Abdou Rachid Thiam
email: thiam@lps.ens.fr

Lionel Forêt

The Physics of Lipid Droplet Nucleation, Growth and Budding

Lipid droplets (LDs) are intracellular oil-in-water emulsion droplets, covered by a phospholipid monolayer and mainly present in the cytosol. Despite their important role in cellular metabolism and growing number of newly identified functions, LD formation mechanism from the endoplasmic reticulum remains poorly understood. To form a LD, the oil molecules synthesized in the ER accumulate between the monolayer leaflets and induce deformation of the membrane. This formation process works through three steps: nucleation, growth and budding, exactly as in phase separation and dewetting phenomena. These steps involve sequential biophysical membrane remodeling mechanisms for which we present basic tools of statistical physics, membrane biophysics, and soft matter science underlying them. We aim to highlight relevant factors that could control LD formation size, site and number through this physics description. An emphasis will be given to a currently underestimated contribution of the molecular interactions between lipids to favor an energetically costless mechanism of LD formation.

Highlights

 Lipid droplet biogenesis occurs in three steps: nucleation, growth and budding  Phase separation and dewetting phenomena are determinant for lipid droplet formation  Interactions between lipids allow for spontaneous lipid droplet nucleation and budding

I Introduction

Lipid droplets (LDs) are dynamic organelles found in almost all organisms. They have an oil core, consisting of triglycerides (TG) and sterol esters (SE), surrounded by a phospholipid (PL) monolayer containing proteins [START_REF] Walther | Lipid droplets and cellular lipid metabolism[END_REF][START_REF] Martin | Lipid droplets: a unified view of a dynamic organelle[END_REF][START_REF] Thiele | Cell biology of lipid droplets[END_REF]. From the physical chemistry standpoint, LDs represent an inverted emulsion, meaning a solution of oil-in-water droplets [START_REF] Thiam | The Biophysics and Cell Biology of Lipid Droplets[END_REF]. Despite an important role in energy metabolism and cellular homeostasis [START_REF] Walther | Lipid droplets and cellular lipid metabolism[END_REF], and an expanding list of newly identified functions [START_REF] Welte | Expanding Roles for Lipid Droplets[END_REF], the mechanism of LD formation remains largely unknown [START_REF] Thiele | Cell biology of lipid droplets[END_REF][START_REF] Wilfling | Lipid droplet biogenesis[END_REF][START_REF] Pol | Review: biogenesis of the multifunctional lipid droplet: lipids, proteins, and sites[END_REF][START_REF] Ohsaki | Biogenesis of cytoplasmic lipid droplets: from the lipid ester globule in the membrane to the visible structure[END_REF][START_REF] Brasaemle | Packaging of fat: an evolving model of lipid droplet assembly and expansion[END_REF][START_REF] Sturley | Lipid droplet formation on opposing sides of the endoplasmic reticulum[END_REF]. Under nutrient rich conditions, excess metabolites are transformed mainly into TG and SE molecules, depending on

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ACCEPTED MANUSCRIPT 2 cell type, and encapsulated between the endoplasmic reticulum (ER) monolayer leaflets [START_REF] Pol | Review: biogenesis of the multifunctional lipid droplet: lipids, proteins, and sites[END_REF][START_REF] Brasaemle | Packaging of fat: an evolving model of lipid droplet assembly and expansion[END_REF] (for convenience, we will refer to TG as the group of oil molecules forming LDs). TG molecules then gather in the ER bilayer to form an oil lens that ultimately buds off, mainly towards the cytoplasmic side. The mechanisms of nascent LD advent and growth, and its directional budding preference, are still unclear [START_REF] Pol | Review: biogenesis of the multifunctional lipid droplet: lipids, proteins, and sites[END_REF][START_REF] Ohsaki | Biogenesis of cytoplasmic lipid droplets: from the lipid ester globule in the membrane to the visible structure[END_REF]. Proteins certainly affect these different steps of LD formation, but the lipid chemistry, PLs and oil molecules, play an important role that is still not resolved [START_REF] Thiam | The Biophysics and Cell Biology of Lipid Droplets[END_REF][START_REF] Yang | Controlling the size of lipid droplets: lipid and protein factors[END_REF][START_REF] Penno | Phospholipids and lipid droplets[END_REF]. The relative contribution of proteins and lipids and their coordination for LD budding are also not fully understood [START_REF] Pol | Review: biogenesis of the multifunctional lipid droplet: lipids, proteins, and sites[END_REF]. This review presents a thermodynamics and mechanics view of LD formation [START_REF] Mcmahon | Membrane curvature and mechanisms of dynamic cell membrane remodelling[END_REF][START_REF] Hurley | Membrane budding[END_REF][START_REF] De Gennes | Capillarity and wetting phenomena: drops, bubbles, pearls, waves[END_REF], essentially based on statistical and soft matter physics. This physics standpoint offers a better understanding of LD nucleation, growth, and budding, and protein recruitment to forming LDs, and finally of potential different LD formation mechanisms in the cell.

The following steps of LD biogenesis will be discussed and analyzed: (i) nucleation, i.e. the initial accumulation of oil molecules at places in the ER membrane where they form nascent LDs, (ii) growth, i.e. size expansion, not due to synthesized TG absorption, but by ripening and fusion, and (iii) budding, i.e. the deformation of the ER monolayers leading to the appearance of a nearly spherical oil-filled protrusion. We will emphasize the role in these steps of the molecular interactions between the oil molecules and PLs constituting the ER, a contribution for LD formation that has not yet been considered.

II Nucleation and formation sites of LDs

Individual TG molecules made in the ER by synthesis enzymes [START_REF] Cases | Identification of a gene encoding an acyl CoA : diacylglycerol acyltransferase, a key enzyme in triacylglycerol synthesis[END_REF][START_REF] Coleman | Enzymes of triacylglycerol synthesis and their regulation[END_REF][START_REF] Yen | DGAT enzymes and triacylglycerol biosynthesis[END_REF][START_REF] Turkish | Regulation of triglyceride metabolism. I. Eukaryotic neutral lipid synthesis: "Many ways to skin ACAT or a DGAT[END_REF][START_REF] Buhman | The enzymes of neutral lipid synthesis[END_REF] are encapsulated between the ER monolayers. The first step in the LD formation process is the accumulation of the molecules to form an oil lens in the bilayer [START_REF] Martin | Lipid droplets: a unified view of a dynamic organelle[END_REF][START_REF] Wilfling | Lipid droplet biogenesis[END_REF][START_REF] Pol | Review: biogenesis of the multifunctional lipid droplet: lipids, proteins, and sites[END_REF], Fig. 1. What is the mechanism responsible for the oil lens nucleation or nascent LD appearance? Where do the oil lenses form and how are they regulated?

These are important questions for LD biology [START_REF] Fujimoto | Not just fat: the structure and function of the lipid droplet[END_REF] which can be better addressed by understanding the physics of phase separation and nucleation phenomena.

1) Nucleation by phase separation

The initial nucleation of a TG lens between the two monolayers of the ER membrane could result from a simple phase separation or demixing, a well understood phenomenon in physical chemistry [START_REF] Barrat | Basic concepts for simple and complex liquids[END_REF]. Individual TG molecules dispersed in a PL bilayer can condense to form droplets in order to globally reduce their interaction with PLs or proteins of the membrane, Fig. 1, exactly in the same way as oil or gas molecules in water condense into droplets or bubbles (e.g. in sparkling

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water), to minimize the contact between foreign molecules [START_REF] Barrat | Basic concepts for simple and complex liquids[END_REF]. The (free) energy cost of having a TG molecule in the bilayer instead of in a forming lens is [START_REF] Walther | Lipid droplets and cellular lipid metabolism[END_REF] where is the energy cost for the interaction of a free TG molecule with other ER components (e.g. PLs), is the interaction energy between TG molecules in the lens, and the last term of [START_REF] Walther | Lipid droplets and cellular lipid metabolism[END_REF] arises from the mixing entropy, i.e. the propensity of entropy to disperse TG in the bilayer, with the concentration of free TGs in the bilayer, and the thermal energy. Spontaneous demixing of TG molecules to form nascent LDs occurs if the energy cost for dispersing TG in the bilayer is positive, i.e.

. This condition requires that , which means that the energy cost for the interaction between a TG and a PL molecule is larger than the one between two TG molecules;

this is in general the case, as the mixing of different molecules is often energetically costly. Only when this condition is fulfilled, demixing occurs if the TG concentration in the bilayer exceeds a critical value . The existence of a critical demixing concentration (CDC = ) is common to all phase-separating systems -in the context of detergency it is called the Critical Micellar Concentration (CMC) [START_REF] Chandler | Interfaces and the driving force of hydrophobic assembly[END_REF][START_REF] Safran | Statistical thermodynamics of surfaces, interfaces, and membranes[END_REF]. A TG demixing phenomenon based on energy minimization is supported by in silico studies showing the spontaneous appearance of TG blisters in a bilayer of POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine) above a CDC of around 3% to the POPC concentration [START_REF] Khandelia | Triglyceride blisters in lipid bilayers: implications for lipid droplet biogenesis and the mobile lipid signal in cancer cell membranes[END_REF].

