scholarly journals Localization of capping protein in chicken epithelial cells by immunofluorescence and biochemical fractionation.

1992 ◽  
Vol 118 (2) ◽  
pp. 335-346 ◽  
Author(s):  
D A Schafer ◽  
M S Mooseker ◽  
J A Cooper
Keyword(s):  
Epithelial Cells ◽  
Actin Filaments ◽  
Intestinal Epithelial Cells ◽  
Sensory Epithelium ◽  
Junctional Complex ◽  
Intestinal Epithelial ◽  
Capping Protein ◽  
Pigmented Epithelium ◽  
Biochemical Fractionation ◽  
Junctional Complexes

We have localized capping protein in epithelial cells of several chicken tissues using affinity-purified polyclonal antibodies and immunofluorescence. Capping protein has a distribution in each tissue coincident with proteins of the cell-cell junctional complex, which includes the zonula adherens, zonula occludens, and desmosome. "En face" views of the epithelial cells showed capping protein distributed in a polygonal pattern coincident with cell boundaries in intestinal epithelium, sensory epithelium of the cochlea, and the pigmented epithelium of the retina and at regions of cell-cell contact between chick embryo kidney cells in culture. "Edge-on" views obtained by confocal microscopy of intact single intestinal epithelial cells and of retinal pigmented epithelium showed that capping protein is located in the apical region of the epithelial cells coincident with the junctional complexes. These images do not resolve the individual types of junctions of the junctional complex. Immunolabeling of microvilli or stereocilia was faint or not detectable. Capping protein was also detected in the cytoplasm of intact intestinal epithelial cells and in nuclei of cells in the pigmented retina and in the kidney cell cultures, but not in nuclei of cells of the intestinal epithelium or sensory epithelium. Biochemical fractionation of isolated intestinal epithelial cells shows capping protein in the brush border fraction, which contains the junctional complexes, and in the soluble fraction. These results are consistent with the results of the immunolabeling experiments. Highly purified microvilli of the brush borders also contained capping protein; this result was unexpected based on the low intensity of immunofluorescence staining of microvilli and stereocilia. The microvilli were not contaminated with junctional complexes, as defined by the absence of several markers for cell junctions. The cause and significance of this discrepancy is not certain at this time. Since capping protein binds the barbed end of actin filaments in vitro, we hypothesize that capping protein is bound to the barbed ends of actin filaments associated with one or more of the junctions of the junctional complex.

scholarly journals alpha-Actinin localization in the junctional complex of intestinal epithelial cells.

1979 ◽  
Vol 80 (1) ◽  
pp. 203-210 ◽  
Author(s):  
S W Craig ◽  
J V Pardo
Keyword(s):  
Epithelial Cells ◽  
Intestinal Epithelial Cells ◽  
Sodium Dodecyl ◽  
Junctional Complex ◽  
Intestinal Epithelial ◽  
Membrane Structures ◽  
Apical Portion ◽  
Chicken Gizzard ◽  
Subunit Molecular Weight ◽  
Polygonal Pattern

We have used antibody to chicken gizzard alpha-actinin to identify and localize this molecule in chicken intestinal epithelium. The antibody binds only to alpha-actinin when tested against a crude extract of chicken gizzard. Extracts of purified epithelial cells contain a molecule which has a subunit molecular weight of 100,000 on sodium dodecyl sulphate gels and which is able to inhibit the interaction of alpha-actinin antibody and 125I-labeled chicken gizzard alpha-actinin. By indirect immunofluorescence, alpha-actinin is localized in the apical portion of chicken intestinal epithelial cells. Ethanol-fixed cryostat sections of intestine taken through the apical portion of the epithelial cells and in a plane perpendicular to the long axis of the cells show that alpha-actinin is organized in a polygonal pattern which corresponds to the outlines of the polygonally packed epithelial cells. We interpret the data as indicating that alpha-actinin is a component of the tight junction (zonula occludens) and/or the belt desmosome (zonula adherens), both of which are membrane structures known to encircle the cell and to be confined to its apical portion.

scholarly journals The spectrin-related molecule, TW-260/240, cross-links the actin bundles of the microvillus rootlets in the brush borders of intestinal epithelial cells.

