Dataset Information

Subcellular organelle lipidomics in TLR-4-activated macrophages.

ABSTRACT: Lipids orchestrate biological processes by acting remotely as signaling molecules or locally as membrane components that modulate protein function. Detailed insight into lipid function requires knowledge of the subcellular localization of individual lipids. We report an analysis of the subcellular lipidome of the mammalian macrophage, a cell type that plays key roles in inflammation, immune responses, and phagocytosis. Nuclei, mitochondria, endoplasmic reticulum (ER), plasmalemma, and cytoplasm were isolated from RAW 264.7 macrophages in basal and activated states. Subsequent lipidomic analyses of major membrane lipid categories identified 229 individual/isobaric species, including 163 glycerophospholipids, 48 sphingolipids, 13 sterols, and 5 prenols. Major subcellular compartments exhibited substantially divergent glycerophospholipid profiles. Activation of macrophages by the Toll-like receptor 4-specific lipopolysaccharide Kdo(2)-lipid A caused significant remodeling of the subcellular lipidome. Some changes in lipid composition occurred in all compartments (e.g., increases in the levels of ceramides and the cholesterol precursors desmosterol and lanosterol). Other changes were manifest in specific organelles. For example, oxidized sterols increased and unsaturated cardiolipins decreased in mitochondria, whereas unsaturated ether-linked phosphatidylethanolamines decreased in the ER. We speculate that these changes may reflect mitochondrial oxidative stress and the release of arachidonic acid from the ER in response to cell activation.

SUBMITTER: Andreyev AY

PROVIDER: S-EPMC2918461 | biostudies-literature | 2010 Sep

REPOSITORIES: biostudies-literature

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Subcellular organelle lipidomics in TLR-4-activated macrophages.

Andreyev Alexander Y AY Fahy Eoin E Guan Ziqiang Z Kelly Samuel S Li Xiang X McDonald Jeffrey G JG Milne Stephen S Myers David D Park Hyejung H Ryan Andrea A Thompson Bonne M BM Wang Elaine E Zhao Yihua Y Brown H Alex HA Merrill Alfred H AH Raetz Christian R H CR Russell David W DW Subramaniam Shankar S Dennis Edward A EA

Journal of lipid research 20100623 9

Lipids orchestrate biological processes by acting remotely as signaling molecules or locally as membrane components that modulate protein function. Detailed insight into lipid function requires knowledge of the subcellular localization of individual lipids. We report an analysis of the subcellular lipidome of the mammalian macrophage, a cell type that plays key roles in inflammation, immune responses, and phagocytosis. Nuclei, mitochondria, endoplasmic reticulum (ER), plasmalemma, and cytoplasm ...[more]

PMID: 20574076

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Project description:Compartmentalization of biological processes and the associated cellular components is crucial for cell function. Typically, the location of a component is revealed through a co-localization and/or co-purification with an organelle marker. Therefore, the identification of reliable markers is critical for a thorough understanding of cellular function and dysfunction. We fractionated macrophage-like RAW264.7 cells, both in the resting and endotoxin-activated states, into six fractions representing the major organelles/compartments: nuclei, mitochondria, cytoplasm, endoplasmic reticulum, and plasma membrane as well as an additional dense microsomal fraction. The identity of the first five of these fractions was confirmed via the distribution of conventional enzymatic markers. Through a quantitative liquid chromatography/mass spectrometry-based proteomics analysis of the fractions, we identified 50-member ensembles of marker proteins ("marker ensembles") specific for each of the corresponding organelles/compartments. Our analysis attributed 206 of the 250 marker proteins ( approximately 82%) to organelles that are consistent with the location annotations in the public domain (obtained using DAVID 2008, EntrezGene, Swiss-Prot, and references therein). Moreover, we were able to correct locations for a subset of the remaining proteins, thus proving the superior power of analysis using multiple organelles as compared with an analysis using one specific organelle. The marker ensembles were used to calculate the organelle composition of the six above mentioned subcellular fractions. Knowledge of the precise composition of these fractions can be used to calculate the levels of metabolites in the pure organelles. As a proof of principle, we applied these calculations to known mitochondria-specific lipids (cardiolipins and ubiquinones) and demonstrated their exclusive mitochondrial location. We speculate that the organelle-specific protein ensembles may be used to systematically redefine originally morphologically defined organelles as biochemical entities.

Dataset Information

Subcellular organelle lipidomics in TLR-4-activated macrophages.

Publications

Subcellular organelle lipidomics in TLR-4-activated macrophages.

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