Thin-Layer Chromatography to Separate Phospholipids and Neutral Lipids from Yeast

Knittelfelder, Oskar

doi:10.1101/pdb.prot085456

Cited by 23 publications

(20 citation statements)

References 3 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…2b ). Corroborating the LC-MS/MS findings, thin layer chromatography (TLC) 31 showed a distinct spot for oxidized PS between PS and lyso-PS ( Supplementary Fig. 14 ).…”

Section: Resultsmentioning

confidence: 72%

“…The reaction was quenched as described 29 , and preparative HPLC procedure (see Supplementary Note ) was used to purify the oxidized PS. The purity of oxidized PS was assessed first by thin layer chromatography (TLC) 31 ( Supplementary Fig. 14 ) and 1 H-NMR analysis, and the identity was further confirmed by lipid fragmentation analysis described in the next section, 1 H-NMR and IR spectroscopic analysis (see Supplementary Note ).…”

Section: Methodsmentioning

confidence: 99%

See 1 more Smart Citation

A chemical–genetic screen identifies ABHD12 as an oxidized-phosphatidylserine lipase

et al. 2019

View full text Add to dashboard Cite

Reactive oxygen species (ROS) are transient, highly reactive intermediates or byproducts produced during oxygen metabolism. However, when innate mechanisms are unable to cope with sequestration of surplus ROS, it causes oxidative stress, where excess ROS damage biomolecules. Oxidized phosphatidylserine (PS), a pro-apoptotic “eat me” signal, is produced in response to elevated ROS, yet, little is known of its chemical composition and metabolism. Here, we report a small molecule that generates ROS in different mammalian cells, using which we detect, characterize and study oxidized PS in mammalian cells. We describe a chemical genetic screen to identify enzymes that regulate oxidized PS in mammalian cells, and find that the lipase ABHD12 hydrolyzes oxidized PS. We validate these findings in different physiological settings including primary peritoneal macrophages, and brains from Abhd12–/– knockout mice under inflammatory stress, and in the process functionally annotate an enzyme capable of regulating oxidized PS in vivo.

show abstract

“…2b ). Corroborating the LC-MS/MS findings, thin layer chromatography (TLC) 31 showed a distinct spot for oxidized PS between PS and lyso-PS ( Supplementary Fig. 14 ).…”

Section: Resultsmentioning

confidence: 72%

Section: Methodsmentioning

confidence: 99%

A chemical–genetic screen identifies ABHD12 as an oxidized-phosphatidylserine lipase

et al. 2019

View full text Add to dashboard Cite

show abstract

“…Plates were imaged using a Bio-Rad Gel Doc and bands were quantified using Fiji. Lipids were identified according to Knittelfelder & Kohlwein [126] and by comparing whole lipid extracts to lipid standards (Millipore Sigma, data not shown). Colorimetric band intensities were quantified in Fiji and triglyceride or sterol ester bands were normalized to cholesterol band intensity.…”

Section: Methodsmentioning

confidence: 99%

Section: Thin Layer Chromatographymentioning

confidence: 99%

Adipose triglyceride lipase promotes prostaglandin-dependent actin remodeling by regulating substrate release from lipid droplets

Giedt

Thomalla

Johnson

et al. 2021

Preprint

View full text Add to dashboard Cite

To ensure fertility, it is paramount to understand the factors controlling oocyte quality. One incompletely characterized factor contributing to oocyte quality is lipids. In somatic cells, a key regulator of lipid metabolism is lipid droplets (LDs), the sites of intracellular fat storage. Yet the role of LDs in fertility is poorly understood. Here we use Drosophila oogenesis as a model for uncovering if and how LDs promote egg development. LD accumulation in nurse cells coincides with dynamic actin remodeling necessary for late-stage follicle morphogenesis and fertility. Loss of major LD proteins, including PLIN2, Jabba, and ATGL, disrupts both actin bundle formation and cortical actin integrity; this unusual phenotype is also seen when Pxt, the enzyme responsible for prostaglandin (PG) synthesis, is missing. Further, both pharmacologic and genetic loss of PG synthesis or loss of PLIN2 or Jabba impairs intracellular LD dispersal. These similar phenotypes suggest that PGs and LD proteins act in the same pathway. Dominant genetic interaction studies indicate that there are three actin regulatory pathways: PLIN2 regulates actin remodeling independent of PG signaling, whereas Jabba and ATGL act in two separate PG-dependent pathways to regulate actin remodeling. We find that neither Jabba nor ATGL modulate the levels of Pxt or its localization to the endoplasmic reticulum. As ATGL is a triglyceride lipase, we hypothesize that it may release arachidonic acid (AA), the substrate for PG production, from triglycerides stored in LDs. Indeed, lipidomic analysis reveals the presence of AA-containing triglycerides in ovaries. In addition, exogenous AA is toxic and reduction of ATGL ameliorates toxicity; these observations suggest that ATGL indeed generates free AA. Our studies provide the first evidence that LDs and their associated proteins regulate PG signaling to control actin remodeling. In particular, we propose that ATGL releases AA from LDs to drive PG synthesis necessary for follicle development. We speculate that the same pathways are conserved across organisms to regulate oocyte development and promote fertility.

show abstract

“…Neutral lipid separation and analysis was performed by thin-layer chromatography (TLC) on silica gel 60 plates (Merck), essentially as described before [ 54 ], using CHCl 3 /MeOH/water (32.5:12.5:2) mixture as mobile phase [ 55 , 56 ]. TLC plates were derivatized by dipping into 3.2% H 2 SO 4 and 0.5% MnCl 2 , followed by carbonization at 120 °C for 30 min.…”

Section: Methodsmentioning

confidence: 99%

Porin 1 Modulates Autophagy in Yeast

Broeskamp

Edrich

Knittelfelder

et al. 2021

Cells

View full text Add to dashboard Cite

Autophagy is a cellular recycling program which efficiently reduces the cellular burden of ageing. Autophagy is characterised by nucleation of isolation membranes, which grow in size and further expand to form autophagosomes, engulfing cellular material to be degraded by fusion with lysosomes (vacuole in yeast). Autophagosomal membranes do not bud from a single cell organelle, but are generated de novo. Several lipid sources for autophagosomal membranes have been identified, but the whole process of their generation is complex and not entirely understood. In this study, we investigated how the mitochondrial outer membrane protein porin 1 (Por1), the yeast orthologue of mammalian voltage-dependent anion channel (VDAC), affects autophagy in yeast. We show that POR1 deficiency reduces the autophagic capacity and leads to changes in vacuole and lipid homeostasis. We further investigated whether limited phosphatidylethanolamine (PE) availability in por1∆ was causative for reduced autophagy by overexpression of the PE-generating phosphatidylserine decarboxylase 1 (Psd1). Altogether, our results show that POR1 deficiency is associated with reduced autophagy, which can be circumvented by additional PSD1 overexpression. This suggests a role for Por1 in Psd1-mediated autophagy regulation.

show abstract

Thin-Layer Chromatography to Separate Phospholipids and Neutral Lipids from Yeast

Cited by 23 publications

References 3 publications

A chemical–genetic screen identifies ABHD12 as an oxidized-phosphatidylserine lipase

A chemical–genetic screen identifies ABHD12 as an oxidized-phosphatidylserine lipase

Adipose triglyceride lipase promotes prostaglandin-dependent actin remodeling by regulating substrate release from lipid droplets

Porin 1 Modulates Autophagy in Yeast

Contact Info

Product

Resources

About