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Three-dimensional structure of the glycine-betaine transporter BetP by cryo electron crystallography
(2008)
The soil bacterium Corynebacterium glutamicum has five secondary transporters for compatible solutes allowing it to cope with osmotic stress. The most abundant of them, the transporter BetP, performs a high affinity uptake of glycine-betain when encountering hyperosmotic stress. BetP belongs to the betaine/carnitine/choline/transporter (BCCT) family, and is predicted to have twelve transmembrane helices with both termini facing the cytoplasm. The goal of this thesis is to facilitate understanding of BetP function by determining a three dimensional (3D) model of its structure. Two-dimensional (2D) crystallization of wild-type (WT) BetP has been successfully performed by reconstitution into a mixture of E. coli lipids and bovine cardiolipin, which resulted in vesicular crystals diffracting to 7.5 Å resolution (Ziegler, Morbach et al. 2004). Diffraction patterns of these crystals however showed unfocused spots, generally due to high mosaicity. Better results were obtained by using the constitutively active mutant BetPdeltaC45 in which the first 45 amino acids of the positively charged C-terminus were removed. BetPdeltaC45 crystals obtained under the same conditions for BetP WT were concluded to be pseudo crystals, based on the inconsistence of symmetry. These crystals had BetPdeltaC45 molecules randomly up/downwards inserted into membrane crystals, and cannot be used for structure determination, even though they diffracted up to 7 Å. The problem of pseudo crystal formation could be solved by changing the lipids used for 2D crystallization to a native lipid extract from C. glutamicum cells. This change of lipids improved the crystals to well-ordered packing with exclusive p121_b symmetry. To understand the role of lipids in crystal packing and order, lipids were extracted at different stages during crystallization, and identified by using multiple precursor ion scanning mass spectrometry. The results show that phosphatidyl glycerol (PG) 16:0-18:1 is the most dominant lipid species in C. glutamicum membranes, and that BetP has a preference for the fatty acid moieties 16:0-18:1. Crystallization with synthetic PG 16:0-18:1 proved that an excess of this lipid prevents pseudo crystal formation, but these crystals did not reach the quality as previously achieved by using the C. glutamicum lipids. Apart from the effect of lipids in crystallinity, the concentration and type of salts influenced crystal growth and morphology. High salt conditions (>400 mM LiCl or KCl) yielded tubular crystals, whereas low salt conditions (<300 mM LiCl, NaCl or KCl) led to formation of up to 10 µm large sheet-like crystals. The intermediate concentration gave a mixture of sheet-like and tubular crystals. In terms of resolution, sheets diffracted better than tubes. The sheet-like crystals used for 3D map reconstruction were obtained from a dialysis buffer containing 200 mM NaCl combined with using C. glutamicum lipids. Electron microscopic images were taken from frozen-hydrated crystals using a helium-cooled JEOL 300 SFF microscope or a liquid nitrogen-cooled FEI Tecnai G2 microscope at 300 kV, which allowed optimal data collection and minimized radiation damage to the sample. More than 1000 images of tilt angles up to 50° were taken and evaluated using optical diffraction of a laser beam. The best 200 images were processed with the MRC image processing software package, and 79 images from different tilt angles were merged to the final data set used for calculation of a 3D map at a planar resolution of 8 Å. The structure shows BetPdeltaC45 as a trimer with each monomer consisting of 12 transmembrane alpha-helices. Protein termini and loop regions could not be determined due to the limited resolution of the map. Six of the twelve helices line a central cavity forming a potential substrate-binding chamber. Each monomer shows a central cavity in different sizes and shapes. Thus, the constitutively active BetPdeltaC45 thus forms an unusual asymmetric homotrimer. BetP most likely reflects three different conformational states of secondary transporters: the cytoplasmically open (C), the occluded (O), and the periplasmically open (P) states. The C and O states are similar to BetP WT projection structure, while the P state is discrepant and highly flexible due to the shape and size of the central cavity as well as the lowest intensity of the density. The observation of the P state corresponds well to the constitutively active property of BetPdeltaC45. For the high resolution structure of the C and O states are available, this work presents the first structural information of the P state of a secondary transporter.
