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.

Mammalian cells acquire cholesterol, a major membrane constituent, via low-density lipoprotein (LDL) uptake. However, the mechanisms by which LDL cholesterol reaches the plasma membrane (PM) have remained obscure. Here, we applied LDL labeled with BODIPY cholesteryl linoleate to identify this pathway in living cells. The egress of BODIPY cholesterol (BC) from late endosomal (LE) organelles was dependent on acid lipase and Niemann-Pick C1 (NPC1) protein, as for natural cholesterol. We show that NPC1 was needed to recruit Rab8a to BC-containing LEs, and Rab8a enhanced the motility and segregation of BC- and CD63-positive organelles from lysosomes. The BC carriers docked to the cortical actin by a Rab8a- and Myosin5b (Myo5b)-dependent mechanism, typically in the proximity of focal adhesions (FAs). LDL increased the number and dynamics of FAs and stimulated cell migration in an acid lipase, NPC1, and Rab8a-dependent fashion, providing evidence that this cholesterol delivery route to the PM is important for cell movement.

FAT4 mutations lead to several human diseases that disrupt the normal development of the kidney. However, the underlying mechanism remains elusive. In studying the duplex kidney phenotypes observed upon deletion of Fat4 in mice, we have uncovered an interaction between the atypical cadherin FAT4 and RET, a tyrosine kinase receptor essential for kidney development. Analysis of kidney development in Fat4-/- kidneys revealed abnormal ureteric budding and excessive RET signaling. Removal of one copy of the RET ligand Gdnf rescues Fat4-/- kidney development, supporting the proposal that loss of Fat4 hyperactivates RET signaling. Conditional knockout analyses revealed a non-autonomous role for Fat4 in regulating RET signaling. Mechanistically, we found that FAT4 interacts with RET through extracellular cadherin repeats. Importantly, expression of FAT4 perturbs the assembly of the RET-GFRA1-GDNF complex, reducing RET signaling. Thus, FAT4 interacts with RET to fine-tune RET signaling, establishing a juxtacrine mechanism controlling kidney development.

During energy bursts, neutral lipids fabricated within the ER bilayer demix to form lipid droplets (LDs). LDs bud off mainly in the cytosol where they regulate metabolism and multiple biological processes. They indeed become accessible to most enzymes and can interact with other organelles. How such directional emergence is achieved remains elusive. Here, we found that this directionality is controlled by an asymmetry in monolayer surface coverage. Model LDs emerge on the membrane leaflet of higher coverage, which is improved by the insertion of proteins and phospholipids. In cells, continuous LD emergence on the cytosol would require a constant refill of phospholipids to the ER cytosolic leaflet. Consistent with this model, cells deficient in phospholipids present an increased number of LDs exposed to the ER lumen and compensate by remodeling ER shape. Our results reveal an active cooperation between phospholipids and proteins to extract LDs from ER.

Lipid droplets (LDs) store lipids that can be utilized during times of scarcity via autophagic and lysosomal pathways, but how LDs and autophagosomes interact remained unclear. Here, we discovered that the E2 autophagic enzyme, ATG3, localizes to the surface of certain ultra-large LDs in differentiated murine 3T3-L1 adipocytes or Huh7 human liver cells undergoing prolonged starvation. Subsequently, ATG3 lipidates microtubule-associated protein 1 light-chain 3B (LC3B) to these LDs. In vitro, ATG3 could bind alone to purified and artificial LDs to mediate this lipidation reaction. We observed that LC3B-lipidated LDs were consistently in close proximity to collections of LC3B-membranes and were lacking Plin1. This phenotype is distinct from macrolipophagy, but it required autophagy because it disappeared following ATG5 or Beclin1 knockout. Our data suggest that extended starvation triggers a noncanonical autophagy mechanism, similar to LC3B-associated phagocytosis, in which the surface of large LDs serves as an LC3B lipidation platform for autophagic processes.

Cells convert excess energy into neutral lipids that are made in the endoplasmic reticulum (ER) bilayer. The lipids are then packaged into spherical or budded lipid droplets (LDs) covered by a phospholipid monolayer containing proteins. LDs play a key role in cellular energy metabolism and homeostasis. A key unanswered question in the life of LDs is how they bud off from the ER. Here, we tackle this question by studying the budding of artificial LDs from model membranes. We find that the bilayer phospholipid composition and surface tension are key parameters of LD budding. Phospholipids have differential LD budding aptitudes, and those inducing budding decrease the bilayer tension. We observe that decreasing tension favors the egress of neutral lipids from the bilayer and LD budding. In cells, budding conditions favor the formation of small LDs. Our discovery reveals the importance of altering ER physical chemistry for controlled cellular LD formation.