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D Drenckhahn

Publications and source records attributed to D Drenckhahn.

At least 37 records · Page 2Linked to original sources
PubMed1998

From cytoskeleton to polarity and chemoreception in the gut epithelium.

The membrane surface of polarized epithelial cells can be separated in apical and basolateral domains that differ in molecular composition and function. Components of the cytoskeleton obviously play an important role in both generation and maintenance of epithelial polarity. The microtubular system is uniformly aligned in enterocytes and pancreatic acinar cells with the minus ends of the microtubules located apically and the plus ends basally. Drug-induced disassembly of microtubules results in mistargeting of pancreatic zymogen granules and of apical but not basolateral membrane proteins of enterocytes. Association of zymogen granules with the minus end motor protein, cytoplasmic dynein, and components of its receptor, dynactin, indicates that microtubules are important for vectorial delivery of apical but not basolateral post-Golgi vesicles. The lateral membrane domain of the gut epithelium is scaffolded by a spectrin-based membrane cytoskeleton consisting basically of actin, spectrin, and ankyrin. Ankyrin binds to different integral membrane proteins, such as the sodium pump in glandular and kidney epithelial cells or to the anion exchanger (AE2) of gastric parietal cells, thereby probably playing a critical role in maintenance and/or generation of the polarized distribution of these basolateral membrane proteins. Scattered epithelial cells sharing apical cytoskeletal features of gustatory receptor cells were identified as brush cells (tufted cells). These cells are rich in nitric oxide synthase and contain in their apical brush border the gustatory trimeric G-protein, alpha-gustducin, indicating that brush cells are involved in chemoreceptive signaling.

Animals↗
PubMed1998

Not just scaffolding: plectin regulates actin dynamics in cultured cells.

Plectin, a major linker and scaffolding protein of the cytoskeleton, has been shown to be essential for the mechanical integrity of skin, skeletal muscle, and heart. Studying fibroblast and astroglial cell cultures derived from plectin (-/-) mice, we found that their actin cytoskeleton, including focal adhesion contacts, was developed more extensively than in wild-type cells. Also it failed to show characteristic short-term rearrangments in response to extracellular stimuli activating the Rho/Rac/Cdc42 signaling cascades. As a consequence, cell motility, adherence, and shear stress resistance were altered, and morphogenic processes were delayed. Furthermore, we show that plectin interacts with G-actin in vitro in a phosphatidylinositol-4,5-biphosphate-dependent manner and associates with actin stress fibers in living cells. The actin stress fiber phenotype of plectin-deficient fibroblasts could be reversed to a large degree by transient transfection of full-length plectin or plectin fragments containing the amino-terminal actin-binding domain (ABD). These results reveal a novel role of plectin as regulator of cellular processes involving actin filament dynamics that goes beyond its proposed role in scaffolding and mechanical stabilization of cells.

Actins↗
PubMed1998

Localization and quantification of the cytoskeleton-associated protein adducin in the kidneys of normal and Milan hypertensive rats.

Hypertension and kidney dysfunction in sodium transport observed in the Milan hypertensive strain (MHS) of rats are genetically associated with point mutations of adducin, an actin- and spectrin-binding protein of the membrane cytoskeleton. Polymorphism in the adducin locus has been reported to occur also in cases of human primary hypertension. In this study we show by immunostaining that adducin is localized along the basolateral epithelial membrane surface of the entire proximal and distal tubule with no detectable differences between MHS rats and the normotensive control strain (MNS). However, the total amount of adducin in kidney homogenates is reduced by about 45% in MHS rats as determined by quantitative immunoblotting. In erythrocyte membranes of MHS rats, adducin is reduced approximately 10%. The reduction of renal adducin in MHS rats is mainly caused by a reduction of the adducin pool that is loosely associated with kidney membranes and can be released by the non-ionic detergent, Triton X-100. The Triton-resistant, tightly membrane-bound pool of renal adducin differed by approximately 10% between MHS and MNS rats. Since several ion transporters have been shown to be tethered to the membrane cytoskeleton, we suppose that the reduction of the dynamic, loosely bound pool of adducin in MHS rats might interfere with the normal turnover and incorporation of yet unknown transporters involved in kidney sodium transport. However, the Na+,K+-ATPase appears to be not involved, as indicated by normal distribution and amounts of NA+,K+-ATPase in the kidney of MHS rats revealed by immunostaining and immunoblotting.

