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Revisiting the Gregarious Lipid Droplet: Maintaining contacts to regulate energy homeostasis in the cell and beyond-Review

Contact Volume 3: 1–16 Seipin-Mediated Contacts as Gatekeepers ! The Author(s) 2020 Article reuse guidelines: of Lipid Flux at the Endoplasmic sagepub.com/journals-permissions DOI: 10.1177/2515256420945820 Reticulum–Lipid Droplet Nexus journals.sagepub.com/home/ctc

Veijo T. Salo1,2 , Maarit Holtt€ a-Vuori€ 1,2 and Elina Ikonen1,2

Abstract Lipid droplets (LDs) are dynamic cellular hubs of lipid metabolism. While LDs contact a plethora of organelles, they have the most intimate relationship with the endoplasmic reticulum (ER). Indeed, LDs are initially assembled at specialized ER subdomains, and recent work has unraveled an increasing array of proteins regulating ER-LD contacts. Among these, seipin, a highly conserved lipodystrophy protein critical for LD growth and adipogenesis, deserves special attention. Here, we review recent insights into the role of seipin in LD biogenesis and as a regulator of ER-LD contacts. These studies have also highlighted the evolving concept of ER and LDs as a functional continuum for lipid partitioning and pinpointed a role for seipin at the ER-LD nexus in controlling lipid flux between these compartments.

Keywords seipin, endoplasmic reticulum–lipid droplet contacts, lipid droplet biogenesis

Lipid droplets (LDs) are intracellular storage organelles mutations in seipin also result in hereditary spastic para- composed of a core of hydrophobic neutral lipids (NLs), plegias (Windpassinger et al., 2004) and a severe form of mainly triglycerides (TAG) and sterol esters, surrounded encephalopathy (Guillen-Navarro et al., 2013). Seipin is by a phospholipid (PL) monolayer (Henne et al., 2018). evolutionary conserved, and perturbation of seipin or its Their main function is to store excess energy in the form homologs results in aberrant LD morphology in numer- of TAGs, but LDs also play instrumental roles in many ous model systems (Szymanski et al., 2007; Fei et al., other cellular processes, including maintaining endoplas- 2008; Boutet et al., 2009; Cai et al., 2015; Salo et al., mic reticulum (ER) homeostasis (Welte, 2015; Olzmann 2016; Wang et al., 2016; Kornke and Maniak, 2017; and Carvalho, 2019). LDs are formed in the ER, and Coradetti et al., 2018; Cao et al., 2019). In accordance they retain a close physical and functional relationship with its role in lipodystrophy syndrome in humans, with it after their formation. The lipid and protein ﬂuxes seipin is essential for adipogenesis in various organisms (Payne et al., 2008; Chen et al., 2009; Victoria et al., between these compartments are controlled at ER-LD 2010; Cui et al., 2011; Tian et al., 2011; Ebihara et al., contacts. Recent studies have begun to shed light on the 2015; Mori et al., 2016). properties of LD forming ER subdomains and ER-LD contacts. In this review, we discuss recent insights into LD biogenesis and ER-LD contacts with a special focus on the lipodystrophy protein seipin. 1Department of Anatomy and Stem Cells and Metabolism Research Program, Faculty of Medicine, University of Helsinki Seipin Structure Gives Clues to Its 2Minerva Foundation Institute for Medical Research, Helsinki, Finland Function Received April 7, 2020. Revised June 6, 2020. Accepted June 30, 2020. Seipin in an oligomeric ER transmembrane (TM) pro- Corresponding Author: tein originally identiﬁed to be mutated in Berardinelli– Elina Ikonen, Department of Anatomy and Stem Cells and Metabolism Research Program, Faculty of Medicine, University of Helsinki, Helsinki, Seip Congenital Lipodistrophy Type 2 (BSCL2) congen- Finland. ital lipodystrophy (Magre et al., 2001). Distinct Email: [email protected]

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The LD phenotype in the absence of seipin is consis- Structurally seipin contains two transmembrane tent among examined systems, with a proliferation of domains, an evolutionary conserved ER luminal loop tiny and some supersized LDs in an apparently stochas- and variable N- and C-terminal cytoplasmic regions tic manner (Cartwright and Goodman, 2012; Chen and (Lundin et al., 2006; Yang et al., 2013). The luminal Goodman, 2017). Several hypotheses have been pro- loop and its flanking two transmembrane domains are posed for seipin in relation to LDs: a role in controlling critical for seipin function in LD biogenesis, as they ER PL metabolism, sequestering NLs or regulating ER- alone are sufficient to rescue defective LD formation in LD contact sites. These hypotheses are not mutually seipin-deficient cells (Yang et al., 2013; Wang et al., exclusive, and the structure of seipin is in principle com- 2016). Seipin forms homo-oligomers, each containing patible with any of these. In addition to its role in LD 11 (human) or 12 (Drosophila) seipin monomers (Binns formation, seipin has been linked to cytoskeleton remod- et al., 2010; Sim et al., 2012; Sui et al., 2018; Yan et al., eling, calcium handling, and lipolysis (Chen et al., 2012; 2018). In yeast, seipin forms foci localizing to ER-LD Bi et al., 2014; Yang et al., 2014; Ding et al., 2018; Li contact sites (Szymanski et al., 2007; Fei et al., 2008). In et al., 2019b) with a growing number of identified inter- mammalian cells, endogenously fluorescently tagged action partners (Table 1). seipin appears as homogenous, discrete, and mobile foci in the ER, a subset of which are stably associated

Table 1. Seipin Interacting Proteins.

