Zooming into lipid droplet biology through the lens of electron microscopy.

Electron microscopy (EM), in its various flavors, has significantly contributed to our understanding of lipid droplets (LD) as central organelles in cellular metabolism. For example, EM has illuminated that LDs, in contrast to all other cellular organelles, are uniquely enclosed by a single phospholipid monolayer, revealed the architecture of LD contact sites with different organelles, and provided near-atomic resolution maps of key enzymes that regulate neutral lipid biosynthesis and LD biogenesis. In this review, we first provide a brief history of pivotal findings in LD biology unveiled through the lens of an electron microscope. We describe the main EM techniques used in the context of LD research and discuss their current capabilities and limitations, thereby providing a foundation for utilizing suitable EM methodology to address LD-related questions with sufficient level of structural preservation, detail, and resolution. Finally, we highlight examples where EM has recently been and is expected to be instrumental in expanding the frontiers of LD biology.

Genetically encoded multimeric tags for subcellular protein localization in cryo-EM.

Cryo-electron tomography (cryo-ET) allows for label-free high-resolution imaging of macromolecular assemblies in their native cellular context. However, the localization of macromolecules of interest in tomographic volumes can be challenging. Here we present a ligand-inducible labeling strategy for intracellular proteins based on fluorescent, 25-nm-sized, genetically encoded multimeric particles (GEMs). The particles exhibit recognizable structural signatures, enabling their automated detection in cryo-ET data by convolutional neural networks. The coupling of GEMs to green fluorescent protein-tagged macromolecules of interest is triggered by addition of a small-molecule ligand, allowing for time-controlled labeling to minimize disturbance to native protein function. We demonstrate the applicability of GEMs for subcellular-level localization of endogenous and overexpressed proteins across different organelles in human cells using cryo-correlative fluorescence and cryo-ET imaging. We describe means for quantifying labeling specificity and efficiency, and for systematic optimization for rare and abundant protein targets, with emphasis on assessing the potential effects of labeling on protein function.

ATPase activity of DFCP1 controls selective autophagy.

Cellular homeostasis is governed by removal of damaged organelles and protein aggregates by selective autophagy mediated by cargo adaptors such as p62/SQSTM1. Autophagosomes can assemble in specialized cup-shaped regions of the endoplasmic reticulum (ER) known as omegasomes, which are characterized by the presence of the ER protein DFCP1/ZFYVE1. The function of DFCP1 is unknown, as are the mechanisms of omegasome formation and constriction. Here, we demonstrate that DFCP1 is an ATPase that is activated by membrane binding and dimerizes in an ATP-dependent fashion. Whereas depletion of DFCP1 has a minor effect on bulk autophagic flux, DFCP1 is required to maintain the autophagic flux of p62 under both fed and starved conditions, and this is dependent on its ability to bind and hydrolyse ATP. While DFCP1 mutants defective in ATP binding or hydrolysis localize to forming omegasomes, these omegasomes fail to constrict properly in a size-dependent manner. Consequently, the release of nascent autophagosomes from large omegasomes is markedly delayed. While knockout of DFCP1 does not affect bulk autophagy, it inhibits selective autophagy, including aggrephagy, mitophagy and micronucleophagy. We conclude that DFCP1 mediates ATPase-driven constriction of large omegasomes to release autophagosomes for selective autophagy.

Seipin-still a mysterious protein?

Cells store excess energy in the form of lipid droplets (LDs), a specialized sub-compartment of the endoplasmic reticulum (ER) network. The lipodystrophy protein seipin is a key player in LD biogenesis and ER-LD contact site maintenance. Recent structural and in silico studies have started to shed light on the molecular function of seipin as a LD nucleator in early LD biogenesis, whilst new cell biological work implies a role for seipin in ER-mitochondria contact sites and calcium metabolism. In this minireview, I discuss recent insights into the molecular function of seipin.

Cholesterol esters form supercooled lipid droplets whose nucleation is facilitated by triacylglycerols.

