Papers by Roland Nitschke
Journal of Biological Chemistry, Apr 1, 1999
Enhanced osmotic water permeability has been observed in Xenopus oocytes expressing cystic fibros... more Enhanced osmotic water permeability has been observed in Xenopus oocytes expressing cystic fibrosis transmembrane conductance regulator (CFTR) protein. Subsequent studies have shown that CFTR activates an endogenous water permeability in oocytes, but that CFTR itself is not the water channel. Here, we show CFTR-dependent activation of endogenous water permeability in normal but not in cystic fibrosis human airway epithelial cells. Cell volume was measured by novel confocal x-z laser scanning microscopy. Glycerol uptake and antisense studies suggest CFTR-dependent regulation of aquaporin 3 (AQP3) water channels in airway epithelial cells. Regulatory interaction was confirmed by coexpression of CFTR and AQP3 cloned from human airways in Xenopus oocytes and of CFTR and rat AQP3 in Chinese hamster ovary cells. These findings indicate that CFTR is a regulator of AQP3 in airway epithelial cells.
Nature Methods, Jan 3, 2022
In the version of this article initially published, the affiliation for author Roland Nitschke wa... more In the version of this article initially published, the affiliation for author Roland Nitschke was incorrectly formulated. The affiliation has been corrected to read "
<strong>This an XLS Spreadsheet and three Entity-Relationship Schemas representing the late... more <strong>This an XLS Spreadsheet and three Entity-Relationship Schemas representing the latest version (v02.01) of the 4DN-BINA-OME (NBO) tiered system of Microscopy Metadata Specifications extending the OME Data Model.</strong> <em>Note: v2.01 is a minor release that corrects minor errors that were found in v2.00.</em> While the power of modern microscopy techniques is undeniable, rigorous record-keeping and quality control are required to ensure that imaging data may be properly interpreted (quality), reproduced (reproducibility), and used to extract reliable information and scientific knowledge which can be shared for further analysis (value). In the absence of agreed guidelines, it is inherently difficult for scientists to create comprehensive records of imaging experiments and ensure the quality of resulting image data or for manufacturers to incorporate standardized reporting and performance metrics. To solve this problem, the 4D Nucleome (4DN) Initiative and BioImaging North America (BINA) here propose light Microscopy Metadata specifications that scale with experimental intent and with the complexity of the instrumentation and analytical requirements. They consist of a set of three extensions of the latest version of the Open Microscopy Environment (OME) Data Model, which was released in 2016. Because of their scalable nature, the 4DN-BINA-OME Microscopy Metadata Specifications clearly specify which provenance and quality control metadata should be recorded for a given experiment. Last but not least, this endeavor is closely aligned with the undertakings of the recently established global community initiative dedicated to Quality Assessment and Reproducibility in Light Microscopy (QUAREP-LiMi; quarep.org). As a result, the ensuing flexible 4DN-BINA-OME (NBO) framework represents a turning point towards achieving community-driven Microscopy Metadata standards that would increase data fidelity, improve repeatability and reproducibility, ease future analysis and facilitate the verifiable comparison of differe [...]
<em>Note: v2.01 is a minor release that corrects minor errors that were found in v2.00.<... more <em>Note: v2.01 is a minor release that corrects minor errors that were found in v2.00.</em> 4D<strong>N</strong>-<strong>B</strong>INA-<strong>O</strong>ME (NBO) Microscopy Metadata Specifications v2.0 is the result of a proposal put forth by the 4D Nucleome (4DN) Imaging Standards Working Group and by the Bioimaging North America (BINA) Quality Control and Data Management Working Group (QC-DM-WG) for a suite of tiered <strong>Microscopy Metadata Specifications</strong> that extend the October 2016 version of the OME Data Model schema. v2.0 supersedes and replaces v1.07 of the guidelines, which were called Microscopy Metadata 4DN Guidelines. The next version of the <strong>NBO Microscopy Metadata Specifications</strong> will be released as part of a community outreach effort conducted in collaboration with the Quality Assessment and Reproducibility for Light Microscopy (QUAREP-LiMi) initiative. <strong>Development continues!</strong> Everyone is encouraged to contribute to the next release v02-10 draft of the specifications. Click <strong>here</strong> for a shortcut to the model directory. To create an issue, click on Issues, the "New Issue" button, and select whether you are submitting a proposed new feature/element or want to propose a change to elements of the current proposal.
