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Updated: Jul 15, 2025

A Fluorescence-based Assay for Characterization and Quantification of Lipid Droplet Formation in Human Intestinal Organoids
Published on: October 13, 2019
ATG14 plays a critical role in hepatic lipid droplet homeostasis
Menghao Huang1, Yang Zhang1, Jimin Park1
1Department of Biochemistry and Molecular Biology, Indiana University School of Medicine, Indianapolis, IN, USA.
Background & Aims:
Autophagy-related 14 (ATG14) is a key regulator of autophagy. ATG14 is also localized to lipid droplet; however, the function of ATG14 on lipid droplet remains unclear. In this study, we aimed to elucidate the role of ATG14 in lipid droplet homeostasis.
Methods:
ATG14 loss-of-function and gain-of-function in lipid droplet metabolism were analyzed by fluorescence imaging in ATG14 knockdown or overexpression hepatocytes. Specific domains involved in the ATG14 targeting to lipid droplets were analyzed by deletion or site-specific mutagenesis. ATG14-interacting proteins were analyzed by co-immunoprecipitation. The effect of ATG14 on lipolysis was analyzed in human hepatocytes and mouse livers that were deficient in ATG14, comparative gene identification-58 (CGI-58), or both.
Results:
Our data show that ATG14 is enriched on lipid droplets in hepatocytes. Mutagenesis analysis reveals that the Barkor/ATG14 autophagosome targeting sequence (BATS) domain of ATG14 is responsible for the ATG14 localization to lipid droplets. Co-immunoprecipitation analysis illustrates that ATG14 interacts with adipose triglyceride lipase (ATGL) and CGI-58. Moreover, ATG14 also enhances the interaction between ATGL and CGI-58. In vitro lipolysis analysis demonstrates that ATG14 deficiency remarkably decreases triglyceride hydrolysis.
Conclusions:
Our data suggest that ATG14 can directly enhance lipid droplet breakdown through interactions with ATGL and CGI-58.
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