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Horst Vogel

Publications and source records attributed to Horst Vogel.

At least 19 recordsLinked to original sources
PubMed2006

Fabrication and functionalization of nanochannels by electron-beam-induced silicon oxide deposition.

We report on the fabrication and electrical characterization of functionalized solid-state nanopores in low stress silicon nitride membranes. First, a pore of approximately 50 nm diameter was drilled using a focused ion beam technique, followed by the local deposition of silicon dioxide. A low-energy electron beam induced the decomposition of adsorbed tetraethyl orthosilicate resulting in site-selective functionalization of the nanopore by the formation of highly insulating silicon oxide. The deposition occurs monolayer by monolayer, which allows for control of the final diameter with subnanometer accuracy. Changes in the pore diameter could be monitored in real time by scanning electron microscopy. Recorded ion currents flowing through a single nanopore revealed asymmetry in the ion conduction properties with the sign of the applied potential. The low-frequency excess noise observed at negative voltage originated from stepwise conductance fluctuations of the open pore.

Electrons↗
PubMed2006

Monitoring the diffusion of single heterotrimeric G proteins in supported cell-membrane sheets reveals their partitioning into microdomains.

Supported cell-membrane sheets are promising in vitro systems to investigate the properties of membranes with native protein/lipid composition, in particular their sub-compartmentalization and the differential localization of proteins associated to them. While such studies are usually performed using static microscopy techniques, we demonstrate here the potential offered by dynamic diffusion measurements. Whereas the overall fluidity of the lipid bilayer was preserved, the preparation of the membrane sheets led to the selective immobilization of extracellular and transmembrane (TM) glycosylated proteins and the anchored proteins/lipids associated with them. Taking advantage of this, we investigated the association of the G protein Gq with TM proteins, in particular G-protein coupled receptors (GPCRs), by monitoring the changes in diffusion occurring after preparation of the supported membranes. Two fluorescently tagged Galphaq proteins were constructed, which remained either mostly monomeric in the plasma membrane or associated with Gbetagamma in heterotrimers. While both constructs diffused similarly in living cells, the preparation of the supported membranes led to the selective immobilization of the heterotrimers with minimal changes of the diffusion of the monomeric Galphaq. The diverse mobility of monomeric and heterotrimeric Galphaq was a result of their different lipid anchors as demonstrated by monitoring the diffusion of the corresponding anchors alone. We propose that the immobilization of the heterotrimer was caused by its partitioning inside membrane microdomains surrounding GPCRs.

Cell Compartmentation↗
PubMed2006

Modulation of proton-induced current fluctuations in the human nicotinic acetylcholine receptor channel.

The nicotinic acetylcholine receptor (nAChR) is a ligand-gated ion channel that switches upon activation from a closed state to a full conducting state. We found that the mutation delta S268K, located at 12' position of the second transmembrane domain of the delta subunit of the human nAChR generates a long-lived intermediate conducting state, from which openings to a wild-type like conductance level occur on a submillisecond time scale. Aiming to understand the interplay between structural changes near the 12' position and channel gating, we investigated the influence of various parameters: different ligands (acetylcholine, choline and epibatidine), ligand concentrations, transmembrane voltages and both fetal and adult nAChRs. Since sojourns in the high conductance state are not fully resolved in time, spectral noise analysis was used as a complement to dwell time analysis to determine the gating rate constants. Open channel current fluctuations are described by a two-state Markov model. The characteristic time of the process is markedly influenced by the ligand and the receptor type, whereas the frequency of openings to the high conductance state increases with membrane hyperpolarization. Conductance changes are discussed with regard to reversible transfer reaction of single protons at the lysine 12' side chain.

Amino Acid Sequence↗
PubMed2006

Functional asymmetry of transmembrane segments in nicotinic acetylcholine receptors.