Biophysical Journal, Sep 1, 2015
The molecular processes of particle binding and endocytosis are influenced by the locally changin... more The molecular processes of particle binding and endocytosis are influenced by the locally changing mobility of the particle nearby the plasma membrane of a living cell. However, it is unclear how the particle's hydrodynamic drag and momentum vary locally and how they are mechanically transferred to the cell. We have measured the thermal fluctuations of a 1 mm-sized polystyrene sphere, which was placed in defined distances to plasma membranes of various cell types by using an optical trap and fast three-dimensional (3D) interferometric particle tracking. From the particle position fluctuations on a 30 ms timescale, we determined the distance-dependent change of the viscous drag in directions perpendicular and parallel to the cell membrane. Measurements on macrophages, adenocarcinoma cells, and epithelial cells revealed a significantly longer hydrodynamic coupling length of the particle to the membrane than those measured at giant unilamellar vesicles (GUVs) or a plane glass interface. In contrast to GUVs, there is also a strong increase in friction and in mean first passage time normal to the cell membrane. This hydrodynamic coupling transfers a different amount of momentum to the interior of living cells and might serve as an ultra-soft stimulus triggering further reactions.
Microscopy and Microanalysis, Feb 1, 1995
A confocal attachment (Odyssey) to an inverted microscope was modified to better study living cul... more A confocal attachment (Odyssey) to an inverted microscope was modified to better study living cultured epithelial cells stained with fluorescent dyes. Improvements to the instrument included elimination of light leaks, improved electronic shielding, reduction of thermal effects, and use of low dark current detectors. In addition, rapid changes in illumination wavelength and power were accomplished by replacing the original mechanical filter changer by an acousto-optic tunable filter attached to the argon laser light source. The addition of a liquid crystal tunable filter to one of the two photomultiplier detectors also permitted rapid spectral scanning of the fluorescence emission. High-resolution, differential interference contrast transmitted light images were formed simultaneously by replacement of the photodiode-based transmitted light detector with a photomultiplier tube and dichroic mirror assembly. An illumination intensity of only 40 μW/cm2 at the back focal plane of the microscope objective allowed high-quality fluorescence and transmitted light images of living cells at video rates with minimal bleaching and photodynamic damage. Both excitation ratio imaging and emission spectral scanning of living epithelial cells were accomplished. The system performance was evaluated by optical sections of fluorescent beads and thin films.
Medical Microbiology and Immunology, Feb 1, 1987
The molecular weights (mol. wts.) of the two double-stranded (ds) RNA segments of infectious burs... more The molecular weights (mol. wts.) of the two double-stranded (ds) RNA segments of infectious bursal disease virus (IBDV) were determined using previously sequenced reovirus genes M3 and $2 as internal ds RNA reference molecules. Electrophoresis under fully denaturing conditions revealed mol. wts. of 2.26 x 106 daltons and 1.98 • 106 daltons. By direct length measurements under the electron microscope, using two different spreading conditions, the two segments were calculated to be composed of 3274 + 79 base pairs (bp) and 2821 + 59 bp or 3299 + 68 bp and 2830 + 73 bp, resulting in mol. wts. of 2.24-2.26 • 106 daltons and 1.93-1.94 • 106 daltons, respectively. Base pair distances of 2.67 + 0.08 A and 2.71 + 0.11/~, in ds RNA were close to those of the A-RNA form; in ds DNA included as a control, the rise per base pair was 3.18 A, which is consistent with published results. Mol. wts. obtained for IBDV indicate that the RNAs of the other birnaviruses are also smaller than reported.
Mediators of Inflammation, 2015
Specialized microanatomical areas of the bone marrow provide the signals that are mandatory for t... more Specialized microanatomical areas of the bone marrow provide the signals that are mandatory for the maintenance and regulation of hematopoietic stem cells (HSCs) and progenitor cells. A complex microenvironment adjacent to the marrow vasculature (vascular niche) and close to the endosteum (endosteal niche) harbors multiple cell types including mesenchymal stromal cells and their derivatives such as CAR cells expressing high levels of chemokines C-X-C motif ligand 12 and early osteoblastic lineage cells, endothelial cells, and megakaryocytes. The characterization of the cellular and molecular networks operating in the HSC niche has opened new perspectives for the understanding of the bidirectional cross-talk between HSCs and stromal cell populations in normal and malignant conditions. A structural and functional remodeling of the niche may contribute to the development of myeloproliferative neoplasms (MPN). Malignant HSCs may alter the function and survival of MSCs that do not belong to the neoplastic clone. For example, a regression of nestin + MSCs by apoptosis has been attributed to neuroglial damage in MPN. Nonneoplastic MSCs in turn can promote aggressiveness and drug resistance of malignant cells. In the future, strategies to counteract the pathological interaction between the niche and neoplastic HSCs may offer additional treatment strategies for MPN patients.
