André Nadler

Membrane Lipids Tune Synaptic Transmission by Direct Modulation of Presynaptic Potassium Channels

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.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.

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

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.

PIP3 Induces the Recycling of Receptor Tyrosine Kinases

EGFR is recycled to the cell surface in response to the phosphoinositide PIP3. Recycling Receptors The epidermal growth factor receptor (EGFR) promotes cellular proliferation. Activation of EGFRs by ligand binding typically leads to receptor internalization and then degradation of the receptor, thereby terminating signaling downstream of the receptor. Laketa et al. found that high concentrations of the phosphoinositide PIP3 (phosphatidylinositol 3,4,5-trisphosphate) triggered the internalization of EGFRs and their recycling to the cell surface. Because high concentrations of PIP3 can be generated both physiologically and pathophysiologically, this mechanism could prevent activated EGFRs from degradation, diverting them back to the surface to sustain the cell’s response to EGF. 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.

Phosphatidylinositol 4,5-bisphosphate optical uncaging potentiates exocytosis

Phosphatidylinositol-4,5-bisphosphate [PI(4,5)P2] is essential for exocytosis. Classical ways of manipulating PI(4,5)P2 levels are slower than its 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 may bypass 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.

Tetraspanin microdomains control localized protein kinase C signaling in B cells

The tetraspanin protein CD53 recruits the kinase PKCβ to the plasma membrane to activate signaling in B cells. Kinase recruiting by a tetraspanin The protein kinase C (PKC) family member PKCβ mediates antigen-dependent B cell receptor (BCR) signaling to activate B cells. Mice deficient in the PKCβ isoform have defective antibody responses, whereas PKCβ-specific inhibitors are under investigation for the treatment of B cell malignancies. Using live-cell imaging of mouse and human B cells, Zuidscherwoude et al. showed that, in response to BCR stimulation, PKCβ was specifically and transiently recruited to plasma membrane microdomains enriched in the tetraspanin protein CD53. Mouse or human B cells deficient in CD53, but not other tetraspanins, showed impaired PKCβ activation and reduced phosphorylation of its targets, suggesting that CD53-containing membrane regions act as platforms for the activation of PKCβ in these cells. 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.

Triostin A derived hybrid for simultaneous DNA binding and metal coordination

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.

Influence of Substrate Dideuteration on the Reaction of the Bifunctional Heme Enzyme Psi Factor Producing Oxygenase A (PpoA)

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 (VH/VD) 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 VH/VD but resulted in a different product pattern presumably due to an altered lifetime and partitioning of a reaction intermediate.

Lipid Discovery by Combinatorial Screening and Untargeted LC-MS/MS

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.