Animals↗
PubMed1998

Correlation of endothelial vimentin content with hemodynamic parameters.

In mammalian species, vimentin is the sole intermediate filament protein of endothelial cells lining the chambers of the heart and the inner surface of large blood vessels. Obvious quantitative differences in the vimentin-like immunoreactivity of endothelial cells observed in different vascular segments led us to undertake a systematic survey on the endothelial content of vimentin throughout the heart chambers, the vena cava, the pulmonary trunk, and the aorta of the pig. Immunostaining and immunoblotting showed that vimentin in endothelial cells of cardiovascular segments exposed to high shear stress and blood pressure (pulmonary trunk, aorta, left ventricle) is approximately 2- to -3-fold higher than in endothelial cells exposed to lower levels of hemodynamic stress (vena cava, left and right atria, right ventricle). Throughout the aorta, an approximately 1.5-fold increase in the vimentin contents was observed in a proximal to distal direction. The total endothelial amount of vimentin was determined to be 1.2% (inferior vena cava) and 2-3.5% (aorta) of total cellular protein. These data support the notion that the endothelial vimentin cytoskeleton can adapt to different hemodynamic loads, indicating that vimentin might help endothelial cells to withstand the mechanical forces exerted by blood flow and blood pressure.

Animals↗
PubMed1998

Identification of the taste cell G-protein, alpha-gustducin, in brush cells of the rat pancreatic duct system.

The major pancreatic excretory ducts have been shown to contain a large number of specialized epithelial cells, named brush cells, that are characterized by an apical tuft of stiff microvilli. The function of pancreatic brush cells is unknown. Because of some structural similarities to taste receptor cells of the tongue, we addressed the question whether pancreatic brush cells contain the taste cell-specific GTP-binding protein, alpha-gustducin, and hence might be considered to be involved in intraductal chemoreception. By immunostaining, we show that ductal brush cells of the rat pancreatic duct system contain alpha-gustducin, which is concentrated in the apical tuft of microvilli and is also found along the basolateral cell surface. A further outcome of this study is that brush cells are concentrated in the terminal portions of extralobular ducts and in the major pancreatic duct where brush cells comprise up to 22% of the ductal epithelium. Immunoblotting of the major pancreatic duct revealed a 42-kDa band that comigrated with alpha-gustducin of the rat tongue. In view of our previous observation that the ductal brush cells are particularly rich in nitric oxide synthase-I, there is reason to assume that these cells might play a role in certain aspects of chemoreceptive signalling. Thus, chemosensory control of pancreatic secretion might occur at two independent sites, the intestine and the terminal portions of the excretory duct system.

Animals↗
PubMed1998

Anion exchanger 2 (AE2) binds to erythrocyte ankyrin and is colocalized with ankyrin along the basolateral plasma membrane of human gastric parietal cells.

The hydrochloric acid secreting parietal cells of the human stomach mucosa have been shown to express anion exchanger 2 (AE2). AE2 is restricted to the basolateral membrane domain and is responsible for the basolateral uptake of Cl- and release of HCO3-. It is unknown which mechanism is responsible for the basolateral positioning of AE2 in parietal cells. We raised the question whether AE2 might be immobilized at the cell surface by linkage via ankyrin to the spectrin/actin-based membrane cytoskeleton. In the present study we communicate two observations that support this hypothesis, namely that in parietal cells ankyrin is localized with AE2 along the basolateral cell surface and, secondly, that purified erythrocyte ankyrin binds to the in vitro-translated cytoplasmic domain of AE2. We conclude from these observations that AE2 in parietal cells might be linked via ankyrin to the basolateral membrane cytoskeleton and that this type of linkage might play a role in immobilizing AE2 in a non-random fashion along the basolateral membrane domain.

Anion Transport Proteins↗
PubMed1998

Ischemia-induced phosphorylation and translocation of stress protein alpha B-crystallin to Z lines of myocardium.