Proposed function Protein name Protein function Main evidence for interaction of interaction Reference

Ldb16 Regulator of LD Co-ip: endogenous proteins in Forms a complex Cartwright et al. (2015), morphology yeast with seipin to Wang et al. (2014), and regulate LDs Wolinski et al. (2015) Promethin/LDAF1/ Regulator of LD iden- Co-ip: endogenous proteins Forms a complex Castro et al. (2019), Ldo45 Ldo16 tity (yeast) (yeast and human cells), with seipin to Chung et al. (2019), comigration of endogenously regulate LDs Eisenberg-Bord et al. tagged proteins (human cells) (2018), and Teixeira et al. (2018) GPAT3-4/Gat1-2 Catalyzes G3P to LPA Co-ip: endogenous proteins Seipin negatively Pagac et al. (2016) and Sim conversion (yeast), overexpressed pro- regulates GPAT et al. (2020) teins (murine cells) enzyme activity SERCA ER calcium pump, Co-ip: endogenous proteins Seipin positively Bi et al. (2014) þ transports Ca2 (human cells), overexpressed regulates SERCA into ER lumen proteins (Drosophila cells) activity Lipin-1 Catalyzes PA to DAG Co-ip and AFM: overexpressed Seipin recruits lipin Sim et al. (2012) and conversion proteins (murine cells) to the ER Talukder et al. (2015) membrane AGPAT-2 Catalyzes LPA to PA Co-ip and AFM: overexpressed Scaffolding of lipo- Sim et al. (2012) and conversion proteins (mice cells) genic enzymes Talukder et al. (2015) Serine palmitoyl- Sphingolipid synthesis Co-ip and BiFic of overexpressed Seipin negatively Su et al. (2019) transferase protein (yeast) regulates sphin- Complex, FA- golipid elongase Tsc13 production 14-3-3-b A scaffolding protein, Co-ip: overexpressed proteins Cytoskeleton Yang et al. (2014) signal transduction (murine and human cells) remodeling Perilipin-2 LD coat protein, lipol- Co-ip: overexpressed proteins Seipin regulates Mori et al. (2016) ysis regulator (human cells) perilipin-2 localization to LDs Perilipin-1 LD coat protein, lipol- Co-ip and AFM: overexpressed Seipin regulates Jiao et al. (2019) ysis regulator proteins (human cells) perilipin-1 localization to LDs Reep-1 Regulates ER Co-ip: overexpressed proteins None proposed Renvoise et al. (2016) morphology (murine cells)

ER ¼ endoplasmic reticulum; AGPAT ¼ 1-acylglycerol-3-phosphate-O-acyltransferase; GPAT ¼ Glycerol-3-phosphate acyltransferase; SERCA ¼ sarco/ þ endoplasmic reticulum Ca2 -ATPase ; FA ¼ Fatty acid; G3P ¼ Glycerol 3-phosphate; LPA ¼Lysophosphatidic acid; DAG ¼ diacylglycerol; PA ¼ phosphatidic acid; LD ¼ lipid droplet; AFM ¼ Atomic force microscopy. Salo et al. 3 with ER-LD contacts (Salo et al., 2016; Wang et al., and Thiam, 2020), these seipin helices might recognize 2016). Seipin oligomerization is critical for its function, similar packing defects induced by NL lenses in the ER as evidenced by the fact that the lipodystrophy- (Sui et al., 2018). associated A212P-seipin mutant forms smaller The bulk of the seipin luminal region forms a beta- oligomers and fails to rescue the aberrant LD biogenesis sandwich-like fold, structurally similar to many lipid- of seipin-deficient cells (Binns et al., 2010; Sim et al., binding domains, including those of Niemann-Pick C2 2013; Salo et al., 2016). The same holds true for an olig- (NPC2) and PKC (Sui et al., 2018; Yan et al., 2018).This omerization deficient mutant designed based on the suggests that seipin may bind lipids in the ER lumen. In cryo-electron microscopy (EM) structure of seipin vitro work suggests that both purified full-length seipin (Yan et al., 2018). and a truncated variant containing only the putative The structures of the luminal domain of human and lipid binding domain can bind anionic PLs, such as Drosophila seipins have been solved by cryo-EM (Sui phosphatidic acid (PA; Sui et al., 2018). A mutant har- et al., 2018; Yan et al., 2018; Figure 1). They form a boring amino acid substitutions in the hydrophobic ring-like/disk-like structure of circa 15 nm in diameter, cavity of the beta sandwich was unable to complement with 11 (human) or 12 (Drosophila) seipin monomers the seipin knockout (KO) LD phenotype (Sui et al., closely intertwined. Each monomer displays two notable 2018). However, which lipids seipin interacts with in features: several hydrophobic helices (HHs) in very close intact cells remains to be determined. proximity to the bilayer membrane, and two beta sheets that exhibit an overall fold similar to certain lipid bind- Seipin Forms an LD Assembly Complex ing domains (Sui et al., 2018; Yan et al., 2018). The structure of the TM or cytoplasmic regions could not The most extensively studied binding partners of seipin be solved by cryo-EM in these studies, suggesting con- are Ldb16 and Ldo16 and its splicing isoform Ldo 45/ formational structural flexibility therein. promethin (Table 1). In yeast, seipin forms a stable com- The ER luminal HHs of seipin are able to bind to a plex with Ldb16, an ER TM protein with no known monolayer covering NLs in vitro (Sui et al., 2018) and mammalian homologues.Seipin is required for Ldb16 sta- appear to be widely conserved across species (Chapman bilization, and depletion of either Ldb16 or seipin results et al., 2019). When expressed as individual peptides in in indistinguishable LD phenotypes (Wang et al., 2014; cells, the HHs localize to LDs. This localization is Grippa et al., 2015). These LD defects can be rescued by impaired by mutating three hydrophobic residues to heterologous expression of human seipin, strongly sug- aspartic acid (Sui et al., 2018). A mutant seipin with gesting that human seipin functionally corresponds to corresponding amino acid substitutions still rescues the yeast Ldb16-seipin complex. seipin function in LD biogenesis, but further concomi- Yeast ER-LD-localized protein isoforms Ldo16 and tant deletion of the N-terminus of seipin, shown previ- Ldo45 were recently discovered to be interactors of the ously to localize to LDs when expressed alone (Wang seipin complex (Eisenberg-Bord et al., 2018; Teixeira et al., 2016), results in a dysfunctional seipin. Hence, et al., 2018). Overexpression of Ldo45 leads to prolifer- the presence of either LD-targeting luminal HHs or the ation and clustering of LDs that exhibit aberrant protein cytoplasmic N-terminus is required for seipin function. targeting. This phenotype is similar to seipin KO cells, Considering that the LD PL monolayer harbors packing suggesting that Ldo45 and seipin may play antagonistic defects (Bacle et al., 2017; Prevost et al., 2018; Chorlay roles in LD formation and maintenance (Eisenberg-Bord