In the demixing scenario, the interaction of PLs with TG is crucial (table 1) for the packaging of TG into nascent LDs [START_REF] Yang | Controlling the size of lipid droplets: lipid and protein factors[END_REF][START_REF] Penno | Phospholipids and lipid droplets[END_REF][START_REF] Hsieh | Perilipin family members preferentially sequester to either triacylglycerol-specific or cholesteryl-ester-specific intracellular lipid storage droplets[END_REF][START_REF] Zanghellini | Phospholipid demixing and the birth of a lipid droplet[END_REF]. For example, by inducing squalene accumulation instead of TG, demixing could be inhibited to an extent that LD formation is impeded [START_REF] Spanova | Effect of lipid particle biogenesis on the subcellular distribution of squalene in the yeast Saccharomyces cerevisiae[END_REF]; SE and TG could also demix differently, potentially explaining the packaging of SE and TG into different LDs within the same cell [START_REF] Hsieh | Perilipin family members preferentially sequester to either triacylglycerol-specific or cholesteryl-ester-specific intracellular lipid storage droplets[END_REF]. Inversely, changing the PL composition could change the CDC to inhibit demixing and nucleation. Such contribution of PLs is consistent with the systematic variation of LD size and number upon PL modulation [START_REF] Thiam | The Biophysics and Cell Biology of Lipid Droplets[END_REF][START_REF] Yang | Controlling the size of lipid droplets: lipid and protein factors[END_REF][START_REF] Penno | Phospholipids and lipid droplets[END_REF][START_REF] Guo | Functional genomic screen reveals genes involved in lipid-droplet formation and utilization[END_REF][START_REF] Cohen | Regulation of Lipid Droplet Size in Mammary Epithelial Cells by Remodeling of Membrane Lipid Composition-A Potential Mechanism[END_REF][START_REF] Boutet | Seipin deficiency alters fatty acid Delta 9 desaturation and lipid droplet formation in Berardinelli-Seip congenital lipodystrophy[END_REF].

2) Nucleation sites

Assuming TG demixing induces LD nucleation, basic features of nucleation phenomena presented in box 1 should also describe LD nucleation. After synthesis, individual TG molecules freely diffuse in the ER bilayer, randomly meet and assemble to form transient pre-droplet structures. Most of these structures will be short-lived (a priori much less than a millisecond [START_REF] Khandelia | Triglyceride blisters in lipid bilayers: implications for lipid droplet biogenesis and the mobile lipid signal in cancer cell membranes[END_REF]) and only a very small fraction of them will cross the nucleation barrier to form stable TG lenses (nascent LDs). The

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nucleation rate of the lenses depends on the height of the nucleation barrier [START_REF] Chandler | Interfaces and the driving force of hydrophobic assembly[END_REF][START_REF] Safran | Statistical thermodynamics of surfaces, interfaces, and membranes[END_REF]; it is determined by the relative importance of the physical interactions listed in table 1, in particular the molecular interactions between PL and TG, which are a priori predominant.

On a uniform bilayer, LD nucleation would occur at random places with no specific sites. The lower the nucleation barrier, the higher the number of nucleated LDs. Yet, because of the complex morphology of the ER and the possible heterogeneities of its biochemical composition, favored sites where nucleation is energetically cheaper may exist, as will be discussed in the next section and in Fig. 2. Indeed, owing to the exponential dependency of the nucleation barrier rate with the energy barrier, box 1, the probability to nucleate LDs is considerably increased at ER sites where the nucleation energy is lowered, even slightly. In Cos-1 cells for example, these favorite sites could coincide with the ER tubular network at the cell periphery where the first generation of LDs appears there upon oleate loading [START_REF] Kassan | Acyl-CoA synthetase 3 promotes lipid droplet biogenesis in ER microdomains[END_REF].

3) Regulation of nucleation sites

Physical mechanisms that can locally lower the nucleation barrier and regulate nucleation sites are sketched in Fig. 2. The first mechanism (Fig. 2A) is related to the ER morphology. The unzipping and deformation of the ER monolayers could be energetically cheaper at places of particular topology such as protrusion tips; they are highly bent and display hydrophobic defectsreflected by PL packing defectswhich are energetically favorable for TG accumulation. Three other potential mechanisms (Fig. 2B-D) can be assigned the heterogeneity of the ER membrane composition. The simplest case (Fig. 2B) is the accumulation of TG molecules at the places they are made by diacylglycerol O-acyltransferase 1 or 2 enzymes (DGAT1 and 2). This accumulation can occur if the lateral diffusion of TG molecules (a priori similar to PLs, ca. 1µm 2 /sec) is slow compared to their synthesis rate; the TG concentration will increase around the synthesis sites and quickly reach the CDC to nucleate a LD. In the opposite case, if TG molecules diffused faster, they could reach sites rich in proteins important for LD formation, such as fat storage-inducing transmembrane proteins (FIT), Seipin, Perilipin 1 or 2, long-chain acyl-coenzyme A synthetase (ACSL) [START_REF] Pol | Review: biogenesis of the multifunctional lipid droplet: lipids, proteins, and sites[END_REF][START_REF] Yang | Controlling the size of lipid droplets: lipid and protein factors[END_REF][START_REF] Robenek | Adipophilin-enriched domains in the ER membrane are sites of lipid droplet biogenesis[END_REF][START_REF] Gross | Direct binding of triglyceride to fat storageinducing transmembrane proteins 1 and 2 is important for lipid droplet formation[END_REF][START_REF] Tan | Recent Advances in Understanding Proteins Involved in Lipid Droplet Formation, Growth and Fusion[END_REF][START_REF] Choudhary | A conserved family of proteins facilitates nascent lipid droplet budding from the ER[END_REF]. These proteins could favor LD nucleation by lowering the membrane bending energy (Fig. 2C) or the interfacial energy between TG and the ER monolayers (Fig. 2D); they could achieve this action by locally modulating the ER physical chemistry, for example, by interacting with diacylglycerols required for making TG [START_REF] Skinner | Diacylglycerol enrichment of endoplasmic reticulum or lipid droplets recruits perilipin 3/TIP47 during lipid storage and mobilization[END_REF], or favoring the local recruitment of specific PLs [START_REF] Boutet | Seipin deficiency alters fatty acid Delta 9 desaturation and lipid droplet formation in Berardinelli-Seip congenital lipodystrophy[END_REF][START_REF] Cartwright | Seipin performs dissectible functions in promoting lipid droplet biogenesis and regulating droplet morphology[END_REF][START_REF] Szymanski | The lipodystrophy protein seipin is found at endoplasmic reticulum lipid droplet junctions and is important for droplet morphology[END_REF].

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In the case LD nucleation does not require the mechanical action of proteins (random nucleation, nucleation induced by membrane topology, or nucleation near TG synthesis sites), nucleation may itself induce local changes in membrane curvature [START_REF] Mcmahon | Membrane curvature and mechanisms of dynamic cell membrane remodelling[END_REF], thickness, and hydrophobicity, which could be sensed by proteins to bind nascent LDs [START_REF] Pol | Review: biogenesis of the multifunctional lipid droplet: lipids, proteins, and sites[END_REF][START_REF] Kassan | Acyl-CoA synthetase 3 promotes lipid droplet biogenesis in ER microdomains[END_REF], Fig. 3. Many proteins, including amphipathic helix-binding proteins, found on LDs, have indeed the ability to sense and induce local spontaneous curvatures in membranes [START_REF] Mcmahon | Membrane curvature and mechanisms of dynamic cell membrane remodelling[END_REF][START_REF] Shen | SnapShot: membrane curvature sensors and generators[END_REF][START_REF] Antonny | Mechanisms of membrane curvature sensing[END_REF]. Whether the spontaneous TG demixing mediates protein recruitment to nascent LDs [START_REF] Pol | Review: biogenesis of the multifunctional lipid droplet: lipids, proteins, and sites[END_REF][START_REF] Phillips | Emerging roles for lipids in shaping membrane-protein function[END_REF], or, inversely, proteins determine nascent LD nucleation sites, remains unknown. Both could happen in the ER [START_REF] Phillips | Emerging roles for lipids in shaping membrane-protein function[END_REF], and their dual occurrence could explain how cells achieve LDs of different biological functions [START_REF] Wilfling | Lipid droplet biogenesis[END_REF][START_REF] Pol | Review: biogenesis of the multifunctional lipid droplet: lipids, proteins, and sites[END_REF][START_REF] Wolins | A proposed model of fat packaging by exchangeable lipid droplet proteins[END_REF]. This is an important question of LD biology.