Membrane proteins play vital role in a variety of cellular processes, such as signal transduction, transport and recognition. In turn they are involved in numerous human diseases and currently represent one of the most prevalent drug targets. A comprehensive understanding of the mechanisms mediated by membrane proteins requires information about their structures at near-atomic resolution, although structural studies of membrane proteins remain behind those of soluble proteins. A bottleneck in the study of membrane proteins resides in the difficulties that are encountered during their high-level production in cell based systems. However, many toxic effects attributed to the over production of membrane proteins are eliminated by cell-free expression, as viable host cells are no longer required. Therefore, the objective of this study was to obtain adequate amounts of selected membrane transport proteins for their structural studies using a cell-free expression system. For the establishment of the cell-free system for membrane proteins, the transporters YbgR and YiiP from Salmonella typhimurium LT2, PF0558 and PF1373 from Pyrococcus furiosus, from the cation diffusion family (CDF), BetP from Corynebacterium glutamicum from the betaine/carnitine/choline transporter (BCCT) family and Aq-2030 from Aquifex aeolicus VF5 from the monovalent cation/proton antiporter-2 (CPA2) family were selected. An Escherichia coli S-30 extract based cellfree system was established by generating the best expression constructs of the target proteins, preparing T7 RNA polymerase and an S-30 extract with high translation efficiency. The functionality of the S-30 extract was shown by the cell-free expression of correctly folded Green Fluorescent Protein (GFP). Essential factors of the cell-free system such as the Mg2+ concentration, the bacterial S-30 extract proportion in the reaction mixture and the time-course of cell-free reactions have been optimized. For the cell-free production of membrane proteins in soluble form, the possibility to supplement cell-free reactions with detergents was explored. A wide range of non-ionic or zwitterionic detergents, were found to be compatible with cell-free synthesis, while ionic detergents and non-ionic detergents at high concentrations had an inhibitory effect. Moreover, high concentrations of polyoxyethylene-alkyl-ethers (Brij) detergents were found to have enhancing effect on the production levels as well as on the solubility of cell-free produced proteins. As membrane proteins tend to misfold and aggregate in a membrane-free translation system, the possibility to supplement the cell-free reactions with inner membrane vesicles (IMVs) to obtain correctly folded target transport proteins was explored. All the target proteins were successfully produced in the batch cell-free reactions and were found to be incorporated in the IMVs. A continuous exchange cell-free (CECF) system was established, where consumable substrates (amino acids, nucleotides and energy regenerating compounds) were supplied to the cell-free reaction mixture through a dialysis membrane, which in consequence resulted in high-level production of target proteins compared to the batch system. The osmosensing and osmoregulated sodium-coupled symporter BetP from C. glutamicum was chosen for the large scale production in CECF set-up. The protein is easily produced in E. coli and is functional as assayed by its transport activity, after purification and reconstitution in liposomes. It is therefore possible to compare in-vivo and cell-free production. High-level cell-free production of BetP was achieved in CECF mode in different forms: (i) as precipitate, (ii) as soluble form in detergent, and (iii) incorporated in IMVs. Cell-free production of BetP resulted in the yield of about 0.5 mg of purified BetP from 1 ml of CECF reaction. The yield of purified BetP was increased to 1.6 fold by addition of 1% polyoxyethylene-(20)-cetyl-ether (Brij58) detergent in the reaction mixture. Moreover, the high level cell-free production of BetP (0.5 mg purified BetP/ml reaction mixture) incorporated in IMVs was shown for the first time in this work.However, it was observed that oligomerization of BetP was not efficient in the cell-free system. Factors that can promote the folding of membrane proteins such as lipids and chaperones were investigated. Addition of lipids and molecular chaperone GroE facilitated correct folding of BetP resulting in increased yield and stability of cell-free produced BetP. The results obtained indicate that most of the cell-free produced BetP exists in functional oligomeric form. The possibility of obtaining milligram amounts of BetP, a 12 trans-membrane protein from the cell-free reactions holds promise for structural and functional studies of other membrane proteins. In any case, the strategies adapted in this study should prove extremely valuable for the production of membrane proteins in the E. coli cell-free expression system.
G-protein coupled receptors (GPCRs) comprise the largest superfamily of cell surface receptors and possess a signature motif of seven transmembrane helices. The endothelin B (ETB) receptor is a member of rhodopsin like GPCR family. It plays an important role in vasodilation and is found in the membranes of the endothelial cells enveloping blood vessels. Knowledge of the three-dimensional structure of G-protein coupled receptors in general would significantly add to our understanding of their molecular mechanisms and would be useful in the search for new specific drugs. However, three-dimensional structural analysis will require milligram quantities of pure and homogeneous protein. This dissertation is a study of the production, biochemical characterization and preliminary structural studies of the human ETB G-protein coupled receptor. The present work aimed at elucidating the structure and mechanistic details of function of the receptor by using a combination of X-ray crystallographic and NMR methods