André Nadler Group

The study of biological processes involving live microscopy techniques requires adequate temperature control to respect the physiology of the organism under study. We present here a design strategy for a microscope temperature stage based on thermoelectric elements. The design allows the user to access a range of temperatures below and above room temperature and can accommodate samples of different geometries. In addition, by cooling simultaneously the sample insert and the objective, we minimize the temperature gradients along the sample for large magnification objectives requiring immersion oil. We illustrate how this design can be used to study the physiology of the zebrafish embryo over the temperature tolerance of this species. We envision that this device could benefit the communities using model and non-model organisms with physiological temperatures different from typical mammalian cell culture incubation in biomedical research.

Studying the role of molecularly distinct lipid species in cell signaling remains challenging due to a scarcity of methods for performing quantitative lipid biochemistry in living cells. We have recently used lipid uncaging to quantify lipid-protein affinities and rates of lipid trans-bilayer movement and turnover in the diacylglycerol signaling pathway. This approach is based on acquiring live-cell dose-response curves requiring light dose titrations and experimental determination of uncaging photoreaction efficiency. We here aimed to develop a methodological approach that allows us to retrieve quantitative kinetic data from uncaging experiments that 1) require only typically available datasets without the need for specialized additional constraints and 2) should in principle be applicable to other types of photoactivation experiments. Our new analysis framework allows us to identify model parameters such as diacylglycerol-protein affinities and trans-bilayer movement rates, together with initial uncaged diacylglycerol levels, using noisy single-cell data for a broad variety of structurally different diacylglycerol species. We find that lipid unsaturation degree and side-chain length generally correlate with faster lipid trans-bilayer movement and turnover and also affect lipid-protein affinities. In summary, our work demonstrates how rate parameters and lipid-protein affinities can be quantified from single-cell signaling trajectories with sufficient sensitivity to resolve the subtle kinetic differences caused by the chemical diversity of cellular signaling lipid pools.

Cytotoxic T lymphocytes (CTLs) kill virus-infected and cancer cells through T cell receptor (TCR) recognition. How CTLs terminate signaling and disengage to allow serial killing has remained a mystery. TCR activation triggers membrane specialization within the immune synapse, including the production of diacylglycerol (DAG), a lipid that can induce negative membrane curvature. We found that activated TCRs were shed into DAG-enriched ectosomes at the immune synapse rather than internalized through endocytosis, suggesting that DAG may contribute to the outward budding required for ectocytosis. Budding ectosomes were endocytosed directly by target cells, thereby terminating TCR signaling and simultaneously disengaging the CTL from the target cell to allow serial killing. Thus, ectocytosis renders TCR signaling self-limiting.

