Project Area A

Methodological developments

The common aim of the five projects in project area A is to elaborate and/or optimize high-end light microscopy methods designed to learn more about the function of membrane receptors. The methods grouped in this area appear at first sight heterogenous, but this heterogeneity of approaches and views is expected to provide the CRC/TR a particular impulse to gain essentially new and multifaceted information about receptor function.

A1

Black-lipid-membrane platform with diffusion suppression for confocal single-molecule FLIM-FRET analysis of gated membrane receptors and transporters.

The G-protein coupled neurotensin receptor type 1 (NTSR-1) is expressed in bacteria, purified and labeled with two fluorophores for single-molecule Förster resonance energy transfer (smFRET). Reconstituted in specific liposomes, NTSR-1 or HCN2 ion channels will be fused with a planar black-lipid-membrane (BLM). Dynamic conformational changes are studied using time-resolved confocal as well as widefield smFRET microscopy in microfluidic chips with adjustable membrane potentials. Diffusion of membrane proteins in the BLM will be suppressed by an Annexin-V network for extended observation times.

A2

Label-free investigation of structure-dynamic relations of single membrane receptors using a tip-enhanced Raman scattering approach

This project will target specifically the development for a nanoscale and label-free structure-investigation of membrane receptors, potentially even more specific membrane receptor surfaces. By adapting tip-enhanced Raman spectroscopy (TERS) towards the specific requirements of biological membranes, a structural investigation based on vibrational spectroscopy will allow to identify single receptors and eventually unravel structure-function relations once these receptors undergo externally triggered changes. Ultimately, channeling events at a single receptor level can be tracked down under controlled environmental conditions.

A3

Improved Optogenetic Tools: Characterization of new rhodopsins and engineering of novel features into existing photoreceptors

In this project we plan to develop improved optogenetic tools with larger charge transfer per molecule and lower light intensity requirement, based on engineering of established optogenetic tools and characterization of new photoreceptors. Particularly we plan to test engineered channelrhodopsin mutants and to engineer and test fusion constructs of nucleotidyl cyclases and cyclic nucleotide-gated ion channels.

A4

Multidimensional super-resolution imaging of membrane receptors

In order to visualize and quantify the spatial distribution of more than ten different target molecules in a single experiment, we aim to develop a new method for multidimensional super-resolution imaging that runs cycles of fluorescence tagging and super-resolution imaging using the same fluorophore. In order to remove the fluorophore from the sample before the next round of fluorescence tagging and imaging, we will develop and optimize different approaches including photobleaching, photocleaving of fluorophore linkers, and transient binding of short fluorescently labeled oligonucleotides.

A5

Relating the binding of single ligands to activation in single HCN and CNG channels

From single cyclic nucleotide-gated channels (CNGA2) the binding of single labelled cGMP molecules and the single-channel activity will be simultaneously recorded. In addition, a single-molecule approach will be developed for supported membranes to quantify the dwell time of single labelled cAMP molecules at the four cyclic-nucleotide binding domains of individual homotetrameric HCN2 channels. Novel cGMP and cAMP derivatives will be synthesized. The results should help to unravel the interaction of the four subunits in CNGA2 and HCN2 channels and how this interaction is controlled by voltage in HCN2 channels.

Project Area B

Ligand-gated ion channels

In project area B eight projects are summarized that use high-end light microscopy to unravel the function of ligand-gated ion channels, i.e. ionotropic receptors. Five of these projects focus on the analysis of specific channels in the synapse (B2, B3, B4, B5, B6), a biological structure whose dimension is too small for analysis with conventional light microscopy. Therefore, super-resolution microscopy will be applied as a powerful tool to analyze morphological properties in synapses. Although its resolution is still inferior to electron microscopy, the labeling for super-resolution microscopy is easier and it also offers the possibility to label with different fluorophores emitting light at different wavelengths. Moreover, super-resolution light microscopy bears the potential to generate data in vital preparations, although this issue is also challenging because of thermal movement of the structures to be studied. In detail, the synaptic channels are NMDA receptors, GABAA receptors, glutamate receptors of the neuromuscular junction, and presynaptic glutamate receptors of the kainate subtype as well as glycine receptors of the spinal chord.

B1

Elucidating assembly, ligand binding and gating of heterotetrameric CNG channels by FRET

Cyclic nucleotide-gated channels (CNG) constitute the last step of the phototransduction cascade. In this project optical and electrophysiological techniques will be used to elucidate elementary steps in the activation of the channel, such as the sequence of ligand binding to the individual subunits and their conformational rearrangement. Another part of the project will focus on the mechanisms by which the cell ensures that the channels are assembled of subunits in a fixed stoichiometry.