for collecting structural data. To obtain homogenous and monodisperse receptor protein preparation for structural and functional studies, we implemented the baculovirus expression system for the production of ETB receptor for the present work. The two step affinity purification ensured capture of full-length receptor. Silver stained SDS-PAGE of the purified receptor-ligand complex indicated greater than 90% protein purity. Based on previous reports, we used the high affinity ligand (endothelin -1) binding to the receptor for co-crystallization of receptor-ligand complex by locking the receptor in the activated conformation. As a prerequisite for 3D crystallization trials, the stability of the detergent solubilized receptor-ligand complex was assessed with respect to pH, temperature and time. Receptor-ligand complex did not show any degradation and aggregation over 6 days at 4°C and 18°C. Interestingly, change of pH suggested that receptor-ligand complex is unstable at lower pH due to possible charge induced conformational changes. In our work, we introduced the idea of using fluorophore labeled ligand for simple visual recognition of the receptor-ligand complex during purification and crystallization. On the other hand, we alternatively used biotinylated endothelin-1 to produce an adequate amount of ligand bound receptor complex, thus ensuring homogeneity of the purified complex for use in structural studies. Thus far, preliminary crystals have been obtained for both the unlabelled ET-1 and fluorophore labeled ET-1 complexed with ETB receptor. Moreover, we performed the systematic investigation of the protein/peptide binding partner for the receptor-ligand complex with the chief aims of stabilizing structure and increasing the possibilities of 3D-crystal contacts. Thus subsequent to formation of receptor-ligand complex, the additional in vitro formation of a ternary arrestin-receptor-ligand complex was also attempted for use in structural studies. We successfully demonstrated that arrestin mutant (R169E) forms a tight complex with ETB receptor regardless of its phosphorylation state. A second approach to get insight into the ETB receptor ligand binding site relied on the use of spin isotope labeled ET-1 ligand peptide by employing solid state MAS NMR method. Preliminary data provided compelling evidence that the C-terminal region of the peptide is immobilized in an ordered environment and presumably bound to the receptor. This indicates that the approach is feasible, although there are difficulties in sample preparation for further spectral measurements and data collection which are currently being discussed in ongoing investigations. At this point of our research work, we initiated a collaborative effort to obtain high yields of pure, active receptor without post translational modifications, from an E. coli cell lysate based in vitro expression system. We successfully optimized the production of homogenous and monodisperse endothelin B receptor in mg amounts. Thus this could potentially provide an alternative source of high quality receptor production in large quantities for immediate crystallization trials. Thus we hope that the results from these investigations can be applied in a more general sense to the production and crystallization of other G protein-coupled receptors.
The mitochondrial respiratory chain consists of NADH:ubiquinone oxidoreductase (Complex-I), succinate:ubiquinone reductase (Complex-II), ubiquinol:cytochrome c reductase (Complex-III), cytochrome c oxidase (Complex-IV) and cytochrome c as an electron mediator between Complex-III and Complex-IV. Paracoccus denitrificans membranes were used as a model system for the association of the mitochondrial respiratory chain. More than 50 years ago, a model was given for a supercomplex assembly formed by stable associations between these complexes. This model gradually shifted by the model of random diffusion given by Hackenbrock et al. 1986 Different independent approaches were used to further analyze this situation in a native membrane environment, thus avoiding any perturbation caused by detergent solubilization: (a) measuring the distance and orientation of the different complexes by multi-frequency EPR Spectroscopy we started to analyze simple system, the interaction between CuA fragment derived from P. denitrificans and various c type cytochrome by Pulsed X band and G band (180 GHz) EPR. Partner proteins for the CuA (excess negative surface charge) were (i) horse heart cytochrome c which contain a large number of positive charges in heme crevice,(ii) the cytochrome c552 soluble fragment (physiological electron donor and have positive charges), and as a control (iii) the cytochrome c1 soluble fragment (negative surface potential, derived from bc1 complex) The measurements were performed at several magnetic field positions varying temperature between 5 to 30 K. Both the X band and the high-field measurements show the existence of a strong relaxation enhancement of the CuA by the specific binding of the P. denitrificans cytochrome c552 and horse heart cytochrome c. This relaxation enhancement is dependent on temperature and provides information about the distance and relative orientation of the two interacting spins within this protein-protein complex. (b) For quantitative information about lateral diffusion of cytochrome c oxidase in the native membrane Fluorescence Correlation Spectroscopy (FCS) was used. In this experiment, diffusion coefficients for oxidase differ in the case of supercomplex for wild type membrane and for two deletion mutants lacking either Complex-I or Complex-III. (c) The optical absorption spectroscopy at microsecond level resolution was tried for the translational mobility of oxidase in membrane vesicles. Due to the presence of different hemes in the native membrane, carbon monoxide (CO) used as a probe for the experiment. The optimization of the experimental conditions were carried out to get the optimal signal.