Box 1: Basics of nucleation phenomena

Insert here Fig.box1

Nucleation of droplets in a fluid mixture undergoing phase separation is a stochastic process.

Individual molecules randomly diffuse in their environment, occasionally come into contact and assemble to form transient small pre-droplet structures [START_REF] Barrat | Basic concepts for simple and complex liquids[END_REF][START_REF] Chandler | Interfaces and the driving force of hydrophobic assembly[END_REF][START_REF] Safran | Statistical thermodynamics of surfaces, interfaces, and membranes[END_REF]. The large majority of these predroplets rapidly re-disperse and only a very small fraction of them survives and keeps growing.

This nucleation barrier feature is the consequence of the characteristic "bell" shape of the droplet (free) energy, function of the droplet volume, Fig. box1. The maximum of this energy is reached at and is called the nucleation energy, . Since the system will spontaneously evolve to decrease the free energy, large pre-droplets ( ) will grow while small pre-droplets ( )

will collapse [START_REF] Barrat | Basic concepts for simple and complex liquids[END_REF]. With the help of the molecular (thermal) agitation, some pre-droplets can nevertheless "climb the hill" and reach , from which they will keep growing by absorbing free molecules diffusing around. The nucleation rate goes as ~ : the higher the nucleation energy barrier, the less probable is the nucleation of a new droplet.

The bell shape of , and hence the existence of a nucleation energy, results from the competition of two contributions.

(

) 2
The parameter reflects the energy gain of a free molecule absorbed by a droplet; it is given by equation ( 1) in the case of LDs and dependent on TG-TG and TG-PL interactions; is the density

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of molecules. The term of E promotes droplet growth and dominates when . The second term (V) is an energy cost due to the droplet interface and mainly depends on TG-PL interaction in the case of forming LDs. This term opposes droplet formation and dominates when . In the case of LDs, additional effects should contribute to : the unzipping of the bilayer and the deformation of the monolayers surrounding the droplet, table 1.

III Communication between formation sites for LD growth

From cell to cell, LD number and size vary. The biological relevance of this diversity remains unclear [START_REF] Wolins | A proposed model of fat packaging by exchangeable lipid droplet proteins[END_REF][START_REF] Suzuki | Lipid droplets: size matters[END_REF]. For different cells, this variation may originate from the communication between LD nucleation sites, reducing the number of formed LDs.

TG molecules made in the ER can diffuse to the formation sites to grow nascent LDs. However two cases can be discriminated depending on the TG synthesis rate and mobility. In the case of an overall rapid synthesis rate (which is dependent on the synthesis enzymes expression levels and the synthesis reaction speed), nascent LDs are ignorant of each other, not necessarily communicating, because they are equally fed with TG molecules, grown, budded, and eventually detached. If instead TG diffusion is fast, the volume of nascent LDs will vary differently in time and their Laplace pressures will also change ( [START_REF] Thiam | The Biophysics and Cell Biology of Lipid Droplets[END_REF] for review). In this situation, a TG flux can be established in the ER bilayer from very small droplets (at the pre-droplets stage to nascent LDs; large Laplace pressure) to bigger LDs (smaller Laplace pressure), Fig. 4A. This phenomenon is called ripening and is responsible for the transfer of TG during adipocyte differentiation from smaller to bigger cytosolic LDs, and mediated by fat-specific protein p27 [START_REF] Gong | Fsp27 promotes lipid droplet growth by lipid exchange and transfer at lipid droplet contact sites[END_REF]. Occurrence of ripening allows for the communication between forming LDs and can introduce variability on forming LD growth rate, size and number. Ripening is a slow process, driven by the differences in Laplace pressures and allowed by the diffusion of molecules from the volume of smaller droplets towards bigger droplets; sometimes it is confusingly referred to as fusion, which is a different process, featuring rapid surface and volume mixing of two droplets [START_REF] Thiam | The Biophysics and Cell Biology of Lipid Droplets[END_REF]. In fusion, two pre-droplets or nascent LDs could laterally approach and coalesce when close enough, Fig. 4B. The fusion of two pre-droplets will increase the resulting pre-droplet volume that can become a nascent LD, i.e.

. Nascent LDs can fuse with pre-droplets, or with other nascent LDs, to form bigger LDs.

Fusion events can rapidly generate more thermodynamically stable nascent LDs. Finally, both fusion and ripening could generate supersized LDs in the ER.

Proteins can potentially regulate ripening and fusion within the ER membrane. FIT proteins can bind TG molecules [START_REF] Gross | Direct binding of triglyceride to fat storageinducing transmembrane proteins 1 and 2 is important for lipid droplet formation[END_REF] and could modulate communication between LD sites by shuttling TG to These potential functions are consistent with the frequently observed number and size variations of formed LDs when the proteins are knocked down [START_REF] Yang | Controlling the size of lipid droplets: lipid and protein factors[END_REF][START_REF] Kassan | Acyl-CoA synthetase 3 promotes lipid droplet biogenesis in ER microdomains[END_REF][START_REF] Tan | Recent Advances in Understanding Proteins Involved in Lipid Droplet Formation, Growth and Fusion[END_REF][START_REF] Brasaemle | Thematic review series: adipocyte biology. The perilipin family of structural lipid droplet proteins: stabilization of lipid droplets and control of lipolysis[END_REF].

IV LD budding

Accumulating TG molecules is necessary but not sufficient to form a LD. A TG phase could demix but simply form a film or a flat lens between the two ER monolayers, growing mainly in a planar direction. LD biogenesis requires that the oil phase forms a nearly spherical droplet enclosed in a spherical protrusion of one of the ER monolayers, protrusion that eventually undergoes fission from the parent membrane to release the LD. The membrane deformation process prior to LD release is called budding.

1) Budding mechanisms in biology and LD formation

Membrane budding is ubiquitous in biology, especially in vesicular trafficking. The biogenesis of bilayer vesicles from the plasma, Golgi or ER membranes are often carried out by coat protein machineries, respectively Clathrin, complex protein I and II (COPI and COPII), which assemble on the membranes and polymerize to form a spherical shell enclosing the budded vesicles [START_REF] Hurley | Membrane budding[END_REF]. The size of these vesicles is generally in the range of 100 nm [START_REF] Hurley | Membrane budding[END_REF]. For LD biogenesis, with a size a priori superior to this value [START_REF] Kassan | Acyl-CoA synthetase 3 promotes lipid droplet biogenesis in ER microdomains[END_REF], budding is not known to be mediated by similar protein machineries; in such eventuality, COPI would be a good candidate. Among the known coat machineries, COPI was mainly shown to be important for LD formation and functionality [START_REF] Guo | Functional genomic screen reveals genes involved in lipid-droplet formation and utilization[END_REF][START_REF] Beller | COPI complex is a regulator of lipid homeostasis[END_REF][START_REF] Soni | Coatomer-dependent protein delivery to lipid droplets[END_REF][START_REF] Wilfling | Arf1/COPI machinery acts directly on lipid droplets and enables their connection to the ER for protein targeting[END_REF][START_REF] Thiam | COPI buds 60-nm lipid droplets from reconstituted waterphospholipid-triacylglyceride interfaces, suggesting a tension clamp function[END_REF].

COPI is able to bud 60 nm nano-LD from artificial and purified LDs [START_REF] Wilfling | Arf1/COPI machinery acts directly on lipid droplets and enables their connection to the ER for protein targeting[END_REF][START_REF] Thiam | COPI buds 60-nm lipid droplets from reconstituted waterphospholipid-triacylglyceride interfaces, suggesting a tension clamp function[END_REF]; whether it can assemble on specific sites of the ER to locally bud nano-LDs for LD formation is not known and cannot be excluded. Other protein candidates for LDs budding can be speculatively proteins having the ability to deform membranes; these proteins include caveolin-related proteins [START_REF] Cohen | Role of caveolin-1 in the modulation of lipolysis and lipid droplet formation[END_REF], or oleosins in plants. Caveolins deform bilayer membranes to form vesicles; they localize to LDs,

