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Rab18 is not necessary for lipid droplet biogenesis or turnover in human mammary carcinoma cells

ABSTRACT: Rab GTPases recruit peripheral membrane proteins and can define organelle identity. Rab18 localizes to the ER but also to the surfaces of lipid droplets (LDs), where it has been implicated in effector protein recruitment and in defining LD identity. Here, we studied Rab18 localization and function in SUM159 cells. Rab18 localized to the ER and to LD membranes upon LD induction, with the latter depending on the Rab18 activation state. In cells lacking Rab18, LDs were modestly reduced in size and numbers, but we found little evidence for Rab18 function in LD formation, LD turnover upon cell starvation, or the targeting of several proteins to LDs. We conclude that Rab18 is not a general, necessary component of the protein machinery involved in LD biogenesis or turnover, but instead likely plays a specialized role in specific cell types

INSTRUMENT(S): Q Exactive

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Breast, Cell Culture

DISEASE(S): Breast Cancer

SUBMITTER: Zon Weng Lai

LAB HEAD: Tobias Walther

PROVIDER: PXD009683 | Pride | 2018-06-06

REPOSITORIES: Pride

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RAB18 impacts autophagy via lipid droplet-derived lipid transfer and is rescued by ATG9A

Project description:Autophagy is a lysosomal degradation pathway that mediates protein and organelle turnover and maintains cellular homeostasis. Autophagosomes transport cargo to lysosomes and their formation is dependent on an appropriate lipid supply. Here, we show that the knockout of the RAB GTPase RAB18 interferes with lipid droplet (LD) metabolism, resulting in an impaired fatty acid mobilization. The reduced LD-derived lipid availability influences autophagy and provokes adaptive modifications of the autophagy network, which include increased ATG2B expression and ATG12-ATG5 conjugate formation as well as enhanced ATG2B and ATG9A phosphorylation. Phosphorylation of ATG9A directs this transmembrane protein to the site of autophagosome formation and this particular modification is sufficient to rescue autophagic activity under basal conditions in the absence of RAB18. However, it is incapable of enabling an increased autophagy under inductive conditions. Thus, we illustrate the role of RAB18 in connecting LDs and autophagy, further emphasize the importance of LD-derived lipids for the degradative pathway, and characterize an ATG9A phosphorylation-dependent autophagy rescue mechanism as an adaptive response that maintains autophagy under conditions of reduced LD-derived lipid availability.

2020-01-29 | PXD013719 | Pride

Identification of molecular machinery mediating ER to lipid droplet protein targeting

Project description:Lipid droplets (LDs) are organelles that store lipids and contain important metabolic proteins at their surfaces. The pathway for membrane-embedded hydrophobic proteins to reach the LD surface from the ER is largely unknown. Here, we find that many proteins, including key lipid synthesis and degradation enzymes, are excluded from forming LDs in the ER by the seipin oligomeric complex and target LDs via ER-LD membrane continuities that are established well after LD formation. We utilized systematic, unbiased approaches to identify key components of this ER-to-LD (ERTOLD) pathway. We find that specific membrane-fusion machinery, including membrane fusion regulators (Trs20 and Rab1), a tether (Rint1), and effectors (Syx5, membrin, Bet1, Ykt6), are required for late ERTOLD targeting of GPAT4 and other proteins that utilize this pathway. The fusion machinery components localize to the interface of LDs and the ER, and within the ER, appear to be organized at ER exit sites. Our data therefore uncover a novel mechanism for membrane protein trafficking for ERTOLD targeting via heterotypic organelle fusion between the ER and LDs.

2022-06-27 | PXD027283 | Pride

Lipid droplets protect human β-cells from lipotoxic-induced stress and cell identity changes

Project description:Free fatty acids (FFAs) are often stored in lipid droplet (LD) depots for eventual metabolic and/or synthetic use in many cell types, such a muscle, liver, and fat. In pancreatic islets, overt LD accumulation was detected in humans but not mice. LD buildup in islets was principally observed after roughly 11 years of age, increasing throughout adulthood under physiologic conditions, and also enriched in type 2 diabetes. To obtain insight into the role of LDs in human islet β cell function, the levels of a key LD scaffold protein, perilipin2 (PLIN2), were manipulated by lentiviral-mediated knock-down (KD) or over-expression (OE) in EndoCβH2-Cre cells, a human cell line with adult islet β-like properties. Glucose stimulated insulin secretion was blunted in PLIN2KD cells and improved in PLIN2OE cells. An unbiased transcriptomic analysis revealed that limiting LD formation induced effectors of endoplasmic reticulum (ER) stress that compromised the expression of critical β cell function and identity genes. These changes were essentially reversed by PLIN2OE or using the ER stress inhibitor, tauroursodeoxycholic acid. These results strongly suggest that LDs are essential for adult human islet β cell activity by preserving FFA homeostasis.

