The versatility of microvascular pericytes: from mesenchyme to smooth muscle?
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Animals↗
Biomedical subjects
D Drenckhahn
Publications and source records attributed to D Drenckhahn.
Molecular heterogeneity of the actin filament cytoskeleton associated with microvilli of photoreceptors, Müller's glial cells and pigment epithelial cells of the retina.
The present study addressed the question as to whether the four different actin-associated proteins that are associated with the actin core bundle in intestinal microvilli (i.e. villin, fimbrin, myosin I and ezrin) are essential components of all microvilli of the body. The retina provides an excellent example of a tissue supplied with three different sets of microvilli, namely those of Müller's glial cells (Müller baskets), photoreceptors (calycal processes), and pigment epithelial cells. The main outcome of this study is that none of these microvilli contain all four actin-associated proteins present in intestinal microvilli. Müller cell microvilli contain villin, ezrin and myosin I (95 kDa isoform) but not fimbrin. Calycal processes of photoreceptors contain fimbrin but not villin, myosin I and ezrin. Finally, microvilli of pigment epithelial cells are positive for ezrin but not for villin, fimbrin and myosin I. Because of limited cross-reactivities of the antibodies to myosin I and ezrin, the myosin I data refer to the chicken retina whereas the findings with anti-ezrin were obtained with the rat retina. A further outcome of this study is that the actin filament core bundles in microvilli of chicken pigment epithelial cells are presumed to contain a crosslinking protein, which is not immunologically related to either villin, fimbrin or myosin I of the intestinal brush border.
Actins↗
Distribution of actin in cone photoreceptor synapses.
The invaginated photoreceptor terminal harbours a great number of postsynaptic dendrites that contact the photoreceptor terminal at morphologically distinct synapses. In this paper we studied the subcellular distribution of actin in cone photoreceptor synapses of light-adapted goldfish and guppy retinas and its relation to ribbon and spinule synapses. In the outer plexiform layer, fluorescent staining for actin by antibodies and also by fluorescently labelled phalloidin was discontinuous and showed large dot-like immunoreactive structures (3-5 microns in diameter). Immunogold electron microscopy of light-adapted retinas isolated 3 h after the onset of light demonstrated that these dot-like structures corresponded to dendritic profiles of bipolar and horizontal cells within the synaptic cavity of cones. A much less intense staining was observed within the presynaptic cone photoreceptor terminal itself. Immunogold particles were also found overlying the tips of horizontal and bipolar dendrites that form the triadic postsynaptic complex of the ribbon synapse. In contrast to the triads light-induced terminal outgrowings of horizontal cells, also known as spinules, remained largely unlabelled. These observations suggest that actin plays a role in maintenance and/or certain steps of the formation of the postsynaptic dendritic complex.
Actins↗
Production of polyclonal antibodies against proteins and peptides.
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Adjuvants, Immunologic↗
Improved in vitro rheological system for studying the effect of fluid shear stress on cultured cells.
A rheological in vitro system has been developed to study and quantify cellular adhesion under precisely defined external shear forces. The system is similar to a cone-and-plate viscosimeter. A rotating transparent cone produces both steady and pulsatile flow profiles on cultured cells. Direct visualization of cells by phase-contrast or fluorescence optics and connection of the optical system to a computer-controlled x/y-linear stage allows automatic recording of any point of the cell cultures. With the use of up to 12 individual rheological units, this setup allows the quantitative analysis of cell substrate adhesion by determination of cell detachment kinetics. Two examples of application of this rheological system have been studied. First, we show that the extracellular matrix protein laminin strongly increases endothelial cell adhesion under fluid shear stress. In a second approach, we obtained further support for the concept that shear stress-induced formation of actin filament stress fibers is important for endothelial cells to resist the fluid shear stress; inhibition of stress fiber formation by doxorubicin resulted in significant detachment of endothelial cells exposed to medium levels of fluid shear stress (5 dyn/cm2). No detachment was seen under resting conditions.
