de Physique Statistique, Ecole Normale Supérieure de Paris, Université Pierre et Marie Curie, Université Paris Diderot, Centre National de la Recherche Scientiﬁque, 75005 Paris, France; cInstitut de Pharmacologie Moléculaire et Cellulaire, Université de Nice Sophia Antipolis and Centre National de la Recherche Scientiﬁque, 06560 Valbonne, France; and dHeidelberg University Biochemistry Center, Heidelberg University, 69120 Heidelberg, Germany

Edited by William F. DeGrado, School of Pharmacy, University of California, San Francisco, CA, and approved July 3, 2013 (received for review April 23, 2013) Intracellular trafficking between organelles is achieved by coat induces the formation of 60-nm “nano” LDs from the mother LD. protein complexes, coat protomers, that bud vesicles from bilayer This budding process increases the surface tension, which makes the membranes. Lipid droplets are protected by a monolayer and thus mother LD more reactive with its environment, such as soluble seem unsuitable targets for coatomers. Unexpectedly, coat protein enzymes or membranes, and thereby can explain how COPI is in- complex I (COPI) is required for lipid droplet targeting of some volved in the targeting of enzymes to a natural LD surface. proteins, suggesting a possible direct interaction between COPI and lipid droplets. Here, we find that COPI coat components can Results bud 60-nm triacylglycerol nanodroplets from artificial lipid droplet Arf1 Binds TAG/Buffer Interface in a GTP-Dependent Manner. On lipid (LD) interfaces. This budding decreases phospholipid packing of bilayer membranes, COPI assembles in two steps: binding of Arf1 the monolayer decorating the mother LD. As a result, hydrophobic to the membrane in a GTP-dependent manner, followed by en bloc