Cellular Physiology and Biochemistry, 2007
K ATP channel activity influences beta cell Ca 2+ homeostasis by regulating Ca 2+ influx through ... more K ATP channel activity influences beta cell Ca 2+ homeostasis by regulating Ca 2+ influx through L-type Ca 2+ channels. The present paper demonstrates that loss of K ATP channel activity due to pharmacologic or genetic ablation affects Ca 2+ storage in intracellular organelles. ATP depletion, by the mitochondrial inhibitor FCCP, led to Ca 2+ release from the endoplasmic reticulum (ER) of wildtype beta cells. Blockade of ER Ca 2+ ATPases by cyclopiazonic acid abolished the FCCP-induced Ca 2+ transient. In beta cells treated with K ATP channel inhibitors FCCP elicited a significantly larger Ca 2+ transient. Cyclopiazonic acid did not abolish this Ca 2+ transient suggesting that non-ER compartments are recruited as additional Ca 2+ stores in beta cells lacking K ATP channel activity. Genetic ablation of K ATP channels in SUR1KO mice produced identical results. In INS-1 cells transfected with a mitochondrial-targeted Ca 2+-sensitive fluorescence dye (ratiometric pericam) the increase in mitochondrial Ca 2+ evoked by tolbutamide was 5fold larger compared to 15 mM glucose. These data show that genetic or pharmacologic ablation of K ATP channel activity conveys Ca 2+ release from a non-ER store. Based on the sensitivity to FCCP and the property of tolbutamide to increase mitochondrial Ca 2+ it is suggested that mitochondria are the recruited store. The change in Ca 2+ sequestration in beta cells treated with insulinotropic antidiabetics may have implications for beta cell survival and the therapeutic use of these drugs.
... Burhoff, I. [ &amp;amp;gt; &amp;amp;gt; ]. Masereel, B. [ &amp;amp;gt; &amp;a... more ... Burhoff, I. [ &amp;amp;gt; &amp;amp;gt; ]. Masereel, B. [ &amp;amp;gt; &amp;amp;gt; ]. Pirotte, Bernard mailto [Université de Liège - ULg &amp;amp;gt; Département de pharmacie &amp;amp;gt; Chimie pharmaceutique &amp;amp;gt;]. Schlatter, E. [ &amp;amp;gt; &amp;amp;gt; ]. Delarge, J. [ &amp;amp;gt; &amp;amp;gt; ]. Lang, HJ [ &amp;amp;gt; &amp;amp;gt; ]. Englert, HC [ &amp;amp;gt; &amp;amp;gt; ]. Salomonsson, M. [ &amp;amp;gt; &amp;amp;gt; ]. Persson, AEG [ &amp;amp;gt; &amp;amp;gt; ]. Eidelman, O. [ &amp;amp;gt; ...
Pflügers Archiv: European Journal of Physiology, Mar 1, 1989
Piretanide blocks the Na + 2CI-K + cotransporter protein in the thick ascending limb (TAL) of the... more Piretanide blocks the Na + 2CI-K + cotransporter protein in the thick ascending limb (TAL) of the loop of Henle reversibly. When tested from the luminal side in isolated perfused cTAL segments it leads to a half maximal inhibition (ICon) of the equivalent short =circuit current (I#r) at a concentration of 10-u mol/l. From#he basola~gral side it has no effect on l~c up to 10-~ mol/l. The present study was designed-to search for high affinity blockers of the Na + 2CI-K + cotransporter with large molecular weight in an attempt to use these macromolecules for antibody-labelling or affinity separation of this transport-protein. Amino-ethyldextran or amino-ethyl-polyethylene glycol (M.W. 5kd) were coupled to isothiocyanato-piretanide (ISO-PIR) at room temperature in DMSO. The resulting compounds dextran-sulfonylurea-piretanide (PIR-DEX) and polyethylene glycol-sulfonylurea-piretanide (PIR-PEG) (M.W. 5.38kd) were purified and tested in isolated perfused cTAL segments. IC50 values for ISO-PIR, PIR-DEX and PIR-PEG were estimated from dose response curves after their addition to the lumen or bath perfusate, respectively. ISO-PIR, ~IR-DEX6and PIR-PE~ acted from the lumen side at 3"10-, 6"10-and 2"10mol/l. The inhibitory effect was easily reversible. From the basolateral4side no effect for any compound was seen at up to I0-mol/l. In clearance experiments PIR-DEX was given to female Wistar rats as an i.v. bolus (25 ~mol/kg) and the diuretic urine was collected. After dialysis (exclusion limit 2.5kd) the dialysed urine and the dialysate were tested in isolated perfused cTAL segments. The dialysates had no effect on l~r, but the dialysed urine inhibited l~r by 35% from the-luminal side. The present data show:~Righ molecular derivatives of piretanide with dextran or polyethylene glycol moieties block the Na + 2Cl-K + cotransporter in cTAL segments at roughly the same low concentration as piretanide itself. Our data exclude a metabolism of these piretanide compounds in the kidney. Since these macromolecular probes can probably not enter the cell their inhibitory effect indicates that the binding site for piretanide diuretics on the Na + 2CI-K + cotransporter is exposed on the surface of the luminal cell membrane.