It is becoming clear that stress proteins play a role in various aspects of postischemic myocardial recovery and that the cytoskeleton of cardiac myocytes is an important determinant for cellular survival during ischemia and energy depletion. In the present study, we addressed the question of whether the cytoskeleton-binding stress protein alpha B-crystallin may be involved in early cellular responses of rat and porcine myocardium to ischemia. Immunostaining and subcellular fractionation revealed a rapid ischemia-induced redistribution of alpha B-crystallin from a cytosolic pool to intercalated disks and Z lines of the myofibrils. This striking translocation of alpha B-crystallin from the cytosol to sites of the myofibrillar system that are known to be sensitive to ischemia-reperfusion injury was accompanied by a rapid shift of a fraction of alpha B-crystallin to a more acidic isoelectric point. This shift is caused by alpha B-crystallin phosphorylation, as identified by its augmentation in the presence of phosphatase inhibitors (vanadate, fluoride) and comigration of the acidic alpha B-crystallin form with the phosphorylated B1 form of lenticular alpha B-crystallin. In view of the chaperone-like function of alpha B-crystallin in conjunction with its high level of constitutive expression in the myocardium (1-2% of soluble protein content), we consider alpha B-crystallin an excellent candidate to play a role in early aspects of the protection of the myocardial contractile apparatus against ischemia-reperfusion injury.

Animals↗
PubMed1998

Targeting of the myosin-I myr 3 to intercellular adherens type junctions induced by dominant active Cdc42 in HeLa cells.

Myr 3, a member of the myosin-I family from rat, is shown in this study to be localized at adherens-type intercellular junctions in epithelial and nonepithelial tissues. Formation of intercellular junctions and the accompanying recruitment of myr 3 to these junctions involves signaling by the Rho subfamily of small GTP-binding proteins. This conclusion is based on studies with HtTA-1 HeLa cells that were induced by overexpression of constitutively active Cdc42Hs to form typical adherens-type intercellular junctions enriched in cadherins (N-cadherin), beta-catenin, filamentous actin and myr 3. Recruitement of myr 3 to Cdc42-induced adherens junctions in HeLa cells was dependent on a short region of the tail domain and a functional myosin motor domain, but was independent of its myosin-I tail homology and SH3 regions. Overexpression of constitutively active Rac1 induced a distinct type of adherens junction in HeLa cells that was characterized by elaborate intercellular interdigitations enriched in N-cadherin, beta-catenin and F-actin. Myr 3 was often present, but not specifically enriched in the intercellular junctions induced by constitutively active Rac1.

Amino Acid Sequence↗
PubMed1997

Cytoskeleton-membrane connections in the human erythrocyte membrane: band 4.1 binds to tetrameric band 3 protein.

Band 4.1 provides, besides ankyrin, the main linkage between the erythrocyte membrane and its cytoskeleton. Its predominant binding sites in the membrane are located on the glycophorins. However, the cytoplasmic domain of band 3 can also bind band 4.1. We have studied which of the different band 3 oligomers observed (monomers, dimers, tetramers) can act as band 4.1 binding sites, by equilibrium sedimentation experiments on mixtures of purified band 3 and dye-labelled band 4.1 in solutions of a nonionic detergent. At low molar ratios of band 4.1 and band 3, the sedimentation equilibrium distributions obtained could all be perfectly fitted assuming that only two dye-labelled particles were present: uncomplexed band 4.1 and a complex formed between one band 4.1 molecule and one band 3 tetramer. The presence of small amounts of complexes containing band 3 monomers or dimers could not be completely ruled out but is unlikely. On the other hand, stabilized band 3 dimers effectively bound band 4.1. At higher molar band 4.1/band 3 ratio, the band 3 tetramer apparently could bind up to at least four band 4.1 molecules. The band 4.1/band 3 tetramer complex was found to be unstable. The results described, together with those reported previously, point at a prominent role of tetrameric band 3 in ligand binding.

Anion Exchange Protein 1, Erythrocyte↗
PubMed1997

Localization of dystrophin and beta-dystroglycan in bovine retinal photoreceptor processes extending into the postsynaptic dendritic complex.