triacylglycerol molecules become more exposed to the aqueous recruitment of coatomer by Arf1–GTP (16, 17). We investigate the SCIENCES environment, increasing LD surface tension. In vivo, this surface possibility of this stepwise assembly on artificial LD surfaces. APPLIED PHYSICAL tension increase may prime lipid droplets for reactions with We tested Arf1 binding to LDs with two complementary ap- neighboring proteins or membranes. It provides a mechanism proaches: flotation assay and microfluidics. We prepared TAG fundamentally different from transport vesicle formation by COPI, droplets that were surrounded by a monolayer of a phospholipid likely responsible for the diverse lipid droplet phenotypes associ- mixture (PL) of the same composition as that used to prepare ated with depletion of COPI subunits. control liposomes (PL composition is similar to that of natural LDs) (18). Arf1 binds to such droplets in a GTP-dependent regulator | membrane tension | lipid droplet targetting | buffer-in-oil drops manner and with a similar efficiency as on liposomes (Fig. 1A). We confirmed Arf1 binding to buffer/TAG interfaces using a BIOCHEMISTRY fl he dynamic behavior of cells requires a constant trafficking micro uidic setup allowing direct visualization of protein inter- Tbetween organelles, which is largely achieved by vesicles. On actions. We produced micrometric buffer drops in a stream of oil bilayer-bound organelles, protein coats drive budding of trans- containing the phospholipids. The buffer/TAG interface is then port vesicles (1). The coat protein complexes II (COPII) and I coated with a monolayer of PL, as attested by the change in (COPI) generate vesicles from the endoplasmic reticulum and surface tension (see Fig. 4). In each buffer drop, biochemical Golgi apparatus, respectively, whereas clathrin coats use various reactions taking place at the buffer/TAG interface can be ob- fl adaptor complexes to generate vesicles from the trans-Golgi served by uorescence microscopy. The small buffer volume network, endosomes, and the plasma membrane. Coatomer is minimizes the amount of coatomer and Arf1 required, a decisive a cytosolic complex that forms the building blocks of the COPI advantage compared with the inverse geometry where oil drop- B coat. At the Golgi apparatus, coatomer is recruited en bloc to the lets are produced in a stream of buffer. Fig. 1 shows images of bilayer by Arf1 in a GTP-dependent manner (1–3). All known buffer droplets containing Cy3-labeled Arf1 and, alternatively, – coat proteins act on phospholipid bilayer membranes. GDP or GTP. In agreement with the biochemical assay, Cy3 Thus, it is surprising that COPI depletion affects lipid droplets Arf1 accumulates in a GTP-dependent manner at the TAG/ fi (LDs) that are bounded by a single monolayer of phospholipids buffer interface decorated with a monolayer of PL, con rming coating an organic phase of neutral lipids such as triacylglycerols that Arf1 is able to bind to the LD lipid monolayer surface. (TAGs) (4–6). LDs expand and shrink during times of energy COPI Machinery Buds Particles from TAG/Buffer Interface. Next, we excess or scarcity (7). LD-bound proteins, including lipases and tested the ability of coatomer to be recruited to Arf1-decorated neutral lipid synthesis enzymes (8–12), mediate these processes. LDs. We added Alexa 647-labeled coatomer to Cy3–Arf1 and For instance, COPI depletion leads to mistargeting of adipose GTP to the buffer-in-oil drops. Under these conditions, the triglyceride lipase (ATGL), the enzyme catalyzing the first step fluorescent proteins did not only cover the TAG/buffer interface. of TAG lipolysis, to LDs, which results in TAG overstorage in cells (5, 6). How COPI mediates its effect on the targeting of LD proteins Author contributions: A.R.T., B.A., F.W., T.C.W., R.B., J.E.R., and F.P. designed research; is unknown, but evidence from proteomic and microscopy ex- A.R.T., B.A., J.W., J.D., and F.P. performed research;A.R.T.,B.A.,and F.P. analyzed data; periments suggests COPI might act directly on LDs (4–6, 13–15). and A.R.T., B.A., and F.P. wrote the paper. Interaction of COPI with a monolayer membrane has never been The authors declare no conflict of interest. shown. Here we demonstrate that COPI machinery directly This article is a PNAS Direct Submission. assembles at the TAG surface and propose a simple mechanism 1To whom correspondence may be addressed. E-mail: [email protected] or pincet@ by which this machinery may regulate protein targeting to LDs. lps.ens.fr. We show that Arf1 and COPI can associate directly with the mono- This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. layer of an artificial mother TAG LD and that this association 1073/pnas.1307685110/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1307685110 PNAS Early Edition | 1of6 Downloaded by guest on September 25, 2021 spots can clearly be identified as nano LDs with coat polymer visible at their surface (Fig. 3A, white arrows and Inset). The coat polymer disappears after treatment with ArfGAP3 (Fig. 3B). The size distribution of these nanodroplets shows that they are mono- disperse with a typical diameter of 60 ± 15 nm (Fig. 3C). This distribution is consistent with the estimated size obtained from the diffusion coefficients measured by fluorescence cross-correlation spectroscopy (∼90 nm, Fig. S2A) and by independent direct tracking of the particles collected from the buffer drops (∼100 nm, Fig. S2B). Taken together, these results show that the COPI machinery is able to function on LDs in the same manner as on lipid bilayers by inducing the budding and fission of 60-nm TAG nanodroplets very close to the size of COPI vesicles (2, 3).

