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H Oberleithner

Publications and source records attributed to H Oberleithner.

At least 19 recordsLinked to original sources
PubMed2001

Plasma membrane protein clusters appear in CFTR-expressing Xenopus laevis oocytes after cAMP stimulation.

Membrane trafficking of the cystic fibrosis transmembrane conductance regulator (CFTR) is supposed to be an important mechanism controlled by the intracellular messenger cAMP. This has been shown with fluorescence techniques, electron microscopy and membrane capacitance measurements. In order to visualize protein insertion we applied atomic force microscopy (AFM) to inside-out oriented plasma membrane patches of CFTR-expressing Xenopus laevis oocytes before and after cAMP-stimulation. In a first step, oocytes injected with CFTR-cRNA were voltage-clamped, verifying successful CFTR expression. Water-injected oocytes served as controls. Then, plasma membrane patches were excised, placed (inside out) on glass and scanned by AFM. Before cAMP-stimulation plasma membranes of both water-injected and CFTR-expressing oocytes contained about 200 proteins per micron 2. Molecular protein masses were estimated from molecular volumes measured by AFM. Before cAMP-stimulation, protein distribution showed a peak value of 11 nm protein height corresponding to 475 kDa. During cAMP-stimulation with 1 mM isobutylmethylxanthine (IBMX) plasma membrane protein density increased in water-injected oocytes to 700 proteins per micron 2 while the peak value shifted to 7 nm protein height corresponding to 95 kDa. In contrast, CFTR-expressing oocytes showed after cAMP-stimulation about 400 proteins per micron 2 while protein distribution exhibited two peak values, one peak at 10 nm protein height corresponding to 275 kDa and another one at 14 nm corresponding to 750 kDa. They could represent heteromeric protein clusters associated with CFTR. In conclusion, we visualized plasma membrane protein insertion upon cAMP-stimulation and quantified protein distribution with AFM at molecular level. We propose that CFTR causes clustering of plasma membrane proteins.

Animals↗
PubMed2001

Electrical dimension of the nuclear envelope.

Eukaryotic chromosomes are confined to the nucleus, which is separated from the rest of the cell by two concentric membranes known as the nuclear envelope (NE). The NE is punctuated by holes known as nuclear pore complexes (NPCs), which provide the main pathway for transport of cellular material across the nuclear-cytoplasmic boundary. The single NPC is a complicated octameric structure containing more than 100 proteins called nucleoporins. NPCs function as transport machineries for inorganic ions and macromolecules. The most prominent feature of an individual NPC is a large central channel, ~7 nm in width and 50 nm in length. NPCs exhibit high morphological and functional plasticity, adjusting shape to function. Macromolecules ranging from 1 to >100 kDa travel through the central channel into (and out of) the nucleoplasm. Inorganic ions have additional pathways for communication between cytosol and nucleus. NE can turn from a simple sieve that separates two compartments by a given pore size to a smart barrier that adjusts its permeabiltiy to the metabolic demands of the cell. Early microelectrode work characterizes the NE as a membrane barrier of highly variable permeability, indicating that NPCs are under regulatory control. Electrical voltage across the NE is explained as the result of electrical charge separation due to selective barrier permeability and unequal distribution of charged macromolecules across the NE. Patch-clamp work discovers NE ion channel activity associated with NPC function. From comparison of early microelectrode work with patch-clamp data and late results obtained by the nuclear hourglass technique, it is concluded that NPCs are well-controlled supramolecular structures that mediate transport of macromolecules and small ions by separate physical pathways, the large central channel and the small peripheral channels, respectively. Electrical properties of the two pathways are still unclear but could have great impact on the understanding of signal transfer across NE and gene expression.

Aldosterone↗
PubMed2000

Nuclear pore function viewed with atomic force microscopy.

