Biology subjects

Ejsing, C. S.

Publications and source records attributed to Ejsing, C. S..

6 recordsLinked to original sources

bioRxiv2025

Live-cell imaging and lipidomics of low density lipoprotein containing intrinsically fluorescent cholesteryl esters

Low density lipoprotein (LDL) delivers cholesterol to cells in the body in the form of cholesteryl esters (CEs), and dysfunction of this pathway is associated with various diseases. Due to the lack of suitable tools, our understanding of the intracellular transport and hydrolysis of CEs is limited. We present a novel approach for studying LDL-derived CEs in cells, using fatty acyl chain conjugates of the intrinsically fluorescent cholestatrienol (CTL). We demonstrate that CTL esters reconstituted into LDL particles are hydrolyzed in late endosomes and lysosomes (LE/LYSs) by acid lipase, while an LDL-derived CTL ether analog cannot leave LE/LYSs. Using live-cell imaging, lipidomics, and multimodal Bayesian modeling, we discover a sequential biphasic transport of LDL-derived CTL to LE/LYSs matching the kinetics of the lysosomal hydrolysis of LDL-associated CTL-ester with a half-time of 3.0 hours. Hydrolyzed CTL derived from LDL-associated CTL esters is rapidly re-esterified with a similar half-time and stored in lipid droplets, demonstrating efficient sterol transport to the endoplasmic reticulum (ER). The latter is supported by the detection of a faint CTL staining in the ER and by extensive contact formation between endo-lysosomes containing LDL and ER tubules. Using lipidomics and Bayesian kinetic modeling, we also track LDL-derived CEs and triacylglycerols in cells and determine the uptake kinetics for each lipid species individually. Our novel approach allows for precise measurement of post-endocytic trafficking and metabolism of LDL-derived cholesterol and other lipids in living cells.

biochemistry↗

bioRxiv2024

Caging of membrane-to-cortex attachment proteins can trigger cellular symmetry breaking

To migrate, divide, and change shape, cells must regulate the mechanics of their periphery. The cell surface is a complex structure that consists of a thin, contractile cortical actin network tethered to the plasma membrane by specialized membrane-to-cortex attachment (MCA) proteins. This active and constantly fluctuating system maintains a delicate mechanochemical state which permits spontaneous polarization and shape change when needed. Combining in silico, in vitro, and in vivo experiments we show how membrane viscosity and MCA protein length regulate cortical dynamics. We reveal a novel mechanism whereby caging of linker proteins in the actin cortex allows for the amplification of small changes in these key parameters, leading to major alterations of cortical contractility. In cells, this mechanism alone gives rise to symmetry breaking phenomena, suggesting that local changes in lipid composition, in combination with the choice of MCA proteins, contribute to the regulation of cellular morphogenesis and function.

biophysics↗

bioRxiv2023

A High-Throughput Data-Independent Acquisition Workflow for Deep Characterisation of the sn-Isomer Lipidome

We report a workflow based on ozone-induced dissociation for untargeted characterization of hundreds of sn-resolved glycerophospholipid isomers from biological extracts in under 20 minutes, coupled with an automated data analysis pipeline. It provides an order of magnitude increase in the number of sn-isomer pairs identified compared to previous reports, reveals that sn-isomer populations are tightly regulated and significantly different between cell lines, and enables identification of rare lipids containing ultra-long chain monounsaturated acyl chains.

cell biology↗

bioRxiv2022

Lipotype acquisition during neural development in vivo is not recapitulated in stem cell-derived neurons

During development, different tissues acquire distinct lipotypes that are coupled to tissue function and homeostasis. In the brain, where complex membrane trafficking systems are required for neural function, specific glycerophospholipids, sphingolipids, and cholesterol are highly abundant, and defective lipid metabolism is associated with abnormal neural development and neurodegenerative disease. Notably, the production of tissue-specific lipotypes requires appropriate programming of the underlying lipid metabolic machinery, but when and how this occurs is unclear. To address this, we used high-resolution mass spectrometry-based (MSALL) lipidomics to perform a quantitative and comprehensive analysis of mouse brain development covering early embryonic and postnatal stages. We discovered a distinct bifurcation in the establishment of the neural lipotype, whereby the canonical brain lipid biomarkers 22:6-glycerophospholipids and 18:0-sphingolipids begin to be produced in utero, whereas cholesterol attains its characteristic high levels after birth. In contrast, when profiling rodent and human stem cell-derived neurons, we observed that these do not acquire a brain lipotype per se. However, upon probing the lipid metabolic wiring by supplementing brain lipid precursors, we found that the stem cell-derived neurons were partially able to establish a brain-like lipotype, demonstrating that the cells are partially metabolically committed. Altogether, our report provides an extensive lipidomic resource for brain development and highlights a potential challenge in using stem cell-derived neurons for mechanistic studies of lipid biochemistry, membrane biology and biophysics that can be mitigated by further optimizing in vitro differentiation protocols. Significance StatementWe report an extensive time-resolved resource of lipid molecule abundances across mouse brain development, starting as early as 10 days post-fertilization. The resource reveals a bifurcation in the establishment of the neural lipotype where the canonical 22:6-glycerophospholipid and 18:0-sphingolipid biomarkers are attained in utero, whereas cholesterol is attained after birth. Furthermore, we uncover that the neural lipotype is not established in rodent and human stem cell-derived neurons in vitro.