COPI Budding Exclusively Occurs at Interfaces with Low Tension. Because natural LDs undergo shrinking and growth phases, we decided to probe the effect of the state of the LD surface on the efficiency of the COPI-induced budding process. We performed microfluidic experiments with increasing amounts of PL (Fig. S3). As shown in Fig. 4A, the number of COPI-induced nanodroplets formed in the buffer drops dramatically increases between 0.1% and 1% PL per TAG (wt/wt), suggesting that the COPI–coat machinery acts preferentially on a packed PL monolayer. When covering the interface, PLs, thanks to their amphiphilic nature, decrease the surface tension by shielding TAG molecules from the aqueous buffer. Because an interface with a low surface tension is more deformable, a packed PL monolayer should facilitate the budding of COPI nano LDs (20). Following this hypothesis, we used a micromanipulation ap- proach (Fig. S4) to measure the surface tension of LDs at various PL concentrations. Strikingly, the surface tension decreased sharply from ∼20 mN/m to a vanishing surface tension (below 0.5 mN/m, the detection limit of the technique) exactly in the range of PL Fig. 1. GTP-specific binding of Arf1 to LDs. (A) Arf1 binds LDs or liposomes – fi concentration (0.1 1% wt/wt PL/TAG) at which COPI reaches with the same ef ciency and in a GTP-dependent manner. Extruded liposomes its optimum budding efficiency (Fig. 4A). The COPI machinery and TAG droplets containing the same amount of exposed PL (0.5 mM) were incubated during 30 min with Arf1–GDP (500 nM) and, when indicated, with acts mainly at low surface tension, below a threshold of 2 mN/m. EDTA (2 mM) and GTP (100 μM) to promote activation of Arf1. After separation of the free proteins from the liposomes or TAG droplets in a sucrose gradient, Budding Nanodroplets Increases the Surface Tension. The main re- the amount of Arf1 bound to the membrane was revealed by SDS-PAGE gel sult of nanodroplet formation should be a decrease in PL stained with SYPRO Orange staining. The amount of Arf1 bound to liposomes packing on the mother LD as nanodroplets “capture” relatively and TAG droplets is similar (lanes 2 and 4). No binding is observed in the ab- more surface than volume from the mother LD (Fig. S5). Put sence of GTP (lanes 1 and 3). Lane 5 represents the amount of Arf1 input in the differently, nanodroplet budding consumes the PL monolayer solutions. (B)Specific binding of Arf1 to TAG/buffer interface in buffer drops covering the surface of the mother LD, inducing a decrease in fl fl watched in epi uorescence imaging. In a micro uidics channel (Upper), the PL/TAG ratio. Energetically, this consumption will increase micrometric buffer droplets are produced in a stream of oil containing the phospholipids. Consecutive buffer drops containing fluorescent Arf1–Cy3 (30 the surface tension of the mother LD. With a Langmuir trough nM), ARNO (200 nM), and alternately GTP (50 μM) or nucleotide-free control we measured the variation of PL packing with the surface tension are produced with this setup. Arf1 only labels the aqueous/TAG interface when (and therefore PL/TAG ratio, Fig. S6). Remarkably, a 10% de- GTP is present (center buffer drop). Limited labeling of the contour is observed crease in the PL packing from the maximum pressure (corre- in the control drops (outer buffer drops). The Arf1–Cy3 signal looks lower at the sponding to maximum lipid packing for which the surface tension interface between buffer drops because they are adherent and the interface is is below the detection, 0.5 mN/m) is sufficient to raise the surface not vertical, which lowers the integrated intensity. (Scale bar, 50 μm.) tension above the 2 mN/m threshold for COPI budding of nanodroplets. A 10% decrease in the PL packing from the maximum density is achieved by budding off approximately five Instead, Arf1 and coatomer formed mobile spots in the aqueous nanodroplets from a 500-nm mother LD (Fig. S7), a typical size volume and at the buffer/TAG interface (Fig. 2A and Movie S1) B of physiological LDs. Hence, a very limited action of COPI is in a GTP-dependent manner (Fig. 2 ). To determine the con- sufficient to induce a substantial change in surface tension. tent of these spots, we performed the experiment with unlabeled Arf1, labeled coatomer, and oil containing a fluorescent dye LDs with High Surface Tension Can Sense Their Environment. Upon (Bodipy). Again, colocalized spots were observed (Fig. 2C), surface tension increase induced by COPI, a mother LD will suggesting a budding process in which small oil droplets are probably become more prone to react with its environment (e.g., detached from the buffer/TAG interface by the COPI coat. After with soluble proteins or membranes). First, we used α-synuclein addition of ArfGAP3, which promotes GTP hydrolysis of Arf1 as a model of soluble proteins, because it is known to bind and reverses the coating process (19), the coat dissociated from natural LDs (21) and is able to sense phospholipid packing on these spot particles (Fig. 2D). bilayers (22). We found that binding of α-synuclein to LDs is highly dependent on the PL packing at the interface. When the Budded Particles Are 60-nm COPI-Coated LDs. To determine the monolayer is not fully packed α-synuclein binds to the LD sur- characteristics of the newly formed particles, we isolated them face, whereas at full PL coverage no binding occurs (Fig. 4B). from the buffer drops (Fig. S1) and observed them by EM. The Second, we tested the ability of LDs of various PL compositions