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especially under cholesterol rich conditions in adipocytes [START_REF] Fujimoto | Not just fat: the structure and function of the lipid droplet[END_REF][START_REF] Lay | Cholesterol-induced caveolin targeting to lipid droplets in adipocytes: a role for caveolar endocytosis[END_REF], and could therefore physically contribute to the budding of LDs. Likewise, oleosins, crucial for the good packaging of plant lipid bodies (i.e. LDs) [START_REF] Brown | Lipid droplets: proteins floating on a pool of fat[END_REF], could also physically contribute to budding [START_REF] Murphy | Mechanisms of lipid-body formation[END_REF]. Both protein types display hairpin motifs and a high positive curvature, and could naturally induce the ER outer monolayer deformation for budding [START_REF] Parton | Biogenesis of caveolae: a structural model for caveolin-induced domain formation[END_REF][START_REF] Ariotti | Molecular Characterization of Caveolin-induced Membrane Curvature[END_REF]. Many other LD proteins (e.g. DGAT2, Glycerol-3-phosphate Acyltransferase Isoform-4, Human Lysophosphatidylcholine Acyltransferases 1 and 2) [START_REF] Ohsaki | Open questions in lipid droplet biology[END_REF][START_REF] Wilfling | Triacylglycerol synthesis enzymes mediate lipid droplet growth by relocalizing from the ER to lipid droplets[END_REF][START_REF] Moessinger | Human lysophosphatidylcholine acyltransferases 1 and 2 are located in lipid droplets where they catalyze the formation of phosphatidylcholine[END_REF] display hairpin motifs that can potentially induce LD budding. Finally, similarly to apolipoproteins that are able to bud tiny TG particles by binding through amphipathic helices to the ER luminal monolayer [START_REF] Saito | Contributions of domain structure and lipid interaction to the functionality of exchangeable human apolipoproteins[END_REF][START_REF] Mei | Lipid-free apolipoprotein AI structure: insights into HDL formation and atherosclerosis development[END_REF], proteins binding LDs with amphiphathic helices to LDs can have a natural curvature that imposes the budding shape of LDs [START_REF] Robenek | Adipophilin-enriched domains in the ER membrane are sites of lipid droplet biogenesis[END_REF][START_REF] Rowe | Conserved amphipathic helices mediate lipid droplet targeting of perilipins 1-3[END_REF]. Among them are Perilipins, major LD components [START_REF] Wolins | A proposed model of fat packaging by exchangeable lipid droplet proteins[END_REF].

However, the knockdown of most LD proteins does not prevent LD formation [START_REF] Guo | Functional genomic screen reveals genes involved in lipid-droplet formation and utilization[END_REF][START_REF] Beller | COPI complex is a regulator of lipid homeostasis[END_REF]. Although, one cannot exclude the existence of yet unknown and redundant protein machineries mediating LD budding, this observation rather suggest that proteins essentially have a regulatory role such as controlling LD nucleation site [START_REF] Pol | Review: biogenesis of the multifunctional lipid droplet: lipids, proteins, and sites[END_REF] (Fig. 2), or size [START_REF] Suzuki | Lipid droplets: size matters[END_REF], or protein content [START_REF] Guo | Functional genomic screen reveals genes involved in lipid-droplet formation and utilization[END_REF][START_REF] Beller | COPI complex is a regulator of lipid homeostasis[END_REF][START_REF] Wilfling | Arf1/COPI machinery acts directly on lipid droplets and enables their connection to the ER for protein targeting[END_REF]; other factors may be consequently more deterministic for LD budding. While lack of protein involvement is uncommon in biology, the topology of LDs is also uncommon [START_REF] Thiam | The Biophysics and Cell Biology of Lipid Droplets[END_REF]: all other cellular organelles are bilayer-bounded, but LDs are bounded by a PL monolayer covering an oil phase. In the next section, we propose that the molecular interactions between oil molecules and PLs of the ER membrane are, in addition to nucleation, able to mediate LD budding without any energy input from proteins. In this picture, we will then discuss the possible role of proteins in the regulation of budding.

2) From droplet dewetting to LD budding

During LD biogenesis, a cytosolic and luminal ER PL monolayers cover TG molecules forming an oil lens of few tens of nm in diameter. This situation can be envisioned as three-phase wetting phenomenon [START_REF] Kusumaatmaja | Droplet-induced budding transitions of membranes[END_REF], box 2. The three phases are in this case the cytosol, the ER lumen and the oil phase. The shape of the nascent LD should thus depend to a large extent on the surface tension of the interfaces, or membranes, separating the different phases, i.e. the surface energy cost for generating an interface between two fluids ( [START_REF] Thiam | The Biophysics and Cell Biology of Lipid Droplets[END_REF] for review on surface tension). The surface tension of the ER bilayer membrane has been determined around mN/m [START_REF] Upadhyaya | Tension in tubulovesicular networks of Golgi and endoplasmic reticulum membranes[END_REF]. The surface tensions of the TG-cytosolic and TG-ER luminal monolayers are probably similar and can be expected to be of the order of mN/m, which is the surface tension of

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reconstituted LD monolayer surface fully covered by PLs [START_REF] Thiam | COPI buds 60-nm lipid droplets from reconstituted waterphospholipid-triacylglyceride interfaces, suggesting a tension clamp function[END_REF][START_REF] Thiam | The Energy of COPI for Budding Membranes[END_REF]. These surface tension values are mainly determined by the PL composition of the different membranes and the oil phase [START_REF] Thiam | The Biophysics and Cell Biology of Lipid Droplets[END_REF].

LD budding to the cytosol corresponds to a partial or complete dewetting of the oil phase from the ER luminal PL monolayer (Fig. 5A(3,4)), and could in principle be solely driven by the interfacial forces (i.e. originating from surface tension). By analogy with wetting droplets [START_REF] Bonn | Wetting and spreading[END_REF], box 2 and Fig. 5A, the shape of a forming LD (Fig. 5B) can be characterized by the contact angle , determined (at least in part) by the surface tensions [START_REF] Bonn | Wetting and spreading[END_REF][START_REF] Hotrum | Monitoring entering and spreading of emulsion droplets at an expanding air/water interface: a novel technique[END_REF]. Small values of the contact angle , i.e. close to 0° (Fig. 5A(3,4)), correspond to budding.

The value of the angle has other important biological consequences besides budding. Small angles reflect the propensity of LDs to remain connected with the ER, and thereby the capacity of proteins to reach them from the latter [START_REF] Wilfling | Arf1/COPI machinery acts directly on lipid droplets and enables their connection to the ER for protein targeting[END_REF][START_REF] Thiam | COPI buds 60-nm lipid droplets from reconstituted waterphospholipid-triacylglyceride interfaces, suggesting a tension clamp function[END_REF][START_REF] Wilfling | Triacylglycerol synthesis enzymes mediate lipid droplet growth by relocalizing from the ER to lipid droplets[END_REF][START_REF] Jacquier | Lipid droplets are functionally connected to the endoplasmic reticulum in Saccharomyces cerevisiae[END_REF]. For large contact angles, LD growth would be mainly by spreading; budding would eventually occur at larger size due to other constraints arising from ER topology or from proteins that could limit or prevent spreading. In cells, although LDs can appear in most cases perfectly spherical, they could be in a non-dewetting state; measuring the real contact angle in this context is challenging.

Proteins especially implicated in LD formation could influence the value of the contact angle to favor budding by modulating the different tensions. Seipin could localize to LD formation sites and modulate membrane tension, e.g. by locally mediating specific PL recruitment [START_REF] Cartwright | Seipin performs dissectible functions in promoting lipid droplet biogenesis and regulating droplet morphology[END_REF][START_REF] Szymanski | The lipodystrophy protein seipin is found at endoplasmic reticulum lipid droplet junctions and is important for droplet morphology[END_REF][START_REF] Grippa | The seipin complex Fld1/Ldb16 stabilizes ER-lipid droplet contact sites[END_REF]. Other enzymes implicated in PL fabrication or modification could also change membrane surface tension by fabricating specific PLs [START_REF] Moessinger | Human lysophosphatidylcholine acyltransferases 1 and 2 are located in lipid droplets where they catalyze the formation of phosphatidylcholine[END_REF][START_REF] Andersson | PLD1 and ERK2 regulate cytosolic lipid droplet formation[END_REF][START_REF] Gubern | Lipid droplet biogenesis induced by stress involves triacylglycerol synthesis that depends on group VIA phospholipase A2[END_REF][START_REF] Krahmer | Phosphatidylcholine synthesis for lipid droplet expansion is mediated by localized activation of CTP:phosphocholine cytidylyltransferase[END_REF][START_REF] Shi | Regulation of lipid droplet size and phospholipid composition by stearoyl-CoA desaturase[END_REF][START_REF] Aitchison | Nuclear-localized CTP:phosphocholine cytidylyltransferase alpha regulates phosphatidylcholine synthesis required for lipid droplet biogenesis[END_REF][START_REF] Adeyo | The yeast lipin orthologue Pah1p is important for biogenesis of lipid droplets[END_REF][START_REF] O'hara | Control of phospholipid synthesis by phosphorylation of the yeast lipin Pah1p/Smp2p Mg2+-dependent phosphatidate phosphatase[END_REF]. Finally, the direct binding of proteins to nascent LDs, such as Perilipins or ASCLs [START_REF] Pol | Review: biogenesis of the multifunctional lipid droplet: lipids, proteins, and sites[END_REF][START_REF] Kassan | Acyl-CoA synthetase 3 promotes lipid droplet biogenesis in ER microdomains[END_REF], could also vary the tension of the TG-cytosolic monolayer.

