The actin assembly machinery coordinates a variety of membrane-remodeling processes. This paper shows that an inherited mutation in the WHAMM gene causes severe defects in autophagy and that the WHAMM protein normally binds to PI(3)P and nucleates actin to promote LC3 incorporation during autophagosome biogenesis.
Small G-proteins regulate the recruitment and activation of WASP-family actin nucleation factors at the plasma membrane. The G-protein Rab1 interacts with the nucleation factor WHAMM to remodel internal membranes into tubules. Unlike other G-proteins that recruit nucleation factors, Rab1 inhibits actin assembly.
Lipid droplets (LDs) are lipid storage organelles that consist of a central core of neutral lipids surrounded by a phospholipid monolayer decorated with a unique set of integral and peripheral proteins. Invariably, at least one member of the perilipin family of proteins (PLIN1-5) associates with LDs in all cell types. Despite key roles of PLIN2 in governing hepatic lipid metabolism, the mechanisms that regulate PLIN2 levels remain incompletely understood. Here, we develop a set of genome-edited PLIN2 reporter cell lines that facilitate the analysis of genes that regulate PLIN2 and LD abundance. Leveraging these reporter cells in a series of CRISPR-Cas9 loss-of-function screens, we generate a comprehensive inventory of genes that influence PLIN2 levels under different metabolic conditions. Moreover, we uncouple their effects on PLIN2 expression and post-translational stability. Identified genetic modifiers include canonical genes that control LD metabolism (e.g., ACSL3, DGAT2, PNPLA2, ABHD5) as well as genes with less characterized roles in PLIN2 and LD regulation such as ubiquitination machinery (e.g., MARCH6, UBE2J2), transcription regulators (e.g., HNF4A, HDAC3), mitochondrial pathways (e.g., electron transport chain and mitochondrial fatty acid synthesis), and others. These CRISPR screens, and several published screens that focus on different aspects of lipid metabolism, provide the foundation for CRISPRlipid (http://crisprlipid.org), a versatile, online data commons for lipid-related functional genomics data. Together, our study uncovers new mechanisms of PLIN2 regulation and provides an extensive, phenotype-rich resource for the exploration of LD biology and lipid metabolism.
Human cells rely on the actin cytoskeleton to properly organize, shape, and move their membrane‐bound organelles. However, the nucleation factors responsible for assembling actin filaments have not been well characterized in relation to human disease. Nephrocerebellar syndrome (NCS) is a recently‐described developmental disorder in which patients encode truncated mutants of the WD40‐repeat protein WDR73 and the actin nucleation factor WHAMM. Here we show that cells from NCS patients exhibit cytoskeletal irregularities, possess enlarged ER‐Golgi intermediate compartments, and have severe defects in the cytoplasmic degradation process of autophagy. Re‐introduction of wild type WHAMM into patient cells restored autophagosome biogenesis, whereas depletion or genetic inactivation of WHAMM in healthy human cell lines impaired both the lipidation of the autophagosomal protein LC3 and the clearance of ubiquitinated protein aggregates. Wild type WHAMM localized to subdomains of nascent autophagosomes, and proper WHAMM function involved binding to the phospholipid PI(3)P, the small G‐protein Rab1, and actin. These results reveal a cytoskeletal mechanism for controlling autophagosome remodeling that is perturbed in cells from NCS patients.
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