2of6 | www.pnas.org/cgi/doi/10.1073/pnas.1307685110 Thiam et al. Downloaded by guest on September 25, 2021 Fig. 2. COPI produces nanodroplets from LDs. (A) Particles containing Arf1 and coatomer appear in the buffer drops in the presence of the COPI machinery (Left is a full image of a buffer drop; the other three panels are large magnifications to better see the particles). Less than 2 min after making the buffer drops containing Arf1–Cy3 (30 nM), coatomer (15 nM) labeled with Alexa 647, GTP (50 μM), and ARNO (200 nM), homogenous Arf1–Cy3 and coat–Alexa 647 spots appear in the aqueous volume and at the buffer/TAG interface. Arf1 (green) and coat (red) spots are colocalized, moving together in the buffer drop (Movie S1). The spots are slightly separated because of the time delay to switch laser in the setup. (Scale bar, 5 μm.) (B) The formation of particles is GTP-dependent. In the controls without GTP, coatomer or Arf1, the amount of spots per area is significantly reduced compared with the experiment with 50 μM GTP, 30 nM Arf1, and 15 nM coatomer (Left). (C) The particles are TAG nanodroplets. Same experiment as in A with unlabeled Arf1 (100 nM) and Bodipy dye (1% wt/wt) in the μ TAG. After collection of the buffer drops as indicated in Fig. S1, colocalized Bodipy/Alexa 647 spots are observed. (Scale bars, 10 m.) (D) Loss of colocalization SCIENCES over time after ArfGAP3 addition. The sample, recovered as shown in Fig. S1, is split in three vials. The amount of particles is quantified as described in Fig. S3. ArfGAP3 is added in two of the samples at different concentrations (50 and 10 nM) corresponding to fractions equal to 0.5 and 0.1 of the Arf1 concentration. APPLIED PHYSICAL Colocalization of coat–Alexa 647 and TAG–Bodipy is lost over time compared with the control.

to fuse with neighboring bilayer membranes by incubating them Hyperreactivity of LDs after COPI action could have several with model membranes [giant unilamellar vesicles (GUVs), Fig. physiological implications. Cytosolic proteins may directly bind 4C, or tensionless micrometer-scale membranes resembling or- to the mother LD surface. Alternatively, LDs might fuse with

ganelles with various curvatures, Fig. 4D]. At vanishing surface surrounding organelles, such as other LDs or bilayer membranes. BIOCHEMISTRY tension [PL/TAG ratio larger than 1% (wt/wt)], LDs remained This mechanism may explain the COPI dependence of targeting intact and did not interact with any of the bilayer membranes. In ATGL-related LD proteins (Fig. S8). For instance, COPI- contrast, at nonzero surface tension [PL/TAG ratio below 0.3% induced budding of nanodroplets and the resulting decrease in (wt/wt)], the LDs quickly got imbedded in the GUV membrane PL packing might lead to the formation of connections between to form visible lenses between the leaflets of the bilayer (Fig. 4C LD and other bilayer membranes, typically endoplasmic reticulum, and Movie S2) or fuse with the tensionless membranes that through which proteins can exchange. These connections have D quickly form a monolayer around them (Fig. 4 and Movie S3). been suggested in various systems by fluorescence recovery after This demonstrates that LDs with lower PL surface packing in- photobleaching and EM experiments (10, 23, 24). Such a mech- duced, for instance, by COPI will become hyperreactive and will anism would probably entail a regulation of the action of COPI interact with neighboring membranes or amphiphilic molecules, so that it is active only whenever the targeting of a specific leading to a remodeling of the composition of the LD surface enzyme is required. through the relocation of new molecules. Materials and Methods Discussion Proteins. Fluorescently labeled Arf1 was generated by using an Arf1 variant, The fact that COPI machinery is able to work on monolayers and where the single cysteine residue of Arf1 was replaced with serine, and the bud oil droplets is a unique and apparently an intrinsic capability C-terminal lysine was replaced with cysteine (Arf1-C159S-K181C) (25). Human of the machinery. Because of the specificity of a monolayer Arf1-C159S-K181C and yeast N-myristoyltransferase were coexpressed in compared with a bilayer, we propose that the COPI machinery Escherichia coli supplied with BSA-loaded myristate. Cell lysates were sub- performs a previously unrecognized function, clamping the sur- jected to 35% (vol/vol) ammonium sulfate, and the precipitate, enriched in face tension of the monolayer at a buffer/oil interface by pre- myristoylated Arf1, was further purified by DEAE-ion exchange. Eluted frac- venting it from dropping down to low tensions, below 2 mN/m. tions of interest were concentrated in spin-column filters with a 10-kDa cutoff At the molecular level, this action of COPI prevents the LD (Millipore) and fluorescently labeled using Cy3-maleimide (GE Healthcare) ’ surface from being fully covered by PLs and provides more ac- according to the manufacturer s protocol. To remove excess dye, samples fi fi cessibility to TAG for binding/reacting with other components. were puri ed by gel ltration using a Superdex 75 column (GE Healthcare). Recombinant coatomer protein was expressed and purified as described Consistent with the specificity of LD phenotypes associated with (26). Sf9 insect cells were infected with baculovirus encoding for heptameric depletion of COPI subunits (4, 6), this is likely the only protein coatomer. Coatomer complexes were isolated from the soluble protein coat that can perform this function: COPII depends on a trans- fraction by nickel-affinity purification, concentrated in spin-column filters membrane guanine nucleotide exchange factor, Sec12, whereas with a 250-kDa cutoff (Millipore), and fluorescently labeled using Alexa- ArfGEFs are peripheral proteins that might directly bind to LDs; Fluor-647-NHS (Molecular Probes) according to the manufacturer’s protocol. clathrin coats assemble on membranes rich in anionic lipids, Excess imidazole and dye was removed by gel filtration using a Superose whereas the surface of LDs is very poor in such lipids (18). 6 column (GE Healthcare).