ConspectusTraditional cell biological techniques are not readily suitable for studying lipid signaling events because genetic perturbations are much slower than the interconversion of lipids in complex metabolic networks. For this reason, novel chemical biological approaches have been developed. One approach is to chemically modify a lipid with a so-called "caging group" that renders it inactive, but this cage can be removed photochemically inside cells to release the bioactive molecule. These caged compounds offer unique advantages for studying the kinetics of cellular biochemistry and have been extensively used in the past. However, a limitation of conventional caged compounds is their ability to diffuse freely inside the cell, which does not permit localized activation below optical precision. This poses a challenge for studying lipid signaling as lipid function inside cells is tightly linked to their parent membrane. An ideal lipid probe should, therefore, be restricted to a single organelle membrane or preferentially to a single leaflet. We first demonstrated the plasma-membrane-specific photorelease of fatty acids by employing sulfonated caging groups. Using these caged fatty acid probes we demonstrated that lipid localization determines signaling outcome. Generalizing this approach, we designed a so-called "click cage" that can be coupled to lipids and offers the possibility to attach organelle targeting groups via click chemistry. Using this strategy, we have synthesized plasma membrane, lysosomal, mitochondria, and endoplasmic-reticulum-targeted lipids that can be used to dissect organelle-specific signaling events. To reduce the synthetic effort associated with generating caged compounds, we designed a coumarin triflate reagent that allows the direct functionalization of phosphate- or carboxylate-containing compounds. With this novel reagent, we synthesized a small library of photocaged G-protein-coupled receptor (GPCR) ligands to study the underlying lipid signaling dynamics. Most recently, we have focused on quantifying the kinetics of lipid signaling for different diacylglycerol (DAG) species using plasma-membrane-targeted caged DAGs. Using this approach, we quantitatively measured lipid-protein affinities and lipid transbilayer dynamics in living cells. After analyzing DAGs with different acyl chain length and saturation degree, we discovered that affinities can vary by up to an order of magnitude. This finding clearly shows that cells are able to distinguish between individual DAG species, thereby demonstrating that lipid diversity matters in cellular signal processing. Although the recent advances have yielded valuable tools to study lipid signaling, challenges remain on specifically targeting the different leaflets of organelle membranes. Furthermore, it is necessary to simplify the experimental approaches required for parametrizing and corroborating quantitative kinetic models of lipid signaling. In the future, we envision that the application of leaflet-specific caged lipids to model membrane systems will be of crucial importance for understanding lipid asymmetry.

Lipids exert their cellular functions in individual organelles, in some cases on the scale of even smaller, specialized membrane domains. Thus, the experimental capacity to precisely manipulate lipid levels at the subcellular level is crucial for studying lipid-related processes in cell biology. Photo-caged lipid probes which partition into specific cellular membranes prior to photoactivation have emerged as key tools for localized and selective perturbation of lipid concentration in living cells. In this review, we provide an overview of the recent advances in the area and outline which developments are still required for the methodology to be more widely implemented in the wider membrane biology community.

Adenosine triphosphate (ATP) production and utilization is critically important for animal development. How these processes are regulated in space and time during tissue growth remains largely unclear. We used a FRET-based sensor to dynamically monitor ATP levels across a growing tissue, using the Drosophila larval wing disc. Although steady-state levels of ATP are spatially uniform across the wing pouch, inhibiting oxidative phosphorylation reveals spatial differences in metabolic behavior, whereby signaling centers at compartment boundaries produce more ATP from glycolysis than the rest of the tissue. Genetic perturbations indicate that the conserved Hedgehog signaling pathway can enhance ATP production by glycolysis. Collectively, our work suggests the existence of a homeostatic feedback loop between Hh signaling and glycolysis, advancing our understanding of the connection between conserved developmental patterning genes and ATP production during animal tissue development.

It has long been proposed that phase-separated compartments can provide a basis for the formation of cellular precursors in prebiotic environments. However, we know very little about the properties of coacervates formed from simple peptides, their compatibility with ribozymes or their functional significance. Here we assess the conditions under which functional ribozymes form coacervates with simple peptides. We find coacervation to be most robust when transitioning from long homopeptides to shorter, more pre-biologically plausible heteropeptides. We mechanistically show that these RNA-peptide coacervates display peptide-dependent material properties and cofactor concentrations. We find that the interspacing of cationic and neutral amino acids increases RNA mobility, and we use isothermal calorimetry to reveal sequence-dependent Mg2+ partitioning, two critical factors that together enable ribozyme activity. Our results establish how peptides of limited length, homogeneity and charge density facilitate the compartmentalization of active ribozymes into non-gelating, magnesium-rich coacervates, a scenario that could be applicable to cellular precursors with peptide-dependent functional phenotypes.

Compartmentalized oscillations of second messengers affect global cellular signaling.