Animals↗
Anion exchanger 1 in human kidney and oncocytoma differs from erythroid AE1 in its NH2 terminus.
Acid-secreting intercalated cells of the kidney collecting duct and tumor cells of renal oncocytoma express an anion exchanger that is immunologically related but not identical to the chloride-bicarbonate anion exchanger of erythrocytes (AE1). In this study, we have mapped the binding site of a monoclonal antibody against erythroid AE1 that does not react with either intercalated cells or oncocytoma. The epitope is located close to the NH2 terminus of AE1, indicating that AE1 in intercalated cells and oncocytoma differs in its NH2 terminus from erythroid AE1. This conclusion was supported by an antibody directed against residues 1-14 of erythroid AE1 that does not react with intercalated cells in oncocytoma. Polymerase chain reaction performed with mRNA from a human kidney revealed that the sequence containing the codons for Met-1 and Met-33 in erythroid mRNA is missing in the kidney transcript, whereas the sequence coding for Met-66 is present. DNA sequence data derived from cloning the 5' end of the human kidney AE1 mRNA clearly showed that the 5' untranslated region comprises part of intron 3, the complete exon 4 that is followed by exon 5 containing Met-66 as the site of translation initiation. Altogether, the results indicate that AE1 in the human kidney is an amino-terminally truncated form of erythroid AE1 that is restricted to the basolateral membrane domain of the acid-secreting intercalated cells of the collecting duct and is also expressed in oncocytoma.
Adenoma, Oxyphilic↗
Replication of Marburg virus in human endothelial cells. A possible mechanism for the development of viral hemorrhagic disease.
Marburg and Ebola virus, members of the family Filoviridae, cause a severe hemorrhagic disease in humans and primates. The disease is characterized as a pantropic virus infection often resulting in a fulminating shock associated with hemorrhage, and death. All known histological and pathophysiological parameters of the disease are not sufficient to explain the devastating symptoms. Previous studies suggested a nonspecific destruction of the endothelium as a possible mechanism. Concerning the important regulatory functions of the endothelium (blood pressure, anti-thrombogenicity, homeostasis), we examined Marburg virus replication in primary cultures of human endothelial cells and organ cultures of human umbilical cord veins. We show here that Marburg virus replicates in endothelial cells almost as well as in monkey kidney cells commonly used for virus propagation. Our data support the concept that the destruction of endothelial cells resulting from Marburg virus replication is a possible mechanism responsible for the hemorrhagic disease and the shock syndrome typical of this infection.
Base Sequence↗
Different sequences of expression of band 3, spectrin, and ankyrin during normal erythropoiesis and erythroleukemia.
Expression of the erythrocyte anion exchanger band 3, and ankyrin and spectrin, two cytoskeletal proteins of the red blood cell membrane, was studied by immunofluorescence using: 1) smears of human bone marrow from healthy donors and from a patient with erythroleukemia, 2) human red blood cell precursors grown in cell culture, and 3) murine erythroleukemia cells grown in cell culture. Double immunostaining with antibodies to band 3 in combination with spectrin or ankyrin revealed that these proteins become expressed synchronously during normal human erythropoiesis. In contrast, both murine erythroleukemia cells (induced by fibronectin and dimethyl sulfoxide to differentiate in vitro) and erythroblasts from a patient suffering from erythroleukemia displayed distinct asynchronicity in expression of these proteins, ie, ankyrin and spectrin were synthesized first, followed by band 3 at a later stage of erythroid development. After the onset of band 3 expression in human erythroleukemia cells, an increase of membrane-associated fluorescence was detectable for both ankyrin and spectrin, supporting the general view that band 3 promotes assembly of the membrane cytoskeleton. These findings indicate that the current concept of a sequential expression of spectrin/ankyrin and band 3 is valid only for erythroleukemia cells or transformed erythropoietic cell lines but does not occur in normal erythropoiesis, during which these proteins become expressed simultaneously.