Top view Side view

Cytosol Membrane- associated helices 160 Å

Transmembrane helices ER lumen Beta-sheets

Figure 1. Seipin Oligomer Structure. The TM regions were modeled into the cryo-EM structure of the luminal domains of human seipin (PDB: 6ds5; Yan et al., 2018) using PyMol software. ER ¼ endoplasmic reticulum. 4 Contact et al., 2018; Teixeira et al., 2018). Ldo16 and Ldo45 were surface tension and PL intrinsic curvature, while the also found to be regulators of a subpopulation of LDs directionality of LD budding to the cytosol may arise that reside in close proximity to the yeast nucleus-vacu- due to ER membrane asymmetry (Ben M’barek et al., ole junctions (NVJs).During nutritional stress, this site 2017; Choudhary et al., 2018; Chorlay et al., 2019; Zoni expands and serves as a site for LD biogenesis under et al., 2020). It therefore seems likely that the LD form- these conditions (Hariri et al., 2018).Indeed, the relative ing ER subdomain harbors a distinct PL milieu, abundance of Ldo16/Ldo45 is regulated by cellular although direct evidence for this is lacking. growth conditions, and Ldo45 is necessary for recruiting LD formation may also be controlled by the overall Pdr16, a lipid transfer protein, to NVJ-associated LDs ER architecture. LD biogenesis appears to occur at (Eisenberg-Bord et al., 2018; Teixeira et al., 2018). peripheral ER tubules in mammalian cells (Kassan Based on remote homology searches, promethin (also et al., 2013; Joshi et al., 2018; Santinho et al., 2020). In known asTMEM159 and recently renamed as Lipid accordance, many ER shaping proteins have been impli- Droplet Assembly Factor 1; LDAF1 ) was proposed as cated in LD formation (Klemm et al., 2013; Falk et al., a homolog of Ldo45 (Eisenberg-Bord et al., 2018). 2014; Papadopoulos et al., 2015). Conversely, proteins Promethin is upregulated during adipogenesis, localizes participating in LD biogenesis such as Rab18 and fat to the ER and LDs, and was found to interact with storage-inducing transmembrane 2 (FIT2) have been seipin (Castro et al., 2019). When expressed heterolo- shown to impact ER morphology (Gerondopoulos gously in yeast, human promethin was able to co- et al., 2014; Hayes et al., 2017; Becuwe et al., 2018). immunoprecipitate the yeast seipin complex, suggesting Changes in ER ultrastructure have been observed also an evolutionary conserved interaction (Castro et al., in seipin-deficient cells (Grippa et al., 2015; Salo et al., 2019). In human cells, endogenously tagged promethin 2016), and many proposed seipin collaborators, such as forms distinct foci in the ER and a high fraction of these Pex30/MCTP2, Ldo45/Promethin, Reep-1, and Aradopsis comigrate with seipin foci (Chung et al., 2019). LDs were LDIP (Table 1), have been suggested to act as ER curva- found to be formed at sites typically occupied by both ture sensors/inducers (Renvoise et al., 2016; Pyc et al., seipin and promethin. Upon subsequent growth of the 2017; Eisenberg-Bord et al., 2018; Joshi et al., 2018; LDs, promethin relocates to LD surface, due to hairpin Teixeira et al., 2018; Wang et al., 2018; Chung et al., topology allowing both ER and LD localization, while 2019). seipin remains at the ER-LD junction (Chung et al., The relationship between ER morphology and LD 2019). nucleation was recently investigated (Santinho et al., 2020). LDs were found to be preferentially generated ER Architecture Promoting LD Formation at ER tubules both in the presence and absence of LD biogenesis is a complex stepwise process in the ER seipin. Experiments in cells and model membranes sug- (Pol et al., 2014). Here, only a brief description of the gested that the presence of free TAG in highly curved currently prevailing model of LD biogenesis is presented. membranes (such as ER tubules) is energetically unfa- For detailed discussion, we kindly refer the reader to vorable compared with flat regions (such as ER sheets). recent comprehensive reviews (Chen and Goodman, This leads to either outflow of TAGs from tubules or 2017; Walther et al., 2017; Chapman et al., 2019; Gao their condensation into LDs. Accordingly, LD nucle- et al., 2019b; Jackson, 2019; Henne et al., 2020; Renne ation can be achieved in vitro by increasing membrane et al., 2020). curvature. Together, these data suggest that the During LD assembly, TAGs synthesized by ER- ER membrane curvature can catalyze LD assembly resident diacylglycerol acyltransferase (DGAT) enzymes (Santinho et al., 2020). first accumulate between the ER bilayer leaflets. A major challenge in deciphering the dynamics and However, as PL bilayers can only accommodate kinetics of LD biogenesis is the small size and possible minute concentrations of TAG, rising local concentra- instability of the formed NL clusters. Indeed, while 40- tions will lead to the formation of nm-sized TAG clus- to 60-nm sized lens-like ER-embedded structures were ters through nucleation and phase separation. These detected in yeast (Choudhary et al., 2015), LD-like struc- form lens-like structures as predicted by simulations tures in the size range of 30 nm were observed at and observed in yeast by EM (Khandelia et al., 2010; seipin-marked sites in human A431 cells during LD Choudhary et al., 2015). Subsequent growth of TAG assembly (Salo et al., 2019).Circa 40 to 50 nm vesicular aggregates will lead to deformation of the ER bilayer structures marked by LiveDrop were detected in COS-7 and budding out of a nascent LD, with the monolayer cells (Li et al., 2019a), and LDs in this size range enclosing the LD derived from the cytoplasmic leaflet of (although not necessarily made de novo) were also the ER (Kassan et al., 2013). These early steps are