Thiam et al. PNAS Early Edition | 3of6 Downloaded by guest on September 25, 2021 Because of the wetting properties of the oil to the T connector and the tube, and the ratio of the flow rates (oil rate/buffer rate = 5), 250-μm buffer droplets are generated at the outlet of the T connector (28) and circulate in a transparent Teflon tube where observations were made. The time of reaction represents the time spent by each microreator in the tube; the length of the tube and the flow rate controls it. The flow rate was 1,250 μL/h and the length of the tube was 2 m. The diameter of the tube was 250 μm, so the reaction time was ∼15 min.

GUVs and Micrometer-Scale LDs. GUVs were prepared using an electro- formation technique (29). One microliter of PL mixture in chloroform at 0.3 mM was dried on an indium tin oxide (ITO)-coated glass plate. The lipid film was desiccated for 1 h. The chamber was sealed with another ITO- coated glass plate. The lipids were then rehydrated with a sucrose solution (300 mOsm). The alternative (8 Hz) voltage between the two glass plates was increased by steps every 6 min: 100 mV, 200 mV, 300 mV, 500 mV, 700 mV, 900 mV, and 1.1 V. The last voltage was maintained for at least 1 h. GUVs were either stored in the chamber at 4 °C overnight or directly collected with a Pasteur pipette. The LDs used in experiments with GUVs (Fig. 4C) were prepared by first drying PLs and solubilizing them afterward into TAG to obtain the required PL/TAG ratio. A mixture of 5 μL of this PL/TAG solution and 95 μL buffer was first vortexed and then sonicated using a Branson 2510 sonicator working at 40 kHz for 20 s. The diameter of the resulting droplets is a few hundred nanometers. GUVs and generated LDs were incubated together in buffer for10 min under gentle shaking and subsequently observed under an optical microscope (Fig. 4C is an example of a GUV incubated with LDs at a 0.3% PL/TAG ratio).