B2

Investigating humoral autoimmunity against the NMDA-receptor NR1 subunit by super-resolution fluorescence microscopy: effects on synapse integrity and function

The project aims at unravelling the pathomechanism of antineuronal autoimmunity mediated by human autoantibodies to the NMDA-receptor in the central nervous system. We will apply super-resolution fluorescence imaging together with electrophysiological techniques to investigate effects on biophysical properties of NMDA-receptor ion channel function, on glutamatergic synaptic transmission, and on network activity in vivo. This approach will help to elucidate the general molecular mechanism of antineural autoimmunity with so far unmatched spatial and temporal resolution.

B3

Interaction of GABAA-receptor function and network activity in the developing hippocampus

During early development, the immature brain generates synchronized oscillatory network activities which are strongly promoted by GABAA receptor activation. It currently remains unclear as to which extent these neuronal activity patterns are required for the proper functional and morphological development of GABAergic synaptic transmission. We will address this question using a combination of high-resolution optical, electrophysiological and optogenetic techniques in the immature rodent hippocampus both at the in vitro and in vivo level.

B4

Activity-induced ionotropic glutamate receptor dynamics at super-resolution in vivo

The activity-dependent rearrangement of ionotropic glutamate receptors mediates manifold forms of synaptic plasticity. However, fundamental principles governing receptor dynamics remain incompletely understood. By combining optogenetic effectors with electrophysiology and super-resolution microscopy in Drosophila melanogaster, this project aims to investigate how spatial and temporal activity patterns control the subunit-specific mobility of synaptic glutamate receptors at nanoscopic resolution in the intact organism.

B5

Enlightening inhibitory neurotransmitter receptor organization and dynamics applying high-end microscopy

The regulation of glycine receptor numbers and organization at synapses is essential for the efficacy of inhibition and the control of neuronal excitability in the spinal cord. The project therefore aims at analyzing the dynamics and the organization of glycine receptor clusters and the particular role of syndapin I for receptor trafficking and for receptor clustering and organization applying super-resolution light microscopy. High-end microscopy shall unveil mechanisms that ensure proper formation, maintenance and adaptation of glycine receptor synapses representing key players of inhibitory neurotransmission.

B6

Functional plasticity of glutamate receptor channels on hippocampal mossy fiber terminals

Mossy fiber boutons (MFBs) of hippocampal granule cells are uniquely plastic. MFB plasticity is controlled by ionotropic autoreceptors for presynaptically released glutamate (GluKs). We focus on GluKs, active zones (AZs) and MFB plasticity. AZs couple presynaptic calcium inflow and vesicle fusion. Changes in size, molecular composition, number and function of AZs may contribute to MFB plasticity. We aim to elucidate nanoscopic protein dynamics at the level of individual AZs, the role of GluKs and the impact of both on MFB plasticity and memory.

B7

Relating ligand binding and activation gating in nicotinic acetylcholine receptors

This project aims to analyze the gating behavior of nicotinic acetylcholine receptors (nAChR). Novel ACh analogs will be synthesized to be employed in confocal patch-clamp fluorometry. Simultaneous monitoring of agonist binding and receptor activation will shed light onto the complex desensitization mechanism, taking into account the specific contribution of the two structurally different binding sites. Furthermore, the role of the clustering protein rapsyn in the desensitization process will be studied.

B8

Interaction and ligand gating within Arabidopsis guard cell hormone-receptor anion-channel complex

Project B8 focuses on the guard cell expressed anion channels SLAC1 and SLAH3 that represent master switches within the drought hormone (ABA)-stimulated stomatal closure. Using super-resolution microscopy as well as FRET and FLIM applications in combination with electrophysiology, we intend to quantify and to decipher the ABA-signaling complex with spatio/temporal resolution. This approach will gain insights into the dynamics of i) drought hormone ABA recognition by the guard cell ABA-signalosome, ii) the ABA-dependent activation of SLAC1 and SLAH3, and iii)  the functional heteromeric assembly of SLAC/SLAH-type subunits.

Project Area C

GPCRs and other membrane receptors

In project area C seven projects on metabotropic membrane receptors are summarized, i.e. receptors that do not evoke their effects by opening a pore conductive for ions, but by transferring their conformational change either to other proteins or by activating an enzyme activity in the receptor itself. Projects C1 to C6 comprise projects on the function of G-protein coupled receptors (GPCRs). In animals, GPCRs form the largest family of membrane receptors at all, thereby conferring highly specific sensitivities for hormones and neurotransmitters. GPCRs mediate their effects by activating heterotrimeric G-proteins, which in turn activate or inhibit downstream effectors including enzymes and ion channels. Because GPCRs do not directly alter an ion conductance, measurement of their activation time course is more complicated compared to ligand-gated ion channels.

C1

Spatial organization and dynamics of GPCR signaling as revealed by single-molecule and super-resolution microscopy

G protein-coupled receptors (GPCRs) constitute the largest family of receptors for hormones and neurotransmitters and represent major pharmacological targets. In this project, we are using high-end optical methods such as single-molecule microscopy and super-resolution imaging (dSTORM) to investigate the organization of GPCRs in dynamic nanodomains on the surface of living cells. We expect these experiments to lead to a deeper understanding of the basic mechanisms of GPCR signaling, which might pave the way to innovative and more specific pharmacological therapies for a wide range of human diseases.