Cellular cholesterol can be metabolized to its fatty acid esters, cholesteryl esters (CEs), to be stored in lipid droplets (LDs). With triacylglycerols (TGs), CEs represent the main neutral lipids in LDs. However, while TG melts at ~4 °C, CE melts at ~44 °C, raising the question of how CE-rich LDs form in cells. Here, we show that CE forms supercooled droplets when the CE concentration in LDs is above 20% to TG and, in particular, liquid-crystalline phases when the fraction of CEs is above 90% at 37 °C. In model bilayers, CEs condense and nucleate droplets when the CE/phospholipid ratio reaches over 10-15%. This concentration is reduced by TG pre-clusters in the membrane that thereby facilitate CE nucleation. Accordingly, blocking TG synthesis in cells is sufficient to strongly dampen CE LD nucleation. Finally, CE LDs emerged at seipins, which cluster and nucleate TG LDs in the ER. However, when TG synthesis is inhibited, similar numbers of LDs are generated in the presence and absence of seipin, suggesting that seipin controls CE LD formation via its TG clustering capacity. Our data point to a unique model whereby TG pre-clusters, favorable at seipins, catalyze the nucleation of CE LDs.

DGAT1 activity synchronises with mitophagy to protect cells from metabolic rewiring by iron depletion.

Mitophagy removes defective mitochondria via lysosomal elimination. Increased mitophagy coincides with metabolic reprogramming, yet it remains unknown whether mitophagy is a cause or consequence of such state changes. The signalling pathways that integrate with mitophagy to sustain cell and tissue integrity also remain poorly defined. We performed temporal metabolomics on mammalian cells treated with deferiprone, a therapeutic iron chelator that stimulates PINK1/PARKIN-independent mitophagy. Iron depletion profoundly rewired the metabolome, hallmarked by remodelling of lipid metabolism within minutes of treatment. DGAT1-dependent lipid droplet biosynthesis occurred several hours before mitochondrial clearance, with lipid droplets bordering mitochondria upon iron chelation. We demonstrate that DGAT1 inhibition restricts mitophagy in vitro, with impaired lysosomal homeostasis and cell viability. Importantly, genetic depletion of DGAT1 in vivo significantly impaired neuronal mitophagy and locomotor function in Drosophila. Our data define iron depletion as a potent signal that rapidly reshapes metabolism and establishes an unexpected synergy between lipid homeostasis and mitophagy that safeguards cell and tissue integrity.

Seipin localizes at endoplasmic-reticulum-mitochondria contact sites to control mitochondrial calcium import and metabolism in adipocytes.

Deficiency of the endoplasmic reticulum (ER) protein seipin results in generalized lipodystrophy by incompletely understood mechanisms. Here, we report mitochondrial abnormalities in seipin-deficient patient cells. A subset of seipin is enriched at ER-mitochondria contact sites (MAMs) in human and mouse cells and localizes in the vicinity of calcium regulators SERCA2, IP3R, and VDAC. Seipin association with MAM calcium regulators is stimulated by fasting-like stimuli, while seipin association with lipid droplets is promoted by lipid loading. Acute seipin removal does not alter ER calcium stores but leads to defective mitochondrial calcium import accompanied by a widespread reduction in Krebs cycle metabolites and ATP levels. In mice, inducible seipin deletion leads to mitochondrial dysfunctions preceding the development of metabolic complications. Together, these data suggest that seipin controls mitochondrial energy metabolism by regulating mitochondrial calcium influx at MAMs. In seipin-deficient adipose tissue, reduced ATP production compromises adipocyte properties, contributing to lipodystrophy pathogenesis.

Seipin traps triacylglycerols to facilitate their nanoscale clustering in the endoplasmic reticulum membrane.

Seipin is a disk-like oligomeric endoplasmic reticulum (ER) protein important for lipid droplet (LD) biogenesis and triacylglycerol (TAG) delivery to growing LDs. Here we show through biomolecular simulations bridged to experiments that seipin can trap TAGs in the ER bilayer via the luminal hydrophobic helices of the protomers delineating the inner opening of the seipin disk. This promotes the nanoscale sequestration of TAGs at a concentration that by itself is insufficient to induce TAG clustering in a lipid membrane. We identify Ser166 in the α3 helix as a favored TAG occupancy site and show that mutating it compromises the ability of seipin complexes to sequester TAG in silico and to promote TAG transfer to LDs in cells. While the S166D-seipin mutant colocalizes poorly with promethin, the association of nascent wild-type seipin complexes with promethin is promoted by TAGs. Together, these results suggest that seipin traps TAGs via its luminal hydrophobic helices, serving as a catalyst for seeding the TAG cluster from dissolved monomers inside the seipin ring, thereby generating a favorable promethin binding interface.