developmental biology↗

bioRxiv2020

Molecular species selectivity of lipid transport creates a mitochondrial sink for di-unsaturated phospholipids

Mitochondria depend on the import of phospholipid precursors for the biosynthesis of the non-bilayer lipids phosphatidylethanolamine (PE) and cardiolipin required for proper function, yet the mechanism of lipid import remains elusive. Pulse labeling yeast with stable isotope-labeled serine followed by mass spectrometry analysis revealed that mitochondria preferentially import di-unsaturated phosphatidylserine (PS) for conversion to PE by the mitochondrial PS decarboxylase Psd1p. Several protein complexes tethering mitochondria to the endomembrane system have been implicated in lipid transport in yeast, including the endoplasmic reticulum (ER)-mitochondrial encounter structure (ERMES), ER-mitochondria complex (EMC) and the vacuole and mitochondria patch (vCLAMP). By limiting the availability of unsaturated phospholipids through overexpression of the glycerol-3-phosphate acyltransferase Sct1p, conditions were created to investigate the mechanism of lipid transfer and the contribution of the tethering complexes in vivo. Under these conditions, inactivation of ERMES components or the vCLAMP component Vps39p exacerbated the lipid phenotype, indicating that ERMES and Vps39 contribute to the mitochondrial sink for unsaturated acyl chains by mediating transfer of di-unsaturated phospholipids. The results support the concept that intermembrane lipid flow is rate-limited by molecular species-dependent lipid efflux from the donor membrane and driven by the lipid species concentration gradient between donor and acceptor membrane.

biochemistry↗

bioRxiv2020

Uptake of endogenous serine is important to maintain sphingolipid homeostasis in Saccharomyces cerevisiae

Sphingolipids are abundant and essential molecules in eukaryotes that have crucial functions as signaling molecules and as membrane components. Sphingolipid biosynthesis starts in the endoplasmic reticulum with the condensation of serine and palmitoyl-CoA. Sphingolipid biosynthesis is highly regulated to maintain sphingolipid homeostasis. Even though, serine is an essential component of the sphingolipid biosynthesis pathway, its role in maintaining sphingolipid homeostasis has not been precisely studied. Here we show that serine uptake is an important factor for the regulation of sphingolipid biosynthesis in Saccharomyces cerevisiae. Using genetic experiments, we find the broad-specificity amino acid permease Gnp1 to be important for serine uptake. We confirm these results with serine uptake assays in gnp1{Delta} cells. We further show that uptake of exogenous serine by Gnp1 is important to maintain cellular serine levels and observe a specific connection between serine uptake and the first step of sphingolipid biosynthesis. Using mass spectrometry-based flux analysis, we further observed imported serine as the main source for de novo sphingolipid biosynthesis. Our results demonstrate that yeast cells preferentially use the uptake of exogenous serine to regulate sphingolipid biosynthesis. Our study can also be a starting point to analyze the role of serine uptake in mammalian sphingolipid metabolism. Author SummarySphingolipids (SPs) are membrane lipids globally required for eukaryotic life. In contrast to other lipid classes, SPs cannot be stored in the cell and therefore their levels have to be tightly regulated. Failure to maintain sphingolipid homeostasis can result in pathologies including neurodegeneration, childhood asthma and cancer. However, we are only starting to understand how SP biosynthesis is adjusted according to need. In this study, we use genetic and biochemical methods to show that the uptake of exogenous serine is necessary to maintain SP homeostasis in Saccharomyces cerevisiae. Serine is one of the precursors of long chain bases in cells, the first intermediate of SP metabolism. Our results suggest that the uptake of serine is directly coupled to SP biosynthesis at ER-plasma membrane contact sites. Overall, our study identifies serine uptake as a novel regulatory factor of SP homeostasis. While we use yeast as a discovery tool, these results also provide valuable insights into mammalian SP biology especially under pathological conditions.

genetics↗