RESUMEN
Lipid droplets are dynamic organelles that store neutral lipids, serve the metabolic needs of cells, and sequester lipids to prevent lipotoxicity and membrane damage. Here we review the current understanding of the mechanisms of lipid droplet biogenesis and turnover, the transfer of lipids and metabolites at membrane contact sites, and the role of lipid droplets in regulating fatty acid flux in lipotoxicity and cell death.
Asunto(s)
Gotas Lipídicas , Metabolismo de los Lípidos , Gotas Lipídicas/metabolismo , Homeostasis , Ácidos Grasos/metabolismoRESUMEN
Imbalances in lipid storage and secretion lead to the accumulation of hepatocyte lipid droplets (LDs) (i.e., hepatic steatosis). Our understanding of the mechanisms that govern the channeling of hepatocyte neutral lipids towards cytosolic LDs or secreted lipoproteins remains incomplete. Here, we performed a series of CRISPR-Cas9 screens under different metabolic states to uncover mechanisms of hepatic neutral lipid flux. Clustering of chemical-genetic interactions identified CLIC-like chloride channel 1 (CLCC1) as a critical regulator of neutral lipid storage and secretion. Loss of CLCC1 resulted in the buildup of large LDs in hepatoma cells and knockout in mice caused liver steatosis. Remarkably, the LDs are in the lumen of the ER and exhibit properties of lipoproteins, indicating a profound shift in neutral lipid flux. Finally, remote homology searches identified a domain in CLCC1 that is homologous to yeast Brl1p and Brr6p, factors that promote the fusion of the inner and outer nuclear envelopes during nuclear pore complex assembly. Loss of CLCC1 lead to extensive nuclear membrane herniations, consistent with impaired nuclear pore complex assembly. Thus, we identify CLCC1 as the human Brl1p/Brr6p homolog and propose that CLCC1-mediated membrane remodeling promotes hepatic neutral lipid flux and nuclear pore complex assembly.
RESUMEN
Phenotypic screens involving pooled CRISPR-Cas9 libraries offer a powerful, rapid yet affordable approach to evaluate gene functions on a global scale. Here, we present a protocol for performing pooled CRISPR-Cas9 loss-of-function screens to identify genetic modifiers using either fluorescence-based or cell death phenotypic readouts. We describe steps for designing and amplifying the library and generating and screening cells. We then detail deep sequencing and statistical analysis using cas9 High Throughput maximum Likelihood Estimator. For complete details on the use and execution of this protocol, please refer to Bersuker et al. (2019),1 Li et al. (2022),2 and Roberts et al. (2022).3.
RESUMEN
Despite the key roles of perilipin-2 (PLIN2) in governing lipid droplet (LD) metabolism, the mechanisms that regulate PLIN2 levels remain incompletely understood. Here, we leverage a set of genome-edited human PLIN2 reporter cell lines in a series of CRISPR-Cas9 loss-of-function screens, identifying genetic modifiers that influence PLIN2 expression and post-translational stability under different metabolic conditions and in different cell types. These regulators include canonical genes that control lipid metabolism as well as genes involved in ubiquitination, transcription, and mitochondrial function. We further demonstrate a role for the E3 ligase MARCH6 in regulating triacylglycerol biosynthesis, thereby influencing LD abundance and PLIN2 stability. Finally, our CRISPR screens and several published screens provide the foundation for CRISPRlipid (http://crisprlipid.org), an online data commons for lipid-related functional genomics data. Our study identifies mechanisms of PLIN2 and LD regulation and provides an extensive resource for the exploration of LD biology and lipid metabolism.
Asunto(s)
Sistemas CRISPR-Cas , Gotas Lipídicas , Humanos , Perilipina-2/genética , Perilipina-2/metabolismo , Gotas Lipídicas/metabolismo , Sistemas CRISPR-Cas/genética , Metabolismo de los Lípidos/genética , Línea CelularRESUMEN
Actin nucleation factors function to organize, shape, and move membrane-bound organelles, yet they remain poorly defined in relation to disease. Galloway-Mowat syndrome (GMS) is an inherited disorder characterized by microcephaly and nephrosis resulting from mutations in the WDR73 gene. This core clinical phenotype appears frequently in the Amish, where virtually all affected individuals harbor homozygous founder mutations in WDR73 as well as the closely linked WHAMM gene, which encodes a nucleation factor. Here we show that patient cells with both mutations exhibit cytoskeletal irregularities and severe defects in autophagy. Reintroduction of wild-type WHAMM restored autophagosomal biogenesis to patient cells, while inactivation of WHAMM in healthy cell lines inhibited lipidation of the autophagosomal protein LC3 and clearance of ubiquitinated protein aggregates. Normal WHAMM function involved binding to the phospholipid PI(3)P and promoting actin nucleation at nascent autophagosomes. These results reveal a cytoskeletal pathway controlling autophagosomal remodeling and illustrate several molecular processes that are perturbed in Amish GMS patients.
Asunto(s)
Complejo 2-3 Proteico Relacionado con la Actina/metabolismo , Amish/genética , Mutación del Sistema de Lectura , Proteínas de la Membrana/genética , Proteínas Asociadas a Microtúbulos/genética , Fosfatos de Fosfatidilinositol/metabolismo , Complejo 2-3 Proteico Relacionado con la Actina/genética , Actinas/metabolismo , Autofagosomas/metabolismo , Autofagosomas/fisiología , Células Cultivadas , Citoesqueleto/metabolismo , Efecto Fundador , Hernia Hiatal/genética , Homocigoto , Humanos , Proteínas de la Membrana/metabolismo , Microcefalia/genética , Proteínas Asociadas a Microtúbulos/metabolismo , Modelos Moleculares , Nefrosis/genética , Fosfatos de Fosfatidilinositol/genética , Proteínas/genética , Proteínas/metabolismoRESUMEN
Small G-proteins are key regulatory molecules that activate the actin nucleation machinery to drive cytoskeletal rearrangements during plasma membrane remodeling. However, the ability of small G-proteins to interact with nucleation factors on internal membranes to control trafficking processes has not been well characterized. Here we investigated roles for members of the Rho, Arf, and Rab G-protein families in regulating WASP homologue associated with actin, membranes, and microtubules (WHAMM), an activator of Arp2/3 complex-mediated actin nucleation. We found that Rab1 stimulated the formation and elongation of WHAMM-associated membrane tubules in cells. Active Rab1 recruited WHAMM to dynamic tubulovesicular structures in fibroblasts, and an active prenylated version of Rab1 bound directly to an N-terminal domain of WHAMM in vitro. In contrast to other G-protein-nucleation factor interactions, Rab1 binding inhibited WHAMM-mediated actin assembly. This ability of Rab1 to regulate WHAMM and the Arp2/3 complex represents a distinct strategy for membrane remodeling in which a Rab G-protein recruits the actin nucleation machinery but dampens its activity.