RcsB is a central transcriptional regulator in enteric bacteria involved in exopolysaccharide (EPS) biosynthesis, in cell division, in the expression of osmoregulated genes, and regulates at least 20 other genes and operons. It is a member of a phosphorelay system and signal transfer is mediated by phosphorylation through the RcsC/YojN phosphorelay. RcsB proteins modified with the phosphorylation mimic BeF3- as shown by its conformational changes and DNA binding properties and resulted phosphorylated RcsB derivatives with sufficient stability. Both, the wild type RcsB protein and the mutant RcsBD11A could be modified with BeF3-. Non-phosphorylated RcsB has been shown to bind as a heterodimer with the coinducer RcsA at the conserved RcsAB box in Rcs regulated promoters. In this study, it has been shown that the modification of RcsB by BeF3 - (I) has a negative effect on its homodimerization, (II) abolishes the complex formation of RcsAB with the RcsAB box as shown by the EMSA and SPR technique. All the effects were found to be reversible by increasing the NaF concentration in the assays presumably leading to the formation of the inactive BeF4 2- salt. This hypothesis of RcsB being modified by BeF3- was also supported by other phosphodonors like ATP and acetyl phosphate, both of them showed the same negative effect on DNA binding by RcsAB heterodimer giving evidence that BeF3- could be used as a phosphorylation mimic. In addition, the phosphorylation mimic BeF3- was found to be a better phosphorylating agent than ATP and acetyl phosphate. This is the first evidence that phosphorylation of RcsB might have a negative effect on the activation of RcsAB regulated operons. Autophosphorylation of RcsB proves that it has the ability to take up phosphoryl groups and the mutant protein also become autophosphorylated with less efficiency or stability than the wild type protein. RcsB probably takes up phosphoryl groups through RcsC -> YojN -> RcsB phosphorelay pathway. To study the interaction among the proteins in this pathway, fluorescence spectroscopy, NMR spectroscopy, and an in vivo ß galactosidase assay were performed by using two domains of RcsC (T-RcsC and R-RcsC), HPt domain of the protein YojN, and RcsB. The interactions between R-RcsC/YojN-HPt and YojN-HPt/RcsB supports the proposed pathway of phosphorylating RcsB. RcsB might also be phosphorylated by YojN-HPt that is phosphorylated by other sensor kinase other than RcsC in a cross-talk mechanism. The phosphorylation of RcsB by YojN-HPt probably has the same negative effect on cps induction as obtained with BeF3 - effect on DNA binding by RcsAB heterodimer.
The transcriptional regulator RcsB controls the expression of a minimum of 20 different genes having diverse functionalities and biosynthetic operons in the family of Enterobacteriaceae. While in the heterodimeric complex with the co activator RcsA, the RcsAB box consensus is recognized, DNA binding sites for RcsB without RcsA have also been identified. The conformation of RcsB might therefore be modulated upon interaction with various co activators, resulting in recognition of different DNA targets. In this study the interaction of RcsB with some of these DNA targets have been analysed by a diverse array of techniques including gel shift assay and SPR. The solution structure of the C-terminal DNA-binding domain of RcsB from Erwinia amylovora spanning amino acid residues 129-215 has been solved in this study by heteronuclear NMR spectroscopy. The C-terminal domain is composed of four α-helices where the two central helices of the H-T-H motif are similar to the structures of the regulatory proteins GerE, NarL and TraR. The DNA-binding activity of the C-terminal domain alone is established for the first time in this study and was specified by fluorescence spectroscopy, SPR and NMR titration experiments. The molecular interaction between the individual RcsB domains was analysed by cross-linking experiments and heteronuclear NMR spectroscopy and the amino acid residues of the C-terminal domain involved in this interaction were identified precisely. Another important part of this project was the cell-free production of different Trp analogue labelled RcsB protein. RcsB protein was produced in quite a good yield with different Trp analogue having spectrally enhanced properties. The isolated RcsB alloproteins proved to be ideal for protein interaction studies by fluorescence spectroscopy and the very first evidence of an oligomerization of RcsB due to molecular association has been put forth from these studies. The phosphorylated state of the RcsB protein was mimicked by a beryllofluoride complex in order to study its role in transcriptional regulation. It was found that RcsB alone could bind to DNA targets upon this modification by the beryllofluoride complex. Thus the phosphorylation of the protein that involves the Asp 56 residue induces a structural change of the protein followed probably by a domain movement also, so that the C-terminal domain having the H-T-H DNA binding motif that was previously eclipsed by the N-terminal domain is relieved of this constraint.