Nicotinic acetylcholine receptors are heteropentameric ion channels that open upon activation to a single conducting state. The second transmembrane segments of each subunit were identified as channel-forming elements, but their respective contribution in the gating process remains unclear. Moreover, the detailed impact of variations of the membrane potential, such as occurring during an action potential, on the transmembrane domains, is unknown. Residues at the 12' position, close to the center of each second transmembrane segment, play a key role in channel gating. We examined their functional symmetry by substituting a lysine to that position of each subunit and measuring the electrical activity of single channels. For 12' lysines in the alpha, gamma and delta subunits rapid transitions between an intermediate and large conductance appeared, which are interpreted as single lysine protonation events. From the kinetics of these transitions we calculated the pK (a) values of respective lysines and showed that they vary differently with membrane hyperpolarization. Respective mutations in beta or epsilon subunits gave receptors with openings of either intermediate or large conductance, suggesting extreme pK (a) values in two open state conformations. The results demonstrate that these parts of the highly homologous transmembrane domains, as probed by the 12' lysines, sense unequal microenvironments and are differently affected by physiologically relevant voltage changes. Moreover, observation of various gating events for mutants of alpha subunits suggests that the open channel pore exists in multiple conformations, which in turn supports the notion of functional asymmetry of the channel.

Amino Acid Sequence↗
PubMed2006

Characterization of an extended receptive ligand repertoire of the human olfactory receptor OR17-40 comprising structurally related compounds.

Molecular properties of odorant compounds essential for activation of the human olfactory receptor hOR17-40 were investigated using a collection of 23 variants of its cognate ligand helional. Coupling receptor activation to an optically detectable intracellular Ca(2+) ion flux allowed dose-dependent screening of different odorant molecules in human embryonic kidney (HEK)293 cells. We found an extended collection of activating ligands and provide first evidence for hOR17-40-specific antagonists. The C-terminal fusion of enhanced green fluorescent protein to the hOR17-40 retained full receptor function and permitted the selection of cells with defined receptor expression levels, which was an essential step for optimizing our screening protocol. Interestingly, cells with a low EGFP fluorescence intensity exhibited efficient hOR17-40 cell surface targeting and odorant-evoked signal transduction; in contrast, highly fluorescent cells displayed mainly incorrectly targeted, intracellular receptors. Fluorescence-activated cell sorting was used to separate hOR17-40-expressing cells on the basis of their endogenous EGFP fluorescence intensity, thereby increasing the fraction of odorant-responsive cells to up to 80% of the total cell number.

Calcium↗
PubMed2006

Covalent labeling of cell-surface proteins for in-vivo FRET studies.

Fluorescence resonance energy transfer (FRET) is a powerful technique to reveal interactions between membrane proteins in live cells. Fluorescence labeling for FRET is typically performed by fusion with fluorescent proteins (FP) with the drawbacks of a limited choice of fluorophores, an arduous control of donor-acceptor ratio and high background fluorescence arising from intracellular FPs. Here we show that these shortcomings can be overcome by using the acyl carrier protein labeling technique. FRET revealed interactions between cell-surface neurokinin-1 receptors simultaneously labeled with a controlled ratio of donors and acceptors. Moreover, using FRET the specific binding of fluorescent agonists could be monitored.

Acyl Carrier Protein↗
PubMed2006

FRET imaging reveals that functional neurokinin-1 receptors are monomeric and reside in membrane microdomains of live cells.

The lateral organization of a prototypical G protein-coupled receptor, the neurokinin-1 receptor (NK1R), was investigated in living cells by fluorescence resonance energy transfer (FRET) microscopy, taking advantage of the recently developed acyl carrier protein (ACP) labeling technique. The NK1R was expressed as fusion protein with ACP to which small fluorophores were then covalently bound. Our approach allowed the recording of FRET images of receptors on living cells with unprecedented high signal-to-noise ratios and a subsequent unequivocal quantification of the FRET data owing to (i) the free choice of optimal fluorophores, (ii) the labeling of NK1Rs exclusively on the cell surface, and (iii) the precise control of the donor-acceptor molar ratio. Our single-cell FRET measurements exclude the presence of constitutive or ligand-induced homodimers or oligomers of NK1Rs. The strong dependence of FRET on the receptor concentration further reveals that NK1Rs tend to concentrate in microdomains, which are found to constitute approximately 1% of the cell membrane and to be sensitive to cholesterol depletion.

Acyl Carrier Protein↗
PubMed2006

Cell membranes suspended across nanoaperture arrays.