Changes in cell metabolism and plasma membrane potential have been linked to shifts between tissue growth and differentiation, and to developmental patterning. How such changes mediate these effects is poorly understood. Here, we use the developing wing of Drosophila to investigate the interplay between cell metabolism and a key developmental regulator-the Hedgehog (Hh) signalling pathway. We show that reducing glycolysis both lowers steady-state levels of ATP and stabilizes Smoothened (Smo), the 7-pass transmembrane protein that transduces the Hh signal. As a result, the transcription factor Cubitus interruptus accumulates in its full-length, transcription activating form. We show that glycolysis is required to maintain the plasma membrane potential and that plasma membrane depolarization blocks cellular uptake of N-acylethanolamides-lipoprotein-borne Hh pathway inhibitors required for Smo destabilization. Similarly, pharmacological inhibition of glycolysis in mammalian cells induces ciliary translocation of Smo-a key step in pathway activation-in the absence of Hh. Thus, changes in cell metabolism alter Hh signalling through their effects on plasma membrane potential.

Every cell produces thousands of distinct lipid species, but insight into how lipid chemical diversity contributes to biological signaling is lacking, particularly because of a scarcity of methods for quantitatively studying lipid function in living cells. Using the example of diacylglycerols, prominent second messengers, we here investigate whether lipid chemical diversity can provide a basis for cellular signal specification. We generated photo-caged lipid probes, which allow acute manipulation of distinct diacylglycerol species in the plasma membrane. Combining uncaging experiments with mathematical modeling, we were able to determine binding constants for diacylglycerol-protein interactions, and kinetic parameters for diacylglycerol transbilayer movement and turnover in quantitative live-cell experiments. Strikingly, we find that affinities and kinetics vary by orders of magnitude due to diacylglycerol side-chain composition. These differences are sufficient to explain differential recruitment of diacylglycerol binding proteins and, thus, differing downstream phosphorylation patterns. Our approach represents a generally applicable method for elucidating the biological function of single lipid species on subcellular scales in quantitative live-cell experiments.

Photorelease of caged compounds is among the most powerful experimental approaches for studying cellular functions on fast timescales. However, its full potential has yet to be exploited, as the number of caged small molecules available for cell biological studies has been limited by synthetic challenges. Addressing this problem, a straightforward, one-step procedure for efficiently synthesizing caged compounds was developed. An in situ generated benzylic coumarin triflate reagent was used to specifically functionalize carboxylate and phosphate moieties in the presence of free hydroxy groups, generating various caged lipid metabolites, including a number of GPCR ligands. By combining the photo-caged ligands with the respective receptors, an easily implementable experimental platform for the optical control and analysis of GPCR-mediated signal transduction in living cells was developed. Ultimately, the described synthetic strategy allows rapid generation of photo-caged small molecules and thus greatly facilitates the analysis of their biological roles in live cell microscopy assays.

Quantitative bottom-up shotgun lipidomics relies on molecular species-specific "signature" fragments consistently detectable in tandem mass spectra of analytes and standards. Molecular species of glycerophospholipids are typically quantified using carboxylate fragments of their fatty acid moieties produced by higher-energy collisional dissociation of their molecular anions. However, employing standards whose fatty acids moieties are similar, yet not identical, to the target lipids could severely compromise their quantification. We developed a generic and portable fragmentation model implemented in the open-source LipidXte software that harmonizes the abundances of carboxylate anion fragments originating from fatty acid moieties having different sn-1/2 positions at the glycerol backbone, length of the hydrocarbon chain, and number and location of double bonds. The postacquisition adjustment enables unbiased absolute (molar) quantification of glycerophospholipid species independent of instrument settings, collision energy, and employed internal standards.

Lipid messengers exert their function on short time scales at distinct subcellular locations, yet most experimental approaches for perturbing their levels trigger cell-wide concentration changes. Herein, we report on a coumarin-based photocaging group that can be modified with organelle-targeting moieties by click chemistry and thus enables photorelease of lipid messengers in distinct organelles. We show that caged arachidonic acid and sphingosine derivatives can be selectively delivered to mitochondria, the ER, lysosomes, and the plasma membrane. By comparing the cellular calcium transients induced by localized uncaging of arachidonic acid and sphingosine, we show that the precise intracellular localization of the released second messenger is crucial for the signaling outcome. Ultimately, we anticipate that this new class of caged compounds will greatly facilitate the study of cellular processes on the organelle level.