In this review we focus on studies using atomic force microscopy (AFM) to describe the function of nuclear pore complexes (NPC). After a short introduction of AFM we follow the route of cargo molecules from the cytosol into the nucleus. AFM visualizes cargo before translocation into the nucleoplasm, cargo docking at the cytoplasmic NPC surface, cargo passing through the NPC and changes in NPC conformation in response to ATP, Calcium and pH. We discuss AFM experiments on nuclear envelopes on the basis of previous data obtained with more conventional techniques such as electron microscopy, confocal microscopy and other imaging techniques. Finally we draw attention to the recently developed nuclear hourglass technique that serves as a new electrophysiological approach to studying the structure-function relationship of NPC in combination with AFM at a molecular level.

Adenosine Triphosphate↗
PubMed2000

High-resistance MDCK-C7 monolayers used for measuring invasive potency of tumour cells.

We describe an electrophysiological method for evaluating the intrinsic invasive potency of tumour cells using renal cells as an in vitro assay system. A high-resistance clone of Madin-Darby canine kidney cells (MDCK-C7) was grown to confluency in a filter cup. Transepithelial electrical resistance across the MDCK-C7 monolayer was measured in a commercially available electrode chamber. After a transepithelial electrical resistance of about 4,000 omega cm2 had been reached, human melanoma or pancreatic carcinoma cells were co-cultivated with the MDCK-C7 monolayer. Both carcinoma cell lines induced resistance breakdown measured after 24 h or later depending on seeding density and cell type. Seeding carcinoma cells on the basolateral surface of MDCK-C7 cells caused a similar decrease in transepithelial resistance of the MDCK-C7 monolayer. Resistance breakdown indicates opening of tight junctions prior to tumour cell invasion. In conclusion, the high-resistance MDCK-C7 cell clone could serve as a valuable biological assay system to determine electrically the metastatic potency of tumour cells in vitro.

Animals↗
PubMed2000

Volume dynamics in migrating epithelial cells measured with atomic force microscopy.

Migration of transformed renal epithelial cells (transformed Madin-Darby canine kidney cells, MDCK-F cells) relies on the activity of a Ca(2+)-sensitive K+ channel (IK channel) that is more active at the rear end of these cells. We have postulated that intermittent IK channel activity induces local cell shrinkage at the rear end of migrating MDCK-F cells and thereby supports the cytoskeletal mechanisms of migration. However, due to the complex morphology of MDCK-F cells we have not yet been able to measure volume changes directly. The aim of the present study was to devise a new technique employing atomic force microscopy (AFM) to measure the volume of MDCK-F cells in their physiological environment and to demonstrate its dependence on IK channel activity. The spatial (x, y' and z) co-ordinates of each pixel of the three-dimensional image of MDCK-F cells allow calculation of the volume of the column "underneath" a given pixel. Thus, total cell volume is the sum of all pixel-defined columns. The mean volume of 17 MDCK-F cells was 2500+/-300 fl. Blockade of the IK channel with the specific inhibitor charybdotoxin (CTX) increased cell volume by 17+/-4%; activation of IK by elevating the intracellular [Ca2+] with the Ca2+ ionophore ionomycin decreased cell volume by 19+/-3%. Subtraction images (experimental minus control) reveal that swelling and shrinkage occur predominantly at the rear end of MDCK-F cells. In summary, our experiments show that AFM allows the measurement not only of total cell volume of living cells in their physiological environment but also the tracing of local effects induced by the polarized distribution of K+ channel activity.

Animals↗
PubMed2000

Nuclear pores collapse in response to CO2 imaged with atomic force microscopy.

Nuclear pore complexes (NPCs) are the rate-limiting barriers for the exchange of macromolecules (e.g. transcription factors or mRNA) between the nuclear and cytosolic compartments. NPC conformation determines movement of cargo in either direction and thus controls gene expression. ATP and calcium are known to induce an NPC shape change (increase in height and decrease in diameter) indicating pore contraction. Here we report a CO2-induced shape change which is different to the ATP/calcium response. Experiments were performed on the isolated nuclear envelope of Xenopus laevis oocytes. The nuclear envelope was spread on glass and the native cytoplasmic surface was imaged with atomic force microscopy (AFM). The preparation was scanned in a water-saturated 100% O2 atmosphere at room temperature. Exposure to 5% CO2 (95%O2) led over a time course of minutes to a dramatic NPC shape change (decrease in height and decrease in diameter) indicating pore closure. NPCs turned flat and central channel openings virtually disappeared. The CO2 response was only slowly reversible. We conclude that NPCs apparently collapse in response to CO2, a structural change that could lead to the functional isolation of the cell nucleus.