Dystrophin is an actin-binding protein of the membrane cytoskeleton that binds to dystroglycan, an integral membrane protein of the plasma membrane that is posttranslationally cleaved into a transmembrane dystrophin-binding beta-moiety and an extracellular laminin- and agrin-binding alpha-moiety. Mutations of dystrophin may not only cause Duchenne muscular dystrophy but may also be associated with abnormal electroretinograms assumed to result from disturbed neurotransmission between retinal photoreceptors and bipolar cells. Here we show by confocal laser microscopy and immunogold electron microscopy that dystrophin and beta-dystroglycan are colocalized in bovine rod photoreceptor synaptic complexes distal from the ribbon-containing active synaptic zones. Both proteins are restricted to a microdomain of the photoreceptor plasma membrane that forms the lateral wall of the synaptic cavity and projects with finger-like extensions into the postsynaptic dendritic complex. Within the cavity these processes eventually come into close contact with bipolar cell dendritic endings. We speculate that the dystrophin-dystroglycan complex of the cavital plasma membrane stabilizes the elaborate synaptic morphology or plays a role in the immobilization of still unknown transporters and receptors involved in certain aspects of neurotransmission to bipolar cells. A further outcome of this study is that dystrophin and dystroglycan are located along the vitread membrane surface of Müller cell endfeet where this protein complex may be important for the attachment of the retina to the basal lamina and the vitreous.

Animals↗
PubMed1997

Dystrophin in the retina.

Dystrophin is a plasma membrane-associated cytoskeletal protein of the spectrin superfamily. The dystrophin cytoskeleton has been first characterized in muscle. Muscular 427 kDa dystrophin binds to subplasmalemmal actin filaments via its amino-terminal domain. The carboxy-terminus of dystrophin binds to a plasma membrane anchor, beta-dystroglycan, which is associated on the external side with the extracellular matrix receptor, alpha-dystroglycan, that binds to the basal lamina proteins laminin-1, laminin-2, and agrin. In the muscle, the dystroglycan complex is associated with the sarcoglycan complex that consists of several glycosylated, integral membrane proteins. The absence or functional deficiency of the dystrophin cytoskeleton is the cause of several types of muscular dystrophies including the lethal Duchenne muscular dystrophy (DMD), one of the most severe and most common genetic disorders of man. The dystrophin complex is believed to stabilize the plasma membrane during cycles of contraction and relaxation. Muscular dystrophin and several types of dystrophin variants are also present in extramuscular tissues, e.g. in distinct regions of the central nervous systems including the retina. Absence of dystrophin from these sites is believed to be responsible for some extramuscular symptoms of DMD, e.g. mental retardation and disturbances in retinal electrophysiology (reduced b-wave in electroretinograms). The reduced b-wave in electroretinograms indicated a disturbance of neurotransmission between photoreceptors and ON-bipolar cells. At least two different dystrophin variants are present in photoreceptor synaptic complexes. One of these dystrophins (Dp260) is virtually exclusively expressed in the retina. In the neuroretina, dystrophin is found in significant amounts in the invaginated photoreceptor synaptic complexes. At this location dystrophin colocalizes with dystroglycan. Agrin, an extracellular ligand of alpha-dystroglycan, is also present at this location whereas the proteins of the sarcoglycan complex appear to be absent in photoreceptor synaptic complexes. Dystrophin and dystroglycan are located distal from the ribbon-containing active synaptic zones where both proteins are restricted to the photoreceptor plasma membrane bordering on the lateral sides of the synaptic invagination. In addition, some neuronal profiles of the postsynaptic complex also contain dystrophin and beta-dystroglycan. These profiles appear to belong at least in part to projections of the photoreceptor terminals into the postsynaptic dendritic complex. In view of the abnormal neurotransmission between photoreceptors and ON-bipolar cells in DMD patients the dystrophin/beta-dystroglycan-containing projections of photoreceptor presynaptic terminals into the postsynaptic dendritic plexus might somehow modify the ON-bipolar pathway. Another retinal site associated with dystrophin/beta-dystropglycan is the plasma membrane of Müller cells where dystrophin/beta-dystroglycan appear to be present at particular high concentrations. At this location the dystrophin/dystroglycan complex may play a role in the attachment of the retina to the vitreous, and, under pathological conditions, in traction-induced retinal detachment.