Animals↗
Human neutrophil elastase increases permeability of cultured pulmonary endothelial cell monolayers.
Polymorphonuclear leukocytes (PMN) contribute to increased pulmonary vascular permeability in inflammatory lung injury, but the mechanism of their action is complex. In the present study we examined possible effects of PMN-derived proteases on the permeability of pulmonary endothelial cell monolayers grown on polycarbonate filter membranes and exposed continuously to a hydrostatic pressure of 10 cm H2O. Cell- and serum free PMN-supernatants (human PMN, stimulated with 30 ng/ml phorbol-myristate acetate for 30 min, presence of catalase, were centrifuged, the supernatants were passed through a 0.45 micron filter) dose-dependently (calculated PMN: endothelial cell ratio of 2:1 and more) increased hydraulic conductivity of endothelial cell monolayers ten- to twentyfold within 20-70 min. At the same time the dextran reflection coefficient decreased from 0.8 to 0.1. Phase contrast and scanning electronmicroscopy showed a widening of intercellular gaps. The effects of the postsecretory PMN-supernatant were blocked dose-dependently by inhibitors of human neutrophil elastase (HNE) but not of cathepsin G. On quantitative grounds highly purified HNE was similarly active as postsecretory PMN supernatant. The effects of HNE were inhibited by pretreatment with eglin-c or heat, but not with heparin. The data suggest that HNE is an effective and sufficient neutrophil-derived mediator to increase endothelial permeability. HNE appears to act primarily enzymatically and not as a cationic protein.
Amino Acid Sequence↗
Cultured rat mesangial cells contain smooth muscle alpha-actin not found in vivo.
A monoclonal antibody against smooth muscle alpha-actin (SM alpha-actin) was used to study the expression of SM alpha-actin in kidney sections and mesangial cell (MC) cultures. In the tissue sections, indirect immunofluorescence revealed intense labeling of vascular smooth muscle cells and precapillary pericytes for SM alpha-actin. Glomerular cells including MC were negative, with the exception of scattered smooth muscle cells in the wall of the intraglomerular segment of the efferent arteriole. In contrast, in MC cultures 50 to 95% of the cells displayed bright fluorescence. Immunoreactivity for SM alpha-actin first appeared 3 days after explanation of glomeruli and increased until the primary culture reached subconfluence. In each subculture (1 to 10) expression of SM alpha-actin was weak on day 1 and pronounced at subconfluence. Growth arrest of subconfluent cultures for 1 to 7 days in serum-free medium did not alter the percentage of cells positive for SM alpha-actin. However, exposure of MC to serum-free medium beginning on the first day of subculture curtailed expression of SM alpha-actin. Double-labeling with antibodies against proliferating cell nuclear antigen and SM alpha-actin revealed SM alpha-actin-positive filaments in both replicating and resting cells. In summary, our results demonstrate that some process or processes associated with cell proliferation and cell growth of MC are accompanied by de novo expression of SM alpha-actin. The relevance to the contractile behavior of the difference in SM alpha-actin expression under in vitro and in vivo conditions is unknown.
Actins↗
Identification of the binding interface involved in linkage of cytoskeletal protein 4.1 to the erythrocyte anion exchanger.
Linkages of the cytoskeleton to integral membrane proteins of the plasma membrane have been shown to be important for diverse cellular functions. The erythrocyte membrane provides the best studied example of how the spectrin-actin based membrane cytoskeleton is linked via two proteins, ankyrin and protein 4.1, to the anion exchanger (anion exchanger 1, AE1). Although these and other types of cytoskeleton-membrane connections have been well documented by in vitro binding studies it has not been possible to establish any of such interactions by defining the binding interface at the amino acid level. In the present study we have performed binding studies between protein 4.1 and AE1 using peptides and corresponding idiotypic and anti-idiotypic antibodies to show that arginine-rich clusters of the cytoplasmic domain of AE1 (IRRRY/LRRRY) serve as a major binding site for a motif with opposite charge and identical hydrophobicity present on the membrane-binding domain of protein 4.1 (LEEDY). Both motifs appear to be highly conserved during evolution and may also be involved in other types of cytoskeleton-membrane association, i.e. in binding of protein 4.1 to the glycophorins.