2021-09-15 | GSE184016 | GEO

The large GTPase Sey1/atlastin mediates lipid droplet- and FadL-dependent intracellular fatty acid metabolism of Legionella pneumophila

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2023-06-07 | PXD038200 | Pride

Systematic genome perturbation reveals coordination of lipid droplets and metabolic gene regulation

Project description:Lipid droplets (LDs) store lipids for metabolic eneergy and are central to cellular lipid homeostasis. The mechanisms coordinating lipid storage in LDs with cellular metabolism are unclear but relevant to obesity-related diseases. Here we utilized genome-wide screening to identify genes that modulate lipid storage in human macrophages, a cell type relevant to metabolic diseases. Among ~550 genes regulating lipid storage, we identify MLX, a basic helix-loop-helix leucine-zipper transcription factor that regulates multiple metabolic processes. We show that MLX and family members MLXIP/MondoA and MLXIPL/ChREBP bind LDs via C-terminal amphipathic helices. When LDs increase in cells, LD binding of MLX, MLXIP/MondoA, and MLXIPL/ChREBP reduces their transcriptional activity, whereas the absence of LDs results in hyperactivation. Our findings uncover an unexpected component to a lipid storage response, in which binding of MLX transcription factors to the LD surface modulates their activity, adjusting the expression of metabolic genes to lipid storage levels.

2021-09-08 | PXD012640 | Pride

Functional multi-organelle units control inflammatory lipid metabolism of macrophages

Project description:Eukaryotic cells contain several membrane-separated organelles to compartmentalize distinct metabolic reactions. However, it has remained unclear how these organelle systems are coordinated, when cells adapt metabolic pathways to support their development, survival or effector functions. Here we present OrgaPlexing, a multispectral organelle imaging approach for the comprehensive mapping of six key metabolic organelles and their interactions. We use this analysis on macrophages, immune cells that undergo rapid metabolic switches upon sensing bacterial and inflammatory stimuli. Our results identify lipid droplets (LDs) as primary inflammatory responder organelle, which forms three- and four-way interactions with other organelles. While clusters with endoplasmic reticulum (ER) and mitochondria (M-ER-LD unit) help supply fatty acids for LD growth, the additional recruitment of peroxisomes (M-ER-P-LD unit) supports fatty acid efflux from LDs. Interference with individual components of these units has direct functional consequences for inflammatory lipid synthesis. Together, we show that macrophages form functional multi-organellar units (MOUs) to support metabolic adaptation, and provide an experimental strategy to identify organelle-metabolic signaling hubs.

2024-05-03 | GSE235484 | GEO

BirA*-RAB18 proximity biotinylation in HeLa cells

Project description:Proximity labelling using transient expression of the same exogenous BirA*-RAB18 construct in wild-type HeLa cells and in otherwise isogenic RAB3GAP1-, RAB3GAP2- and TRAPPC9-null HeLa cell lines.

2024-01-26 | PXD016336 | Pride

Arabidopsis EARLY RESPONSIVE TO DEHYDRATION 7 associates with lipid droplets via its senescence domain

Project description:Lipid droplets (LDs) are neutral lipid-containing organelles found in all kingdoms of life and are decorated with proteins that carry out a variety of functions. Despite the similar subcellular location, the LD proteins that carry out these functions vary between kingdoms, and only a few LD proteins share obvious amino acid sequence identity between plants and mammals. To better understand the many cellular roles of LDs in plants, a more comprehensive inventory and characterization of LD proteins is required. To this end, we analyzed proteomics data of isolated LDs and identified EARLY RESPONSIVE TO DEHYDRATION 7 (ERD7) as a putative LD protein. mCherry-tagged ERD7 localized to both LDs and to the cytosol when transiently expressed in Nicotiana benthamiana leaves, whereas a fragment of ERD7 containing a “senescence domain” (SD) localized exclusively to LDs with no appreciable accumulation in the cytosol. Similarly, the SDs of the two Arabidopsis ERD7 homologs, here termed SENESCENCE-ASSOCIATED A2 (SENA2) and SENA3, localized exclusively to LDs as did the SD of a phylogenetically distant SD-containing protein, human spartin. In contrast, SDs of the Arabidopsis SENESCENCE-ASSOCIATED B (SENB) family proteins did not localize to LDs and instead localized to mitochondria. Arabidopsis erd7 mutants did not display any observable LD phenotypes in seeds, seedlings or senescent leaves, perhaps due to genetic redundancy of the SENA protein family. A yeast-two-hybrid screen revealed that the S4 family of MYB transcription factors interacts with ERD7, providing a potential avenue for the exploration of ERD7 function. In summary, we here report a previously unappreciated family of plant LD-associated proteins, SENA, which contain SDs that localize to LDs.

2021-03-29 | PXD023047 | Pride

A fluorescent perilipin 2 knock-in mouse model reveals a high abundance of lipid droplets in the developing and adult brain

Project description:Lipid droplets (LDs) are dynamic lipid storage organelles. They are tightly linked to metabolism and can exert protective functions, making them important players in health and disease. Most LD studies in vivo rely on staining methods, providing only a snapshot. We therefore developed a LD-reporter mouse by endogenously labelling the LD coat protein perilipin 2 (PLIN2) with tdTomato, enabling staining-free fluorescent LD visualisation in living and fixed tissues and cells. Here we validate this model under standard and high-fat diet conditions and demonstrate that LDs are highly abundant in various cell types in the healthy brain, including neurons, astrocytes, ependymal cells, neural stem/progenitor cells and microglia. Furthermore, we also show that LDs are abundant during brain development and can be visualized using live-imaging of embryonic slices. Taken together, our tdTom-Plin2 mouse serves as a novel tool to study LDs and their dynamics under both physiological and diseased conditions in all tissues expressing Plin2.

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Lipid droplet availability affects neural stem/progenitor cell metabolism and proliferation

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2021-11-10 | GSE187470 | GEO

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