3) Influence of membrane rigidity

Surface tension mainly influences the shape of the LD bud, as discussed previously. However, at the nascent LD stage, the rigidity of the ER PL monolayers has a non-negligible effect: it opposes membrane bending and flattens the LD. As the latter grows, its monolayer curvature decreases, reducing thereupon the influence of rigidity. Due to this competition between membrane bending rigidity and surface tension, nascent LD shape may progressively evolve from a flat lens after nucleation, due to the monolayer rigidity, to a spherical shape at large size, dictated by surface tension [START_REF] Foret | Shape and energy of a membrane bud induced by protein coats or viral protein assembly[END_REF]. This competition could explain the existence of a typical LD budding size.

4) Directional budding and the possible roles of proteins

A C C E P T E D M A N U S C R I P T ACCEPTED MANUSCRIPT 10
The origin of the directional budding of LDs, mainly occurring towards the cytosol, remains poorly understood. From a biophysics standpoint, budding directionality requires an asymmetry between the physical properties of the ER monolayers, which should originate from the different protein and lipid composition of the two monolayers, or from a difference in pressure between the ER lumen and the cytosol.

An asymmetry of tension can lead to directional budding by dewetting, box 2. If the tension of the cytosolic monolayer is lower than that of the luminal one, partial or complete dewetting of the oil phase from the luminal monolayer is favored (cases in Fig. 5A(3,4) and Fig. 5B(2,3)). This situation happens for example if there is an excess PL in the ER cytosolic monolayer compared to its luminal one. This imbalance could happen with phosphatidyl choline whose synthesis enzymes reside in the cytosol [START_REF] Moessinger | Human lysophosphatidylcholine acyltransferases 1 and 2 are located in lipid droplets where they catalyze the formation of phosphatidylcholine[END_REF][START_REF] Krahmer | Phosphatidylcholine synthesis for lipid droplet expansion is mediated by localized activation of CTP:phosphocholine cytidylyltransferase[END_REF][START_REF] Moessinger | Two different pathways of phosphatidylcholine synthesis, the Kennedy Pathway and the Lands Cycle, differentially regulate cellular triacylglycerol storage[END_REF], or in the nucleus [START_REF] Krahmer | Phosphatidylcholine synthesis for lipid droplet expansion is mediated by localized activation of CTP:phosphocholine cytidylyltransferase[END_REF][START_REF] Aitchison | Nuclear-localized CTP:phosphocholine cytidylyltransferase alpha regulates phosphatidylcholine synthesis required for lipid droplet biogenesis[END_REF][START_REF] Kory | Protein Crowding Is a Determinant of Lipid Droplet Protein Composition[END_REF], where LDs bud off [START_REF] Aitchison | Nuclear-localized CTP:phosphocholine cytidylyltransferase alpha regulates phosphatidylcholine synthesis required for lipid droplet biogenesis[END_REF][START_REF] Uzbekov | Nuclear lipid droplets identified by electron microscopy of serial sections[END_REF].

An asymmetry in the bending properties of the ER monolayers could also contribute to the budding directionality. An intrinsic positive curvature of the outer monolayer of the ER membrane would favor budding toward the cytosol [START_REF] Sturley | Lipid droplet formation on opposing sides of the endoplasmic reticulum[END_REF] (or to the nucleus [START_REF] Uzbekov | Nuclear lipid droplets identified by electron microscopy of serial sections[END_REF][START_REF] Ohsaki | PML isoform II plays a critical role in nuclear lipid droplet formation[END_REF]). The binding to nascent LDs of cytosolic proteins with positive curvature [START_REF] Shen | SnapShot: membrane curvature sensors and generators[END_REF] (e.g. by the insertion of amphipathic helix, or by the presence of bar-like domain), or an excess of PLs having a positive curvature in the ER cytosolic monolayer (e.g. Lyso-PLs), would favor bending of the ER monolayer and budding into the cytosol [START_REF] Mcmahon | Membrane curvature and mechanisms of dynamic cell membrane remodelling[END_REF][START_REF] Mcmahon | Membrane curvature at a glance[END_REF][START_REF] Stachowiak | A cost-benefit analysis of the physical mechanisms of membrane curvature[END_REF]. Transmembrane proteins could also generate positive curvature. Recent electron microscopy imaging suggest for example the implication of FIT1 in LD directional budding, as its deletion caused the ER luminal localization of LDs [START_REF] Choudhary | A conserved family of proteins facilitates nascent lipid droplet budding from the ER[END_REF]. FIT1 proteins have indeed transmembrane multi-spanning domains and its N and C termini sitting in the cytosol, and therefore could predeform the ER cytosolic monolayer. Other proteins involved in TG synthesis or in LD formation can have similar effects. Finally, LD budding in the cytosol could arise from a protein crowding mechanism due to a protein concentration imbalance between each side of the ER membrane [START_REF] Stachowiak | A cost-benefit analysis of the physical mechanisms of membrane curvature[END_REF][START_REF] Derganc | Membrane bending: the power of protein imbalance[END_REF]. Peripheral proteins bound to a bilayer or a monolayer membrane diffuse, collide and repel [START_REF] Kory | Protein Crowding Is a Determinant of Lipid Droplet Protein Composition[END_REF][START_REF] Stachowiak | Membrane bending by protein-protein crowding[END_REF], generating thereby a surface pressure. This pressure induces membrane curvature resulting for example to the appearance of bilayer protrusions toward the side of higher protein concentration [START_REF] Stachowiak | Membrane bending by protein-protein crowding[END_REF][START_REF] Segrest | Surface Density-Induced Pleating of a Lipid Monolayer Drives Nascent High-Density Lipoprotein Assembly[END_REF]. Since nascent LDs are a priori asymmetrically bound by proteins, as most of their proteins bind from the cytosol, a spontaneous curvature of LD towards the cytosolic side could be favored by such crowding effect.

A C C E P T E D M A N U S C R I P T ACCEPTED MANUSCRIPT

11 Directional budding could also arise from ER topology. The ER has a small luminal volume and a high concentration of protein building blocks, which cause an excess pressure that may limit luminal budding by "pushing" forming LD toward the cytosol.

As a remark, all these mechanisms of membrane asymmetry could be involved in LD budding in the nucleus [START_REF] Uzbekov | Nuclear lipid droplets identified by electron microscopy of serial sections[END_REF][START_REF] Ohsaki | PML isoform II plays a critical role in nuclear lipid droplet formation[END_REF].

Finally, in the particular case of lipoprotein (which can be seen as a particular type of LD), budding occurs towards the lumen. Since cytosolic budding is favored under normal circumstances, an energy input is required for inducing lipoprotein formation. This energy is possibly provided by microsomal triglyceride transfer proteins apolipoproteins. Apolipoprotein bind with nanodisks-forming amphipathic helices that induce membrane curvature [START_REF] Saito | Contributions of domain structure and lipid interaction to the functionality of exchangeable human apolipoproteins[END_REF][START_REF] Mei | Lipid-free apolipoprotein AI structure: insights into HDL formation and atherosclerosis development[END_REF]. By their association with the ER luminal monolayer [START_REF] Ohsaki | Lipid droplets are arrested in the ER membrane by tight binding of lipidated apolipoprotein B-100[END_REF], they could locally impose a curvature to the luminal monolayer, compensating the effect of asymmetry factors mediating cytosolic budding.

Box 2: Wetting basic principles

The equilibrium shape of a fluid drop (phase "o") at the interface between two liquids (phase "c" and "l") is determined by the surface tensionthe interfacial energy per unit area [START_REF] Thiam | The Biophysics and Cell Biology of Lipid Droplets[END_REF] of the three interfaces, , , and . These tensions are determined by the molecular interactions between the components of the phases in contact [START_REF] Thiam | The Biophysics and Cell Biology of Lipid Droplets[END_REF][START_REF] De Gennes | Capillarity and wetting phenomena: drops, bubbles, pearls, waves[END_REF][START_REF] Bonn | Wetting and spreading[END_REF][START_REF] Hotrum | Monitoring entering and spreading of emulsion droplets at an expanding air/water interface: a novel technique[END_REF]. The propensity of the drop to spread along the interface, or to dewet, can be described using the tension-dependent parameters defined in Fig. 5, (called spreading parameter) and (empirically called the entering parameter, being here the budded shape). Three situations can be met: total wetting (Fig. 5A1), partial wetting (Fig. 5A(2,3)) and dewetting (Fig. 5A4) [START_REF] Thiam | The Biophysics and Cell Biology of Lipid Droplets[END_REF][START_REF] Bonn | Wetting and spreading[END_REF][START_REF] Hotrum | Monitoring entering and spreading of emulsion droplets at an expanding air/water interface: a novel technique[END_REF]. In the case of partial wetting, the shape of the drop is essentially characterized by the contact angle in Fig. 5, defined by the different surface tensions:

(

For large values of , the drop forms a flattened lens (Fig. 5B); when is small, i.e. close to 0°, the drop is nearly spherical (Fig. 5A(3,4)). 1), partial wetting (2 and 3), and complete dewetting (4). In the case of partial wetting, the shape is characterized by the angle determined by the value of the tensions: is large in case 2 and small in case 3 where the droplet undergoes partial dewetting. B) A nascent LD can be considered as an oil phase in between the cytosol and the ER lumen. Following the physics of wetting phenomena, different shapes are possible in principle (B1 analogous to partial wetting A2, B2 analogous to partial dewetting A3 and B3 analogous to complete dewetting A4), depending on the tensions of the ER membrane and of the monolayers at the interfaces of the different phases.