Adenosine Triphosphate↗
PubMed2000

Quantitative morphodynamics of endothelial cells within confluent cultures in response to fluid shear stress.

To evaluate shear stress-induced effects on cultured cells we have extended the mechanical setup of a multichannel in vitro rheological system and developed software allowing entire processing control and image data analysis. The values of cell motility, degree of orientation (alignment), and cell elongation were correlated as a function of time (morphodynamics). Collective and individual endothelial cells within confluent cultures displayed a shear stress-dependent characteristic phase behavior of the following time course: resting conditions (phase I), change of motility (phase II), onset of alignment (phase III), and finally cell elongation (phase IV). Especially cell motility was characterized by a randomized zigzag movement around mean trajectories (fluctuations) together with mean cell locomotion. Onset of shear stress caused a down-regulation of fluctuations of 30% within <10 min and simultaneously increased locomotion velocities preferring the flow direction (phase II). After a lag period of 10 to 20 min cells orientated in the direction of flow (phase III) without significant cell elongation, which finally occurs within hours (phase IV). These data provide first evidence that cells within confluent endothelial monolayers respond to shear stress with a characteristic phase behavior.

Animals↗
PubMed2000

Endothelial barrier function under laminar fluid shear stress.

It has been suggested that increasing levels of shear stress could modify endothelial permeability. This might be critical in venous grafting and in the pathogenesis of certain vascular diseases. We present a novel setup based on impedance spectroscopy that allows online investigation of the transendothelial electrical resistance (TER) under pure laminar shear stress. Shear stress-induced change in TER was associated with changes in cell motility and cell shape as a function of time (morphodynamics) and accompanied by a reorganization of catenins that regulate endothelial adherens junctions. Confluent cultures of porcine pulmonary trunk endothelial cells typically displayed a TER between 6 and 15 ohms cm2 under both resting conditions and low shear stress levels (0.5 dyn/cm2). Raising shear stress to the range of 2 to 50 dyn/cm2 caused a transient 2% to 15% increase in TER within 15 minutes that was accompanied by a reduction in cell motility. Subsequently, TER slowly decreased to a minimum of 20% below the starting value. During this period, acceleration of shape change occurred. In the ensuing period, TER values recovered, reaching control levels within hours and associated with an entire deceleration of shape change. A heterogeneous distribution of alpha-, beta-, and gamma-catenin, main components of the endothelial adherens type junctions, was also observed, indicating a differentiated regulation of shear stress-induced junction rearrangement. Additionally, catenins were partly colocalized with beta-actin at the plasma membrane, indicating migration activity of these subcellular parts. Shear stress, even at peak levels of 50 dyn/cm2, did not cause intercellular gap formation. These data show that endothelial monolayers exposed to increased levels of laminar shear stress respond with a shear stress-dependent regulation of permeability and a reorganization of junction-associated proteins, whereas monolayer integrity remains unaffected.

Animals↗
PubMed2000

Pushing, pulling, dragging, and vibrating renal epithelia by using atomic force microscopy.

Renal physiologists focus on events that take place on and around the surfaces of cells. Various techniques have been developed that follow transport functions at the molecular level, but until recently none of these techniques has been capable of making the behavior of molecular structures visible under physiological conditions. This apparent gap may be filled in the future by the application of atomic force microscopy. This technique produces an image not by optical means, but by "feeling" its way across a surface. Atomic force microscopy can, however, be modified in a number of ways, which means that besides producing a high-resolution image, it is possible to obtain several types of data on the interactions between the ultrastructural components of cell membranes (such as proteins) and other biologically active molecules (such as ATP). In this review we describe the recent use of the atomic force microscope in renal physiology, ranging from experiments in intact cells to those in isolated renal transport protein molecules, include examples of these extended applications of the technique, and point to uses that the microscope has recently found in other areas of biology that should prove fruitful in renal physiology in the near future.