Biochemical and Biophysical Research Communications, 2020
Autosomal dominant polycystic kidney disease (ADPKD) is caused by mutations in PKD1 or PKD2, the ... more Autosomal dominant polycystic kidney disease (ADPKD) is caused by mutations in PKD1 or PKD2, the genes encoding polycystin 1 (PC1) and polycystin 2 (PC2), respectively. PC1 and PC2 localize to the primary cilium and form a protein complex, which is thought to regulate signaling events. PKD1 mutations are associated with a stronger phenotype than PKD2, suggesting the existence of PC1 specific functions in renal tubular cells. However, the evidence for diverging molecular functions is scant. The bending of cilia by fluid flow induces a reduction in cell size through a mechanism that involves the kinase LKB1 but not PC2. Here, using different in vitro approaches, we show that contrary to PC2, PC1 regulates cell size under flow and thus phenocopies the loss of cilia. PC1 is required to couple mechanical deflection of cilia to mTOR in tubular cells. This study pinpoints divergent functions of the polycystins in renal tubular cells that may be relevant to disease severity in ADPKD.
Nephron Experimental Nephrology, Sep 15, 2000
In most epithelial tissues Cl– transport relies on the cystic fibrosis transmembrane conductance ... more In most epithelial tissues Cl– transport relies on the cystic fibrosis transmembrane conductance regulator (CFTR) which has dual function as a Cl– channel and as a regulator of other ion channels. More than 900 different mutations in the CFTR gene are the cause for defective transport of Cl– and Na+ and impaired secretion or absorption of electrolytes in cystic fibrosis. However, the CFTR mutation ΔF508 is the most common reason for the frequently inherited disease among the Caucasian population. Maturation and processing of ΔF508-CFTR is defective which leads to expression of only very little but functional CFTR in the cell membrane. Understanding the processing and trafficking of CFTR may give a clue to the question as to how the expression and residual function of ΔF508-CFTR can be enhanced, and may lead to the development of new pharmacological tools for the treatment of cystic fibrosis.
Molecular & Cellular Proteomics, Jun 1, 2016
Protein arginylation is a posttranslational modification of both N-terminal amino acids of protei... more Protein arginylation is a posttranslational modification of both N-terminal amino acids of proteins and sidechain carboxylates and can be crucial for viability and physiology in higher eukaryotes. The lack of arginylation causes severe developmental defects in moss, affects the low oxygen response in Arabidopsis thaliana and is embryo lethal in Drosophila and in mice. Although several studies investigated impact and function of the responsible enzyme, the arginyl-tRNA protein transferase (ATE) in plants, identification of arginylated proteins by mass spectrometry was not hitherto achieved. In the present study, we report the identification of targets and interaction partners of ATE in the model plant Physcomitrella patens by mass spectrometry, employing two different immuno-affinity strategies and a recently established transgenic ATE:GUS reporter line (Schuessele et al., 2016 New Phytol.,
PLOS ONE, May 1, 2013
The microtubular motor Kinesin-2 and its subunit Kif3a are essential for the formation of primary... more The microtubular motor Kinesin-2 and its subunit Kif3a are essential for the formation of primary cilia, an organelle implicated in a wide spectrum of developmental abnormalities. Outside cilia, Kinesin-2 mediated transport has been implicated in vesicle and N-cadherin transport, but it is unknown if and how extraciliary Kif3a affects basic cellular functions such as migration or the formation of multicellular structures. Here we show that tetracycline inducible depletion of Kif3a in MDCK cells slows epithelial cell migration. Microtubules at the leading edge of Kif3a depleted cells failed to grow perpendicularly into the leading edge and microtubular dynamics were dampened in Kif3a depleted cells. Loss of Kif3a retarded lateral membrane specification and completely prevented the formation of three-dimensional spheres in collagen. These data uncover that Kif3a regulates the microtubular cytoskeleton in the cell periphery and imply that extra-ciliary Kif3a has an unexpected function in morphogenesis.