Conclusion

A C C E P T E D M A N U S C R I P T ACCEPTED MANUSCRIPT 12

  Seipin, present on LD formation sites[START_REF] Cartwright | Seipin performs dissectible functions in promoting lipid droplet biogenesis and regulating droplet morphology[END_REF][START_REF] Szymanski | The lipodystrophy protein seipin is found at endoplasmic reticulum lipid droplet junctions and is important for droplet morphology[END_REF][START_REF] Fei | Fld1p, a functional homologue of human seipin, regulates the size of lipid droplets in yeast[END_REF], could stabilize nascent LDs and prevent their disappearance by preventing TG leakage; Seipin could also behave as a physical barrier that prevent lateral fusion between nascent LDs; finally, proteins such as ASCL3 or Perilipins can physically modulate the Laplace pressure of forming LDs, preventing small nascent LDs ( barely over ) from shrinking and mature LDs () from growing faster. The exact role of proteins in modulating LD ripening or fusion in the ER is not known to date. Nonetheless, the potential actions listed above of the proteins would result in the regulation of the distribution of TG in forming LDs, by inhibiting or reinforcing communication between nascent LDs or pre-droplets.

Figure 1

 1 Figure 1 TG demixing within the ER membrane. A) Triglyceride (TG) molecules can diffuse freely in the ER bilayer membrane. They interact with the bilayer components, in particular phospholipids (PL), the main ER component. B) TG molecules condensed in a LD mainly interact with other TGs. The energy gain from A and B mediates the spontaneous TG demixing and the formation of a TG lens in the bilayer.

Figure 2

 2 Figure 2 Different mechanisms can locally lower the LD nucleation energy. A) A local membrane deformation can favor LD nucleation. The case of a protrusion tip is shown.Accumulation of TG molecules at the tip of the protrusion is favored for nucleating a lens, because part of the energy required for deforming a monolayer during LD budding is already provided by the pre-deformation. Additionally, hydrophobic defects at the tip of the protrusion reflect a higher membrane tension than in the flat bilayer; therefore, accommodating TG molecules at the tip decreases the overall energy of the system. B) The local increase of the TG density near synthesis site will force the unzipping of the bilayer and the nucleation of a LD. C) The presence of curvature-inducing proteins, e.g. positively curved proteins, can decrease the energy cost for deforming the PL monolayer by TG accumulation. D) The presence of proteins and/or lipids interacting with TG, can decrease the nucleation energy by decreasing the interfacial energy of the droplet ( in box 1) and favoring the condensation of TGs ( in box 1).

Figure 3

 3 Figure 3 Nucleation of a LD induces a strong curvature or thickness increase of the membrane, and proteins can sense these physical membrane changes to bind nascent LD.

Figure 4 A

 4 Figure 4 A) Droplet ripening: the difference of Laplace pressures can induce a flux of TG from small forming droplets (Pre-droplets or nascent LDs) to larger LDs within the ER bilayer. B) Predroplets and/or nascent LDs in the ER bilayer can fuse to former a bigger and more stable nascent LDs.

Figure 5 A

 5 Figure 5 A) Basics of a three fluid phases wetting situation. The shape of the yellow liquid droplet (o) in between two fluids (c) and (l) is determined by the tensions , and of the interfaces between the different phases o, l, and c. Three cases can happen depending on the values of surface tensions: total wetting (1), partial wetting (2 and 3), and complete dewetting (4). In the case of partial wetting, the shape is characterized by the angle determined by the value of the tensions: is large in case 2 and small in case 3 where the droplet undergoes partial dewetting. B) A nascent LD can be considered as an oil phase in between the cytosol and the ER lumen. Following the physics of wetting phenomena, different shapes are possible in principle (B1 analogous to partial wetting A2, B2 analogous to partial dewetting A3 and B3 analogous to complete dewetting A4), depending on the tensions of the ER membrane and of the monolayers at the interfaces of the different phases.

Table 1

 1 Principal contributions (checked boxes) to the nucleation energy barrier of the different types of molecular interactions between Triglycerides (TG), Phospholipids (PL) and proteins, localized to LD formation sites. The nucleation energy has two contributions (see box 1): (i) the demixing free energy gained by the TG molecules forming a dense phase ( term in box 1) and (ii) the cost associated to the creation of the monolayer at the oil-water interface ( term in box 1). The later comprises two contributions: the monolayer interfacial energy, which depends on the molecular interactions between the monolayer components and TG, and the membrane deformation energy, which depends on the monolayer composition.A C C E P T E DM A N U S C R I P T

  LDs, coated by a PL monolayer, can have a much higher budding energy as compared to bilayer vesicles[START_REF] Thiam | The Energy of COPI for Budding Membranes[END_REF]; it is hence plausible that cells develop/use alternative mechanisms for LD budding compared to the generation of bilayer vesicles. This review offers new orientations for studying LD formation. It can serve as a guideline for better studying LD formation and subsequent protein trafficking. Although speculative from the biology background, all the physics ingredients described, based on nucleation and dewetting phenomena, are relevant for LD biogenesis. We highlight overlooked ER physical chemistry properties, especially the interaction between lipids, which are probably fundamental for LD formation. In vitro experiments based on emulsion droplets should be of particular interest, as they allow a precise estimation of the contribution of lipids and proteins. What is the contribution of PLs in LD formation? Are there specific PLs facilitating LD nucleation? What is the contribution of dewetting in LD biogenesis? Do proteins regulate LD nucleation or does nucleation drive instead protein recruitment to nascent LDs? Among the proposed physical mechanisms resulting in membrane asymmetry, which ones are relevant for the cytosolic budding of LDs? Using a combination of in vitro and cell biology approaches will enable addressing these important questions of LD biology raised by the review.

Acknowledgments

Authors gratefully thank Dr. Luca Monticelli and Steve Donaldson for the critical read of the manuscript. This work was supported by the ATIP-Avenir program and the Programme Emergence de la ville de Paris to A.R.T.

A C C E P T E D

M A N U S C R I P T


Alexis Descatha
email: alexis.descatha@inserm.fr

Céline Dagrenat

Pascal Cassan

Daniel Jost

Thomas Loeb

Michel Baer

Prof Alexis Descatha

Cardiac arrest in the

Keywords: cardiac arrest, location, public, work, occupational, review, meta-analysis, prevention

à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction.Out-of-hospital cardiac arrest (OHCA) in the workplace appears to be managed more effectively than OHCA occurring in other places. A systematic review and metaanalysis of the available epidemiological data was performed, comparing the rate of survival for OHCA in the workplace, versus survival in other locations.

Methods.Four databases (Pub-Med, Scopus, Web of science, "Base de Données de Santé Publique", BDSP, i.e. the French Public Health Database) were searched since 2000, using the key words: ("Cardiac arrest") and ("occupational" OR "workplace" OR "public location"). A two stage process with two independent readers was used to select relevant papers. Numbers of subjects who suffered fromOHCA in the workplace versus other locations were extracted when possible, as well as their respective outcomes (admitted alive to the hospital, discharged alive, good neurological outcome). Metarisks were calculated using the generic variance approach (meta-odds ratiosmetaOR).

Results

After full-text reading, 17papers were included, from 9 countries, mostly published after 2005, and coming mostly from prospective registers. "Workplace" was defined differently in different studies, mostly in terms of industrial sites and offices. The workplace was an exceptional location for occurrences of OHCA (from 0.3% to 4.7 % of all OHCA, from 1.3 to 23.8 events per million peoplePeryear), based on 2077 OHCA.In thequantitative analyses (survival available, 10 studies), MetaOR were found to be relatively consistent and high (form 1.9 (1.5-2.3) to 5.9(2.7-13.0)). When OHCA occurring at workplaces were compared to other public sites, no significant differences were found.

Conclusion.

There is sufficient evidence to support the view that there will be better outcomes for OHCA cases that occur in the workplace than for those occurring elsewhere.

Requirements for occupational health and safety should include prevention of such major (albeit rare) events.

