Parallel CRISPR-Cas9 screens identify mechanisms of PLIN2 and lipid droplet regulation

Roberts, Melissa A.; Deol, Kirandeep K.; Mathiowetz, Alyssa J.; Lange, M.; Leto, Dara; Stevenson, Julian; Hashemi, Seyed Mohammad; Morgens, David W.; Easter, Emilee; Heydari, Kartoosh; Nalls, Mike A.; Beer, M; Kampmann, Martin; Kopito, Ron R.; Faghri, Faraz; Olzmann, James A.

doi:10.1016/j.devcel.2023.07.001

Cited by 11 publications

(9 citation statements)

References 70 publications

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“…We first eliminated these concerns by demonstrating that an endogenous lipid droplet protein, perilipin 2 (PLIN 2) remained on lipid droplets decorated with overexpressed 6xHp (Figure 2I). In addition, overexpressing 6xHp at low and moderate levels (similar levels for all imaging experiments) minimally affected lipid droplet biogenesis (Figure 2J), lipid droplet breakdown following inhibition of long chain acyl-CoA synthase by Triacsin C treatment (Figure 2K)(Hartman et al, 1989; Roberts et al, 2023; Tomoda et al, 1987), and lipid droplet consumption incubation with Hanks’ balanced salt solution (Figure S1B). In conclusion, we found that 6xHp is suitable for targeting lipid droplets with negligible perturbation to the functions of these organelles.…”

Section: Resultsmentioning

confidence: 76%

A fluorogenic complementation tool kit for interrogating lipid droplet-organelle interaction

Li,

Gamuyao,

et al. 2023

Preprint

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Contact sites between lipid droplets and other organelles are essential for cellular lipid and energy homeostasis. Detection of these contact sites at nanometer scale over time in living cells is challenging. Here, we developed a tool kit for detecting contact sites based onFluorogen-ActivatedBimolecular complementation atCONtact sites, FABCON, using a reversible, low affinity split fluorescent protein, splitFAST. FABCON labels contact sites with minimal perturbation to organelle interaction. Via FABCON, we quantitatively demonstrated that endoplasmic reticulum (ER)- and mitochondria (mito)-lipid droplet contact sites are dynamic foci in distinct metabolic conditions, such as during lipid droplet biogenesis and consumption. An automated analysis pipeline further classified individual contact sites into distinct subgroups based on size, likely reflecting differential regulation and function. Moreover, FABCON is generalizable to visualize a repertoire of organelle contact sites including ER-mito. Altogether, FABCON reveals insights into the dynamic regulation of lipid droplet-organelle contact sites and generates new hypotheses for further mechanistical interrogation during metabolic switch.

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Section: Resultsmentioning

confidence: 76%

A fluorogenic complementation tool kit for interrogating lipid droplet-organelle interaction

Li,

Gamuyao,

et al. 2023

Preprint

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“…Moreover, the human E2 enzyme Ube2J2, orthologous to yeast’s Ubc6, works in tandem with MARCH6, affecting proteins including SQLE 32 – 34 . Recent findings expand MARCH6’s role to several other metabolic pathways 34 – 39 . The importance of MARCH6’s role in metabolic regulation is underscored by sterols stimulating ubiquitin-dependent degradation of SQLE 31 .…”

Section: Introductionmentioning

confidence: 65%

“…7a ). When probing the same cell lines for endogenous levels of the lipid droplet protein PLIN2—another protein whose amount is regulated by MARCH6, although potentially not as a direct ubiquitylation target 38 , 39 —we observed similar PLIN2 stabilization in some of the more robust mutants but weaker or even opposite effects in other positions (Supplementary Fig. 7b ).…”

Section: Resultsmentioning

confidence: 90%

Doa10/MARCH6 architecture interconnects E3 ligase activity with lipid-binding transmembrane channel to regulate SQLE

Botsch,

Junker,

Sorgenfrei

et al. 2024

Nat Commun

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Transmembrane E3 ligases play crucial roles in homeostasis. Much protein and organelle quality control, and metabolic regulation, are determined by ER-resident MARCH6 E3 ligases, including Doa10 in yeast. Here, we present Doa10/MARCH6 structural analysis by cryo-EM and AlphaFold predictions, and a structure-based mutagenesis campaign. The majority of Doa10/MARCH6 adopts a unique circular structure within the membrane. This channel is established by a lipid-binding scaffold, and gated by a flexible helical bundle. The ubiquitylation active site is positioned over the channel by connections between the cytosolic E3 ligase RING domain and the membrane-spanning scaffold and gate. Here, by assaying 95 MARCH6 variants for effects on stability of the well-characterized substrate SQLE, which regulates cholesterol levels, we reveal crucial roles of the gated channel and RING domain consistent with AlphaFold-models of substrate-engaged and ubiquitylation complexes. SQLE degradation further depends on connections between the channel and RING domain, and lipid binding sites, revealing how interconnected Doa10/MARCH6 elements could orchestrate metabolic signals, substrate binding, and E3 ligase activity.

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“…Unsaturated FAs like oleate are readily packaged into LDs and thus thought to be tolerated more by cells. On the other hand, studies have shown that treatment of cultured cells with palmitate increased the amount of SFAs specifically in glycerophospholipids which reduces membrane fluidity and trigger an ER stress response [12–14]. Interestingly, addition of oleate rescued palmitate‐induced toxicity in cells by promoting TAG synthesis and LD biogenesis.…”

Section: Fatty Acid‐induced Lipotoxicitymentioning

confidence: 99%

“…This is because these factors are involved in the early steps of glycerolipid biosynthesis, and they also play a role in incorporating SFAs into phospholipids. An increase in SFA‐containing glycerophospholipids affects membrane fluidity and promotes SFA‐induced toxicity [12–14]. Therefore, these results further highlight the competing pathways for channeling SFAs into TAG or membrane phospholipids which can have different functional outcomes on cells.…”

Section: Fatty Acid‐induced Lipotoxicitymentioning

confidence: 99%

Lipid droplets and fatty acid‐induced lipotoxicity: in a nutshell

Obaseki,

Adebayo,

Bandyopadhyay

et al. 2024

FEBS Letters

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Lipid droplets (LDs) are fat storage organelles that are conserved from bacteria to humans. LDs are broken down to supply cells with fatty acids (FAs) that can be used as an energy source or membrane synthesis. An overload of FAs disrupts cellular functions and causes lipotoxicity. Thus, by acting as hubs for storing excess fat, LDs prevent lipotoxicity and preserve cellular homeostasis. LD synthesis and turnover have to be precisely regulated to maintain a balanced lipid distribution and allow for cellular adaptation during stress. Here, we discuss how prolonged exposure to excess lipids affects cellular functions, and the roles of LDs in buffering cellular stress focusing on lipotoxicity.

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Parallel CRISPR-Cas9 screens identify mechanisms of PLIN2 and lipid droplet regulation

Cited by 11 publications

References 70 publications

A fluorogenic complementation tool kit for interrogating lipid droplet-organelle interaction

A fluorogenic complementation tool kit for interrogating lipid droplet-organelle interaction

Doa10/MARCH6 architecture interconnects E3 ligase activity with lipid-binding transmembrane channel to regulate SQLE

Lipid droplets and fatty acid‐induced lipotoxicity: in a nutshell

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