mod- recently observed by cryo-electron tomography ulated by the ER PL composition via effects on ER (Mahamid et al., 2019).Thus, the size limit of the Salo et al. 5 smallest forming LDs is yet unknown and may vary phenotype, arguing for more redundancy in mammalian between cell types. systems (Joshi et al., 2018). Seipin might control ER PL metabolism, especially Seipin Defines LD Formation Sites that of PA, via protein–protein interactions of major lipid synthesizing enzymes, such as AGPAT2, lipin-1, Several lines of evidence suggest that seipin is a key and GPATs (Sim et al., 2012; Talukder et al., 2015; player in early LD biogenesis and may define the ER Pagac et al., 2016; Gao et al., 2019b; Sim et al., 2020). subdomain primed for LD formation. Seipin foci are Indeed, GPAT activity was altered in a number of seipin localized at LD forming sites in the ER during the ear- depleted systems, suggesting that seipin negatively regu- liest observable steps of LD biogenesis (Wang et al., lates GPAT activity to inhibit aberrant expansion of 2016; Salo et al., 2019; Choudhary et al., 2020). LDs (Pagac et al., 2016). In line with this, defective adi- Fluorescently labeled model ER-LD peptides harboring pogenesis due to seipin-depletion was partially restored LD-targeting motifs, such as HPos and LiveDrop (Wang by GPAT inhibition (Pagac et al., 2016; Gao et al., et al., 2016), have been used to visualize nascent LDs 2020). However, in a recent study, GPAT3 deficiency prior to their detection with traditional hydrophobic failed to rescue adipogenesis in seipin-deficient differen- dyes such as BODIPY. In our hands, the bright lipophilic tiating adipocytes (Sim et al., 2020). Based on these find- dye LD540 (Spandl et al., 2009) stains nascent LDs at ings, the significance of seipin-GPAT interaction least as early as HPos or LiveDrop. However, it is not remains to be determined, but increasing evidence sug- clear whether any of these markers detect lenses or only gests that seipin can act as a scaffold for lipogenic budded-out LDs. enzymes. In A431 cells, seipin motility was found to be In the absence of seipin, PA has been suggested to decreased prior to accumulation of LD540, LiveDrop, accumulate in the ER and play an inhibitory role on or endogenously tagged acyl-CoA synthetase 3 (ACSL3) adipogenic transcriptional program (Liu et al., 2014; (Salo et al., 2019), suggesting that seipin is stabilized at Gao et al., 2019b). This notion is supported by the LD-forming sites prior to other known actors. Similar observation of increased PA levels in several seipin results were reported in SUM159 cells, where LDs depleted systems (Fei et al., 2011; Tian et al., 2011; marked by endogenously tagged Perilipin-3 emerged at Jiang et al., 2014; Liu et al., 2014; Pagac et al., 2016; seipin-defined sites (Chung et al., 2019). Seipin can also Cao et al., 2019). However, other studies have failed to spatially define LD formation sites in the ER. detect differences in cellular PA levels even in the pres- Relocalization of seipin to a subdomain of the ER, the ence of a robust LD phenotype, arguing that global PA nuclear envelope, was sufficient to relocate LD biogen- handling may not be the culprit in seipin deficiency esis to this new site (Salo et al., 2019). Analogous results (Grippa et al., 2015; Wang et al., 2016). Rather, seipin were reported when seipin was relocalized to ER-plasma might alter PA metabolism at a distinct subdomain of membrane junctions (Chung et al., 2019). These obser- the ER. This would be in line with the observation that vations not only demonstrate that seipin can define PA-sensing fluorescent probes accumulate at LD- where a LD starts to develop but also suggest consider- associated ER foci in yeast seipin KO cells, suggesting able spatial malleability in LD formation sites. localized PA accumulation (Grippa et al., 2015; Han et al., 2015; Wolinski et al., 2015). Seipin and Regulation of ER PL Metabolism In this regard, the putative PA-binding site of seipin is in the ER lumen (Yan et al., 2018) proposes a topolog- Local PL levels, especially those of PA and DAG, have ical puzzle. While at least some of the PA-generating been proposed to be vital for LD emergence (Adeyo GPAT and AGPAT enzymes may have their active et al., 2011; Cartwright et al., 2015; Wolinski et al., sites in the ER lumen or within the ER bilayer 2015; Ben M’barek et al., 2017; Choudhary et al., (Yamashita et al., 2014), the PA-utilizing lipin enzymes 2018). It is conceivable that seipin somehow directly are soluble, cytosolic proteins (Zhang and Reue, 2017), modulates local PLs at LD forming sites (Yan et al., which would necessitate PA flipping to access seipin. 2018). In line with this, LD budding is almost completely Intriguingly, there are indications that FIT2 might act abolished in a yeast mutant harboring double deletion of as a lipid phosphate phosphatase in the ER luminal side seipin and a putative ER-shaping protein Pex30, result- (Hayes et al., 2017; Becuwe et al., 2018) and that the ing in accumulation of toxic levels of TAG in the ER protein is localized at seipin-marked LD formation (Wang et al., 2018). Moreover, this phenotype was par- sites (Choudhary et al., 2020). In this scenario, seipin tially restored by modulating the ER PL composition might function to trap PA to promote its efficient catal- toward permissive for LD budding. However, codeple- ysis into DAG at the ER-LD contact. However, this tion of seipin and Pex30’s putative mammalian homo- view is challenged by the lack of robust defects in lipid logue, MCTP2 in human cells did not display the same biosynthesis upon acute seipin loss (Wang et al., 2016; 6 Contact