Animals↗
PubMed1997

Role of cadherins and plakoglobin in interendothelial adhesion under resting conditions and shear stress.

The role of cadherins and the cadherin-binding cytosolic protein plakoglobin in intercellular adhesion was studied in cultured human umbilical venous endothelial cells exposed to fluid shear stress. Extracellular Ca2+ depletion (< 10(-7) M) caused the disappearance of both cadherins and plakoglobin from junctions, whereas the distribution of platelet endothelial cell adhesion molecule 1 (PECAM-1) remained unchanged. Cells stayed fully attached to each other for several hours in low Ca2+ but began to dissociate under flow conditions. At the time of recalcification, vascular endothelial (VE) cadherin and beta-catenin became first visible at junctions, followed by plakoglobin with a delay of approximately 20 min. Full fluid shear stress stability of the junctions correlated with the time course of the reappearance of plakoglobin. Inhibition of plakoglobin expression by microinjection of antisense oligonucleotides did not interfere with the junctional association of VE-cadherin, PECAM-1, and beta-catenin. The plakoglobin-deficient cells remained fully attached to each other under resting conditions but began to dissociate in response to flow. Shear stress-induced junctional dissociation was also observed in cultures of plakoglobin-depleted arterial endothelial cells of the porcine pulmonary trunk. These observations show that interendothelial adhesion under hydrodynamic but not resting conditions requires the junctional location of cadherins associated with plakoglobin. beta-Catenin cannot functionally compensate for the junctional loss of plakoglobin, and PECAM-1-mediated adhesion is not sufficient for monolayer integrity under flow.

Antigens, CD↗
PubMed1997

Sorting of actin isoforms in chicken auditory hair cells.

Most nonmuscle cells of higher vertebrates contain two different actin isoforms, beta- and gamma-cytoplasmic actin. The beta-isoform is with few exceptions the predominant isoform in nonmuscle cells and tissues. Perturbation of the beta:gamma ratio has been shown to affect the organization of bundled actin filaments indicating that the beta- and gamma-genes encode functionally distinct cytoarchitectural information. In the present study we localized by immunostaining beta- and gamma-actin in chicken auditory hair cells. These highly specialized cells serve as model system for studying certain developmental and structural aspects of a complex actin filament system with high architectural precision. We show that gamma-actin is the predominant actin isoform in auditory hair cells with an apparent beta:gamma ratio of approximately 1:2. gamma-Actin is not sorted and occurs in all three actin assemblies of the hair border, i.e. the cores of sensory hairs (stereocilia), the subjacent gel-like actin filament meshwork (cuticular plate) and the zonula adherens ring. In contrast to gamma-actin, the beta-isoform is specifically sorted to the actin filament core bundle of stereocilia that is extensively crosslinked by fimbrin. In view of recent studies showing that L-plastin, the leukocyte homolog of fimbrin, has a higher binding affinity for beta-actin than for gamma-actin, a mechanism is proposed for how hair cells might restrict formation of actin filament bundles to a single cellular site (i.e. the stereocilia). The limited level of expression of beta-actin in hair cells may help to prevent ectopic bundle formation in other cellular compartments.

Actins↗
PubMed1996

Purification of synaptic ribbons, structural components of the photoreceptor active zone complex.

Synaptic ribbons are plasma membrane-associated structural elements in photoreceptor synaptic terminals. They seem to act as high capacity "docking sites" of synaptic vesicles that provide the fusion sites of the photoreceptor synapse ("active zones"), with a large supply of immobilized synaptic vesicles rapidly available for exocytosis. Synaptic ribbons are regarded as a specialized type of presynaptic densities found in virtually all synapses. The molecular composition of presynaptic densities and synaptic ribbons is unknown. The aim of this study was the isolation of synaptic ribbons from photoreceptor synapses. For this purpose, we first isolated a membrane fraction from the bovine retina that was strongly enriched in photoreceptor synapses. From this fraction, a Triton X-100-resistant subfraction was purified that consisted mainly of synaptic ribbons and their disassembly products. The high enrichment of synaptic ribbons was verified by electron microscopy and immunolabeling using an antibody that specifically binds to synaptic ribbons. SDS-PAGE analysis of this synaptic ribbon fraction displayed several major polypeptide bands migrating at approximately 240, 60, 55, 43, and 30 kDa. The purification procedure described here is a first promising step toward the identification of the yet unknown constituents of synaptic ribbons from photoreceptor synapses and possibly also of presynaptic densities from other synapses.

