Christoph Biskup

Nuclear Activity of MLA Immune Receptors Links Isolate-Specific and Basal Disease-Resistance Responses

Plant immune responses are triggered by pattern recognition receptors that detect conserved pathogen-associated molecular patterns (PAMPs) or by resistance (R) proteins recognizing isolate-specific pathogen effectors. We show that in barley, intracellular mildew A (MLA) R proteins function in the nucleus to confer resistance against the powdery mildew fungus. Recognition of the fungal avirulence A10 effector by MLA10 induces nuclear associations between receptor and WRKY transcription factors. The identified WRKY proteins act as repressors of PAMP-triggered basal defense. MLA appears to interfere with the WRKY repressor function, thereby de-repressing PAMP-triggered basal defense. Our findings reveal a mechanism by which these polymorphic immune receptors integrate distinct pathogen signals.

Relating ligand binding to activation gating in CNGA2 channels

Cyclic nucleotide-gated (CNG) ion channels mediate sensory signal transduction in photoreceptors and olfactory cells. Structurally, CNG channels are heterotetramers composed of either two or three homologue subunits. Although it is well established that activation is a cooperative process of these subunits, it remains unknown whether the cooperativity is generated by the ligand binding, the gating, or both, and how the subunits interact. In this study, the action of homotetrameric olfactory-type CNGA2 channels was studied in inside-out membrane patches by simultaneously determining channel activation and ligand binding, using the fluorescent cGMP analogue 8-DY547-cGMP as the ligand. At concentrations of 8-DY547-cGMP < 1 μM, steady-state binding was larger than steady-state activation, whereas at higher concentrations it was smaller, generating a crossover of the steady-state relationships. Global analysis of these relationships together with multiple activation time courses following cGMP jumps showed that four ligands bind to the channels and that there is significant interaction between the binding sites. Among the binding steps, the second is most critical for channel opening: its association constant is three orders of magnitude smaller than the others and it triggers a switch from a mostly closed to a maximally open state. These results contribute to unravelling the role of the subunits in the cooperative mechanism of CNGA2 channel activation and could be of general relevance for the action of other ion channels and receptors.

Live-cell imaging of endogenous Ras-GTP illustrates predominant Ras activation at the plasma membrane.

Ras‐GTP imaging studies using the Ras‐binding domain (RBD) of the Ras effector c‐Raf as a reporter for overexpressed Ras have produced discrepant results about the possible activation of Ras at the Golgi apparatus. We report that RBD oligomerization provides probes for visualization of endogenous Ras‐GTP, obviating Ras overexpression and the side effects derived thereof. RBD oligomerization results in tenacious binding to Ras‐GTP and interruption of Ras signalling. Trimeric RBD probes fused to green fluorescent protein report agonist‐induced endogenous Ras activation at the plasma membrane (PM) of COS‐7, PC12 and Jurkat cells, but do not accumulate at the Golgi. PM illumination is exacerbated by Ras overexpression and its sensitivity to dominant‐negative RasS17N and pharmacological manipulations matches Ras‐GTP formation assessed biochemically. Our data illustrate that endogenous Golgi‐located Ras is not under the control of growth factors and argue for the PM as the predominant site of agonist‐induced Ras activation.

FRET between cardiac Na + channel subunits measured with a confocal microscope and a streak camera

When and where proteins associate is a central question in many biomolecular studies. Förster resonance energy transfer (FRET) measurements can be used to address this question when the interacting proteins are labeled with appropriate donor and acceptor fluorophores. We describe an improved method to determine FRET efficiency that uses a mode-locked laser, a confocal microscope and a streak camera. We applied this method to study the association of α and β1 subunits of the human cardiac sodium channel. The subunits were tagged with the cyan and yellow variants of the green fluorescent protein (GFP) and expressed in human embryonic kidney (HEK293) cells. Pronounced FRET between the channel subunits in the endoplasmic reticulum (ER) suggested that the subunits associate before they reach the plasma membrane. The described method allows simultaneous measurement of donor and acceptor fluorescence decays and provides an intrinsically validated estimate of FRET efficiency.

FRET Measurements by TCSPC Laser Scanning Microscopy

We use a two-photon laser scanning microscope with a new Time-Correlated Single Photon Counting (TCSPC) imaging technique to obtain combined intensity-lifetime images for FRET measurements in living cells. Single photon pulses from a photomultiplier and signals from the scanning head are used to record the three-dimensional photon density over the time- and image coordinates. Double exponential decay analysis delivers the lifetime components of the quenched and the unquenched molecules in all pixels of the image. We use the ratio of the intensity coefficients of the fast and slow decay component to create images that show the size of the FRET effects in different parts of the cell.

Interdependence of Receptor Activation and Ligand Binding in HCN2 Pacemaker Channels

HCN pacemaker channels are tetramers mediating rhythmicity in neuronal and cardiac cells. The activity of these channels is controlled by both membrane voltage and the ligand cAMP, binding to each of the four channel subunits. The molecular mechanism underlying channel activation and the relationship between the two activation stimuli are still unknown. Using patch-clamp fluorometry and a fluorescent cAMP analog, we show that full ligand-induced activation appears already with only two ligands bound to the tetrameric channel. Kinetic analysis of channel activation and ligand binding suggests direct interaction between the voltage sensor and the cyclic nucleotide-binding domain, bypassing the pore. By exploiting the duality of activation in HCN2 channels by voltage and ligand binding, we quantify the increase of the binding affinity and overall free energy for binding upon channel activation, proving thus the principle of reciprocity between ligand binding and conformational change in a receptor protein.