Researchers striving to convert biology into an exact science foremost rely on structural biology and biochemical reconstitution approaches to obtain quantitative data. However, cell biological research is moving at an ever-accelerating speed into areas where these approaches lose much of their edge. Intrinsically unstructured proteins and biochemical interaction networks composed of interchangeable, multivalent, and unspecific interactions pose unique challenges to quantitative biology, as do processes that occur in discrete cellular microenvironments. Here we argue that a conceptual change in our way of conducting biochemical experiments is required to take on these new challenges. We propose that reconstitution of cellular processes in vitro should be much more focused on mimicking the cellular environment in vivo, an approach that requires detailed knowledge of the material properties of cellular compartments, essentially requiring a material science of the cell. In a similar vein, we suggest that quantitative biochemical experiments in vitro should be accompanied by corresponding experiments in vivo, as many newly relevant cellular processes are highly context-dependent. In essence, this constitutes a call for chemical biologists to convert their discipline from a proof-of-principle science to an area that could rightfully be called quantitative biochemistry in living cells. In this essay, we discuss novel techniques and experimental strategies with regard to their potential to fulfill such ambitious aims.

Phosphatidylinositol-4,5-bisphosphate [PI(4,5)P2] is essential for exocytosis. Classical ways of manipulating PI(4,5)P2 levels are slower than metabolism, making it difficult to distinguish effects of PI(4,5)P2 from those of its metabolites. We developed a membrane-permeant, photoactivatable PI(4,5)P2, which is loaded into cells in an inactive form and activated by light, allowing sub-second increases in PI(4,5)P2 levels. By combining this compound with electrophysiological measurements in mouse adrenal chromaffin cells, we show that PI(4,5)P2 uncaging potentiates exocytosis and identify synaptotagmin-1 (the Ca2+ sensor for exocytosis) and Munc13-2 (a vesicle priming protein) as the relevant effector proteins. PI(4,5)P2 activation of exocytosis did not depend on the PI(4,5)P2-binding CAPS-proteins, suggesting that PI(4,5)P2 uncaging bypasses CAPS-function. Finally, PI(4,5)P2 uncaging triggered the rapid fusion of a subset of readily-releasable vesicles, revealing a rapid role of PI(4,5)P2 in fusion triggering. Thus, optical uncaging of signaling lipids can uncover their rapid effects on cellular processes and identify lipid effectors.

Activation of B cells by the binding of antigens to the B cell receptor (BCR) requires the protein kinase C (PKC) family member PKCβ. Because PKCs must translocate to the plasma membrane to become activated, we investigated the mechanisms regulating their spatial distribution in mouse and human B cells. Through live-cell imaging, we showed that BCR-stimulated production of the second messenger diacylglycerol (DAG) resulted in the translocation of PKCβ from the cytosol to plasma membrane regions containing the tetraspanin protein CD53. CD53 was specifically enriched at sites of BCR signaling, suggesting that BCR-dependent PKC signaling was initiated at these tetraspanin microdomains. Fluorescence lifetime imaging microscopy studies confirmed the molecular recruitment of PKC to CD53-containing microdomains, which required the amino terminus of CD53. Furthermore, we showed that Cd53-deficient B cells were defective in the phosphorylation of PKC substrates. Consistent with this finding, PKC recruitment to the plasma membrane was impaired in both mouse and human CD53-deficient B cells compared to that in their wild-type counterparts. These data suggest that CD53 promotes BCR-dependent PKC signaling by recruiting PKC to the plasma membrane so that it can phosphorylate its substrates and that tetraspanin-containing microdomains can act as signaling hotspots in the plasma membrane.

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.