Animals↗
PubMed1996

Taste receptor-like cells in the rat gut identified by expression of alpha-gustducin.

The alpha-subunit of the trimeric G-protein complex specific for taste receptor cells of the tongue, alpha-gustducin, is described here to be also expressed in the stomach and intestine. The alpha-gustducin-containing cells were identified as brush cells that are scattered throughout the surface epithelium of the gut and share structural features of taste receptor cells of the tongue. These findings provide clues to the long-sought molecular and cellular basis for chemoreception in the gut.

Animals↗
PubMed1996

Cytoskeletal differences between stereocilia of the human sperm passageway and microvilli/stereocilia in other locations.

BACKGROUND: Stereocilia of the human ductus epididymidis and ductus deferens display unique features in that they arise from an apical cell protrusion (hillock) and contain thick stem portions which are interconnected by cytoplasmic bridges. The molecular basis for this unique fusion and branching pattern is hitherto unknown. These morphologic specialties led us to study the cytoskeleton of male spermway stereocilia with respect to the major proteins that constitute the supportive cytoskeleton of intestinal microvilli and inner ear stereocilia. METHODS: Samples of the human epididymidis and ductus deferens were studied by immunoblotting and immunocytochemistry at the light and electron microscope levels. RESULTS: Spermway stereocilia are supported by an internal actin filament bundle crosslinked by fimbrin and associated with the membrane linker molecule ezrin. The stem portions and hillock area are supplied with the crossbridge forming molecule alpha-actinin. Spermway stereocilia differ from brush border microvilli of the intestine, kidney, and ductuli efferentes by the lack of the second bundling protein villin and the unusual expression of alpha-actinin in the stem region. They resemble inner ear stereocilia by the presence of fimbrin and absence of villin, but differ from them by expression of ezrin and alpha-actinin. Thus, the main molecular difference between spermway stereocilia and stereocilia/microvilli of other locations is the presence of alpha-actinin in their stem portion and the hillock area. CONCLUSIONS: Since alpha-actinin can form crossbridges between adjacent actin filaments (bundles) at longer distances than the other crosslinker of the stereocilium core bundle, fimbrin, we assume that alpha-actinin is essential for both the formation of the stem portions of spermway stereocilia and for the generation of their striking branching pattern. A developmentally regulated temporal sequence of expression of fimbrin and alpha-actinin might control the unique architecture of spermway stereocilia.

Actins↗
PubMed1996

Astrocytes and Bergmann glia as an important site of nitric oxide synthase I.

In the central nervous system nitric oxide appears to be critically involved in a number of physiological and pathological processes. Although there is convincing evidence for expression of nitric oxide synthase in cultured glial cells, demonstration of this enzyme in glial cells in situ remained largely unsatisfactory. In the present study we applied immunostaining to freeze-dried sections of snap-frozen hippocampi and cerebella of rats and to sections of freeze-dried brain tissue in order to minimize diffusion artefacts and thus to obtain more precise information about the true in situ localization of nitric oxide synthase. Here we show that astrocytes and Bergmann glia react strongly with antibodies raised against cerebellar nitric oxide synthase and against a type I nitric oxide synthase-specific C-terminal peptide, respectively. This finding was further substantiated by histochemical localization of NADPH-diaphorase activity in astrocytes and Bergmann glia as well as by immunoreactivity of both types of glia cells with antibodies to the NADPH-delivering enzyme glucose-6-phosphate dehydrogenase. We conclude, that astrocytes are important sites of nitric oxide synthase I in brain, suggesting that these cells might use nitric oxide as gaseous messenger molecule for various aspects of glia-neuron signalling.

Animals↗