Salo et al., 2019) and that PA appears to locally accu- formation, but promethin may aid seipin in decreasing mulate in some seipin-deficient systems. the barrier for LD formation. Therefore, in the absence Besides PA, studies have also linked seipin to sphin- of promethin, as LDs are assembled by seipin alone, the golipid metabolism and defects in acyl chain saturation flux to these LDs keeps the ER TAG concentration low (Boutet et al., 2009; Amine et al., 2017; Su et al., 2019). enough to prevent aberrant LD formation, but not high Further work is needed to define how these relate to enough to trigger the assembly of new LDs by seipin. This the LD biogenesis and adipogenesis defects of seipin would then lead to the formation of fewer but larger LDs. loss. The recent discovery that polyunsaturated fatty acids were found to facilitate seipin recruitment to Morphology of Seipin-Mediated ER-LD ER-LD contacts in C. elegans is highly interesting in this regard (Cao et al., 2019). Contacts After their formation, LDs remain functionally con- Seipin May Sequester TAGs at LD nected to the ER via ER-LD contacts, which function to facilitate cargo exchange between the two organ- Formation Sites elles. Morphologically, ER-LD contacts can be broad- Emerging studies suggest that seipin may directly control ly categorized into two overlapping classes: membrane TAG distribution in the ER and its partitioning into nascent bridges, wherein the LD monolayer appears continu- LDs. In a yeast system where NL synthesis was held con- ous with the ER bilayer; and areas of close ER-LD stant, LD biogenesis was severely delayed in seipin-deficient proximity without direct membrane continuity cells with a relative accumulation of TAG in ER mem- (Figure 2A and B; Salo and Ikonen, 2019). In addition, branes (Cartwright et al., 2015). Similarly, upon LD induc- proteinaceous tethers between the two organelles have tion in mammalian cells in the absence of seipin, numerous also been observed by EM (Wang et al., 2016; Salo tiny and growth-abortive LDs emerged from the ER, with a et al., 2019; Figure 2C). Although the functional sig- subset of these completely detaching from the ER, a phe- nificance of these different contact zones is not yet nomenon not observed in wild-type cells (Salo et al., 2016; understood, seipin appears to be primarily involved Wang et al., 2016). These tiny LDs may reflect spontaneous in the formation and maintenance of the membrane phase separation or oiling out of excess TAG, as has been bridges between ER and LDs. postulated to occur in protein-free systems (Deslandes et al., Considering the persistent localization of seipin at 2017). Thus, seipin could function to attract and foster ER-LD contact sites (Salo et al., 2016; Wang et al., TAG aggregates in the ER for controlled LD formation. 2016), the morphological features of seipin-mediated How could seipin sequester TAG at LD formation ER-LD contacts were recently characterized. Using sites? The membrane-anchored HHs may be important correlative light and EM, seipin-mediated contacts for this, as they have affinity for a monolayer enclosing were found to harbor a strikingly uniform, neck-like NLs (Sui et al., 2018). Furthermore, seipin appears to architecture (Salo et al., 2019). At these ER-LD control TAG condensation in the ER (Santinho et al., necks, the LD monolayer appeared to fuse with the 2020) and copurified seipin-promethin complexes con- ER lumen, indicating membrane continuity between tained TAG molecules (Chung et al., 2019). Therefore, the outer leaflet of the ER and the LD monolayer seipin may initially attract TAG molecules in the ER (Figure 2C and D). The diameter of the region in bilayer and foster TAG aggregates to grow into a nascent touch with the LD was circa 15 nm, a size consistent LD. This is in line with the recently reported DGAT1 with the structure of oligomeric seipin resolved with the structure placing the active TAG-generating site within cryo-EM (Sui et al., 2018; Yan et al., 2018). This sug- the ER bilayer (Sui et al., 2020; Wang et al., 2020). In gests that seipin may physically limit the diameter of the absence of seipin, efficient TAG phase separation in the ER-LD neck. High curvature of the neck implicates the ER would be delayed, leading to an increase in the ER a specific PL composition, and the lipid-binding TAG concentration, as reported (Cartwright et al., 2015; domain of seipin might be involved in stabilizing the Gao et al., 2017). This increased bilayer TAG concentra- neck. Indeed, such seipin-mediated ER-LD necks were tion eventually leads to the aberrant oiling out of LDs. disrupted by acute removal of seipin via auxin-induced The precise role of promethin in this scenario needs to degradation (Salo et al., 2019). be established, as such oiling out of LDs was not observed A morphologically similar ER-LD contact of circa in promethin KO cells. Instead, promethin KO cells gen- 15 nm in diameter, with apparent membrane continuity, erated fewer, but larger LDs, which still contained seipin was also recently observed by cryo-EM tomography of at their ER-LD contact (Chung et al., 2019). This may HeLa cells (Figure 3E of Mahamid et al., 2019) and suggest that seipin is de facto required to keep ER TAG similar neck-like structures were previously observed concentration low enough to prevent aberrant LD in, for example, Drosophila and Cos-1 cells (Kassan Salo et al. 7