Thrombin-mediated hepatocellular carcinoma cell migration: Cooperative action via proteinase-activated receptors 1 and 4

Proteinase‐activated receptor‐1 (PAR1), a thrombin receptor and the prototype of a newly discovered G‐protein‐coupled receptor subfamily, plays an important role in tumor development and progression. In this study, we documented the expression of the thrombin receptors PAR1, PAR3, and PAR4 in permanent hepatocellular carcinoma (HCC) cell lines and primary HCC cell cultures. Stimulation of HCC cells with thrombin and the PAR1‐selective activating peptide, TFLLRN‐NH2, increased transmembrane migration across a collagen barrier. This effect was blocked by the PAR1 antagonist SCH 79797, confirming that the PAR1 thrombin receptor subtype is involved in regulating hepatoma cell migration. In addition, the PAR4‐selective agonist, AYPGKF‐NH2, also stimulated HCC cell migration whilst the PAR4 antagonist, trans‐cinnamoyl‐YPGKF‐NH2, attenuated the effect of thrombin on HCC cell migration. PAR1‐ and PAR4‐triggered HCC cell migration was blocked by inhibiting a number of key mediators of signal transduction, including G proteins of the Gi/Go family, matrix metalloproteinases, ERK/MAPKinase, cyclic AMP‐dependent protein kinase, Src tyrosine kinase, and the EGF receptor kinase. Our data point to a cooperative PAR1/PAR4 signaling network that contributes to thrombin‐mediated tumor cell migration. We suggest that a combined inhibition of coagulation cascade serine proteinases, the two PARs and their complex signaling pathways may provide a new strategy for treating hepatocellular carcinoma. J. Cell. Physiol. 211: 699–707, 2007.

Development and critical evaluation of fluorescent chloride nanosensors.

In this study, we describe the preparation and evaluation of new fluorescent sensor nanoparticles for the ratiometric measurement of chloride concentrations. Both a chloride-sensitive dye (lucigenin) and a reference dye (sulforhodamine derivative) were incorporated into polyacrylamide nanoparticles via inverse microemulsion polymerization and investigated for their response to chloride ions in buffered suspension as well as in living cells. The fluorescence intensity of lucigenin reversibly decreased in the presence of chloride ions due to a collisional quenching process, which can be described with the Stern-Volmer equation. The determined Stern-Volmer constant K SV for the quenching of lucigenin incorporated into particles was found to be 53 M (-1) and is considerably smaller than the Stern-Volmer constant for quenching of free lucigenin ( K SV = 250 M (-1)) under the same conditions. To test the nanosensors in living cells, we incorporated them into Chinese hamster ovary cells and mouse fibroblasts by using the conventional lipofectamin technique and monitored the response to changing chloride concentrations in the cell.

Interaction of PSD-95 with potassium channels visualized by fluorescence lifetime-based resonance energy transfer imaging

Resonance energy transfer (RET) has been extensively used to estimate the distance between two different fluorophores. This study demonstrates how protein-protein interactions can be visualized and quantified in living cells by time-correlated single-photon counting (TCSPC) imaging techniques that exploit the RET between appropriate fluorescent labels. We used this method to investigate the association of the potassium inward rectifier channel Kir2.1 and the neuronal PDZ protein PSD-95, which has been implicated in subcellular targeting and clustering of ion channels. Our data show that the two proteins not only colocalize within clusters but also interact with each other. Moreover, the data allow a spatially resolved quantification of this protein-protein interaction with respect to the relative number and the proximity between interacting molecules. Depending on the subcellular localization, a fraction of 20 to 60% of PSD-95 molecules interacted with Kir2.1 channels, approximating their fluorescent labels by less than 5 nm.

Anoxia generates rapid and massive opening of KATP channels in ventricular cardiac myocytes

OBJECTIVE The aim was to improve the measurement of both the time course and amplitude of anoxia-induced KATP-channel current (IKATP) in isolated heart cells to specify the role of these channels in the time course of K+ accumulation in the ischemic myocardium. METHODS Ionic currents in isolated ventricular heart cells of the mouse were measured with a patch clamp technique under normoxic conditions (atmospheric pO2), during wash-out of oxygen, and under anoxic conditions (pO2 < 0.2 mmHg). During the measurement, the actual pO2 in the close proximity of the cell was determined with an optical technique by exciting Pd-meso-tetra(4-carboxyphenyl)porphin with light flashes of 508-570 nm and evaluating the quenching kinetics of the emitted phosphorescence signal at 630-700 nm. These quenching kinetics steeply depend on pO2 and can be evaluated best at pO2 values near 0 mmHg. RESULTS Out of 28 cells, 23 cells started to develop IKATP at pO2 values between 0 and 0.4 mmHg, i.e. in the range of the level of half maximum activity of the cytochrome oxidase. The remaining five cells developed IKATP between 0.4 and 1.8 mmHg. With respect to the time course, 18 out of 27 cells started to develop IKATP within the first minute after pO2 had decreased to values below 0.2 mmHg. The amplitude of IKATP induced by anoxia and various metabolic inhibitors was large, 29 +/- 12 and 48 +/- 21 nA (+40 mV), respectively. The anoxia-induced IKATP was significantly smaller than IKATP induced by metabolic inhibitors. During the pulses of 50 ms duration to +40 mV, the amplitude of IKATP decayed and, after clamping back to -80 mV, IKATP generated large tail currents. This suggests a notable change in the concentration gradient of K+ ions in the time range of tens of milliseconds. CONCLUSIONS The results in isolated myocytes indicate that KATP channels open sufficiently rapidly after starting anoxia and generate sufficiently large conductance at maintained anoxia to explain both the time course and magnitude of the ischemic K+ accumulation if an appropriate counter-ion flux is available.