Bhapkar, A. | Search bioRxiv

bioRxiv2026

Seipin and perilipin 3 promote distinct early steps of nascent lipid droplet biogenesis

Lipid droplets (LDs) are lipid storage organelles with critical roles in metabolism. Cells form LDs in response to various metabolic stresses (1). LD biogenesis occurs in the endoplasmic reticulum (ER) membrane and begins when triglyceride (TG) in the membrane phase separates (2-4). After nucleation, nascent LDs grow by absorbing TG from the membrane (5,6). How cells determine when TG in the ER membrane is added to existing LDs or is used to form new LDs or is not known. Here, we developed the first LD formation assay that allows the synthesis of TG in membranes, mimicking what occurs in cells. LDs arise stochastically after TG production begins. LD nucleation is affected by TG production rate and membrane phospholipid composition. Membrane-embedded LDs suppress nascent LD formation by sequestering TG and destabilizing nascent LDs by Ostwald ripening. We show that cells overcome this suppression in two ways. The ER membrane protein seipin accelerates LD nucleation where it localizes in a membrane, probably by increasing the kinetics of TG phase separation. The LD coat perilipin 3 inhibits nascent droplet dissipation by ripening. Together, our findings reveal a minimal LD formation machinery, and the mechanisms cells use to control whether TG in the ER drives new LD formation.

cell biology↗

bioRxiv2026

ER cholesteryl ester phase separation underlies switch-like cholesterol sensing

Cellular lipid homeostasis requires mechanisms that detect subtle changes in lipid abundance and trigger rapid, coordinated responses. The INSIG/SCAP/SREBP2 pathway provides a central feedback system linking endoplasmic reticulum (ER) cholesterol levels to the transcriptional control of cholesterol genes, yet the origin of its remarkable cooperative switch mechanism remains unclear. Here we identify cholesterol esterification as a physical mechanism that amplifies sterol sensing and generates switch-like pathway regulation. We show that cholesteryl oleate (CE), produced by SOAT1 and opposed by NCEH1-mediated hydrolysis, undergoes a cooperative phase transition within the ER membrane to form transient CE-rich domains. These lipid assemblies create a threshold-dependent platform that concentrates SCAP and promotes formation of the SCAP-INSIG retention complex, thereby coupling ER lipid organization to SREBP inhibition. Because CE domain formation is nucleation-driven, variations in cholesterol availability are converted into an abrupt transition between distinct membrane states. Perturbing CE metabolism uncouples cholesterol abundance from pathway activity: SOAT1 inhibition prevents CE domain formation, releases SCAP from the ER, and activates SREBP2 despite cholesterol sufficiency, whereas NCEH1 inhibition stabilizes the domains and reduces SREBP2 activation under cholesterol-limiting conditions. Thus, the balance between esterification and hydrolysis determines a membrane physical state that serves as the functional output sensed by the cholesterol regulatory machinery. Our findings reveal ER lipid phase transitions as a general principle for creating ultrasensitive control in cellular homeostasis and establish cholesterol esterification as an active regulatory process rather than a passive storage pathway.

cell biology↗