Animals↗
PubMed2000

Differential localization of human nongastric H(+)-K(+)-ATPase ATP1AL1 in polarized renal epithelial cells.

The human H(+)-K(+)-ATPase, ATP1AL1, belongs to the subgroup of nongastric, K(+)-transporting ATPases. In concert with the structurally related gastric H(+)-K(+)-ATPase, it plays a major role in K(+) reabsorption in various tissues, including colon and kidney. Physiological and immunocytochemical data suggest that the functional heteromeric ion pumps are usually found in the apical plasma membranes of renal epithelial cells. However, the low expression levels of characteristic nongastric ion pumps makes it difficult to verify their spatial distribution in vivo. To investigate the sorting behavior of ATP1AL1, we expressed this pump by stable transfection in MDCK and LLC-PK(1) renal epithelial cell lines. Stable interaction of ATP1AL1 with either the endogenous Na(+)-K(+)-ATPase beta-subunit or the gastric H(+)-K(+)-ATPase beta-subunit was tested by confocal immunofluorescence microscopy and surface biotinylation. In cells transfected with ATP1AL1 alone, the alpha-subunit accumulated intracellularly, consistent with its inability to assemble and travel to the plasma membrane with the endogenous Na(+)-K(+)-ATPase beta-subunit. Cotransfection of ATP1AL1 with the gastric H(+)-K(+)-ATPase beta-subunit resulted in plasma membrane localization of both pump subunits. In cotransfected MDCK cells the heteromeric ion pump was predominantly polarized to the apical plasma membrane. Functional expression of ATP1AL1 was confirmed by (86)Rb(+) uptake measurements. In contrast, cotransfected LLC-PK(1) cells accumulate ATP1AL1 at the lateral membrane. The distinct polarization of ATP1AL1 indicates that the alpha-subunit encodes sorting information that is differently interpreted by cell type-specific sorting mechanisms.

Animals↗
PubMed2000

Plasma membrane plasticity of Xenopus laevis oocyte imaged with atomic force microscopy.

Proteins are known to form functional clusters in plasma membranes. In order to identify individual proteins within clusters we developed a method to visualize by atomic force microscopy (AFM) the cytoplasmic surface of native plasma membrane, excised from Xenopus laevis oocyte and spread on poly-L-lysine coated glass. After removal of the vitelline membrane intact oocytes were brought in contact with coated glass and then rolled off. Inside-out oriented plasma membrane patches left at the glass surface were first identified with the lipid fluorescent marker FM1-43 and then scanned by AFM. Membrane patches exhibiting the typical phospholipid bilayer height of 5 nm showed multiple proteins, protruding from the inner surface of the membrane, with heights of 5 to 20 nm. Modelling plasma membrane proteins as spherical structures embedded in the lipid bilayer and protruding into the cytoplasm allowed an estimation of the respective molecular masses. Proteins ranged from 35 to 2,000 kDa with a peak value of 280 kDa. The most frequently found membrane protein structure (40/microm2) had a total height of 10 nm and an estimated molecular mass of 280 kDa. Membrane proteins were found firmly attached to the poly-L-lysine coated glass surface while the lipid bilayer was found highly mobile. We detected protein structures with distinguishable subunits of still unknown identity. Since X. laevis oocyte is a generally accepted expression system for foreign proteins, this method could turn out to be useful to structurally identify specific proteins in their native environment at the molecular level.

Animals↗
PubMed2000

Aldosterone and nuclear volume cycling.

