Lipid droplets (LDs) are emerging cellular organelles that of crucial importance in cell biology and human diseases. In this study, we present our screen ∼4,700 Saccharomyces cerevisiae mutants for abnormalities the number morphology LDs; identify 17 fld (few LDs) 116 mld (many mutants. One (fld1) is caused by deletion YLR404W, a previously uncharacterized open reading frame. Cells lacking FLD1 contain strikingly enlarged (supersized) LDs, LDs from fld1Δ cells demonstrate significantly...

The biosynthesis and storage of triglyceride (TG) is an important cellular process conserved from yeast to man. Most mammalian cells accumulate TG in lipid droplets, most prominent adipocytes, which are specialized store large amounts the over long periods. In this study, we followed targeting by fluorescence imaging living 3T3-L1 adipocytes COS7 fibroblasts. Key findings were (i) not only but also its direct metabolic precursor diacylglycerol, DG, accumulates on droplets; (ii) essential...

Phosphatidylserine (PS) plays a central role in cell signaling and the biosynthesis of other lipids. To date, however, subcellular distribution transmembrane topology this crucial phospholipid remain ill-defined. We transfected cells with GFP-tagged C2 domain lactadherin to detect by light electron microscopy PS exposed on cytosolic leaflet plasmalemma organellar membranes. Cytoplasmically was found be clustered plasma membrane, associated caveolae, trans-Golgi network, endocytic organelles...

Triglyceride cycling is the process of continuous degradation and re-synthesis triglyceride in cellular stores. We show 3T3-L1 adipocytes that triglycerides are subject to rapid turnover re-arrangement fatty acids with an estimated half-life 2-4 h. develop a tracing technology can simultaneously quantitatively follow metabolism multiple study futile substrate cycle directly molecular species resolution. Our approach based on alkyne acid tracers mass spectrometry. The connected modification...

Phosphatidylcholine (PC) is synthesized by two different pathways, the Lands cycle and Kennedy pathway. The recently identified key enzymes of cycle, lysophosphatidylcholine acyltransferase 1 2 (LPCAT1 -2), were reported to localize endoplasmic reticulum function in lung surfactant production inflammation response. Here, we show various mammalian cell lines that both additionally lipid droplets (LDs), which consist a core neutral lipids surrounded monolayer phospholipid, mainly PC. This dual...

Fatty acids are abundant constituents of all biological systems, and their metabolism is important for normal function at levels an organism. Aberrations in fatty acid associated with pathological states have become a focus current research, particularly due to the interest metabolic overload diseases. Here we present click-chemistry-based method that allows tracing virtually any system. It combines high sensitivity excellent linearity fast sample turnover. Since it free radioactivity, can...

Lipid droplets (LDs), the major intracellular storage sites for neutral lipids, consist of a lipid core surrounded by phospholipid monolayer membrane. In addition to their function in storage, LDs participate biosynthesis and recently were implicated proteasomal protein degradation autophagy. To identify components machinery on LDs, we studied several candidates identified previous LD proteome analyses. Here, demonstrate that highly conserved broadly expressed ancient ubiquitous 1 (AUP1)...

1-Deoxysphingolipids (deoxySLs) are atypical sphingolipids that elevated in the plasma of patients with type 2 diabetes and hereditary sensory autonomic neuropathy 1 (HSAN1). Clinically, diabetic HSAN1 very similar, suggesting involvement deoxySLs pathology both diseases. However, little is known about biology these lipids underlying pathomechanism. We synthesized an alkyne analog 1-deoxysphinganine (doxSA), metabolic precursor all deoxySLs, to trace metabolism localization deoxySLs. Our...

Cholesterol is an important lipid of mammalian cells and plays a fundamental role in many biological processes. Its concentration the various cellular membranes differs tightly regulated. Here, we present novel alkyne cholesterol analog suitable for tracing both metabolism localization. This probe can be detected by click chemistry employing reporter azides. Alkyne accepted enzymes from different species (Brevibacterium, yeast, rat, human) these include oxidases, hydroxylases, acyl...

Click chemistry is evolving as a powerful tool in biological applications because it allows the sensitive and specific detection of compounds with alkyne or azido groups. Here we describe use lipids substrates for vitro enzymatic assays lipid modifying enzymes. The small moiety introduced synthetically at terminus hydrocarbon chain various substrate lipids. After assay, label click-reacted azide-bearing fluorogenic dye 3-azido-7-hydroxycoumarin, followed by separation mix thin-layer...

The grey and white matter regions of the mammalian brain consist both neurons neuroglial cells. Among neuroglia, two macroglia oligodendrocytes astrocytes are most abundant cell types. While major function is formation lipid-rich myelin structure, heterogeneous group fulfils a multitude important roles in cerebral development homeostasis. Brain lipid homeostasis involves synthesis specific lipidome by local metabolism. In this study we have investigated fatty acid uptake biosynthesis murine...