Membrane Curvature Catalyzes Lipid Droplet Assembly.

Lipid droplet (LD) biogenesis begins in the endoplasmic reticulum (ER) bilayer, but how the ER topology impacts this process is unclear. An early step in LD formation is nucleation, wherein free neutral lipids, mainly triacylglycerols (TGs) and sterol esters (SEs), condense into a nascent LD. How this transition occurs is poorly known. Here, we found that LDs preferably assemble at ER tubules, with higher curvature than ER sheets, independently of the LD assembly protein seipin. Indeed, the critical TG concentration required for initiating LD assembly is lower at curved versus flat membrane regions. In agreement with this finding, flat ER regions bear higher amounts of free TGs than tubular ones and present less LDs. By using an in vitro approach, we discovered that the presence of free TGs in tubules is energetically unfavorable, leading to outflow of TGs to flat membrane regions or condensation into LDs. Accordingly, in vitro LD nucleation can be achieved by the sole increase of membrane curvature. In contrast to TGs, the presence of free SEs is favored at tubules and increasing SE levels is inhibitory to the curvature-induced nucleation of TG LDs. Finally, we found that seipin is enriched at ER tubules and controls the condensation process, preventing excessive tubule-induced nucleation. The absence of seipin provokes erratic LD nucleation events determined by the abundance of ER tubules. In summary, our data indicate that membrane curvature catalyzes LD assembly.

Author Correction: An efficient auxin-inducible degron system with low basal degradation in human cells.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

An efficient auxin-inducible degron system with low basal degradation in human cells.

Auxin-inducible degron technology allows rapid and controlled protein depletion. However, basal degradation without auxin and inefficient auxin-inducible depletion have limited its utility. We have identified a potent auxin-inducible degron system composed of auxin receptor F-box protein AtAFB2 and short degron miniIAA7. The system showed minimal basal degradation and enabled rapid auxin-inducible depletion of endogenous human transmembrane, cytoplasmic and nuclear proteins in 1 h with robust functional phenotypes.

Seipin Facilitates Triglyceride Flow to Lipid Droplet and Counteracts Droplet Ripening via Endoplasmic Reticulum Contact.

Seipin is an oligomeric integral endoplasmic reticulum (ER) protein involved in lipid droplet (LD) biogenesis. To study the role of seipin in LD formation, we relocalized it to the nuclear envelope and found that LDs formed at these new seipin-defined sites. The sites were characterized by uniform seipin-mediated ER-LD necks. At low seipin content, LDs only grew at seipin sites, and tiny, growth-incompetent LDs appeared in a Rab18-dependent manner. When seipin was removed from ER-LD contacts within 1 h, no lipid metabolic defects were observed, but LDs became heterogeneous in size. Studies in seipin-ablated cells and model membranes revealed that this heterogeneity arises via a biophysical ripening process, with triglycerides partitioning from smaller to larger LDs through droplet-bilayer contacts. These results suggest that seipin supports the formation of structurally uniform ER-LD contacts and facilitates the delivery of triglycerides from ER to LDs. This counteracts ripening-induced shrinkage of small LDs.

Moving out but keeping in touch: contacts between endoplasmic reticulum and lipid droplets.

The formation of neutral lipid filled and phospholipid monolayer engulfed lipid droplets (LDs) from the bilayer of the endoplasmic reticulum (ER) is an active area of investigation. This process harnesses the biophysical properties of the lipids involved and necessitates cooperation of protein machineries in both organelle membranes. Increasing evidence suggests that once formed, LDs keep close contact to the mother organelle and that this may be achieved via several, morphologically distinct and potentially functionally specialized connections. These may help LDs to dynamically respond to changes in lipid metabolic status sensed by the ER. In this review, we will discuss recent progress in understanding how LDs interact with the ER.

Role for formin-like 1-dependent acto-myosin assembly in lipid droplet dynamics and lipid storage.

