# Mechanisms of Lipid Droplet Formation

> **NIH NIH R01** · HARVARD UNIVERSITY D/B/A HARVARD SCHOOL OF PUBLIC HEALTH · 2022 · $61,566

## Abstract

PROJECT SUMMARY
Lipid droplets (LDs) are ubiquitous monolayer-bound organelles that function in cellular lipid storage (for metabolic energy
or membrane synthesis). LDs form from the ER, but how LDs are formed remains unknown and is a central question for the
field. The current model indicates that neutral lipids, such as triacylglycerols (TG), are synthesized in the ER and released
into the bilayer. At a critical concentration, TGs de-mix from the phospholipid bilayer in a phase transition that forms
nascent LDs that bud toward the cytosol. We hypothesize that proteins are essential to ensure this process occurs in a
defined manner and to prevent the formation of “ectopic” and potentially dysfunctional LDs, disrupting ER and cell
function. Specifically, two ER proteins – seipin and lipid droplet assembly factor 1 (LDAF1) – operate in the lipid droplet
assembly complex (LDACs) in the ER to form LDs. Both proteins form an oligomeric assembly with seipin forming a ring
of 10-12 subunits and an equal number of LDAF1 occupying the middle of the ring. While we have identified components
of the LD formation machinery and gained some insight into their structures, how these proteins function to facilitate LD
formation remains mostly a mystery. Here we propose to utilize the latest tools and approaches, including biochemistry,
structural biology, molecular simulations, and cell biology, to address the following questions: How and where is TG made
relative to LDACs? What are the molecular structures of the seipin/LDAF1 LDACs? How do these oligomeric complexes
assemble/disassemble? Where do LDACs localize in cells? How do they function to organize LD formation? We will
address these questions by completing four specific aims. Aim 1 will address the mechanism of TG synthesis in the ER by
the DGAT1 enzyme. We will expand on our recent elucidation of the molecular structure of human DGAT1, combining
molecular dynamics and biochemical experiments to elucidate the precise mechanism of TG generation and determine how
TG is released into the ER membrane for LD formation. Aim 2 will determine how and where LD assembly complexes
assemble in cells to form LDs. We will determine the relationship of TG synthesis to LDACs, whether seipin/LDAF1
LDACs localize to ER tubules and how they assemble. Aim 3 will focus on elucidating the molecular structure of the
seipin/LDAF1 LDAC in vitro and in cells. We will utilize cell and structural biology approaches, including cryo-EM and
cryo-ET to test the hypothesis that seipin and LDAF1 form a ring structure with LDAF1 in center and that these LDACs
form at areas of membrane curvature (tubules) where the structure may adopt dynamic conformations and activate of the
complex. Aim 4 will determine the molecular function of the seipin/LDAF1 LDAC in vitro and in molecular dynamics
simulations. We will reconstitute LD formation to test the hypothesis that the seipin/LDAF1 LDAC catalyzes phase
transition of TG in the membrane, ensuring L...

## Key facts

- **NIH application ID:** 10475248
- **Project number:** 5R01GM124348-06
- **Recipient organization:** HARVARD UNIVERSITY D/B/A HARVARD SCHOOL OF PUBLIC HEALTH
- **Principal Investigator:** ROBERT V FARESE
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2022
- **Award amount:** $61,566
- **Award type:** 5
- **Project period:** 2017-09-01 → 2022-09-01

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10475248

## Citation

> US National Institutes of Health, RePORTER application 10475248, Mechanisms of Lipid Droplet Formation (5R01GM124348-06). Retrieved via AI Analytics 2026-05-24 from https://api.ai-analytics.org/grant/nih/10475248. Licensed CC0.

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