The response of target cells to the steroid hormone aldosterone has been divided into acute nongenomic (< 10 min) and sustained genomic (> 10 min) action. In the light of recent experiments this nomenclature does not hold anymore and should be abandoned. By applying atomic force microscopy (AFM) we observed in living endothelial cells that aldosterone induces cell volume increase in less than 10 minutes. The cell nucleus was identified as the swelling site. Hormone-induced nuclear swelling can reach 15 to 28% of total cell volume dissipating within 30 minutes. This phenomenon could have functional impact on flow resistance in small blood vessels. AFM-investigation of the intracellular signal pathway in nuclear envelope of aldosterone-injected Xenopus laevis oocytes visualizes putative intracellular receptors (40 kD granules) bound to nuclear pores 2 minutes after hormone injection, with subsequent macromolecule translocation into the nucleus. 15 minutes later macromolecules (800 kD plugs) appear in the central channels of the nuclear pores. The plugs resemble ribonucleoproteins that carry the aldosterone-induced mRNA to the ribosomes. We postulate that steroid-induced nuclear swelling is caused by a shift of receptors/transcription factors from cytoplasm into nucleoplasm followed by gene transcription. Nuclear volume returns to normal when mRNA export through the nuclear pores is finished. Thus, steroid-induced net-movements of macromolecules between intracellular compartments initiate shifts in cell volume compensated by volume regulatory transporters and ion channels in the plasma membrane.

Aldosterone↗
PubMed1999

Nuclear hourglass technique: an approach that detects electrically open nuclear pores in Xenopus laevis oocyte.

Nuclear pore complexes (NPCs) mediate both active transport and passive diffusion across the nuclear envelope (NE). Determination of NE electrical conductance, however, has been confounded by the lack of an appropriate technical approach. The nuclear patch clamp technique is restricted to preparations with electrically closed NPCs, and microelectrode techniques fail to resolve the extremely low input resistance of large oocyte nuclei. To address the problem, we have developed an approach for measuring the NE electrical conductance of Xenopus laevis oocyte nuclei. The method uses a tapered glass tube, which narrows in its middle part to 2/3 of the diameter of the nucleus. The isolated nucleus is sucked into the narrow part of the capillary by gentle fluid movement, while the resulting change in electrical resistance is monitored. NE electrical conductance was unexpectedly large (7.9 +/- 0.34 S/cm(2)). Evaluation of NPC density by atomic force microscopy showed that this conductance corresponded to 3.7 x 10(6) NPCs. In contrast to earlier conclusions drawn from nuclear patch clamp experiments, NPCs were in an electrically "open" state with a mean single NPC electrical conductance of 1.7 +/- 0.07 nS. Enabling or blocking of active NPC transport (accomplished by the addition of cytosolic extracts or gp62-directed antibodies) revealed this large NPC conductance to be independent of the activation state of the transport machinery located in the center of NPCs. We conclude that peripheral channels, which are presumed to reside in the NPC subunits, establish a high ionic permeability that is virtually independent of the active protein transport mechanism.

Animals↗
PubMed1999

Continuous detection of extracellular ATP on living cells by using atomic force microscopy.

Atomic force microscopy is a powerful technique used to investigate the surface of living cells under physiological conditions. The resolution of the instrument is mainly limited by the softness of living cells and the interactions with the scanning tip (cantilever). Atomic force microscopy, in combination with myosin-functionalized cantilevers, was used in the detection of ATP concentrations in solution and on living cells. Functionally active tips were used to scan the surface of cells in culture and to show that the CFTR+ cell line (S9) had a basal surface ATP concentration that could be detected with atomic force microscopy (n = 10). ATP-dependent signals were not detectable in cells scanned with noncoated or heat-inactivated enzyme-coated tips (n = 9). Enzymatically active tips may serve as a model for future development of atomic force microscopy biosensors that can simultaneously detect topographical and biologically important compounds at the surface microenvironment of living cells.

Adenosine Triphosphate↗
PubMed1998

ATP-Induced shape change of nuclear pores visualized with the atomic force microscope.