Sodium proton antiporters are ubiquitous membrane proteins found in the cytoplasmic and organelle membranes of cells of many different origins, including plants, animals and microorganisms. They are involved in cell energetics, and play primary roles in the homeostasis of intracellular pH, cellular Na+ content and cell volume. Adaptation to high salinity and/or extreme pH in plants and bacteria or in human heart muscles requires the action of such Na+/H+ antiporters. NhaA is the essential Na+/H+ antiporter for pH and Na+ homeostasis (at alkaline pH) in Escherichia coli and many other enterobacteria. NhaA is an electrogenic Na+/H+ antiporter that exchanges 2H+ for 1Na+ (or Li+). NhaA shares with many other prokaryotic and eukaryotic antiporters a very strong dependence on pH. In order to achieve three-dimensional structure of NhaA, the previously described NhaA protein preparation was modified: (i) the wild type bacterial strain (TA16) used for homologous over-expression of NhaA was replaced with a delta nhaA strain (RK20). As a result, the purity and homogeneity of the sample was significantly improved; (ii) the previously two-step purification procedure was shortened to a single step affinity chromatography purification; (iii) a wide-range screening of crystallisation conditions, more than 20,000, was performed; (iv) a Seleno-L-methionine (SeMet) NhaA derivative was produced in order to solve the phases during structure determination. In parallel, attempts of production and crystallisation of co-complexes composed of NhaA and antibody fragments have been made. Four different monoclonal antibodies were available against NhaA. Selected antibody fragments were produced and the stability of the complex analysed. Here, the crystal structure of the pH down-regulated secondary transporter NhaA of Escherichia coli is presented at 3.45 Å resolution. A negatively charged ion funnel opens to the cytoplasm and ends in the middle of the membrane at the putative ion-binding site. There, a unique assembly of two pairs of short helices connected by crossed, extended chains creates a balanced electrostatic environment. A possible mechanism is proposed: the binding of charged substrates causes electric imbalance inducing movements, which allow for a rapid alternating access mechanism. This ion exchange machinery is regulated by a conformational change elicited by a pH signal perceived at the cytoplasmic funnel entry. The structure represents a novel fold that provides two major insights: it reveals the structural basis for the mechanism of Na+/H+ exchange and its unique regulation by pH in NhaA and in many other similar antiporters. Furthermore, it is also important for the understanding of the architecture of membrane proteins in general. However, although many aspects of the ion-translocation mechanism and pH regulation are clarified by the NhaA structure, higher resolution structures with Li+ or Na+ bound are required for understanding the ligand binding and the translocation mechanism at the atomic level. The alkaline pH-induced conformation is essential to further understand the pH-control and proton access to the binding site.
The adaptive immune system of jawed vertebrates is based on recognition and elimination of cells that are either invaded by intracellular pathogens or malignantly transformed. One essential component of these processes is the cell surface presentation of antigenic peptides via major histocompatibility complex (MHC) class I molecules to cytotoxic T-cells (CTLs). Cells degrade defective ribosomal products and misfolded or unwanted proteins by the ubiquitin-proteasome pathway. The resulting degradation products are recognized and translocated by the transporter associated with antigen processing (TAP) into the endoplasmic reticulum (ER) lumen, where they are loaded onto MHC I molecules. Assembled peptide-MHC complexes are then shuttled by the secretory pathway to the cell surface for antigen presentation to CTLs, leading in the case of viral infection or malignant transformation to lysis and apoptosis of the target cell. Due to the fact that the TAP complex represents a key control point within the antigen presentation pathway, several viruses have evolved sophisticated strategies to evade immune surveillance by interfering with TAP function.
Detailed studies of the TAP mechanism or its viral inhibition have been severely impeded by difficulties in expressing sufficient amounts of functional heterodimeric TAP complex. Thus, the overexpression of TAP in the methylotrophic yeast Pichia pastoris was established for functional analysis of this important ABC complex. Biomass production was scaled up by fermentation using classical batch and feed methods. Extensive screening of optimal solubilization and purification conditions allowed the isolation of the heterodimeric transport complex. Notably, only the very mild detergent digitonin preserved TAP function. Hereby, the optimal solubilization and purification strategy yielded in 30 mg TAP transporter per liter culture. Remarkably, the protein amount was 50-fold increased compared to previously described expression/purification in cultured insect cells.
The high yield and quality of TAP produced in P. pastoris allowed an extensive analysis of substrate binding and transport kinetics of the transport complex in the membrane, its solubilized and purified state, as well as the reconstituted state. Thereby, a strong and direct effect of the lipid bilayer on ATP hydrolysis and peptide transport was discovered. These important results were extended further by successful functional reconstitution of the antigen translocation machinery in different lipid environments. For the first time, a stimulation of the transport activity by phosphatidylinositol (PI) and phosphatidylethanolamine (PE) was observed, whereas cholesterol was identified as an inhibitor of TAP activity.
Purification of TAP and subsequent thin-layer chromatography (TLC)/liquid chromatography Fourier transform-mass spectrometry (LC FT-MS) fingerprinting of residual lipids exhibited specifically associated glycerophospholipids; mainly PC, PE, and PI species. Strikingly, these lipids not only represent the primary class of phospholipids of the ER but were also shown to be essential for functional reactivation of delipidated, and thus inactive, TAP. The results demonstrate that transport of antigenic peptides by the ABC transporter TAP strictly requires specific glycerophospholipids.
In addition to the biochemical characterization of heterologous produced TAP, the soluble domain of the viral inhibitor US6 from human cytomegalovirus was expressed in E. coli. Optimization of the purification and refolding strategy yielded in functional protein, with a 35-fold increased protein amount compared to previous purification procedures. Protein activity was analyzed by specific inhibition of ATP binding to TAP. Furthermore, high protein yields allowed detailed investigation of TAP-dependent spatial and mechanistic separation of MHC I restricted cross-presentation in professional antigen presenting cells (pAPC).
