Biomedical subjects

Hermann Schillers

Publications and source records attributed to Hermann Schillers.

12 recordsLinked to original sources

PubMed2006

Ethanol alters access to the cell nucleus.

Ethanol is the most frequently used drug among humans. We tested the hypothesis whether ethanol, at clinically relevant concentrations modifies, signaling across the nuclear envelope (NE). In cell nuclei isolated from Xenopus oocytes, we measured NE electrical resistance and NE macromolecule permeability 1 to 20 h after addition of ethanol (0.05 to 0.2%). Furthermore, with atomic force microscopy, nuclear pores of the NE were imaged after exposure to ethanol. We found that NE permeability decreased within hours of ethanol exposure. In parallel, nuclei swell and nuclear pores form clusters in the NE. Force measurements on individual nuclear pores indicate that pores found in clusters are stiffer than those found randomly distributed in the NE. Application of a transcription blocker (actinomycin D) or RNase treatment of isolated nuclei in vitro after ethanol exposure prevents the permeability changes. In conclusion, ethanol, at commonly used concentrations, changes NE structure by transcriptional processes in the cell nucleus. Within hours, the NE becomes less permeable for diffusible ions and macromolecules. This could explain altered signaling to and communication with the cell nucleus in the pathophysiology of alcohol abuse.

Animals↗

PubMed2006

An extracellular loop of the human non-gastric H,K-ATPase alpha-subunit is involved in apical plasma membrane polarization.

The human non-gastric H,K-ATPase, ATP1AL1, belongs to the gene family of P-type ATPases. Consistent with their physiological roles in ion transport, members of this group, including the Na,KATPase and the gastric and non-gastric H,K-ATPases, are differentially polarized to either the basolateral or apical plasma membrane in epithelial cells. However, their polarized distribution is highly complex and depends on specific sorting signals or motifs which are recognized by the subcellular targeting machinery. For the gastric H,K-ATPase it has been suggested that the 4(th) transmembrane spanning domain (TM4) and its flanking regions induce conformational sorting motifs which direct the ion pump exclusively to the epithelial apical membrane. Here, we show in transfected Madin-Darby canine kidney (MDCK) cells that the related non-gastric H,KATPase, ATP1AL1, does contain similar sorting motifs in close proximity to TM4. A short extracellular loop between TM3 and TM4 is critical for this pump's apical delivery. A single point mutation in the corresponding region redirects ATP1AL1 to the basolateral membrane. In conclusion, our work provides further evidence that the cellular distribution of P-type ATPases is determined by conformational sorting motifs.

Amino Acid Sequence↗

PubMed2006

Differential action of steroid hormones on human endothelium.

The action of glucocorticoids on vascular permeability is well established. However, little is known about the action of mineralocorticoids on the structure and function of blood vessels. As endothelial cells are targets for both glucocorticoids and mineralocorticoids, we exposed human umbilical vein endothelial cells to both types of steroids. Aldosterone (mineralocorticoid) and dexamethasone (glucocorticoid) were applied for 3 days in culture before measurements of transendothelial ion and macromolecule permeability, apical cell surface and cell stiffness were taken. Transendothelial ion permeability was measured with electrical cell impedance sensing, macromolecule permeability with fluorescence-labeled dextran and apical cell membrane surface by three-dimensional AFM imaging. Cell stiffness was measured using the AFM scanning tip as a mechanical nanosensor. We found that aldosterone increased both apical cell surface and apical cell stiffness significantly, while transendothelial permeability remained unaffected. By contrast, dexamethasone significantly decreased ion and macromolecule permeability, while apical cell surface and cell stiffness did not change. Specific receptor antagonists for dexamethasone (RU486) and aldosterone (spironolactone) prevented the observed responses. We conclude that glucocorticoids strengthen cell-to-cell contacts (;peripheral action'), whereas mineralocorticoids enlarge and stiffen cells (;central action'). This could explain the dexamethasone-mediated retention of fluid in the vascular system, and endothelial dysfunction in states of hyperaldosteronism.

Aldosterone↗

PubMed2006

Single plasma membrane K+ channel detection by using dual-color quantum dot labeling.

K+ channels are widely expressed in eukaryotic and prokaryotic cells, where one of their key functions is to set the membrane potential. Many K+ channels are tetramers that share common architectural properties. The crystal structure of bacterial and mammalian K+ channels has been resolved and provides the basis for modeling their three-dimensional structure in different functional states. This wealth of information on K+ channel structure contrasts with the difficulties to visualize single K+ channel proteins in their physiological environment. We describe a method to identify single Ca2+-activated K+ channel molecules in the plasma membrane of migrating cells. Our method is based on dual-color labeling with quantum dots. We show that >90% of the observed quantum dots correspond to single K+ channel proteins. We anticipate that our method can be adopted to label any other ion channel in the plasma membrane on the single molecule level.

Animals↗

PubMed2006

Physiological concept for a blood based CFTR test.