Amino Acid Sequence↗
Identification of brush cells in the alimentary and respiratory system by antibodies to villin and fimbrin.
Brush cells represent a population of epithelial cells with unknown function, which are scattered throughout the epithelial lining of both the respiratory system and the alimentary system. These cells are reliably distinguished from other epithelial cells only at the ultrastructural level by the presence of an apical tuft of stiff microvilli and extremely long microvillar rootlets that may project down to the perinuclear space. In the present study we show that brush cells can be identified in tissue sections even at the light microscopic level by immunostaining with antibodies against villin and fimbrin, two proteins that crosslink actin filaments to form bundles. In brush cells, villin and fimbrin are not only present in the actin filament core bundles of apical microvilli and their long rootlets but, in addition, both proteins are also associated with microvilli extending from the basolateral cell surface of the brush cells. Basolateral immunostaining specific for villin and fimbrin does not occur in any other epithelial cell type of the respiratory and alimentary tract. Thus immunostaining with antibodies against both proteins allows unequivocal identification of individual brush cells even in sectional planes that do not contain the brightly stained apical tuft of microvilli and their long rootlets.
Animals↗
Pericyte involvement in capillary sprouting during angiogenesis in situ.
To investigate the participation of microvascular pericytes in the process of capillary sprouting, we examined whole-mount preparations of the rat mesentery by use of a double immunofluorescence approach. Angiogenesis was induced by intraperitoneal injections of either the mast cell-degranulating substance compound 48/80 or tumor cell-conditioned medium. Capillary sprouts were visualized by staining with rhodamine-conjugated phalloidin and pericytes were simultaneously stained by an antibody to the intermediate filament protein desmin. Developing pericytes were negative for the smooth-muscle isoform of alpha-actin, but were clearly reactive for desmin. Pericytes appear to be involved in the earliest stages of capillary sprouting. Pericytes were regularly found lying at and in front of the advancing tips of endothelial sprouts. At many sites pericytes were seen to bridge the gap between the leading edges of opposing endothelial sprouts, which were apparently preparing to merge, suggesting that pericytic processes may serve as guiding structures aiding outgrowth of endothelial cells.
Animals↗
Structure and assembly of hemagglutinin mutants of fowl plague virus with impaired surface transport.
Five temperature-sensitive mutants of influenza virus A/FPV/Rostock/34 (H7N1), ts206, ts293, ts478, ts482, and ts651, displaying correct hemagglutinin (HA) insertion into the apical plasma membrane of MDCK cells at the permissive temperature but defective transport to the cell surface at the restrictive temperature, have been investigated. Nucleotide sequence analysis of the HA gene of the mutants and their revertants demonstrated that with each mutant a single amino acid change is responsible for the transport block. The amino acid substitutions were compared with those of mutants ts1 and ts227, which have been analyzed previously (W. Schuy, C. Will, K. Kuroda, C. Scholtissek, W. Garten, and H.-D. Klenk, EMBO J. 5:2831-2836, 1986). With the exception of ts206, the changed amino acids of all mutants and revertants accumulate in three distinct areas of the three-dimensional HA model: (i) at the tip of the 80-A (8-nm)-long alpha helix, (ii) at the connection between the globular region and stem, and (iii) in the basal domain of the stem. The concept that these areas are critical for HA assembly and hence for transport is supported by the finding that the mutants that are unable to leave the endoplasmic reticulum at the nonpermissive temperature do not correctly trimerize. Upon analysis by density gradient centrifugation, cross-linking, and digestion with trypsin and endoglucosaminidase H, two groups can be discriminated among these mutants: with ts1, ts227, and ts478, the HA forms large irreversible aggregates, whereas with ts206 and ts293, it is retained in the monomeric form in the endoplasmic reticulum. With a third group, comprising mutants ts482 and ts651 that enter the Golgi apparatus, trimerization was not impaired.