Lysophosphatidic acid (LPA) is a serum-borne lipid mediator that binds to a variety of different G protein-coupled receptors to trigger an exceptionally wide range of biological effects, including cell survival and differentiation, cancer cell migration, and embryonic development. Here we synthesized caged LPA (cgLPA), a "photolysable" coumarin-masked derivative of LPA. We demonstrate that illumination of cgLPA with 405 nm light liberates bioactive LPA on a subsecond scale to evoke Ca(2+) signaling, Rho activation, and cytoskeletal contraction. In addition, we developed an "optotaxis" assay to attract melanoma cells through a stable chemotactic gradient by repeated liberation of LPA through local photolysis of extracellular cgLPA. We expect that this method of light-controlled chemotaxis will be generally applicable to a large variety of small molecules that drive cellular migration or other responses.

We present a method for the systematic identification of picogram quantities of new lipids in total extracts of tissues and fluids. It relies on the modularity of lipid structures and applies all-ions fragmentation LC-MS/MS and Arcadiate software to recognize individual modules originating from the same lipid precursor of known or assumed structure. In this way it alleviates the need to recognize and fragment very low abundant precursors of novel molecules in complex lipid extracts. In a single analysis of rat kidney extract the method identified 58 known and discovered 74 novel endogenous endocannabinoids and endocannabinoid-related molecules, including a novel class of N-acylaspartates that inhibit Hedgehog signaling while having no impact on endocannabinoid receptors.

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.

Lipid derivatives that can be activated by light, often referred to as 'caged' lipids, are useful tools to manipulate intact cells non-invasively. Here we focus on experimental approaches that have made use of caged lipids. Apart from summarizing the recent advances and available tools in the field, we strive to highlight the experimental challenges that arise from lipid-specific biophysical properties and the abundance of an enormous diversity of distinct molecular lipid species in cells. This article is part of a Special Issue entitled Tools to study lipid functions.

Voltage-gated potassium (Kv) channels are involved in action potential (AP) repolarization in excitable cells. Exogenous application of membrane-derived lipids, such as arachidonic acid (AA), regulates the gating of Kv channels. Whether membrane-derived lipids released under physiological conditions have an impact on neuronal coding through this mechanism is unknown. We show that AA released in an activity-dependent manner from postsynaptic hippocampal CA3 pyramidal cells acts as retrograde messenger, inducing a robust facilitation of mossy fiber (Mf) synaptic transmission over several minutes. AA acts by broadening presynaptic APs through the direct modulation of Kv channels. This form of short-term plasticity can be triggered when postsynaptic cell fires with physiologically relevant patterns and sets the threshold for the induction of the presynaptic form of long-term potentiation (LTP) at hippocampal Mf synapses. Hence, direct modulation of presynaptic Kv channels by activity-dependent release of lipids serves as a physiological mechanism for tuning synaptic transmission.

Down-regulation of receptor tyrosine kinases such as the epidermal growth factor receptor (EGFR) is achieved by endocytosis of the receptor followed by degradation or recycling. We demonstrated that in the absence of ligand, increased phosphatidylinositol 3,4,5-trisphosphate (PIP3) concentrations induced clathrin- and dynamin-mediated endocytosis of EGFR but not that of transferrin or G protein (heterotrimeric guanine nucleotide-binding protein)-coupled receptors. Endocytosis of the receptor in response to binding of EGF resulted in a decrease in the abundance of the EGFR, but PIP3-induced internalization decreased receptor ubiquitination and phosphorylation and resulted in recycling of the receptor to the plasma membrane. An RNA interference (RNAi) screen directed against lipid-binding domain-containing proteins identified polarity complex proteins, including PARD3 (partitioning defective 3), as essential for PIP3-induced receptor tyrosine kinase recycling. Thus, PIP3 and polarity complex proteins regulate receptor tyrosine kinase trafficking, which may enhance cellular responsiveness to growth factors.