In Nervensystemen werden zahlreiche Informationen wahrgenommen und verarbeitet um ein adäquates Verhalten hervorzurufen. Für die Untersuchung der funktionellen Zusammenhänge hierbei wurden verschiedene Methoden entwickelt, die eine gezielte Manipulation neuronaler Prozesse ermöglichen. Durch Analyse der resultierenden Effekte können dabei synaptische Proteine, einzelne Neuronen oder neuronale Netzwerke funktionell charakterisiert werden. Bisherige Ansätze verfügen jedoch nur über eine geringe zeitliche und räumliche Auflösung oder erlauben lediglich eine eingeschränkte Anwendung im frei beweglichen Tier.
Diese Nachteile können durch die heterologe Expression von lichtgesteuerten, mikrobiellen Rhodopsinen zur gezielten Manipulation des Membranpotentials umgangen werden. So induziert die Photoaktivierung des Kationenkanals Channelrhodopsin 2 (ChR2; (Nagel et al., Curr Biol 2005)) eine Depolarisation, während die Chloridpumpe Halorhodopsin (NpHR; (Zhang et al., Nature 2007)) für die Hyperpolarisation verwendet werden kann. Dabei ermöglichen die schnellen Kinetiken der Rhodopsine eine zeitlich präzise Steuerung des Membranpotentials. Durch Auswahl geeigneter Promotoren ist zudem oftmals eine zell spezifische Expression möglich. Dieser Ansatz wird daher allgemein als Optogenetik bezeichnet.
In der vorliegenden Arbeit wurden zunächst konventionelle Techniken genutzt, um die Funktion von zwei assoziierten Proteinen eines Acetylcholin Rezeptors in C. elegans zu untersuchen. Des Weiteren wurden verschiedene Methoden für den Fadenwurm entwickelt und angewendet, die die Vorteile optogenetischer Techniken für die funktionelle Charakterisierung synaptischer Proteine und neuronaler Netzwerke nutzbar machen. Hierbei erlaubt die Transparenz von C. elegans die optogenetische Stimulation im lebenden Organismus unter nicht invasiven Bedingungen. Weitere Vorteile von C. elegans als neurobiologischem Modellorganismus liegen in seiner einfachen Handhabung (Hope, 1999) und der stereotypen Entwicklung seines Nervensystems mit bekannten anatomischen Ausprägungen (Sulston and Horvitz, Dev Biol 1977; Varshney et al., PLoS Comput Biol 2011; White et al., Philos Trans R Soc Lond B Biol Sci 1986). Durch ihre Häufigkeit und die experimentelle Zugänglichkeit wird hierbei die neuromuskuläre Synapse oftmals zur Erforschung der synaptischen Reizweiterleitung genutzt (Von Stetina et al., Int Rev Neurobiol 2006). Durch pharmakologische (Lewis et al., Neuroscience 1980; McIntire et al., Nature 1993; Miller et al., Proc Natl Acad Sci U S A 1996; Richmond and Jorgensen, Nat Neurosci 1999) und elektrische Stimulation (Richmond and Jorgensen, Nat Neurosci 1999) können dabei Defekte der Transmission hervorgehoben werden, während Verhaltensexperimente oder elektrophysiologische Messungen der post synaptischen Ströme in Muskelzellen eine quantitative Analyse ermöglichen (Richmond and Jorgensen, Nat Neurosci 1999).
Diese Methoden wurden für die funktionelle Charakterisierung von NRA 2 und NRA 4 verwendet, die beide als akzessorische Proteine zusammen mit dem Levamisol sensitiven Acetylcholin Rezeptor der Körperwandmuskelzellen aufgereinigt wurden (Gottschalk et al., EMBO J 2005). Dabei konnte gezeigt werden, dass NRA 2 und NRA 4 im Endoplasmatischen Retikulum (ER) der Muskelzellen einen Komplex bilden, der die Sensitivität von beiden nikotinischen Acetylcholin Rezeptoren gegenüber verschiedenen cholinergen Agonisten verändert. In diesem Zusammenhang wurde auch nachgewiesen, dass die Oberflächenexpression einzelner Untereinheiten der beiden Rezeptoren durch NRA 2/4 beeinflusst wird. Diese Resultate legen die Vermutung nahe, dass beide Proteine die Zusammensetzung der Rezeptoren und somit ihre pharmakologischen Eigenschaften modulieren. Denkbar ist dabei eine regulatorische Funktion bei der Assemblierung verschiedener Untereinheiten zu einem funktionellen Rezeptor oder bei der Kontrolle des ER Austritts von Rezeptoren mit bestimmter Zusammensetzung. In dieser Hinsicht konnte jedoch keine Interaktion von NRA 2/4 mit der Notch Signalkaskade nachgewiesen werden, wie sie für die homologen Proteine nicalin und NOMO in Vertebraten gezeigt wurde (Haffner et al., J Biol Chem 2007; Haffner et al., EMBO J 2004).