Lipid droplets (LDs) are cellular organelles specialized in triacylglycerol (TG) storage undergoing homotypic clustering and fusion. In non-adipocytic cells with numerous LDs this is balanced by poorly understood droplet dissociation mechanisms. We identify non-muscle myosin IIa (NMIIa/MYH-9) and formin-like 1 (FMNL1) in the LD proteome. NMIIa and actin filaments concentrate around LDs, and form transient foci between dissociating LDs. NMIIa depletion results in decreased LD dissociations, enlarged LDs, decreased hydrolysis and increased storage of TGs. FMNL1 is required for actin assembly on LDs in vitro and for NMIIa recruitment to LDs in cells. We propose a novel acto-myosin structure regulating lipid storage: FMNL1-dependent assembly of myosin II-functionalized actin filaments on LDs facilitates their dissociation, thereby affecting LD surface-to-volume ratio and enzyme accessibility to TGs. In neutrophilic leucocytes from MYH9-related disease patients NMIIa inclusions are accompanied by increased lipid storage in droplets, suggesting that NMIIa dysfunction may contribute to lipid imbalance in man.

Seipin regulates ER-lipid droplet contacts and cargo delivery.

Seipin is an endoplasmic reticulum (ER) membrane protein implicated in lipid droplet (LD) biogenesis and mutated in severe congenital lipodystrophy (BSCL2). Here, we show that seipin is stably associated with nascent ER-LD contacts in human cells, typically via one mobile focal point per LD Seipin appears critical for such contacts since ER-LD contacts were completely missing or morphologically aberrant in seipin knockout and BSCL2 patient cells. In parallel, LD mobility was increased and protein delivery from the ER to LDs to promote LD growth was decreased. Moreover, while growing LDs normally acquire lipid and protein constituents from the ER, this process was compromised in seipin-deficient cells. In the absence of seipin, the initial synthesis of neutral lipids from exogenous fatty acid was normal, but fatty acid incorporation into neutral lipids in cells with pre-existing LDs was impaired. Together, our data suggest that seipin helps to connect newly formed LDs to the ER and that by stabilizing ER-LD contacts seipin facilitates the incorporation of protein and lipid cargo into growing LDs in human cells.

Alleviation of seipinopathy-related ER stress by triglyceride storage.

Mutations affecting the N-glycosylation site in Berardinelli-Seip lipodystrophy (BSCL)-associated gene BSCL2/seipin lead to a dominantly inherited spastic paraplegia termed seipinopathy. While the loss of function of seipin leads to severe congenital lipodystrophy, the effects of seipin N-glycosylation mutations on lipid balance in the nervous system are unknown. In this study, we show that expression of seipin N-glycosylation mutant N88S led to decreased triglyceride (TG) content in astrocytoma and motor neuron cell lines. This was corrected by supplementation with exogenous oleic acid. Upon oleic acid loading, seipin N88S protein was relocated from the endoplasmic reticulum (ER) to the surface of lipid droplets and this was paralleled by alleviation of ER stress induced by the mutant protein. This effect was not limited to seipin N88S, as oleic acid loading also reduced tunicamycin-induced ER stress in motor neuron cells. Furthermore, both seipin N88S and tunicamycin-induced ER stress were decreased by inhibiting lipolysis, suggesting that lipid droplets protected neuronal cells from ER stress. In developing zebrafish larvae, seipin N88S expression led to TG imbalance and reduced spontaneous free swimming. Importantly, supplementation with exogenous oleic acid reduced ER stress in the zebrafish head and increased fish motility. We propose that the decreased TG content contributes to the pathology induced by seipin N88S, and that rescuing TG levels may provide a novel therapeutic strategy in seipinopathy.

Zebrafish: gaining popularity in lipid research.

Zebrafish are an increasingly popular vertebrate model organism in which to study biological phenomena. It has been widely used, especially in developmental biology and neurobiology, and many aspects of its development and physiology are similar to those of mammals. The popularity of zebrafish relies on its relatively low cost, rapid development and ease of genetic manipulation. Moreover, the optical transparency of the developing fish together with novel imaging techniques enable the direct visualization of complex phenomena at the level of the entire organism. This potential is now also being increasingly appreciated by the lipid research community. In the present review we summarize basic information on the lipid composition and distribution in zebrafish tissues, including lipoprotein metabolism, intestinal lipid absorption, the yolk lipids and their mobilization, as well as lipids in the nervous system. We also discuss studies in which zebrafish have been employed for the visualization of whole-body lipid distribution and trafficking. Finally, recent advances in using zebrafish as a model for lipid-related diseases, including atherosclerosis, obesity, diabetes and hepatic steatosis are highlighted. As the insights into zebrafish lipid metabolism increase, it is likely that zebrafish as a model organism will become an increasingly powerful tool in lipid research.