IntroductionSince the survival rate decreases exponentially with increases in the interval between out-of-hospital cardiac arrest (OHCA) and the chain of survival, the locations of first responders, their training level, and the location of automated external defibrillators (AED) are particularly important. [START_REF] Nolan | Cardiopulmonary resuscitation[END_REF] Despite its relatively low incidence, it has been suggested that OHCA in the workplace appears to be managed more effectively than OHCA occurring in other places, which encourages the setting up of first-aid programs in certain companies. [START_REF] Descatha | Automated external defibrillators in the workplace[END_REF] .Some studiesthat compare OHCA managed in the workplace versus OHCAmanaged atother locationshave recently been published [START_REF] Muraoka | Location of out-of-hospital cardiac arrests in Takatsuki City: where should automated external defibrillator be placed[END_REF][START_REF] Descatha | Is the workplace a site of cardiac arrest like any other?[END_REF][START_REF] Murakami | Outcomes of out-of-hospital cardiac arrest by public location in the public-access defibrillation era[END_REF] .

The goal of this study was to undertake a systematic review and meta-analysis of the available epidemiological data, comparing the rate of survivalfor OHCA in the workplace, versus survival atother locations. The proportion of workplace OHCA in relation toother locations and its frequencyare also described, if available.

Methods

Literature research

Four databases (Pub-Med, Scopus, Web of science, "Base de Données de Santé Publique", BDSP, i.e. the French Public Health Database) were searched, using the key words: ("Cardiac arrest") and ("occupational" OR "workplace" OR "public location"). No language limitation was added. Papers published before the recommendations ofthe International Liaison Committee on Resuscitation (ILCOR) of 2000 were considered too old [START_REF] Monsieurs | European Resuscitation Council Guidelines 2000 for Automated External Defibrillation. A statement from the Basic Life Support and Automated External Defibrillation Working Group(1) and approved by the Executive Committee of the European Resuscitation Council[END_REF] . The first selection of articles was performed by two independent readers (A.D. and C.D.).The aim, based on the title and abstract, was to include only papers with (i) original studies dealing with cardiac arrest, (ii) sufficient details to distinguish workplace locationsfrom otherlocations (public or private),(iii) outcomes respecting the Utstein recommendations [START_REF] Cummins | Recommended guidelines for uniform reporting of data from out-of-hospital cardiac arrest: the Utstein Style. A statement for health professionals from a task force of the American Heart Association, the European Resuscitation Council, the Heart and Stroke Foundation of Canada, and the Australian Resuscitation Council[END_REF][START_REF] Peberdy | Recommended guidelines for monitoring, reporting, and conducting research on medical emergency team, outreach, and rapid response systems: an Utstein-style scientific statement: a scientific statement from the International Liaison Committee on Resuscitation (American Heart Association, Australian Resuscitation Council, European Resuscitation Council, Heart and Stroke Foundation of Canada, InterAmerican Heart Foundation, Resuscitation Council of Southern Africa, and the New Zealand Resuscitation Council); the American Heart Association Emergency Cardiovascular Care Committee; the Council on Cardiopulmonary, Perioperative, and Critical Care; and the Interdisciplinary Working Group on Quality of Care and Outcomes Research[END_REF] . The second stage included examination of full-text papers based on the same criteria. Studies meeting these criteria were included in the meta-analysis after a review by the independent readers (A.D. and C.D.).

Assessment of methodological quality

Alist evaluating methodological quality in terms of four categories was created, adapted from Utstein style and Prisma recommendations [START_REF] Cummins | Recommended guidelines for uniform reporting of data from out-of-hospital cardiac arrest: the Utstein Style. A statement for health professionals from a task force of the American Heart Association, the European Resuscitation Council, the Heart and Stroke Foundation of Canada, and the Australian Resuscitation Council[END_REF][START_REF] Peberdy | Recommended guidelines for monitoring, reporting, and conducting research on medical emergency team, outreach, and rapid response systems: an Utstein-style scientific statement: a scientific statement from the International Liaison Committee on Resuscitation (American Heart Association, Australian Resuscitation Council, European Resuscitation Council, Heart and Stroke Foundation of Canada, InterAmerican Heart Foundation, Resuscitation Council of Southern Africa, and the New Zealand Resuscitation Council); the American Heart Association Emergency Cardiovascular Care Committee; the Council on Cardiopulmonary, Perioperative, and Critical Care; and the Interdisciplinary Working Group on Quality of Care and Outcomes Research[END_REF][START_REF] Liberati | The PRISMA statement for reporting systematic reviews and meta-analyses of studies that evaluate health care interventions: explanation and elaboration[END_REF] . The five relevant questionswere:(i) was the study design exhaustive? (i.e. with data from a register), (ii) was the workplace location defined precisely?(iii) didthe outcome of OHCA patients include survival at 6months with neurological evaluation? (iv) wasthe proportion of treatable OHCA available ? (i.e. not traumatic, shockable, rhythm, withwitnesses) (v) How recent were the studies? (performed in the last 10 years). Two reviewers (A.D. and C.D.) independently assessed the quality of each study by scoring each criterion as positive or negative. Disagreement was resolved by consensus. The fair quality score was based on a total score of 3 or higher.

Data extraction and analysis

Relevant data were extracted from the articles: the number of workplace OHCA, their frequency,and their survival were extracted, as well as the number of thoserelated to other locations. Frequency of OHCA wasalso extracted, if available. The core findings in each article were expressed using measures of association (odds ratio or OR) with a corresponding 95% confidence interval (CI).Three outcomes were considered: (i) admitted alive to hospital, (ii) discharged alive from hospital or still alive 30 days after the OHCA, (iii) favorable neurological outcome (i.e. Cerebral Performance Category (CPC) 1: return to normal cerebral function and normal living, or CPC 2: cerebral disability, but sufficient function for independent activities of daily living). Whenever possible, these associations were directly extracted from the original article. In articles where this information was not available, associations were calculated when sufficient raw data was provided. Raw data were requested from all authors if needed. Some were unable to give us authorization for access to the raw data. Nevertheless, metarisks(meta-OR) were calculated using the generic variance approach.

The weight given to each studywas the inverse of the variance of the estimated effect.

Heterogeneity was tested with the Q statistic. From the Q statistic, we calculated a summary OR and 95% CI with the random effect method. This approach provides more conservative estimates (broader CI) than a fixed effect model, assuming that the differences between results are solely due to chance. For each outcome, all OHCA were included and only those considered as treatable (excluding OHCA without any witness, with traumatic cause,with non-shockable rhythm, depending on available data). To compare workplaces with other public sites, we also recalculated meta-OR for such treatable OHCA.

We tested the publication bias due to study size using Egger's regression approach. Meta-ORs were run on all studies, and on fair quality methodological studies only.

The meta-analysis was performed using STATA (Version 11.2; Stata Corp., College Station, TX, USA). The PRISMA checklist wasused. [START_REF] Liberati | The PRISMA statement for reporting systematic reviews and meta-analyses of studies that evaluate health care interventions: explanation and elaboration[END_REF]

Results

In the four selected data bases, we found 30 papers corresponding to our first stage (Figure 1).

Only two papers were included due to cross-references (in the reference list and not in the databases), and neither of these? was selected after full-reading. After full-text reading,17 papers were included [START_REF] Pell | Potential impact of public access defibrillators on survival after out of hospital cardiopulmonary arrest: retrospective cohort study[END_REF][START_REF] Descatha | Details of the initial management of cardiac arrest occurring in the workplace in a French urban area[END_REF][START_REF] Eisenburger | Cardiac arrest in public locations--an independent predictor for better outcome?[END_REF][START_REF] Muraoka | Location of out-of-hospital cardiac arrests in Takatsuki City: where should automated external defibrillator be placed[END_REF][START_REF] Reed | Location of cardiac arrests in the public access defibrillation trial[END_REF][START_REF] Iwami | Outcome and characteristics of out-of-hospital cardiac arrest according to location of arrest: A report from a large-scale, population-based study in Osaka, Japan[END_REF][START_REF] Descatha | Is the workplace a site of cardiac arrest like any other?[END_REF][START_REF] Descatha | Cardiac arrest in the workplace: pilot study on the RéAC register[END_REF][START_REF] Murakami | Outcomes of out-of-hospital cardiac arrest by public location in the public-access defibrillation era[END_REF][START_REF] Weisfeldt | Survival after application of automatic external defibrillators before arrival of the emergency medical system: evaluation in the resuscitation outcomes consortium population of 21 million[END_REF][START_REF] Zakaria | Implications for public access defibrillation placement by non-traumatic out-of-hospital cardiac arrest occurrence in Singapore[END_REF][START_REF] Malcom | The location and incidence of out-of-hospital cardiac arrest in Georgia: implications for placement of automated external defibrillators[END_REF][START_REF] Folke | Location of cardiac arrest in a city center: strategic placement of automated external defibrillators in public locations[END_REF][START_REF] Engdahl | Localization of out-of-hospital cardiac arrest in Goteborg 1994-2002 and implications for public access defibrillation[END_REF][START_REF] Moon | Analysis of out-of-hospital cardiac arrest location and public access defibrillator placement in Metropolitan Phoenix, Arizona[END_REF][START_REF] Brooks | Determining risk for out-ofhospital cardiac arrest by location type in a Canadian urban setting to guide future public access defibrillator placement[END_REF][START_REF] Hansen | Automated external defibrillators inaccessible to more than half of nearby cardiac arrests in public locations during evening, nighttime, and weekends[END_REF] . Agreement between the two reviewers was good (kappa 0.90). Selected studies came from nine countries (mostly from the United States, Japan, and France), and were mostly published after 2005 (Table 1).Data were obtained mostly from prospective registers.No discrepancy between the readers was found (considering the simple but robust criteria). "Workplace" was defineddifferently in different studies, mostly in terms of industrial sites and offices. The workplace was an exceptional location for occurrences of OHCA (from 0.3% to 4.7 % of all OHCA, from 1.3 to 23.8 events per million people per year), based on 2077 OHCA.

