Frank Stein

The fatty acid composition of diacylglycerols determines local signaling patterns.

Cellular signals are transduced through vast networks of proteins and small molecule metabolites. Rigorous control of the respective signaling molecules is required to ensure a precise and reproducible outcome. On the protein level this is often accomplished by specific reversible chemical modifications such as phosphorylation or by localization of proteins to defined cellular compartments. 2] Much less is known about cellular mechanisms that control small-molecule-mediated signaling events. This is largely due to the intrinsically more difficult observation of small-molecule turnover and localization in living cells. These difficulties are potentiated when lipid signaling is investigated. The variety of known lipid backbones is fairly comprehensive but the diversity and combinations of fatty acids attached to these backbones provides many thousand possibilities and lipidomics shows that a large portion of this diversity is available in cells. This overwhelming and generally not addressable complexity has led to a situation where lipid signaling events are treated as head-group signaling events and the existing chemical differences between individual species of the same lipid class are widely ignored although a number of in vitro studies suggest significant differences in potency. Along the same lines, the influence of subcellular concentration gradients of defined lipid species on intracellular signaling has not been studied thoroughly so far. We hypothesized that both lipid species diversity and subcellular concentration gradients of distinct lipid species might serve as molecular mechanisms to drive specific lipid-mediated signaling events. Experimentally, both fatty acid diversity and locally elevated levels of a given species may be generated by using photoactivatable lipids in intact cells. We chose to analyze diacylglycerol (DAG) signaling due to its important role in several cellular signaling pathways that include G-protein coupled receptors as well as growth factor triggered and calcium-based signaling networks. Recent lipidomics analyses demonstrated the co-existence of 30–50 DAG species with different fatty acid compositions in mammalian cells. While DAGs are best known to activate various protein kinase C (PKC) isoforms by binding to their C1 domains and recruiting them to cellular membranes, DAG-induced translocation and activation of proteins such as RasGRPs, Munc13, and DGKg have also been described. In addition, DAGs have been shown to directly activate human transient receptor potential C3 (TRPC3) and TRPC6 channels. So far, locally elevated DAG levels have either been experimentally achieved by liberation of a photoactivatable T-cell receptor agonist which causes downstream DAG production or by local uncaging of a nonphysiological DAG analogue. 23] While these approaches have led to important insights into the mechanism of T-cell receptor mediated signaling and microtubule-organizing center (MTOC) polarization in T-cells, they cannot be utilized

Bifunctional Sphingosine for Cell-Based Analysis of Protein-Sphingolipid Interactions

Sphingolipids are essential structural components of cellular membranes and are crucial regulators of cellular processes. While current high-throughput approaches allow for the systematic mapping of interactions of soluble proteins with their lipid-binding partners, photo-cross-linking is the only technique that enables for the proteome-wide mapping of integral membrane proteins with their direct lipid environment. Here, we report the synthesis of a photoactivatable and clickable analog of sphingosine (pacSph). When administered to sphingosine-1-phosphate lyase deficient cells, pacSph allows its metabolic fate and the subcellular flux of de novo synthesized sphingolipids to be followed in a time-resolved manner. The chemoproteomic profiling yielded over 180 novel sphingolipid-binding proteins, of which we validated a number, demonstrating the unique value of this technique as a discovery tool. This work provides an important resource for the understanding of the global cellular interplay between sphingolipids and their interacting proteins.

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.

Trifunctional lipid probes for comprehensive studies of single lipid species in living cells

Significance Some lipids such as sphingosine and diacylglycerol are potent signaling effectors. However, comprehensive investigations of their bioactive actions are often hampered by a lack of tools that can be used in living cells. Here, we present chemically modified lipids that allow investigation of acute lipid signaling, lipid metabolism, lipid−protein interactions, and lipid localization by using a single probe for each target lipid. Equipped with a caging group, the lipid probe is biologically inactive, until activated by a flash of light. A second photoreaction cross-links the probe to protein interactors that may subsequently be analyzed by mass spectrometry or fluorescence/electron microscopy. We envision that this versatile design will be central to unraveling complex lipid signaling networks. Lipid-mediated signaling events regulate many cellular processes. Investigations of the complex underlying mechanisms are difficult because several different methods need to be used under varying conditions. Here we introduce multifunctional lipid derivatives to study lipid metabolism, lipid−protein interactions, and intracellular lipid localization with a single tool per target lipid. The probes are equipped with two photoreactive groups to allow photoliberation (uncaging) and photo–cross-linking in a sequential manner, as well as a click-handle for subsequent functionalization. We demonstrate the versatility of the design for the signaling lipids sphingosine and diacylglycerol; uncaging of the probe for these two species triggered calcium signaling and intracellular protein translocation events, respectively. We performed proteomic screens to map the lipid-interacting proteome for both lipids. Finally, we visualized a sphingosine transport deficiency in patient-derived Niemann−Pick disease type C fibroblasts by fluorescence as well as correlative light and electron microscopy, pointing toward the diagnostic potential of such tools. We envision that this type of probe will become important for analyzing and ultimately understanding lipid signaling events in a comprehensive manner.