We tested the hypothesis that the cystic fibrosis transmembrane conductance regulator (CFTR) could be involved in the volume regulation of human red blood cells (RBC). Experiments were based on two gadolinium (Gd(3+)) sensitive mechanisms, i.e. inhibition of ATP release (thetaATP(i)) and membrane destabilization. RBC of either cystic fibrosis (CF) patients or healthy donors (non-CF) were exposed to KCl buffer containing Gd(3+). A significantly larger quantity of non-CF RBC (2.55 %) hemolyzed as compared to CF RBC (0.89 %). It was found that both of the Gd(3+) mechanisms simultaneously are needed to achieve hemolysis, since either overriding thetaATP(i) by exogenous ATP addition prevented Gd(3+) induced hemolysis, or mimicking thetaATP(i) by apyrase in absence of Gd(3+) could not trigger hemolysis. Additionally, ion driven volume uptake was found to be a prerequisite for Gd3+ induced hemolysis as chloride and potassium channel blockers reduced the Gd(3+) response. The results show that in non-CF RBC Gd(3+) exerts its dual effect leading to hemolysis. On the contrary, in CF RBC, lacking CFTR dependent ATP release, the sole Gd(3+) effect of membrane destabilization is not sufficient to induce hemolysis similar to non-CF. This concept could form the basis of a novel method suitable for testing CFTR function in a blood sample.

Adenosine Triphosphate↗

PubMed2006

IADS, a decomposition product of DIDS activates a cation conductance in Xenopus oocytes and human erythrocytes: new compound for the diagnosis of cystic fibrosis.

DIDS (4,4'-diisothiocyanato-stilbene-2,2'-disulfonic acid) is a commonly used blocker of plasma membrane anion channels and transporters. We observed that DIDS undergoes decomposition while stored in DMSO (dimethyl sulfoxide) forming a biologically active compound. One decomposition product, called IADS, was identified and synthesized. Voltage-clamp and patch clamp experiments on Xenopus laevis oocytes and human erythrocytes revealed that IADS is able to activate a plasma membrane cation conductance in both cell types. Furthermore, we found that IADS induces hemolysis in red blood cells of healthy donors but fails to hemolyze erythrocytes of donors with cystic fibrosis. Thus, IADS stimulated activation of a cation conductance could form the basis for a novel diagnostic test of cystic fibrosis.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗

PubMed2005

The genome of HSV-1 translocates through the nuclear pore as a condensed rod-like structure.

Incoming herpes simplex virus type-1 (HSV-1) capsids are known to dock to the nuclear pore complex (NPC) and release their genome. It has remained elusive, however, how the huge viral DNA translocates through the comparatively small NPC channel. In the present study, the interaction of HSV-1 with NPCs was analyzed by atomic force microscopy. In addition to capsids, smaller subviral structures--most with a diameter of 35-40 nm and a length of 130-160 nm--were visualized at the cytoplasmic side of the NPC. These components differed from capsids in their adhesion and stiffness properties, and were the sole subviral structures translocated through dilated NPCs towards the nucleus. It is presumed that they are the HSV-1 genome, and that a change in NPC conformation allows translocation of this genome as a densely packaged, rodlike structure.

Animals↗

PubMed2005

Steroids dilate nuclear pores imaged with atomic force microscopy.

Macromolecules that act in the cell nucleus must overcome the nuclear envelope (NE). This barrier between cytosol and the nucleus is perforated by nuclear pore complexes (NPCs) that serve as translocation machineries. We visualized the translocation process at the NE surface, applying a nanotechnical approach using atomic force microscopy (AFM). In order to initiate protein targeting to NPCs, dexamethasone (dex) was injected into Xenopus laevis oocytes. Dex is a synthetic steroid of great therapeutic relevance that specifically binds to glucocorticoid receptors and thus triggers an intracellular signal cascade involving the cell nucleus. Ninety and 180 sec after dex injection cell nuclei were isolated, the NEs spread on glass and scanned with AFM. With single molecule resolution we observed that dex initiated proteins (DIPs) first bind to NPC-free areas of the outer nuclear membrane. This causes NPCs to dilate. Then, in a second step, DIPs attach directly to NPCs and enter the dilated central channels. DIPs accumulation and NPC conformational changes were blocked by RU486, a specific glucocorticoid receptor antagonist. In conclusion, dex exposure induces NPC dilation. NPCs change conformation already prior to transport. The NPC dilation signal is most likely transmitted through NPC associated filaments or yet unknown structures in the NE outer membrane. NPC dilation could have significant impact on nuclear targeting of therapeutic macromolecules.

Animals↗

PubMed2004

Human endothelium: target for aldosterone.