Journal of Biological Chemistry, Dec 1, 1998
Loading of HT 29 cells with the Ca 2؉ dye fura-2/AM resulted in an nonhomogeneous intracellular d... more Loading of HT 29 cells with the Ca 2؉ dye fura-2/AM resulted in an nonhomogeneous intracellular distribution of the dye. Cellular compartments with high fura-2 concentrations were identified by correlation with mitochondrial markers, cellular autofluorescence induced by UV, and dynamic measurement of autofluorescence after inhibition of oxidative phosphorylation. Stimulation with carbachol (10 ؊4 mol/liter) increased cytosolic, nuclear, and mitochondrial Ca 2؉ activity ([Ca 2؉ ] c , [Ca 2؉ ] n , and [Ca 2؉ ] m , respectively) measured by UV confocal and conventional imaging. Similar results were obtained with a prototype two-photon microscope (Zeiss, Jena, Germany) allowing for fura-2 excitation. The increase of [Ca 2؉ ] m lagged behind that of [Ca 2؉ ] c and [Ca 2؉ ] n by 10-20 s, and after removing the agonist, [Ca 2؉ ] m also decreased with a delay. A strong increase of [Ca 2؉ ] m occurred only when a certain threshold of [Ca 2؉ ] c (around 1 mol/liter) was exceeded. In a very similar way, ATP, neurotensin, and thapsigargin increased [Ca 2؉ ] c and [Ca 2؉ ] m. Carbonyl cyanide p-trifluoromethoxyphenylhyrdrazone reversibly reduced the increase of [Ca 2؉ ] m. The source of the mitochondrial Ca 2؉ increase had intra-and extracellular components, as revealed by experiments in low extracellular Ca 2؉. We conclude that agonist-induced Ca 2؉ signals are transduced into mitochondria. 1) Mitochondria could serve as a Ca 2؉ sink, 2) mitochondria could allow the modulation of [Ca 2؉ ] c and [Ca 2؉ ] n signals, and 3) [Ca 2؉ ] m may serve as a stimulatory metabolic signal when a cell is highly stimulated.
Medical Microbiology and Immunology, Feb 1, 1986
A virus previously isolated from fledgling budgerigars (Melopsittacus undulatus) suffering from a... more A virus previously isolated from fledgling budgerigars (Melopsittacus undulatus) suffering from an acute disease, has been purified and the structural characteristics have been determined. The virions with a buoyant density of 1.34 g/ml are non-enveloped icosahedral particles with a diameter of about 46-48 nm. Their DNA genome has a molecular weight of about 3.3 X 10(6) d, and exists as supericoiled circular, relaxed circular, and linear molecules. There are eight structural proteins, the most abundant of which has a molecular weight of about 42,000 d. Empty capsid shells with buoyant densities of 1.31 g/ml are similar in size and shape, but lack DNA and histone-like polypeptides. Virus replication in chicken embryo cells results in cytopathic changes characterized by rounding and enlargement of the nucleus, and formation of intranuclear inclusion bodies. All these properties justify classification of the virus as polyoma-like.
Journal of Cell Science, May 1, 2010
The structure and function of the primary cilium as a sensory organelle depends on a motor-protei... more The structure and function of the primary cilium as a sensory organelle depends on a motor-protein-powered intraflagellar transport system (IFT); defective IFT results in retinal degeneration and pleiotropic disorders such as the Bardet Biedl syndrome (BBS) and defective hedgehog (HH) signaling. Protein transport to the cilium involves Rab GTPases. Rab8, together with a multi protein complex of BBS proteins, recruits cargo to the basal body for transport to the cilium. Loss of Rab23 in mice recapitulates the HH phenotype but its function in HH signaling is unknown. Here we established a novel protocol, based on fluorescence recovery after photo-bleaching (FRAP), allowing the quantitative analysis of protein transport into the cilium of MDCK cells. We compared the effect of Rab8, Rab5 and Rab23 on the ciliary transport of the HH-associated transmembrane receptor Smoothened, the microtubular tip protein EB1, and the receptor protein Kim1. Ciliary FRAP confirmed the role of Rab8 in protein entry to the cilium. Dominant negative Rab5 had no impact on the ciliary transport of Smoothened or EB1, but slowed the recovery of the apical protein Kim1 in the cilium. Depletion of Rab23 or expression of dominant-negative Rab23 decreased the ciliary steady state specifically of Smoothened but not EB1 or Kim1, suggesting a role of Rab23 in protein turnover in the cilium.
Journal of Cell Biology, Nov 13, 2006
Zenodo (CERN European Organization for Nuclear Research), Jun 2, 2022
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Papers by Roland Nitschke