A sensitive, expandable AQC-based LC-MS/MS method to measure amino metabolites and sphingolipids in cell and serum samples.

Sphingolipids are a major lipid species found in all eukaryotes. Among structurally complex and diversified lipids, sphingoid bases have been heavily linked to various metabolic diseases. However, most current LC-MS-based methods lack the sensitivity to detect low-abundant sphingoid bases.

PDGFRβ + cell HIF2α is dispensable for white adipose tissue metabolic remodeling and hepatic lipid accumulation in obese mice.

Background: Obesity is associated with extensive white adipose tissue (WAT) expansion and remodeling. Healthy WAT expansion contributes to the maintenance of energy balance in the liver, thereby ameliorating obesity-related hepatic steatosis. Tissue-resident mesenchymal stromal cell populations, including PDGFRβ + perivascular cells, are increasingly recognized pivotal as determinants of the manner in which WAT expands.

An organelle-specific photoactivation and dual-isotope labeling strategy reveals phosphatidylethanolamine metabolic flux.

Phosphatidylethanolamine metabolism plays essential roles in eukaryotic cells but has not been completely investigated due to its complexity. This is because lipid species, unlike proteins or nucleic acids, cannot be easily manipulated at the single molecule level or controlled with subcellular resolution, two of the key factors toward understanding their functions. Here, we use the organelle-targeting photoactivation method to study PE metabolism in living cells with a high spatiotemporal resolution.

Recruitment of a splicing factor to the nuclear lamina for its inactivation.

Precursor messenger RNA splicing is a highly regulated process, mediated by a complex RNA-protein machinery, the spliceosome, that encompasses several hundred proteins and five small nuclear RNAs in humans. Emerging evidence suggests that the spatial organization of splicing factors and their spatio-temporal dynamics participate in the regulation of splicing. So far, methods to manipulate the spatial distribution of splicing factors in a temporally defined manner in living cells are missing.

Vacuole-Specific Lipid Release for Tracking Intracellular Lipid Metabolism and Transport in .

Lipid metabolism is spatiotemporally regulated within cells, yet intervention into lipid functions at subcellular resolution remains difficult. Here, we report a method that enables site-specific release of sphingolipids and cholesterol inside the vacuole in . Using this approach, we monitored real-time sphingolipid metabolic flux out of the vacuole by mass spectrometry and found that the endoplasmic reticulum-vacuole-tethering protein Mdm1 facilitated the metabolism of sphingoid bases into ceramides.

Chemical Biology Tools to Study Lipids and their Metabolism with Increased Spatial and Temporal Resolution.

Lipids are important cellular components providing many essential functions. To fulfill these various functions evolution has selected for a diverse set of lipids and this diversity is seen at the organismal, cellular and subcellular level. Understanding how cells maintain this complex lipid organization is a very challenging problem, which for lipids, is not easily addressed using biochemical and genetic techniques.

Luciferase Controlled Protein Interactions.

Protein trafficking and protein-protein interactions (PPIs) are central to regulatory processes in cells. Induced dimerization systems have been developed to control PPIs and regulate protein trafficking (localization) or interactions. Chemically induced dimerization (CID) has proven to be a robust approach to control protein interactions and localization.

Conserved Functions of Ether Lipids and Sphingolipids in the Early Secretory Pathway.

Sphingolipids play important roles in physiology and cell biology, but a systematic examination of their functions is lacking. We performed a genome-wide CRISPRi screen in sphingolipid-depleted human cells and identified hypersensitive mutants in genes of membrane trafficking and lipid biosynthesis, including ether lipid synthesis. Systematic lipidomic analysis showed a coordinate regulation of ether lipids with sphingolipids, suggesting an adaptation and functional compensation.

Luciferase-Induced Photouncaging: Bioluminolysis.

Bioluminescence resonance energy transfer (BRET) has been widely used for studying dynamic processes in biological systems such as protein-protein interactions and other signaling events. Aside from acting as a reporter, BRET can also turn on functions in living systems. Herein, we report the application of BRET to performing a biorthogonal reaction in living cells; namely, releasing functional molecules through energy transfer to a coumarin molecule, a process termed bioluminolysis.

Optical control of sphingosine-1-phosphate formation and function.

Sphingosine-1-phosphate (S1P) plays important roles as a signaling lipid in a variety of physiological and pathophysiological processes. S1P signals via a family of G-protein-coupled receptors (GPCRs) (S1P) and intracellular targets. Here, we report on photoswitchable analogs of S1P and its precursor sphingosine, respectively termed PhotoS1P and PhotoSph.

Lysosome-targeted photoactivation reveals local sphingosine metabolism signatures.

Lipids are essential components of eukaryotic cell membranes and play crucial roles in cellular signaling and metabolism. While increasing evidence shows that the activities of lipids are dependent upon subcellular localization, tools to study local lipid metabolism and signaling are limited. Herein, we report an approach that enabled us to selectively deliver photo-caged lipids into lysosomes and thereafter to quickly release the lipid molecules by illumination.

Mitochondria-specific photoactivation to monitor local sphingosine metabolism and function.