A ER-LD contact topologies C Seipin NE-trapped cells

inside view 20000 nmnm side view B ER-LD contact examples D CLEM ()end-seipin-GFPx7 / LiveDrop-mCherry

inside view side view

Figure 2. Seipin-Mediated ER-LD Contacts. A: Topologies of ER-LD contacts. Left panel: membranous bridges, mediated by seipin, right panel: zones of ER-LD proximity, likely mediated by various ER-LD tethers. B: Examples of ER-LD contacts from primary human fibroblasts. Left panel is adapted from Salo et al. (2019) with permission of the publisher, and right panel is from the same data set. Scale bar: 50 nm. Orange arrowhead indicates neck-like membranous bridge. C and D: Examples of the architecture of seipin mediated ER-LD contacts, adapted from Salo et al. (2019) with permission of the publisher. Scale bars for C: 100 nm, 50 nm, 20 nm, and 20 nm. Scale bars for D: 10 mm, 50 nm, 50 nm and 20 nm. C: Examplary neck-like ER-LD contact from A431 cells with seipin trapped at the NE ER subdomain. Orange arrowhead indicates neck-like membranous bridge, blue arrowheads indicate fibrous tethers. Right panel is 3D modeling of the LD (brown), NE (blue), and NE-LD contact site (red). D: CLEM of A431 cells with seipin tagged endogenously with GFPx7 and expressing the nascent LD marker LiveDrop-mCherry. The inset of the fluorescence microscopy panel (left) depicts the same LD in the same orientation as the EM images. Orange arrowhead indicates neck-like membranous bridge. Right panel depicts 3D modeling of the LD (brown), ER (yellow) and the ER-LD contact site (red). ER ¼ endoplasmic reticulum; LD ¼ lipid droplet; NE ¼ nuclear envelope; CLEM ¼ correlative light and electron microscopy.

A Seipin antibody B Diﬀerentiated human adipocyte stem cells WT anti-seipin Projection Lipidtox (LDs)

Seipin KO Projection Projection Projection Single slice

Figure 3. Seipin in Human Adipocytes. A: Confocal images of human A431 WT and seipin KO cells stained with anti-seipin antibodies (Salo et al., 2019). Scale bar: 10 mm. B: Airyscan images of human adipocytes derived from visceral fat stem cells. Adipogenic differentiation was induced by a cocktail of transferrin (10 mg/ml final concentration), human insulin (Actrapid; 66 nM), cortisol (100 nM), triiodothyronine (1 nM), 3-isobutyl-1-methylxanthine (500 mM), and rosiglitazone (2 mM). After 3 days, supplements were reduced to transferrin, insulin, cortisol, and triiodothyronine with the aforementioned concentrations. After 14 days of adipogenic differentiation, cells were fixed and stained with anti-seipin antibodies followed by anti-rabbit Alexa 647 and LipidTox Green. Scale bars: 10 mm and 1 mm. LD ¼ lipid droplet; KO ¼ knockout; WT ¼wild type. et al., 2013; Wilfling et al., 2013). Membranous bridges data suggest that seipin localizes to and controls the between inner nuclear membrane and intranuclear LDs formation of such bridges. This would be a logical con- in yeast cells were also reported to be seipin-dependent sequence of early LD biogenesis occurring at seipin- (Romanauska and Kohler,€ 2018). Altogether, these marked sites: if the initial LD budding event were to 8 Contact take place through the seipin disk, this would constrain DGAT2 (LD; Xu et al., 2012); Rab18 (LD) and the the dimensions of the ER-LD neck accordingly. NRZ complex and associated SNARE proteins (ER; In nonadipogenic cell lines studied so far, LDs typi- Xu et al., 2018); and Orp2 (LD) and VAPA (ER; cally harbor a single distinct seipin focus stably associ- Weber-Boyvat et al., 2015).Snx14 is also capable of teth- ated with each LD (Salo et al., 2016; Wang et al., 2016) ering LDs to the ER, by binding to both organelles and seipin-mediated ER-LD necks appear to be long- simultaneously (Datta et al., 2019) and a similar topo- lasting structures (stable at least on timescale of hours; logical arrangement has been proposed for DGAT2 Salo et al., 2019). Moreover, seipin recruitment to pre- (McFie et al., 2018).DFCP1 has also been documented existing LDs has not been reported. These data imply to facilitate ER-LD contact formation, acting in concert that seipin does not act like a canonical membrane con- with Rab18 (Gao et al., 2019a; Li et al., 2019a). In yeast tact site tether but might rather remain at the topologi- cells, Pex30 and the yeast homolog of FIT2 localize to cally unique ER-LD contact site via virtue of LD ER-LD contacts, colocalizing with seipin (Choudhary biogenesis occurring through the seipin disk. et al., 2018; Joshi et al., 2018; Wang et al., 2018). There may, however, be species–specific differences, Newly discovered ER-LD contact site proteins with as overexpressed C. elegans seipin was shown to form putative lipid transfer activity include Orp4, VPS13A, much larger (several hundreds of nanometers in diame- and VPS13C (Kumar et al., 2018; Du et al., 2020). ter) peri-LD cages at sites where the tubular elements of At least in the case of Rab18, Snx14, DFCP1, and the ER are in close proximity to the LD (Cao et al., VPS13A, overexpression has been shown to increase 2019). Overexpression of C. elegans seipin promoted ER-LD proximity, strongly suggesting that these factors the formation of such structures also in Cos-7 cells can facilitate tethering of the two organelles (Kumar et al., and, conversely, heterologously expressed human 2018; Xu et al., 2018; Datta et al., 2019; Li et al., 2019a). seipin localized similarly in tubular peri-LD cages in the The topology of these expanded ER-LD contact zones is C. elegans intestine. Such structures have not been probably not similar to seipin-mediated ER-LD necks but rather reflects a close proximity of the two organelles reported upon overexpression of human or Drosophila without direct membrane continuity (Figure 2A). seipin in nonadipogenic cell lines (Salo et al., 2016; However, these factors are still likely to influence/modu- Wang et al., 2016), but seipin peri-LD cages do resemble late seipin-mediated ER-LD contacts. For example, the localization of overexpressed N-glycosylation defective DFCP1 or Rab18 perturbation decreased ER-LD prox- N88S-seipin mutant, which also accumulates as ring-like imities in seipin depleted Cos-7 cells (Li et al., 2019a). structures flanking LDs (Holtt€ a-Vuorietal.,2013).€ DFCP1 and Rab18 were proposed to form a complex An intriguing possibility is that the peri-LD cages of and were shown to co-immunoprecipitate seipin, suggest- C. elegans intestine, a major fat deposit in these organ- ing that seipin may participate in modulation of isms, are a consequence of LD fusion where individual DFCP1Rab18-mediated ER-LD contacts. The phenotype LDs, initially harboring one seipin-mediated neck, fuse of DFCP1 or Rab18 depletion, however, is distinct from and the resulting structure would contain multiple such that of seipin loss; overall, LD sizes were moderately contact sites in very close proximity. One could envision reduced and LD numbers were increased. However, this to be similar to the case in adipocytes, where a single Rab18 and DFCP1 seem to have cell-type specific func- unilocular LD forms via ripening-mediated coalescence tions, as their perturbation can also induce supersized of a high number of smaller ER-derived LDs (Paar et al., LDs in some cell types such as 3T3-L1 cells, while only 2012; Chu et al., 2014). These giant LDs could thus con- minor alterations in LDs are observed in others (Jayson tain a high number of seipin-mediated contacts. Indeed, et al., 2018; Xu et al., 2018; Li et al., 2019a). Clearly, our immunofluorescence analysis of endogenous seipin further investigations into the interplay between Rab18, localization in differentiated human adipocyte stem cells DFCP1, and seipin are warranted. suggests that this is the case, with numerous seipin foci DFCP1 has also been implicated in early LD juxtaposed to enlarged LDs (Figure 3). Detailed charac- biogenesis. Overexpressed DFCP1 labeled distinct terization of seipin-mediated ER-LD contacts at the EM ER-associated foci that increased in number upon fatty level in adipocytes has not been reported. acid administration (Li et al., 2019a). A subset of these developed into LDs, but DFCP1 labeling preceded that of Modulation of Seipin-Mediated ER-LD early LD marker peptides HPos or LiveDrop, suggesting Contacts by Other Proteins that DFCP1 may detect very early NL accumulations. By EM, these DFCP1 foci appeared as small vesicles or ER- Besides seipin, a number of other proteins act at ER-LD associated papillary structures. Whether seipin localizes contacts (Bohnert, 2020). These include tethering protein to these DFCP1 positive regions prior to their maturation complexes, where one partner binds to the ER while the into HPos or LiveDrop-positive organelles is an interest- other contacts the LD, such as FATP1 (ER) and ing question not yet addressed. Salo et al. 9