We present a method for spreading large (>100 microm(2)) cell membrane fragments across nanoapertures in planar supports. Electron-beam and focused-ion-beam lithography were used to fabricate arrays of 50-600 nm diameter holes in free-standing silicon nitride (SiN) solid films 100-500 nm thick. By pressing adhering live cells onto the nanostructured SiN surface and then removing them, planar cell membrane sheets (CMSs) were transferred in a well-defined orientation onto the SiN support. We demonstrate the accessibility to both extracellular and intracellular surfaces of CMSs by targeting membrane constituents side-specifically with fluorescent markers. Our approach is of interest for studying ligand-receptor interactions using optical, electrical, and scanning probe techniques at the single-molecule level.

Cell Membrane↗
PubMed2006

Organization of nanoparticles on hard substrates using block copolymer films as templates.

We present a technique for the organization of pre-synthesized nanoparticles on hard substrates, using block copolymer films as sacrificial templates. A thin block copolymer film is dip-coated on the substrate of interest and the sample is exposed to a solution containing nanoparticles. Spontaneous preferential adsorption of the nanoparticles on one phase of the block copolymer film results in their lateral organization. An oxygen plasma etch is used to remove the polymer film; the nanoparticles end up organized on the substrate. We demonstrate that this is a general approach for the patterning of inorganic nanoparticles on hard substrates, showing the organization of metal and semiconductor nanoparticles having different chemistries at the particle/solvent and solvent/polymer interfaces. The nanoparticle patterns that we present have typical periodicities in the nanometer scale. In some cases, microcontact printing is used to create a double length scale of organization, on the micrometer and on the nanometer level. The characteristic periodicity of the template is studied with respect to the nanoparticle size in order to optimize the organization. Finally, we describe how to extend this technique for the production of continuous gold nanowires on hard substrates. We expect that the flexibility of this approach and the degree of control that can be obtained over nanoparticle organization should make it a powerful tool for nanoscale fabrication.

Cadmium Compounds↗
PubMed2006

Engineered site-directed labeling of nicotinic acetylcholine receptors using reactive epibatidine derivatives: appraisal of epibatidine-docking models in neuronal and muscular receptors.

We developed an engineered site-directed labeling method (Foucaud et al., 2001) to investigate ligand receptor interactions on the acetylcholine (ACh)- binding site of nicotinic acetylcholine receptors (nAChRs). The method uses cysteine receptor mutants, together with cysteine-reactive ligand analogs, to generate a site-directed covalent reaction within the binding site. We selected epibatidine (EPB) as a prototypical ligand, acting at all types of nAChRs with sufficient affinity to allow this study. Accordingly, we synthesized three cysteine-reactive derivatives, all modified at the C-3 of the pyridine ring of the alkaloid with NCS; -NHCOCH2Cl, and -CH2Cl groups, respectively (Fig. 1). The binding properties have been established on rat brain, alpha7-5HT3 chimera, and Torpedo membranes, respectively, whereas the functional properties were tested on alpha4beta2 and alpha7 receptor expressed in oocytes and Cys-less muscular receptor expressed in HEK cells (Sakr et al., 2005).

Animals↗
PubMed2005

Diffusion-time distribution analysis reveals characteristic ligand-dependent interaction patterns of nuclear receptors in living cells.

Nuclear receptors initiate transcription, interact with regulatory proteins, and are influenced by hormones, drugs, and pollutants. Herein, we discover ligand-specific mobility patterns of human estrogen receptor-alpha (ER) in living cells using diffusion-time distribution analysis (DDA). This novel method, based on fluorescence correlation spectroscopy (FCS), is especially suited to unraveling multiple protein interactions in vivo at native expression levels. We found that ER forms a limited number of distinct complexes with a varying population by dynamic interaction with other nuclear components. Dose-response curves of different ligands could be obtained for each receptor interaction. The potential to identify interacting proteins was demonstrated by comparing DDA of the ER cofactor SRC-3 attached to yellow fluorescent protein (YFP) with those of YFP-ER. Our findings open up new routes to elucidating transcription regulation and to detecting and distinguishing pharmacologically and toxicologically active compounds in vivo. Moreover, DDA provides a general approach to monitoring biochemical networks in individual living cells.

Bacterial Proteins↗
PubMed2005

Pumping of mammalian cells with a nozzle-diffuser micropump.