Photoactivation ('uncaging') is a powerful approach for releasing bioactive small-molecules in living cells. Current uncaging methods are limited by the random distribution of caged molecules within cells. We have developed a mitochondria-specific photoactivation method, which permitted us to release free sphingosine inside mitochondria and thereafter monitor local sphingosine metabolism by lipidomics.

Structure and conserved function of iso-branched sphingoid bases from the nematode .

Sphingolipids are bio-active metabolites that show structural diversity among eukaryotes. They are essential for growth of all eukaryotic cells but when produced in an uncontrolled manner can lead to cell death and pathologies including auto-immune reactions, cancer, diabetes and neurodegeneration. is an important genetic model organism both to find new drug-targets against parasitic nematodes and to study the conserved roles of sphingolipids in animals like their essential functions in very basic cellular processes ranging from maintenance of cell polarity and mitochondrial repair to growth and survival.

A Fluorescent Probe for Neural Stem/Progenitor Cells with High Differentiation Capability into Neurons.

Selection of a specific neural stem/progenitor cells (NSPCs) has attracted broad attention in regenerative medicine for neurological disorders. Here, we report a fluorescent probe, CDg13, and its application for isolating strong neurogenic NSPCs. In comparison to the NSPCs isolated by other biomarkers, CDg13-stained NSPCs showed higher capability to differentiate into neurons.

Exclusive photorelease of signalling lipids at the plasma membrane.

Photoactivation of caged biomolecules has become a powerful approach to study cellular signalling events. Here we report a method for anchoring and uncaging biomolecules exclusively at the outer leaflet of the plasma membrane by employing a photocleavable, sulfonated coumarin derivative. The novel caging group allows quantifying the reaction progress and efficiency of uncaging reactions in a live-cell microscopy setup, thereby greatly improving the control of uncaging experiments.

A single-cell model of PIP3 dynamics using chemical dimerization.

Most cellular processes are driven by simple biochemical mechanisms such as protein and lipid phosphorylation, but the sum of all these conversions is exceedingly complex. Hence, intuition alone is not enough to discern the underlying mechanisms in the light of experimental data. Toward this end, mathematical models provide a conceptual and numerical framework to formally evaluate the plausibility of biochemical processes.

Reversible chemical dimerizer-induced recovery of PIP2 levels moves clathrin to the plasma membrane.

Chemical dimerizers are powerful non-invasive tools for bringing molecules together inside intact cells. We recently introduced a rapidly reversible chemical dimerizer system which enables transient translocation of enzymes to and from the plasma membrane (PM). Here we have applied this system to transiently activate phosphatidylinositol 4,5-bisphosphate (PIP2) breakdown at the PM via translocation of phosphoinositide 5-phosphatase (5Ptase).

T-CrAsH: a heterologous chemical crosslinker.

Copper-free click chemistry is currently the most promising and most rapidly developing technology for performing tailored chemical reactions inside intact living cells and animals. Its potential is particularly intensely explored in the field of live cell imaging, for both proteins and metabolites. Here we expand the application spectrum of click reactions to the chemical crosslinking of two proteins of choice in living cells.

A rapidly reversible chemical dimerizer system to study lipid signaling in living cells.

Chemical dimerizers are powerful tools for non-invasive manipulation of enzyme activities in intact cells. Here we introduce the first rapidly reversible small-molecule-based dimerization system and demonstrate a sufficiently fast switch-off to determine kinetics of lipid metabolizing enzymes in living cells. We applied this new method to induce and stop phosphatidylinositol 3-kinase (PI3K) activity, allowing us to quantitatively measure the turnover of phosphatidylinositol 3,4,5-trisphosphate (PIP3) and its downstream effectors by confocal fluorescence microscopy as well as standard biochemical methods.

Recapture of GFP chromophore fluorescence in a protein host.

When encapsulated by human serum albumin (HSA), certain derivatives of the green fluorescent protein (GFP) chromophore recover their fluorescence due to inhibition of torsional motion. These derivatives show remarkable sensitivity and selectivity as well as favorable spectroscopic properties toward HSA, thus providing selective probes for this and similar proteins and demonstrating the use of GFP chromophores as topological fluorophores.

Accelerating fluorescent sensor discovery: unbiased screening of a diversity-oriented BODIPY library.

Herein, we report the first systematic and unbiased evaluation of the BODIPY fluorophore library against a wide panel of biologically relevant molecules, and discoveries of 2 novel fluorescent probes for BSA and dopamine.

Fluorescence response profiling for small molecule sensors utilizing the green fluorescent protein chromophore and its derivatives.

Using a fluorescence response profile, a systematic examination was performed for synthetic chromophores of the green fluorescent protein (GFP) to discover new small molecule sensors. A group of 41 benzylideneimidazolinone compounds (BDI) was prepared and screened toward 94 biologically relevant analytes to generate fluorescence response profiles. From the response pattern, compounds containing aminobenzyl and heteroaromatic cyclic substructures revealed a pH dependent emission decrease effect, and unlike other fluorescence scaffolds, most BDIs showed fluorescence quenching when mixed with proteins.

Discovery of a green DNA probe for live-cell imaging.

We discovered a DNA-selective probe, and demonstrated its potentials for nucleus imaging and DNA quantification.