C2

How does receptor dynamic influence ligand residence time and ligand efficacy at adenosine receptors

Ligand receptor interaction is often simply seen as a binding event and neglects the conformational dynamic of the target protein and its influence on ligand binding or the efficacy of the ligand. The central question within this project will be to address conformational dynamic of a receptor and monitor ligand binding with high kinetic resolution in living cells at the same time. We will use a fluorescence resonance energy transfer (FRET) based approach to measure ligand on/off-rates and receptor activation/deactivation in real-time to obtain ligand residence time information and ligand efficacy data in living cells.

C3

FRET-based monitoring of Adhesion class G protein-coupled receptor activity

Stimulus modality and signal transduction events of G protein-coupled receptors of the Adhesion class (aGPCR) are poorly defined. Previous work suggests that aGPCRs sense mechanical signals, possibly through intramolecular distance changes between extracellular and transmembrane receptor portions. This project will use FRET measurements to optically quantify such changes upon ligand exposure, and test artificially elongated aGPCR variants for altered receptor activity in an established Drosophila model. Lastly, FRET sensor constructs will be employed to assess the effect of human mutations on aGPCR distance changes.

C4

Kinetics of metabotropic glutamate receptors

The activation time among G protein-coupled receptors (GPCRs) seems to vary by more than five orders of magnitude, whereupon methodological reasons severely limit any analysis. Optimized systems will be developed to accurately measure the activation kinetics of metabotropic GPCRs, including light-induced activation by concentration jumps evoked by either caged agonists or a piezo device as well as photosensitive tethered agonists. The readout for receptor activation will be Förster resonance energy transfer from receptor constructs with appropriate fluorophores. The data will be analyzed by Markovian models.

C5

Real-time imaging of µ-opioid receptor phosphorylation and dephosphorylation

We have recently demonstrated that phosphorylation of the µ-opioid receptor (MOR) in mouse brain in vivo is regulated by drug-selective engagement of distinct G protein-coupled receptor kinases (GRKs). In this proposal we will (I) establish novel FRET-based measurements of the interaction of GRK2, GRK3, GRK5 and GRK6 with MOR, (II) develop FRET-based measurements of the interaction of MOR with different G protein-coupled receptor phosphatases (GRPs) such as PP1α, PP1β and PP1γ, and (III) utilize newly generated phosphosite-specific nanobodies to directly visualize the cellular locus of MOR dephosphorylation using super-resolution microscopy.

C6

Deciphering fast intra- and intermolecular dynamics of G protein-coupled receptors by Fluorescence (Cross) Correlation Spectroscopy

This project aims to analyze the structural dynamics of G protein-coupled receptor (GPCR) activation. We will investigate the hypothesis that there are several rapid (< millisecond) activation steps that together constitute the activation dynamics of GPCRs, and that ligands can influence these dynamics in distinct ways. We intend to resolve these rapid steps with a combined FRET-FCS approach (Fluorescence Resonance Energy Transfer and Fluorescence Correlation Spectroscopy). Advanced FRET sensors will help to delineate the individual elements of the different activation states and learn about the kinetics of their transitions.

C7

Signaling mechanism, subcellular localization and regulation of guanylyl cyclases A and B, the receptors for atrial and C-type natriuretic peptides

The cardiovascular actions of atrial (ANP) and C-type natriuretic peptides (CNP) are mediated by two transmembrane cyclic GMP forming guanylyl cyclase (GC) receptors, GC-A and GC-B. To characterize the mechanism of ligand-dependent activation of cGMP production by these receptors we will use a FRET-FlAsH approach and visualize the dynamics of ligand-induced conformational changes in living cells. In addition we will apply single-molecule microscopy and super-resolution imaging to compare the distinct subcellular localizations of GC-A and GC-B in healthy and hypertrophied cardiomyocytes.

Project Area Z

Central Projects

Besides the administrative project two other central projects are planned that have been designed to cover specific needs of the CRC/TR for the analysis and the management of scientific data. These projects reflect the needs for extensive computer-based analysis of experimental data and their management to guarantee access to the data during the running of the CRC/TR and thereafter.

DATA MANAGEMENT

Integrative Data management and processing

The aim of this project is to design, implement, and maintain a central software platform to store, manage, share, and process data of all CRC/TR projects. The platform will ensure reproducibility and sustainability of research results. It will also allow for an interactive access and will enable a systematic analysis of the large and heterogeneous data. Thus, this project will not only provide participating scientists with a tool to organize and analyze their data, but also foster the exchange of tools and the sharing of knowledge.

COORDINATION

Central administration and project coordination

The aim of this project is the coordination of all activities of this CRC/TR including administration of organizational tasks (central management of budget and staff, reports, gender equality, public relations, establishment of a concept for structured early career support etc.), as well as an efficient communication (internal and external events, meetings and workshops) between all members of the CRC/TR at both locations.