We discuss the successful transport of jurkat cells and 5D10 hybridoma cells using a reciprocating micropump with nozzle-diffuser elements. The effect of the pumping action on cell viability and proliferation, as well as on the damaging of cellular membranes is quantified using four types of well-established biological tests: a trypan blue solution, the tetrazolium salt WST-1 reagent, the LDH cytotoxicity assay and the calcium imaging ATP test. The high viability levels obtained after pumping, even for the most sensitive cells (5D10), indicate that a micropump with nozzle-diffuser elements can be very appropriate for handling living cells in cell-on-a-chip applications.

Adenosine Triphosphate↗
PubMed2005

Kinetics of the initial steps of G protein-coupled receptor-mediated cellular signaling revealed by single-molecule imaging.

We report on an in vivo single-molecule study of the signaling kinetics of G protein-coupled receptors (GPCR) performed using the neurokinin 1 receptor (NK1R) as a representative member. The NK1R signaling cascade is triggered by the specific binding of a fluorescently labeled agonist, substance P (SP). The diffusion of single receptor-ligand complexes in plasma membrane of living HEK 293 cells is imaged using fast single-molecule wide-field fluorescence microscopy at 100 ms time resolution. Diffusion trajectories are obtained which show intra- and intertrace heterogeneity in the diffusion mode. To investigate universal patterns in the diffusion trajectories we take the ligand-binding event as the common starting point. This synchronization allows us to observe changes in the character of the ligand-receptor-complex diffusion. Specifically, we find that the diffusion of ligand-receptor complexes is slowed down significantly and becomes more constrained as a function of time during the first 1000 ms. The decelerated and more constrained diffusion is attributed to an increasing interaction of the GPCR with cellular structures after the ligand-receptor complex is formed.

Cell Line↗
PubMed2005

CD8 kinetically promotes ligand binding to the T-cell antigen receptor.

The mechanism of CD8 cooperation with the TCR in antigen recognition was studied on live T cells. Fluorescence correlation measurements yielded evidence of the presence of two TCR and CD8 subpopulations with different lateral diffusion rate constants. Independently, evidence for two subpopulations was derived from the experimentally observed two distinct association phases of cognate peptide bound to class I MHC (pMHC) tetramers and the T cells. The fast phase rate constant ((1.7 +/- 0.2) x 10(5) M(-1) s(-1)) was independent of examined cell type or MHC-bound peptides' structure. Its value was much faster than that of the association of soluble pMHC and TCR ((7.0 +/- 0.3) x 10(3) M(-1) s(-1)), and close to that of the association of soluble pMHC with CD8 ((1-2) x 10(5) M(-1) s(-1)). The fast binding phase disappeared when CD8-pMHC interaction was blocked by a CD8-specific mAb. The latter rate constant was slowed down approximately 10-fold after cells treatment with methyl-beta-cyclodextrin. These results suggest that the most efficient pMHC-cell association route corresponds to a fast tetramer binding to a colocalized CD8-TCR subpopulation, which apparently resides within membrane rafts: the reaction starts by pMHC association with the CD8. This markedly faster step significantly increases the probability of pMHC-TCR encounters and thereby promotes pMHC association with CD8-proximal TCR. The slow binding phase is assigned to pMHC association with a noncolocalized CD8-TCR subpopulation. Taken together with results of cytotoxicity assays, our data suggest that the colocalized, raft-associated CD8-TCR subpopulation is the one capable of inducing T-cell activation.

Antibodies, Monoclonal↗
PubMed2005

CD8+ cytotoxic T lymphocyte activation by soluble major histocompatibility complex-peptide dimers.

CD8+ cytotoxic T lymphocyte (CTL) can recognize and kill target cells that express only a few cognate major histocompatibility complex class I-peptide (pMHC) complexes. To better understand the molecular basis of this sensitive recognition process, we studied dimeric pMHC complexes containing linkers of different lengths. Although dimers containing short (10-30-A) linkers efficiently bound to and triggered intracellular calcium mobilization and phosphorylation in cloned CTL, dimers containing long linkers (> or = 80 A) did not. Based on this and on fluorescence resonance energy transfer experiments, we describe a dimeric binding mode in which two T cell receptors engage in an anti-parallel fashion two pMHC complexes facing each other with their constant domains. This binding mode allows integration of diverse low affinity interactions, which increases the overall binding and, hence, the sensitivity of antigen recognition. In proof of this, we demonstrated that pMHC dimers containing one agonist and one null ligand efficiently activate CTL, corroborating the importance of endogenous pMHC complexes in antigen recognition.

Amino Acid Sequence↗