Amino Acid Sequence↗
Adducin in erythrocyte precursor cells of rats and humans: expression and compartmentalization.
Adducin is a calmodulin-binding protein involved in the assembly of the erythrocyte membrane skeleton. To investigate the expression of adducin during human erythropoiesis, we performed immunofluorescence studies on smears of cultured human erythroblasts. Adducin immunoreactivity was found in the early stages of erythropoiesis. Proerythroblasts were the first erythroid precursor cells positive for adducin. The adducin signal was very similar to the signal of erythroid beta-spectrin in that both proteins lined the membrane of erythroid precursor cells. Cell fractionation experiments were performed to further analyze the intracellular distribution of adducin in erythroid cells. In erythroblasts, about 60% of total cellular adducin appeared in the Triton-soluble fraction. In reticulocytes, the Triton-soluble fraction decreased to 30% of total reticulocyte adducin. Erythrocytes had no detectable amount of adducin in the Triton-soluble pool. Instead, adducin was quantitatively bound to the Triton-insoluble erythrocyte cytoskeleton. Our results suggest that adducin is expressed early in the development of the erythrocyte membrane skeleton, whereas stable assembly onto the membrane skeleton does not occur before the final stages of mammalian erythropoiesis.
Animals↗
Influence of Ca2+ on synaptic morphology of fish cone photoreceptors.
Isolated fish retinas were incubated with the Ca(2+)-ionophore A23187 at various Ca2+ concentrations. Computer reconstructions of the ultrastructure of cone synapses revealed pronounced reduction of the presynaptic surface (complete loss of the synaptic invaginations) in response to low intracellular Ca2+ (less than 10(-8) M). No obvious effect on synaptic morphology was noticed at Ca2+ greater than 10(-8) M. Ca(2+)-dependent exocytotic activity appears to be a major factor controlling the invaginated morphology of cone synapses.
Animals↗
Immunocytochemical analysis of the cytoskeleton of the human amniotic epithelium.
The amniotic epithelium constitutes a diffusion barrier controlling the passage of solutes and water between the amniotic cavity and maternal circulation. With the present immunocytochemical approach, we have shown that several major components of the cyto-skeleton, i.e., actin, alpha-actinin, spectrin and ezrin, are preferentially associated with the apical and lateral cell surfaces of the human amniotic epithelium. Keratins are distributed throughout the entire cytoplasm, whereas vimentin mainly forms a perinuclear scaffold. These findings indicate a role of the various components of the cytoskeleton in the structural integrity and modulation of cell shape and junctional permeability.
Actinin↗
Plasmodium falciparum: analysis of the interaction of antigen Pf155/RESA with the erythrocyte membrane.
The location of the Plasmodium falciparum vaccine candidate antigen Pf155/RESA in the membrane of infected erythrocytes was analzyed by means of selective surface radioiodination and immunofluorescence of surface-modified cells. The lack of radiolabel in Pf155/RESA as well as its localization by immunofluorescence similar to that of the N-terminal region of erythrocyte band 3 suggests that the antigen is associated with the cytoplasmic phase of the erythrocyte membrane. In concordance with this, Pf155/RESA was detected by immunofluorescence on the surface of inside out membrane vesicles from P. falciparum-infected erythrocytes. Pf155/RESA from spent culture medium also bound to inside out membrane vesicles of normal erythrocytes as well as to cytoskeletal shells of such vesicles, but failed to bind to sealed right-side out membrane vesicles. Depletion of spectrin from the vesicles abolished antigen binding, suggesting that Pf155/RESA association with the erythrocyte cytoskeleton is mediated by spectrin.
Amino Acid Sequence↗