LDs and Membranes Mimicking Organelles. To obtain membranes resembling organelles in terms of shape (curvatures) and tension (low tension), 5 μLPL Fig. 3. COPI-induced nanodroplets are 60 nm in diameter. (A) TAG nano- lipid in chloroform (3 mM) was dried on a coverslip and placed in a desic- droplets with COPI coat observed by negative staining EM. Particles were cator for 1 h. The lipid film was rehydrated in 20 μL of buffer for 10 min. The extracted from buffer drops containing unlabeled Arf1 (100 nM), coatomer solution was collected and injected into a Petri dish. molecules (15 nM), GTP (50 μM), and ARNO (200 nM) in a stream of TAG. To prepare giant LDs, PL mixtures were dried and dissolved into TAG at Budded TAG nanodroplets can be identified because they are surrounded by different concentrations (wt/wt, typically from 0.2 to 5%). Then 5 μL of the oil a layer of dark coats (COPI, white arrows). Two different fields are shown. solution was added to 95 μL of buffer and the mixture was vortexed 10 s (Inset) Magnification of one budded nanodroplet surrounded by a structure using a Fischer Vortex Genie 2 at maximum power. shaped as a coat assembly. Large TAG drops (Left) that are sometimes During the experiment, the LD and the membrane were manipulated and extracted during the process (probably by shear) are not surrounded by brought into contact through two pipettes via aspiration and observed under a coat. (Scale bar, 100 nm.) (B) The sample recovered under the same con- a microscope (bright field and fluorescence). Note that LDs were injected last ditions as in A was treated with a large amount of ArfGAP3 (ArfGAP3/Arf1 = into the Petri dish because they tend to float at the buffer/air interface, where 1) for 10 min. Sixty-nanometer TAG nanodroplets can be identified but are they often spread. no longer surrounded by a layer of coat as in A.(Inset) Magnification view of a droplet without coat. (Scale bar, 100 nm.) (C) Size distribution of 278 μ μ nanodroplets from 20 EM images similar to that in A. The average lipid Flotation Assay. Liposomes (120 L, 1 mM phospholipid) or LDs (120 L, 0.5 mM nanodroplet size measured by EM is 60 ± 15 nm. phospholipid; TAG/buffer 7/93 vol/vol) were mixed with myristoylated Arf1GDP (0.5 μM) in a final volume of 125 μL. When indicated, the suspension was supplemented with 100 μM GTP and 2 mM EDTA to promote GDP to GTP exchange on Arf1. After incubation for 30 min at room temperature, PL Mixture Composition. We chose a lipid composition close to that of natural the sample was adjusted to 30% (wt/vol) sucrose and covered with two LDs (18): dioleoylphsphatidylcholine (DOPC):dioleoylphsphatidylethanolamine cushions of 25% (wt/vol) sucrose (200 μL) and 0% sucrose (50 μL), respectively. (DOPE):cholesterol:lyso-phosphatidylinositol:lyso-phosphatidylethanolamine:lyso- The samples were centrifuged for 80 min at 30,000 rpm in a SW60 rotor phosphatidylcholine (50:20:12:10:5:3). (Beckman). The top (100 μL), medium (200 μL), and bottom (250 μL) fractions Unless specified, PL/TAG is fixed at 0.5% (wt/wt). were collected and analyzed by SDS/PAGE using SYPRO Orange staining.