Für die Untersuchung synaptischer Proteine durch optogenetische Techniken wurde ChR2(H134R) selektiv in cholinergen oder GABAergen Motorneuronen exprimiert, um die akute und lichtgesteuerte Freisetzung des jeweiligen Neurotransmitters zu ermöglichen. Die resultierende Stimulation bzw. Inhibition von Muskelzellen wurde hierbei durch elektrophysiologische Messungen der post synaptischen Ströme und durch Analyse von Kontraktionen respektive Relaxationen untersucht. Dabei wurde gezeigt, dass Störungen der synaptischen Reizweiterleitung die Ausprägung und Dynamik dieser lichtinduzierten Effekte beeinflussen und dadurch charakterisiert werden können. So zeigten beispielsweise Mutanten von Synaptojanin und Endophilin nachlassende Effekte bei anhaltender oder wiederholter Stimulation, was durch die gestörte Regeneration synaptischer Vesikel erklärt werden kann (Harris et al., J Cell Biol 2000; Schuske et al., Neuron 2003; Verstreken et al., Neuron 2003).
Die hohe Sensitivität dieser Methode wurde im Nachfolgenden dazu verwendet, die Inhibition cholinerger Motorneuronen durch den metabotropen GABAB Rezeptor zu untersuchen, der in C. elegans aus den beiden Untereinheiten GBB 1 und GBB 2 gebildet wird (Dittman and Kaplan, J Neurosci 2008; Vashlishan et al., Neuron 2008). Dabei konnte zunächst gezeigt werden, dass diese heterosynaptische Inhibition verschiedene lokomotorische Verhaltensweisen der Tiere beeinflusst. Für die mechanistische Untersuchung wurden anschließend cholinerge Motorneuronen durch ChR2(H134R) photoaktiviert, während resultierende Kontraktionseffekte in Abhängigkeit von GBB 1/2 analysiert wurden. Um hierbei die Funktion von GBB 1/2 durch erhöhte GABA Konzentrationen hervorzuheben, wurden zusätzlich GABAerge Motorneuronen optogenetisch stimuliert oder die Wiederaufnahme von GABA aus dem synaptischen Spalt durch Mutation des Membran ständigen GABA Transporters blockiert. So konnte gezeigt werden, dass GBB 1/2 eine akute Inhibition der cholinergen Motorneuronen bewirken, was vermutlich für die Regulation von Bewegungsabläufen eine wichtige Rolle spielt. Die geringe Dynamik der GBB 1/2 induzierten Effekte deutet allerdings darauf hin, dass die synaptische Aktivität durch den metabotropen Rezeptor kaum nachhaltig moduliert wird.
In nachfolgenden Versuchen wurde die optogenetische Stimulation von Motorneuronen außerdem mit der elektronenmikroskopischen Analyse der präsynaptischen Feinstruktur kombiniert. Dadurch konnte die Dynamik der Exozytose und Endozytose synaptischer Vesikel (SV) in Abhängigkeit von neuronaler Aktivität untersucht werden. So wurde gezeigt, dass synaptische Vesikel nahe der aktiven Zone während einer 30 sekündigen Hyperstimulation nahezu komplett aufgebraucht waren. Die vollständige Regeneration der SV Pools benötigte anschließend etwa 12 Sekunden und erfolgte zunächst in der Peripherie der aktiven Zone, was auf eine laterale Heranführung der Vesikel schließen lässt. Nach etwa 20 Sekunden erholte sich ebenfalls die Wirksamkeit der Stimulation von Muskelzellen durch die Motorneuronen, was durch elektrophysiologische Messungen der photo induzierten post synaptischen Ströme gezeigt wurde. Während der Hyperstimulation bildeten sich außerdem große vesikuläre Strukturen, die sich anschließend nach etwa acht Sekunden wieder aufgelöst hatten. In Analogie zu vergleichbaren Experimenten in anderen Organismen liegt die Vermutung nahe, dass es sich dabei um Zwischenprodukte der so genannten Bulk Phase Endozytose handelt, die das Clathrin abhängige Recycling von synaptischen Vesikeln bei starker neuronaler Aktivität ergänzt (Heuser and Reese, J Cell Biol 1973; Miller and Heuser, J Cell Biol 1984; Richards et al., Neuron 2000). Bemerkenswerterweise war der Abbau der vesikulären Strukturen in Synaptojanin und Endophilin defizienten Tieren stark verzögert. Denkbar ist, dass beide Proteine für die Synthese von synaptischen Vesikeln aus den vesikulären Zwischenprodukten der Bulk Phase Endozytose wichtig sind, analog zur ihrer Funktion bei der Clathrin abhängigen Endozytose an der Plasmamembran.