The interplay of Snx14 and seipin at ER-LD contacts bigger ones through a connecting phase and wherein was also recently investigated (Datta et al., 2019). LDs the directionality is governed by the higher internal pres- were found to be supersized and tiny in Snx14 KO U2OS sure of smaller droplets, imposing a flux of molecules cells, resembling the LD phenotype upon seipin loss. toward lower pressure, that is, bigger droplets (Thiam Overexpressed Snx14 dramatically increased LD-ER et al., 2013; Thiam and Foreˆt, 2016). proximity and increased TAG generation, suggesting Ostwald ripening-mediated fusion is a well-known that Snx14, similarly to its proposed yeast counterpart property of liquid-liquid phase-separated systems. A Mdm1, may regulate fatty acid homeostasis (Datta vast number of recent work has begun to elucidate the et al., 2019; Hariri et al., 2019). Conversely, loss of importance of liquid–liquid phase-separated in organiza- Snx14 decreased LD-ER proximity and Snx14 was thus tion of the cellular milieu, especially with regard to mem- proposed to function as an ER-LD tether, ensuring that braneless organelles (Brangwynne et al., 2009, 2009; the two organelles maintain a connection as ER-produced Alberti et al., 2019), but also in the context of ER- TAG is fluxed into the maturing LD. However, seipin derived organelle biogenesis such as autophagosome for- overexpression failed to rescue the LD phenotype in mation (Fujioka et al., 2020).Ripening-mediated growth Snx14 KO cells and vice versa, and concomitant depletion of LDs via direct LD-LD contacts has also been docu- of both proteins did not aggravate the LD phenotypes. mented (Gong et al., 2011; Ju¨ ngst et al., 2013). At LD- The authors therefore concluded Snx14 to function inde- LD interfaces, a larger LD grows via acquisition of lipids pendently of seipin (Datta et al., 2019). On the other from the smaller LD in a process facilitated by Fsp27/ hand, considering the different topologies of Snx14 and CIDE-C (Gong et al., 2011). Interestingly, mutations in seipin-mediated ER-LD contacts (Snx14 tethering the Fsp27/CIDE-C also lead to congenital lipodystrophy organelles in trans and seipin mediating an ER-LD (Rubio-Cabezas et al., 2009) and CIDE family members neck with membrane continuity), Snx14 might aid in are regulators of adipogenesis (Slayton et al., 2019). the stabilization of seipin-mediated ER-LD necks, espe- However, in contrast to the Fsp27-mediated process, cially during later stages of LD maturation. Indeed, ripening-mediated LD size changes upon acute seipin Snx14 localized to LDs later than seipin, hours after ini- removal take place via the ER bilayer (Salo et al., tiation of LD biogenesis (Datta et al., 2019). 2019). To decipher the role of seipin in this process, heterologous cell fusion experiments were conducted, Seipin-Mediated Lipid Flux to LDs revealing that in a continuous ER network, LDs with seipin at their ER-LD contact site grew, while LDs with The persistent localization of seipin at ER-LD contacts seipin removed from their ER-LD contact shrunk. These and a defined architecture of seipin-mediated ER-LD data imply that seipin at the ER-LD neck facilitates the contacts suggests that seipin has a role in LDs beyond growth of the LD it is associated with (Figure 4A and B). facilitating initial LD assembly. In support of this, deple- This function is critical for preventing the shrinkage of tion of seipin from mature adipocytes in vivo results in smaller LDs (more prone to ripening) and facilitating progressive lipodystrophy and decreased adiposity (Liu their growth (Salo et al., 2019). Recent studies of C. et al., 2014; Zhou et al., 2015). However, due to the elegans seipin also implicate a droplet-autonomous role dramatic effects on initial LD biogenesis in the absence of seipin in promoting LD growth (Cao et al., 2019). of seipin, investigating its role in ER-LD contacts at How is seipin performing this LD growth promoting later stages has been challenging. To overcome this function? Considering the topological arrangement at obstacle, the plant-based auxin-inducible degron seipin-mediated ER-LD necks, an intriguing possibility system (Nishimura et al., 2009; Li et al., 2019c) was is that seipin controls lipid diffusion, possibly by attract- recently utilized to rapidly deplete seipin in human ing TAG molecules from within the ER bilayer and facil- cells. With this system seipin could be acutely removed itating their flux to the LD and/or by controlling PL from preexisting ER-LD contacts, revealing a role for density at the LD surface (Grippa et al., 2015; Salo seipin in LD maintenance (Salo et al., 2019). et al., 2019). Upon acute seipin removal, ER-LD lipid Upon acute seipin removal, LDs rapidly started to flux becomes dominated by biophysical forces favoring become more heterogeneous in size, with larger LDs the growth of larger LDs over smaller ones. In essence, growing and acquiring more lipid cargo from the ER, seipin would thus function as a lipid transfer protein. and smaller LDs shrinking, apparently donating their This is well in line with the proposed role of seipin cargo to the larger LDs. This occurred in the absence during initial LD assembly. of major alterations to overall net fatty acid handling or lipolysis (Salo et al., 2019). The observed LD size changes can be accounted for by a phenomenon called The ER-LD Network as a Joint System Ostwald ripening. It is a molecular diffusion process by The notion that LDs may ripen via the connecting lipid which smaller droplets continuously leak material to phase, the ER bilayer, implies that LDs and ER can be 10 Contact