A high fat Western-style diet leads to hepatic steatosis that can progress steatohepatitis and ultimately cirrhosis or liver cancer. The mechanism the development of upon nutritional overload is complex only partially understood. Using click chemistry-based metabolic tracing microscopy, we study interaction between Kupffer cells hepatocytes ex vivo. In early phase steatosis, alone do not display significant deviations in fatty acid metabolism. However, co-cultures supernatant transfer...

1-Deoxysphingolipids (deoxySLs) are atypical sphingolipids of clinical relevance as they elevated in plasma patients suffering from hereditary sensory and autonomic neuropathy (HSAN1) or type 2 diabetes. Their neurotoxicity is described best but inflict damage to various cell types by an uncertain pathomechanism. Using mouse embryonic fibroblasts alkyne analog 1-deoxysphinganine (doxSA), the metabolic precursor all deoxySLs, we here study impact deoxySLs on macroautophagy/autophagy,...

A series of 2-(diethylamino)thieno1,3ŏxazin-4-ones was synthesized and evaluated in vitro for inhibitory activity toward human leukocyte elastase (HLE). The Gewald thiophene synthesis utilized to obtain several ethyl 2-aminothiophene-3-carboxylates. These precursors were subjected a five-step route thieno2,3-d1,3ŏxazin-4-ones bearing various substituents at positions 5 6. Both thieno2,3-d thieno3,2-d fused oxazin-4-ones possess extraordinary chemical stability, which expressed as rate...

Although the brain controls all main metabolic pathways in whole organism, its lipid metabolism is partially separated from rest of body. Circulating lipids and other metabolites are taken up into areas like hypothalamus locally metabolized sensed involving several hypothalamic cell types. In this study we show that saturated unsaturated fatty acids differentially processed murine hypothalamus. The observed differences involve both distribution metabolism. Key findings were: (i) astrocytes...

Abstract Adipose tissue is an organized endocrine organ with important metabolic and immunological functions immune cell-adipocyte crosstalk known to drive various disease pathologies. Suitable 3D adipose organoid models often lack resident cell populations therefore require the addition of cells isolated from other organs. We have created first model which could contain maintain stromal vascular fraction (SVF) proved be effective in studying biology a convenient manner. Macrophage mast were...

Penicillin-binding protein 3 (PBP3; also called FtsI) is a transpeptidase that catalyzes cross-linking of the peptidoglycan cell wall in division septum Escherichia coli. To determine whether catalytic activity PBP3 activated during division, we assayed acylation with three beta-lactams (cephalexin, aztreonam, and piperacillin) growing cells. Acylation cephalexin, but not aztreonam or piperacillin, appeared to be stimulated by division. Specifically, cephalexin acylated about 50% faster...

The AT1 receptor blocker telmisartan (TEL) prevents diet-induced obesity. Hypothalamic lipid metabolism is functionally important for energy homeostasis, as a surplus of lipids induces an inflammatory response in the hypothalamus, thus promoting development central leptin resistance. However, it unclear to whether TEL treatment affects status hypothalamus. C57BL/6N mice were fed with chow (CONchow) or high-fat diet (CONHFD). HFD-fed gavaged (8 mg/kg/day, 12 weeks, TELHFD). Mice phenotyped...

Mammalian cells synthesize hundreds of different variants their prominent membrane lipid phosphatidylcholine (PC), all differing in the side chain composition. This batch is constantly remodeled by Lands cycle, a metabolic pathway replacing one at time. Using alkyne lyso-phosphatidylpropargylcholine (LpPC), precursor and intermediate PC synthesis remodeling, we study both processes brain endothelial bEND3 cells. A novel method for multiplexed sample analysis mass spectrometry developed that...

1-Deoxysphingolipids (1-deoxySL) are a non-canonical class of sphingolipids linked to genetic and metabolic disorders, including type II diabetes, hereditary sensory autonomic neuropathy 1, macular telangiectasia (MacTel). In MacTel patients, retinal pigment epithelium (RPE) cells exhibit loss apical phagocytic receptors, suggesting potential role for 1-deoxySLs in membrane trafficking. Here we use range lipid chemical biology approaches investigate the effect on properties functions...

Ether lipids are ubiquitous constituents of cellular membranes with no discrete cell biological function assigned yet. Using fluorescent polyene-ether we analyzed their intracellular distribution in living cells by microscopy. Mitochondria and the endoplasmic reticulum accumulated high amounts ether-phosphatidylcholine ether-phosphatidylethanolamine. Both were specifically labeled using corresponding lyso-ether lipids, which established as supreme precursors for lipid tagging. Polyfosine, a...

The demand to study the cellular localization of specific lipids has led recent advances in lipid probes and microscopy. Alkyne bear a small, noninterfering tag can be detected upon click reaction with an azide-coupled reporter. Fluorescent alkyne imaging crucially depends on appropriate azide reporters labeling protocols that allow for efficient therefore sensitive detection. We synthesized several different spacer components tested their suitability fixed cells. implementation...