Durch die zielgerichtete Manipulation der Zellaktivität ermöglichen optogenetische Techniken außerdem die funktionelle Charakterisierung von Neuronen und neuronalen Netzwerken. Um die zelluläre Spezifität dieses Ansatzes zu erhöhen, wurde ein Tracking System entwickelt das die Position frei beweglicher Tiere in Echtzeit bestimmt und nachverfolgt. Dadurch konnte die Photoaktivierung optogenetischer Proteine auf definierte Bereiche der Fadenwürmer und somit auf ausgewählte Neuronen innerhalb der Expressionsmuster von verwendeten Promotoren eingeschränkt werden. Des Weiteren ermöglichte hierbei die Auswertung translatorischer Parameter die Analyse verschiedener lokomotorischer Merkmale wie Geschwindigkeit, Bewegungsbahn oder Ausprägung der Körperbiegungen. Dieses System wurde beispielhaft für die konzertierte Photoaktivierung durch ChR2(H134R) bzw. Photoinhibition durch MAC von zwei verschiedenen Gruppen von Neuronen angewendet, um die Integration mechanosensorischer Informationen durch Command Interneuronen zu untersuchen. In diesem Zusammenhang wurde zudem eine Rekombinase basierte Methode für optogenetische Proteine adaptiert, die die Transkription auf die zelluläre Schnittmenge von zwei verschiedenen Promotoren einschränkt und somit die Spezifität der Expression erhöht. Idealerweise kann dieser Ansatz außerdem mit der gezielten Photoaktivierung kombiniert werden, um die zelluläre Selektivität optogenetischer Anwendungen weiter zu verbessern.
Weiterhin ist die Anwendung optogenetischer Techniken bisher durch intrinsische Eigenschaften der verwendeten Rhodopsine auf die relativ kurzzeitige Manipulation des Membranpotentials von Zellen beschränkt. So benötigt ChR2 durch die schnelle Schließung seines offenen Kanals eine kontinuierliche Photoaktivierung, um eine andauernde Depolarisation hervorzurufen. Dies ist jedoch potentiell mit phototoxischen und – besonders bei C. elegans – phototaktischen Nebeneffekten verbunden. Deswegen wurden diverse Mutanten von ChR2 mit stark verlangsamter Inaktivierung (Berndt et al., Nat Neurosci 2009) für ihren Nutzen zur Langzeit Stimulation von erregbaren Zellen im Nematode getestet. Dabei wurde gezeigt, dass ChR2(C128S) durch einen kurzen Photostimulus mit vergleichsweise niedriger Intensität eine anhaltende Depolarisation über mehrere Minuten auslösen kann. Die wiederholte Stimulation in ASJ Neuronen ermöglichte zudem eine langzeitige Depolarisation über mehrere Tage, wodurch die genetisch veranlagte Entwicklung von Tieren manipuliert werden konnte. Durch gezielte Punktmutation konnten außerdem relevante Eigenschaften von ChR2(C128S) für die Langzeit Stimulation weiter verbessert werden.
Als weiteres optogenetisches Werkzeug wurde zudem die Photoaktivierbare Adenylatzyklase alpha (PACa) aus Euglena gracilis (Iseki et al., Nature 2002; Ntefidou et al., Plant Physiol 2003; Schroder-Lang et al., Nat Methods 2007) für die akute und lichtgetriebene Synthese des sekundären Botenstoffs cAMP in C. elegans etabliert. Die Photoaktivierung von PACa in cholinergen Motorneuronen verstärkte dabei die Neurotransmitterfreisetzung und induzierte hyperlokomotorische Phänotypen, vergleichbar zu Mutanten mit erhöhten cAMP Konzentrationen.
Zusammengefasst wurden diverse optogenetische Techniken für C. elegans entwickelt und optimiert, die die zellspezifische und nicht invasive Manipulation des Membranpotentials beziehungsweise die Synthese des sekundären Botenstoffs cAMP durch Licht im frei beweglichen Tier ermöglichen. Diese Methoden können zur gezielten Störung neuronaler Aktivität angewendet werden, um dadurch neurobiologische Fragestellungen im Fadenwurm zu untersuchen. Dies wurde beispielhaft für die Erforschung der synaptischen Reizweiterleitung und die funktionelle Analyse neuronaler Netzwerke demonstriert. Denkbar ist außerdem, diese für C. elegans etablierten Methoden vergleichbar in anderen Modellorganismen anzuwenden. So sind die Fruchtfliege ebenso wie der Zebrafisch Embryo bereits für optogenetische Techniken erprobt (Arrenberg et al., Proc Natl Acad Sci U S A 2009; Schroll et al., Curr Biol 2006). Für Säugetiere wie die Maus, die Ratte und den Makaken wurden zudem bereits Ansätze entwickelt, die die gezielte Photostimulation in lebenden und frei beweglichen Tieren ermöglichen (Han et al., Neuron 2009; Wentz et al., J Neural Eng 2011; Yizhar et al., Nature 2011; Zhang et al., Nat Rev Neurosci 2007).