Bidirectional transport of molecules between nucleus and cytoplasm through the nuclear pore complexes (NPCs) spanning the nuclear envelope plays a fundamental role in cell function and metabolism. Nuclear import of macromolecules is a two-step process involving initial recognition of targeting signals, docking to the pore and energy-driven translocation. ATP depletion inhibits the translocation step. The mechanism of translocation itself and the conformational changes of the NPC components that occur during macromolecular transport, are still unclear. The present study investigates the effect of ATP on nuclear pore conformation in isolated nuclear envelopes from Xenopus laevis oocytes using the atomic force microscope. All experiments were conducted in a saline solution mimicking the cytosol using unfixed nuclear envelopes. ATP (1 mM) was added during the scanning procedure and the resultant conformational changes of the NPCs were directly monitored. Images of the same nuclear pores recorded before and during ATP exposure revealed dramatic conformational changes of NPCs subsequent to the addition of ATP. The height of the pores protruding from the cytoplasmic surface of the nuclear envelope visibly increased while the diameter of the pore opening decreased. The observed changes occurred within minutes and were transient. The slow-hydrolyzing ATP analogue, ATP-gamma-S, in equimolar concentrations did not exert any effects. The ATP-induced shape change could represent a nuclear pore "contraction."

Adenosine Triphosphate↗
PubMed1998

Migrating transformed MDCK cells are able to structurally polarize a voltage-activated K+ channel.

Cell migration of transformed renal epithelial cells (MDCK-F) depends-in addition to cytoskeletal mechanisms-on the polarized activity of a Ca2+-sensitive K+ channel in the rear part of the cells. However, because of the lack of specific markers for this channel we are not able to determine whether a polarized distribution of the channel protein underlies its functional polarization. To determine whether the migrating MDCK-F cells have retained the ability to target K+ channels to distinct membrane areas we stably transfected the cells with the voltage-dependent K+ channel Kv1.4. Stable expression and insertion into the plasma membrane could be shown by reverse transcription-PCR, genomic PCR, Western blot, and patch-clamp techniques, respectively. The distribution of Kv1.4 was assessed with indirect immunofluorescence by using conventional and confocal microscopy. These experiments revealed that Kv1.4 is expressed only in transfected cells where it elicits the typical voltage-dependent, rapidly inactivating K+ current. The Kv1.4 protein is clustered at the leading edge of protruding lamellipodia of migrating MDCK-F cells. This characteristic distribution of Kv1.4 provides strong evidence that migrating MDCK-F cells are able to insert ion channels into the plasma membrane in an asymmetric way, which reflects the polarization of migrating cells in the plane of movement. These findings suggest that not only epithelial cells and nerve cells, but also migrating cells, can create functionally distinct plasma membrane areas.

Animals↗
PubMed1998

Molecular weights of individual proteins correlate with molecular volumes measured by atomic force microscopy.

Proteins are usually identified by their molecular weights, and atomic force microscopy (AFM) produces images of single molecules in three dimensions. We have used AFM to measure the molecular volumes of a number of proteins and to determine any correlation with their known molecular weights. We used native proteins (the TATA-binding protein Tbp, a fusion protein of glutathione-S-transferase and the renal potassium channel protein ROMK1, the immunoglobulins IgG and IgM, and the vasodilator-stimulated phosphoprotein VASP) and also denatured proteins (the red blood cell proteins actin, Band 3 and spectrin separated by SDS-gel electrophoresis and isolated from nitrocellulose). Proteins studied had molecular weights between 38 and 900 kDa and were imaged attached to a mica substrate. We found that molecular weight increased with an increasing molecular volume (correlation coefficient = 0.994). Thus, the molecular volumes measured with AFM compare well with the calculated volumes of the individual proteins. The degree of resolution achieved (lateral 5 nm, vertical 0.2 nm) depended upon the firm attachment of the proteins to the mica. This was aided by coating the mica with suitable detergent and by imaging using the AFM tapping mode which minimizes any lateral force applied to the protein. We conclude that single (native and denatured) proteins can be imaged by AFM in three dimensions and identified by their specific molecular volumes. This new approach permits detection of the number of monomers of a homomultimeric protein and study of single proteins under physiological conditions at the molecular level.

Anion Exchange Protein 1, Erythrocyte↗