Aldosterone has long been known to control water and electrolyte balance by acting on mineralocorticoid receptors in kidney. However, recent studies demonstrated the presence of these receptors in nonclassical locations, including the cardiovascular system. We tested the hypothesis whether endothelial cells respond to aldosterone with changes in cell volume, a measure for ion-mediated water movement across the cell membrane. By means of atomic force microscopy in fluid, we measured volume of adherent human umbilical venous endothelial cells exposed for 72 hours to 10 nmol/L aldosterone. Over this period of time, cells swell by approximately 18%. Aldosterone-induced swelling is prevented by 100 nmol/L of the mineralocorticoid receptor antagonist spironolactone, added to the primary endothelial cell culture. Aldosterone-treated cells dramatically shrink when 1 micromol/L of the diuretic amiloride is applied. Cells deprived of aldosterone do not respond to amiloride. Our conclusions are: (1) aldosterone leads to sustained cell swelling inhibited by administration of spironolactone or the sodium channel blocker amiloride; (2) cells respond to amiloride after aldosterone exposure; (3) renal diuretics act on endothelial cells; and (4) both amiloride and spironolactone could be useful for medical applications to prevent aldosterone-mediated endothelial dysfunction.

Aldosterone↗

PubMed2004

Imaging CFTR: a tail to tail dimer with a central pore.

The cystic fibrosis transmembrane conductance regulator (CFTR) is a protein that belongs to the superfamily of ATP binding cassette (ABC) transporters. Mutations in the CFTR gene cause cystic fibrosis, an autosomal recessive disorder. The function of CFTR is versatile. It can serve as a regulatory protein, as a membrane transporter and as an ion channel. Dimerization of CFTR is necessary for full ion channel function although structural details of CFTR in native membrane are yet unknown. In order to identify CFTR in native plasma membrane we applied atomic force microscopy (AFM) to inside-out oriented membrane patches of CFTR-expressingXENOPUS LAEVIS oocytes after cAMP stimulation. First, oocytes were injected with CFTR-cRNA and, three days later, voltage-clamped verifying successful CFTR expression and incorporation into the plasma membrane. Then, plasma membrane patches were isolated, placed inside-out on appropriate substrate and incubated with gold-labelled antibodies against the C-terminus of CFTR. Finally, the intracellular surface of the plasma membrane was scanned by AFM. In close vicinity to the immunogold labels we detected ring-like structures with bipartite symmetry. The substructure of the ring, formed by the extramembrane protein domains of CFTR, is consistent with the model of a CFTR dimer. Derived from AFM molecular mass analysis of the intramolecular domains we conclude that two CFTR molecules line up in parallel, tail by tail, forming a pore in its center. This molecular arrangement could represent the CFTR chloride channel configuration, operative in native plasma membrane.

Animals↗

PubMed2004

Evidence for importin alpha independent nuclear translocation of glucocorticoid receptors in Xenopus laevis oocytes.

The glucocorticoid receptor (GR) is a ligand-dependent transcription factor which resides in the cytoplasm as a complex with a number of molecular chaperones. Upon hormonal stimulation, the GR is translocated into the nucleus where it modulates transcription by binding to specific target DNA sequences. GR contains a classical basic nuclear localization signal (NLS) and a second, only poorly characterized NLS. Though the participation of the importin alpha/importin beta pathway in nuclear import of GR has been postulated, relatively little is known about its dynamics and mechanisms. We analyzed the hormone-driven nuclear import of rat GR expressed in Xenopus laevis oocytes by atomic force microscopy (AFM) and western blot analysis. AFM imaging revealed accumulation of macromolecules matching the size of GR at the nuclear envelope beginning 5 min after glucocorticoid hormone injection. In parallel, western blot analysis showed accumulation of GR over the same time scale after glucocorticoid hormone stimulation. Nuclear GR import did not trigger redistribution of importin alpha or importin beta. We conclude that hormone-driven nuclear import of rat GR expressed in Xenopus oocytes is a process with limited capacity and involves mechanisms different from the importin alpha/importin beta pathway. This represents a restriction of nuclear import downstream of import signals.

Active Transport, Cell Nucleus↗

PubMedDate not supplied

Reduced number of CFTR molecules in erythrocyte plasma membrane of cystic fibrosis patients.

Cystic fibrosis (CF), the most common genetic disease among Caucasians, is caused by mutations in the gene encoding CFTR (cystic fibrosis transmembrane conductance regulator). The most frequent mutation, DeltaF508, results in protein misfolding and, as a consequence, prevents CFTR from reaching its final location at the cell surface. CFTR is expressed in various cell types including red blood cells. The functional role of CFTR in erythrocytes is still unclear. Since the number of CFTR copies in a single erythrocyte of healthy donors and CF patients with a homozygous DeltaF508 mutation is unknown, we counted CFTR, localized in erythrocyte plasma membrane, at the single molecule level. A novel experimental approach combining atomic force microscopy with quantum-dot-labeled anti-CFTR antibodies, used as topographic surface markers, was employed to detect individual CFTR molecules. Analysis of erythrocyte plasma membranes taken from healthy donors and CF patients with a homozygous DeltaF508 mutation reveals mean (SEM) values of 698 (12.8) (n=542) and 172 (3.8) (n=538) CFTR molecules per red blood cell, respectively. We conclude that erythrocytes reflect the CFTR status of the organism and that quantification of CFTR in a blood sample could be useful in the diagnosis of CFTR related diseases.

Antibodies↗