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. We synthesized arachidonic acid derivatives bearing the new negatively charged or a neutral, membrane-permeant coumarin caging group to locally induce signalling either at the plasma membrane or on internal membranes in β-cells and brain slices derived from C57B1/6 mice. Uncaging at the plasma membrane triggers a strong enhancement of calcium oscillations in β-cells and a pronounced potentiation of synaptic transmission while uncaging inside cells blocks calcium oscillations in β-cells and causes a more transient effect on neuronal transmission, respectively. The precise subcellular site of arachidonic acid release is therefore crucial for signalling outcome in two independent systems.

Spatiotemporal Analysis of a Glycolytic Activity Gradient Linked to Mouse Embryo Mesoderm Development

Summary How metabolism is rewired during embryonic development is still largely unknown, as it remains a major technical challenge to resolve metabolic activities or metabolite levels with spatiotemporal resolution. Here, we investigated metabolic changes during development of organogenesis-stage mouse embryos, focusing on the presomitic mesoderm (PSM). We measured glycolytic labeling kinetics from 13C-glucose tracing experiments and detected elevated glycolysis in the posterior, more undifferentiated PSM. We found evidence that the spatial metabolic differences are functionally relevant during PSM development. To enable real-time quantification of a glycolytic metabolite with spatiotemporal resolution, we generated a pyruvate FRET-sensor reporter mouse line. We revealed dynamic changes in cytosolic pyruvate levels as cells transit toward a more anterior PSM state. Combined, our approach identifies a gradient of glycolytic activity across the PSM, and we provide evidence that these spatiotemporal metabolic changes are intrinsically linked to PSM development and differentiation.

FluoQ: a tool for rapid analysis of multiparameter fluorescence imaging data applied to oscillatory events.

The number of fluorescent sensors and their use in living cells has significantly increased in the past years. Yet, the analysis of data from single cells or cell populations usually remains a very time-consuming enterprise. Here, we introduce FluoQ, a new macro for the image analysis software ImageJ, which enables fast analysis of multiparameter time-lapse fluorescence microscopy data with minimal manual input. FluoQ provides statistical analysis of all measured parameters and delivers the results in multiple graphic and numeric displays. We demonstrate the power of FluoQ by applying the macro to data analysis in the development and optimization of novel FRET reporters for monitoring the performance of calcium/calmodulin-binding inositol trisphosphate kinases A and B (ITPKA and ITPKB) in HeLa cells. We find that conformational changes in the ITPKA-based sensor follow receptor-mediated calcium oscillations. This indicates that ITPKA contributes to the regulation of intracellular calcium transients by limiting inositol trisphosphate levels.

A novel rearrangement of the carbene ligand in (Z)-(2-dialkylaminoethenyl)carbene complexes

Abstract ( Z )-(2-Dialkylamino)carbenechromium and -tungsten complexes 3a , b , d , 4a and 5c , which are readily available by Michael addition of secondary amines to alkynylcarbene complexes 1a , b , d and 2c rearrange to aminomethylene complexes 7a , b , d , 8a and 9c , when heated to 50–55°C. 3a yielded the chelated tetracarbonyl complex 6a at 65 °C.

Highly Substituted Spiro[4.4]nonatrienes from a -Amino-Substituted ,-Unsaturated Fischer Carbene Complex and Three Molecules of an Arylalkyne

A novel mode of reaction towards arylethynes is shown by the β-trimethylsilyl-substituted α,β-unsaturated Fischer carbene complexes 1. A mixture of the isomeric, highly substituted spiro[4.4]nonatrienes 2 and 3 is formed by the formal insertion of three alkyne molecules and subsequent cyclization (see scheme). Such selective triple insertions of alkynes into ethenylcarbene complexes have not been previously observed.

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). We found that the PIP2 sensor phospholipase C-δ PH domain (PLCδ-PH) is released from the PM upon addition of the reversible chemical dimerizer rCD1. By outcompeting rCD1, rapid release of the 5Ptase from the PM is followed by PIP2 recovery. This permits the observation of the PIP2-dependent clathrin assembly at the PM.