Of the remaining 17 papers, 10for which survival data were available andwere selected in the quantitative analysis review: [START_REF] Pell | Potential impact of public access defibrillators on survival after out of hospital cardiopulmonary arrest: retrospective cohort study[END_REF][START_REF] Descatha | Details of the initial management of cardiac arrest occurring in the workplace in a French urban area[END_REF][START_REF] Eisenburger | Cardiac arrest in public locations--an independent predictor for better outcome?[END_REF][START_REF] Muraoka | Location of out-of-hospital cardiac arrests in Takatsuki City: where should automated external defibrillator be placed[END_REF][START_REF] Reed | Location of cardiac arrests in the public access defibrillation trial[END_REF][START_REF] Iwami | Outcome and characteristics of out-of-hospital cardiac arrest according to location of arrest: A report from a large-scale, population-based study in Osaka, Japan[END_REF][START_REF] Descatha | Is the workplace a site of cardiac arrest like any other?[END_REF][START_REF] Descatha | Cardiac arrest in the workplace: pilot study on the RéAC register[END_REF][START_REF] Murakami | Outcomes of out-of-hospital cardiac arrest by public location in the public-access defibrillation era[END_REF][START_REF] Weisfeldt | Survival after application of automatic external defibrillators before arrival of the emergency medical system: evaluation in the resuscitation outcomes consortium population of 21 million[END_REF] Except for one of them, these studies found a better outcomefor OHCA occurring in workplaces than OHCA occurring elsewhere (n=1383),and metaORswere found to be relatively consistent and high(Table 2), taking into account all outcomes. Sensitivity analyses based on recent and fair quality methodological studies found similar results. There was no significant publication bias (Egger's test, P>0.05).

When OHCA occurring at workplaces were compared to those at other public sites, no significant differences were found.

Discussion

Results reported in the literature from the last fifteen years are consistent, and confirm that the workplace locationseems to bea safer place than any other, with a better rate of survival observed in such locations, althoughsuch OHCA remain an exceptional event. However, improvement of OHCA handling in workplaces seems necessary when compared to outcomes involvingother public places only.

Most of the difficulties with the studies about the management of OHCA in the workplacecome from the heterogeneous definition of "workplace", which ranges from small shops to large factories, construction sites to small businesses etc. Thus, comparing outcomes involving different definitions of the workplace is debatable, given that the term "location elsewhere" is also very vague. Although most of the workplaces described were classified as "industry and business", the definition of such locations remains quite broad. However, all workplaces share similar characteristics, such as relatively young age of the patient and the presence of witnesses, and similar interpretations for what counts as a workplace. Although studies are needed to analyze, in greater detail, differences in thesurvival chain for the differentkinds of workplaces, the homogeneity of our results allows general recommendations to be proposed concerning the prevention and management of cardiac arrest cases in workplaces.

The similarity in results observed for workplaces raises the question of why outcomes are better there than elsewhere. Three explanations are plausible: first, people are usually healthier at work than elsewhere: the "healthy worker effect". This major difference between workplaces and private and/or other public sites, is visible in the younger ages of patients, and the rarity of this type of event, even though the medical condition, usually better, is not reported in these studies. Some studies have tried to minimize the impact of such effects. Two authors used matched controls on age to have similar patternsfor subjects, and were considered in the meta-analysis. However, chronic illnesses were not considered, and the better survival observed is partly explained by the fact that people in workplaces generally are healthier than those in other locations, which would explain the low incidence. Nevertheless, it also is quite plausiblethat improved management of OHCA at workplace locations might be very efficient, taking into accountthe high number of years of life saved in good conditions.

Second, for business and industry, in general, the workplace is also characterized by a higher density of people working together, as with other public locations. However, specific guidelines for workplaces also result in better management of events, with first-aid, and automated external defibrillator implementation, recommended. [START_REF] Descatha | Automated external defibrillators in the workplace[END_REF][START_REF] Rudner | The effects of selected factors on survival of out-of-hospital cardiac arrest victims[END_REF] Third, specific efforts have been made by many employers and occupational health practitioners to improve working conditions and prevent, as much as possible, the occurrence of cardiac arrest (prevention of major injuries, suicide attempts related to occupational psychosocial factors, etc…). Since we were notable to find better outcomesfor workplacesthan for other public sites, improvement in prevention and OHCA management guidelines should be a priority all over the world, again considering the gainin number of years of life under good conditions [START_REF] Chamoux | Place du médecin du travail dans l'organisation des urgences en milieu de travail[END_REF] . Some limitations should be considered concerning the methodology used. First, publication bias should be discussed. Even though the meta-analysis is based on a small number of studies retained, the results of the papers included seem to be coherent and quite homogeneous, and Egger's test do not reveal a major publication bias, such as Funnel plot (results not shown). The choice of the outcomes used to express the results of the metaanalysis may also raise questions. We decided not to include returns to spontaneous circulation as an outcome, and to adopt a broad definition of treatable OHCA. However, these simplifications did allow us to group the data using broad definitions based on different criteria. Furthermore, if raw data had been accessible, better definitions of treatable OHCA would have been available, but would probably have led to higher odds ratios. Pooled meta-Analysis was not possible because some data were not accessible, even though some authors did send us raw data.

In conclusion, there issufficient evidence to support the view that there are better outcomes for OHCA cases that occur in the workplace than for those occurring elsewhere.

Requirements for occupational health and safety should include prevention of such major (albeit rare) events, by requiring first-aid and automated external defibrillators, as some countries already do [START_REF] Papaleo | Organization and management of first aid in workplaces: Critical aspects and innovations to be introduced[END_REF][START_REF] Starr | Automated external defibrillation in the occupational setting[END_REF][START_REF] Rudner | The effects of selected factors on survival of out-of-hospital cardiac arrest victims[END_REF] .Research papers should also distinguish the workplace from other public sites, with a special focus on the details of the workplace environment.

Table 2 .

 2 Summary of meta-odds ratio (Meta-OR) of studies included in the quantitative analysis (with 95% confidence interval or CI95%).

		MetaOR for admission	MetaOR for discharge alive	MetaOR for a good
		alive at hospital (CI95%),	from hospital (Number of	neurological outcome i.e.
		Number of studies	studies) (CI95%), Number of	CPC1/2 (CI95%), Number
		(related references), Q	studies, Q statistic	of studies, Q statistic
		statistic
	All OHCA	2.1 (1.3-3.2), n=3	2.4 (1.3-4.4), n=4	Not enough studies, n=1
		(4,11,15), P=0.136	(10,11,13,14), P=0.051	(15), OR=3.6 (1.5-8.6)
	All treatable OHCA	1.9 (1.5-2.3), n=4	2.4 (1.6-3.7), n=7	5.9(2.7-13.0), n=3 (3,5,15)
		(5,11,12,15), P=0.402	(3,5,10,11,13,14,16),	P=0.037
			P=0.002
	All treatable OHCA, only	2.0 (1.6-2.4), n=2(5,15),	2.9 (1.8-4.7),	5.9(2.7-13.0), n=3 (3,5,15),
	high quality studies	P=0.521	n=5(3,5,13,14,16), P=0.041	P=0.037
	included
	OHCA restricted to	Not enough studies, n=1	1.1 (0.8-1.6),	1.4(0.5-3.9), n=3 (3,5,15) ,
	workplaces or other	(5), OR=1.1 (0.8-1.4)	n=6(3,5,10,3,14,16),P=0.054	P=0.038
	public sites

CPC Cerebral performance category

Figure 2. Related Forrest plots of table 2

Acknowledgments

The authors would like to thank DrFritz Sterz and DrPhilip Eisenburger, and the RéAC study group. We also thank Richard Carter for helpingus to improve the language of this work.

Conflict of interest statement

No relevant conflicts of interest. The authors are paid for their respective affiliations. In addition, Alexis Descathahas received money for his editing work (He is Editor in chief of Archives des maladies professionnelles) from Elsevier Masson.