Buffer. Unless otherwise indicated, experiments were performed in HKM EM. Nano LDs were collected as indicated in Fig. S1. Samples of 5 μL were buffer: 50 mM Hepes, 120 mM Kacetate, and 1 mM MgCl (in Milli-Q water). 2 absorbed to continuous carbon-coated grids (glow discharged) at room temperature for 1 min, rinsed briefly with HKM buffer, and stained with 1% Preparation of Synthetic Liposomes and Droplets for Flotation Experiments. (vol/vol) uranyl acetate for 20 s. Negatively stained samples were imaged μ For synthetic liposomes, a chloroform solution containing 1 mol egg PC under low-dose conditions in an FEI Tecnai12 microscope (120 kV). Micro- and 1.6 nmol Rhodamine-PE was dried under argon gas in a glass tube. The graphs were collected at 26,000× magnification, giving an unbinned pixel fi fi lipid lm was resuspended in 1 mL HKM buffer. After ve cycles of freezing size of 4.2 Å. The diameters of nano LDs were manually measured directly and thawing, the liposome suspension was extruded 19 times through from the micrographs. a 0.4-μm polycarbonate filter. μ μ For synthetic droplets, 70 L TAG was mixed in a glass tube with 0.5 mol Fluorescence Cross-Correlation Spectroscopy. Oil was labeled with 1% vol/vol egg PC and 1.6 nmol Rhodamine-PE from stock solutions in chloroform. The Bodipy (excited at 488 nm). Coatomer was labeled with Alexa 647 (excited at solvent was removed using a stream of argon gas and then 0.93 mL of HKM 632 nm). The product of reaction was recovered as described in Fig. S1 and buffer was added to the TAG/PL mixture. An emulsion was obtained by vortex analyzed with the FCS setup, Confocor2, on the Zeiss microscope LSM510. and extruded nine times through a 1-μm polycarbonate filter. After extrusion, The emission and excitation spectra of the two dyes are separated enough the emulsion becomes extremely turbid. Examination of the suspension under to not cause any cross-talk or FRET. For each channel, autocorrelation curves a microscope shows micrometer-size LDs that were stable for many hours. were recorded simultaneously within 30 s for many runs of different samples (representing 10 different experiments). Similarly, a cross-correlation curve Buffer Drops Preparation. Two syringes were filled, one with oil and the other was simultaneously generated in a third channel. For the cross-correlated with buffer and proteins. Using a syringe pump, streams from both syringes signals, we fit the far-red and green autocorrelation curves, G(τ), with a were allowed to flow into a high-pressure T connector with a 250-μmin- theoretical model comprising three components, giving, therefore, three side diameter constructed of fluorinated ethylene propylene (27) (Fig. S1). diffusion times (30). The choice of such a number of fitting parameters is

4of6 | www.pnas.org/cgi/doi/10.1073/pnas.1307685110 Thiam et al. Downloaded by guest on September 25, 2021 Fig. 4. Consequence of the tension clamp model: LDs become hyperreactive. (A) Relative number of nanodroplets and surface tension for LDs with various PL concentrations. After separation from buffer drops, the nanodroplets were counted for various PL/TAG ratios (Fig. S3). The result was normalized to the maximum value at PL/TAG = 2% (left y axis). The surface tensions of the LDs were also measured by micropipette aspiration (right y axis, Fig. S5). COPI efficiency is optimal at vanishing surface tension (fully packed phospholipid monolayer) and very limited when the surface tension increases over a few millinewtons per meter (low PL packing). (B)Usingthemicrofluidic setup developed in Fig. S1, we form buffer drops containing only GFP-labeled α-synuclein (11 nM). (Left) the oil contains 0.02% α < (wt/wt) PLs or (Right)0.2%PLs. -Synuclein binds to the interface for lower PL concentration ( 0.03), whereas its binding is abolished for higher PL concentration. SCIENCES (C) Micrometer-scale LDs having low phospholipid packing fuse with bilayer membranes. Optical microscopy picture of LDs incubated with a GUV at PL/TAG ratios below 0.3% shows that LDs efficiently fuse with GUVs and are trapped between the two leaflets of the bilayer (Movie S2). The GUV presented here is 35 μmin APPLIED PHYSICAL diameter. When the PL/TAG ratios are larger than 1%, LDs do not fuse with the GUV membrane. (D)Agiantartificial LD (30 μm in diameter, Left)withaPL/TAG ratio close to 0.3% and a fluorescent membrane structure mimicking a neighboring organelle (various curvatures, low surface tension) are brought into contact. Before contact, the LD was not fluorescent. As soon as the membrane touches the LD, they fuse and the membrane spreads around the LD surface (Right), demonstrating the hyperreactivity of the LD. (Insets) The giant LD with an increased contrast to better see the fluorescence. This process is shown in Movie S3.LDs with a PL/TAG ratio larger than 1% did not fuse with the membrane and both entities remained intact after contact for 5 min (Movie S4). (Scale bar, 20 μm.)

