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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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. • The presence of free TG molecules is unfavorable in curved membranes • Membrane curvature facilitates but sterol esters inhibit the assembly of TG LDs • Endoplasmic reticulum topology modulates the number of assembled LDs • Seipin controls the condensation of TGs into LDs at endoplasmic reticulum tubules How triglycerides (TGs) in the ER bilayer condense into lipid droplets (LDs) is poorly known. Santinho et al. show that membrane curvature aids this transition by reducing the critical TG concentration needed for condensing into LDs. Seipin is enriched at tubules and controls this condensation step, preventing uncontrolled LD assembly at ER tubules. [ABSTRACT FROM AUTHOR]

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. • Seipin can determine the site of LD formation • Seipin-mediated ER-LD membrane contacts display a uniform neck-like architecture • Seipin at ER-LD contact facilitates continuous triglyceride transfer to LD • Acute removal of seipin reveals a principle of LD ripening via the ER Salo et al. show that seipin determines where a lipid droplet (LD) forms and establishes a neck between ER and LD. LDs continuously exchange lipids via ER, and biophysically, larger LDs acquire lipids more readily than smaller ones. Seipin facilitates lipid transfer to droplets, helping small LDs to grow. [ABSTRACT FROM AUTHOR]