A definite turn-on: Turning on fluorescence only where successful labeling is happening sounds as desirable as delivering a drug only where the drug target resides. New fluorogenic xanthene derivatives from the Bertozzi research group are getting us closer to "magic bullet" dyes.

Total synthesis of proteins can be challenging despite assembling techniques, such as native chemical ligation (NCL) and expressed protein ligation (EPL). Especially, the combination of recombinant protein expression and chemically addressable solid-phase peptide synthesis (SPPS) is well suited for the redesign of native protein structures. Incorporation of analytical probes and artificial amino acids into full-length natural protein domains, such as the sequence-specific DNA binding zinc-finger motifs, are of interest combining selective DNA recognition and artificial function. The semi-synthesis of the natural 90 amino acid long sequence of the zinc-finger domain of Zif268 is described including various chemically modified constructs. Our approach offers the possibility to exchange any amino acid within the third zinc finger. The realized modifications of the natural sequence include point mutations, attachment of a fluorophore, and the exchange of amino acids at different positions in the zinc finger by artificial amino acids to create additional metal binding sites. The individual constructs were analyzed by circular dichroism (CD) spectroscopy with respect to the integrity of the zinc-finger fold and DNA binding.

The natural product triostin A is known as an antibiotic based on specific DNA recognition. Structurally, a bicyclic depsipeptide backbone provides a well-defined scaffold preorganizing the recognition motifs for bisintercalation. Replacing the intercalating quinoxaline moieties of triostin A by nucleobases results in a potential major groove binder. The functionalization of this DNA binding triostin A analog with a metal binding ligand system is reported, thereby generating a hybrid molecule with DNA binding and metal coordinating capability. Transition metal ions can be placed in close proximity to dsDNA by means of non-covalent interactions. The synthesis of the nucleobase-modified triostin A analog is described containing a propargylglycine for later attachment of the ligand by click-chemistry. As ligand, two [1,4,7]triazacyclononane rings were bridged by a phenol. Formation of the proposed binuclear zinc complex was confirmed for the ligand and the triostin A analog/ligand construct by high-resolution mass spectrometry. The complex as well as the respective hybrid led to stabilization of dsDNA, thus implying that metal complexation and DNA binding are independent processes.

PpoA is a bifunctional enzyme that catalyzes the dioxygenation of unsaturated C18 fatty acids. The products of this reaction are termed psi factors and have been shown to play a crucial role in conferring a balance between sexual and asexual spore development as well as production of secondary metabolites in the fungus Aspergillus nidulans. Studies on the reaction mechanism revealed that PpoA uses two different heme domains to catalyze two subsequent reactions. Initially, the fatty acid substrate is dioxygenated at C8, yielding an 8-hydroperoxy fatty acid at the N-terminal domain. This reaction is catalyzed by a peroxidase/dioxygenase-type domain that exhibits many similarities to prostaglandin H2 synthases and involves a stereospecific homolytic hydrogen abstraction from C8 of the substrate. The C terminus harbors a heme thiolate P450 domain in which rearrangement of the 8-hydroperoxide to the final product, a 5,8-dihydroxy fatty acid, takes place. To obtain further information about the intrinsic kinetics and reaction mechanism of PpoA, we synthesized C5-dideutero- and C8-dideutero-oleic acid by a novel protocol that offers a straightforward synthesis without employing the toxic additive hexamethylphosphoramide (HMPA) during CC coupling reactions or mercury salts upon thioketal deprotection. These deuterated fatty acids were then employed for kinetic analysis under multiple-turnover conditions. The results indicate that the hydrogen abstraction at C8 is the rate-determining step of the overall reaction because we observed a KIE (V(H) /V(D) ) of ∼33 at substrate saturation that suggests extensive nuclear tunneling contributions for hydrogen transfer. Deuteration of the substrate at C5, however, had little effect on V(H) /V(D) but resulted in a different product pattern presumably due to an altered lifetime and partitioning of a reaction intermediate.