driven by the fact we may have signals from the free dye, the budded by image analysis (ImageJ). The suction was carried out using a syringe. The BIOCHEMISTRY particles, and probable aggregates or just large, polluting particles. resulting pressure was measured with a pressure transducer (DP103; Val- idyne Engineering Corp.), the output voltage being monitored with a digital 8 0 11 0 11 0 11 9 <> 2 2 2 => voltmeter. The pressure transducer (range 55 kPa) was calibrated before 1 1 − x − yB 1 C x B 1 C y B 1 C ðτÞ = + @ A + @ A + @ A experiments. G 1 > + τ=τ τ + τ=τ τ + τ=τ τ > N : 1 1 1 + 1 2 1 + 1 3 1 + ; 25τ1 τ2 τ3 Compression Isotherm of PL Monolayer at the TAG/Buffer Interface. The lipid The fraction of each particle is given by (x,y)andN yields the total con- mixture isotherm was carried out using a Teﬂon Langmuir trough (Minimicro;

centration of particles. The diffusion times are τi. For each fitted curve, the KSV) equipped with hydrophilic barriers made of polyoxymethylene (Derlin). access of τi determines the size of the particles by the Stoke–Einstein law, The Whilhelmy pressure sensor (KSV) was coupled with a paper plate. Dimensions of the trough were 165 × 51 mm. Room temperature during the k T radius = B ; experiments was 20.5 ± 0.5 °C. The trough took place on an antivibration 6πη ω2=τ 2 i table in a closed box containing a water-saturated atmosphere to prevent evaporation. Lipid mixture was spread on HKM buffer before gently de- where kB stands for the Boltzmann constant, T the temperature (room positing droplets of TAG solution in chloroform (1% vol/vol). The amount of temperature), η the viscosity of the buffer (1 cSt), and ω the width of the 2 μ fi focal volume (115 nm at 488 excitation and 155 nm at 647 excitation). deposited TAG (0.12 L) was at least vefold higher than the amount of TAG required for the interface to be saturated. The so formed mixed TAG–lipids monolayer, at the interface between the very thin TAG film and HKM sub- Measurement of the Interfacial Tension of LDs with Micropipettes. Phospholi- pids were mixed at the ratio 70% DOPC: 30% DOPE (mol/mol), dried under vacuum phase, relaxed during 5 h before compression. The compression rate was 2· –1· –1 for 1 h, and resuspended in TAG. Ten microliters of this solution was vortexed with held constant (0.5Å min mol ). The pressure was measured with an ac- 200 μL HKM buffer for 30 s. Then 50 μL of this emulsion was injected into a 1-mL curacy of 0.5 mN/m and the molecular area was controlled with 5% accuracy. 2 HKM buffer drop deposited on a coverslip and observed with optical microscopy. The monolayer collapse was observed for 48Å /mol molecular area and 36.6 mN/m pressure values. The pressure owing to pure oil spreading was de- The interfacial tension (IT) of the droplets was measured using a micromanipulation technique. The device was made up of a micromanipulator and termined to be 15.2 mN/m. We carried out independent measurements of – a pipette holder (Narishige). Pipettes were incubated in a 5% (wt/vol) BSA the surface pressure as a function of lipid mixture spread at the TAG HKM fl before use to prevent droplets from adhering to the glass. As shown in Fig. S4, interface formed in a 50-mm-diameter vessel (glass and Te on). Even if micromanipulation of a single droplet enables the IT to be determined similar observations have previously been reported in the literature (32), it is through measurement of pipette diameter, droplet diameters, and minimal worth noting that the results were consistent within the experimental errors pressure at which the droplet was drawn into the pipette (31): with those obtained with the trough regardless of the size of the oil res- ervoir (from oil monolayer up to macroscopic amounts of oil). P IT = suc ; 2 1= − 1= ACKNOWLEDGMENTS. WethankT.Melia,C.Burd,andJ.Bibettefor Rp Rd many helpful discussions. This work was supported by a grant from the Marie Curie Budding and Fusion of Lipid Droplets International Outgoing where Psuc, Rp, and Rd represent the suction pressure, pipette radius, and Fellowship within the Seventh European Community Framework Pro- droplet radius, respectively. The sizes of pipette and droplet were obtained gram (to A.R.T.), a Partner University Funds exchange grant between

Thiam et al. PNAS Early Edition | 5of6 Downloaded by guest on September 25, 2021 the Yale University and Ecole Normale Supérieure laboratories, European Grant R01GM097194 (to T.C.W.). F.W. is a fellow of the Boehringer Research Council Grant 268888 (to B.A.) and National Institutes of Health Ingelheim Fonds.

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