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

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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 nano- droplets formed in the buffer drops dramatically increases be- tween 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.
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