A Seipin mediated TAG ﬂux at the ER-LD contact Both growing Seipin LD growing Bigger growing, smaller shrinking

B Seipin mediated TAG ﬂux and ripening in the ER network

Seipin facilitating Seipin removed: Additional tiny LDs TAG ﬂux to LDs ripening mediated emerge as ER TAG heterogeneous concentration rises growth

C The eﬀect of pre-existing LDs on newly emerging LDs

TAG ﬂux

ER TAG concentration

New LD

Figure 4. Seipin-Mediated TAG Partitioning. A: Schematic of seipin (green ellipse) function in promoting LD growth, adapted from Salo et al.(2019). B: Seipin removal evokes ripening-mediated LD size changes via the ER. C: The effect of preexisting LDs on nascent LD formation sites: seipin-mediated TAG flux to LDs decreases the nearby TAG concentration in the ER, and thus new LDs tend to be formed at sites distant to preexisting LDs. ER ¼ endoplasmic reticulum; LD ¼ lipid droplet; TAG ¼ triglycerides. Salo et al. 11 considered as a joint system. This continuity is evident in providing human visceral fat biopsies and Dr. Xavier the case of many proteins, typically harboring hairpin Prasanna for providing the structures for Figure 1. We motifs, which exhibit dual localization on ER and LDs acknowledge the use of HiLIFE and Biocenter Finland imag- (Kory et al., 2016). However, also NL fluxes through the ing core facilities (Biomedicum Imaging Unit and Electron connecting ER bilayer appear to contribute to LD-LD Microscopy Unit). Figures 1A, 2A and 3C were generated communication. For example, during early LD biogen- with the help of BioRender.com. esis, NLs are likely synthesized at highly dispersed sites along the ER (Poppelreuther et al., 2018). Upon Declaration of Conflicting Interests increased local concentrations, a NL lens may nucleate The author(s) declared no potential conflicts of interest at a site marked by seipin and start to grow. The flux of with respect to the research, authorship, and/or publication NL to the growing LD, enhanced by seipin, may thus of this article. reduce nearby TAG concentration in the ER, disfavoring the formation of other LDs nearby. Thus, a preexisting Funding LD would inhibit nearby LD formation (Figure 4C). The author(s) disclosed receipt of the following financial sup- As a consequence, LDs would be expected to form at port for the research, authorship, and/or publication of this sites more distant to one another than expected by article: This work was supported by Sigrid Juseliuksen chance, which is the case at least in human A431 cells Sa€ati€ o,€ Jane ja Aatos Erkon Sa€ati€ o,€ Helsingin Yliopisto, and (Salo et al., 2019). Indeed, nascent LDs are formed at Academy of Finland (grant no. 307415 and 312491). highly dispersed sites in multiple cell types (Kassan et al., 2013; Chung et al., 2019). ORCID iDs While direct observation of NL fluxes within the ER Veijo T. Salo https://orcid.org/0000-0002-6991-2142 is technically challenging, rapid advances in imaging and Maarit Holtt€ a-Vuori€ https://orcid.org/0000-0002-0488-4486 probe technologies (Valm et al., 2017; Adhikari et al., 2019) are likely to facilitate this in the near future. 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