1983 ◽  
Vol 96 (5) ◽  
pp. 1491-1496 ◽  
Author(s):  
J R Glenney ◽  
P Glenney ◽  
K Weber
Keyword(s):  
Epithelial Cells ◽  
Actin Filaments ◽  
Intestinal Epithelial Cells ◽  
Intestinal Epithelial ◽  
Cross Link ◽  
Actin Bundles ◽  
Terminal Web ◽  
Brush Borders ◽  
Cross Links

Previous studies have shown that molecules related to erythrocyte spectrin are present in the cortical cytoplasm of nonerythroid cells. We report here the localization by immunoelectron microscopy of one such molecule, TW-260/240, in the brush border of intestinal epithelial cells. Using highly specific antibodies against TW-260 and TW-240 as well as antibodies against fodrin, another spectrinlike molecule, we have found that the TW-260/240 molecules are displayed between rootlets at all levels of the terminal web. Occasionally, extended structures appear labeled suggestive of the fine filaments known to cross-link actin bundles. These results are in line with previous in vitro studies showing that TW-260/240 binds to, and cross-links, actin filaments. The results are discussed in terms of a model in which rootlets are immobilized in the terminal web in a matrix of TW-260/240.

scholarly journals Organization of an actin filament-membrane complex. Filament polarity and membrane attachment in the microvilli of intestinal epithelial cells.

1975 ◽  
Vol 67 (3) ◽  
pp. 725-743 ◽  
Author(s):  
M S Mooseker ◽  
L G Tilney
Keyword(s):  
Epithelial Cells ◽  
Actin Filament ◽  
Brush Border ◽  
Actin Filaments ◽  
Functional Organization ◽  
Intestinal Epithelial Cells ◽  
Intestinal Epithelial ◽  
Membrane Complex ◽  
Brush Borders ◽  
Filament Bundles

The association of actin filaments with membranes is now recognized as an important parameter in the motility of nonmuscle cells. We have investigated the organization of one of the most extensive and highly ordered actin filament-membrane complexes in nature, the brush border of intestinal epithelial cells. Through the analysis of isolated, demembranated brush borders decorated with the myosin subfragment, S1, we have determined that all the microvillar actin filaments have the same polarity. The S1 arrowhead complexes point away from the site of attachment of actin filaments at the apical tip of the microvillar membrane. In addition to the end-on attachment of actin filaments at the tip of the microvillus, these filaments are also connected to the plasma membrane all along their lengths by periodic (33 nm) cross bridges. These bridges were best observed in isolated brush borders incubated in high concentrations of Mg++. Their visibility is attributed to the induction of actin paracrystals in the filament bundles of the microvilli. Finally, we present evidence for the presence of myosinlike filaments in the terminal web region of the brush border. A model for the functional organization of actin and myosin in the brush border is presented.

scholarly journals Endocytosis and Recycling of Tight Junction Proteins in Inflammation

2010 ◽  
Vol 2010 ◽  
pp. 1-6 ◽  
Author(s):  
Markus Utech ◽  
Rudolf Mennigen ◽  
Matthias Bruewer
Keyword(s):  
Epithelial Cells ◽  
Intestinal Epithelial Cells ◽  
Junctional Complex ◽  
Intestinal Epithelial ◽  
Tight Junction Proteins ◽  
Critical Function ◽  
Recycling Endosomes ◽  
Inflammatory Conditions ◽  
Apical Junctional Complex ◽  
Junction Proteins

A critical function of the epithelial lining is to form a barrier that separates luminal contents from the underlying interstitium. This barrier function is primarily regulated by the apical junctional complex (AJC) consisting of tight junctions (TJs) and adherens junctions (AJs) and is compromised under inflammatory conditions. In intestinal epithelial cells, proinflammatory cytokines, for example, interferon-gamma (IFN-γ), induce internalization of TJ proteins by endocytosis. Endocytosed TJ proteins are passed into early and recycling endosomes, suggesting the involvement of recycling of internalized TJ proteins. This review summarizes mechanisms by which TJ proteins under inflammatory conditions are internalized in intestinal epithelial cells and point out comparable mechanism in nonintestinal epithelial cells.

scholarly journals The occurrence and distribution of H-2 antigens on mouse intestinal epithelial cells.

1979 ◽  
Vol 27 (3) ◽  
pp. 746-750 ◽  
Author(s):  
W N Kirby ◽  
E L Parr
Keyword(s):  
Epithelial Cells ◽  
Intestinal Epithelial Cells ◽  
Junctional Complex ◽  
Apical Surface ◽  
Intestinal Epithelial ◽  
Histocompatibility Antigens ◽  
Dissociated Cells ◽  
Membrane Components ◽  
Apical Membranes ◽  
Transplantation Antigens

This report describes an immunoferritin labeling study of mouse H-2 histocompatibility antigens on epithelial cells dissociated from the small intestine by EDTA and trypsin. Before cell dissociation, the intestine was prefixed in paraformaldehyde or periodate-lysine-paraformaldehyde in order to preserve the shape of the cells and to immobilize H-2 antigens in their native positions. The results demonstrated the presence of H-2 antigens on the lateral and basal cell membranes at about the same high density that was observed at the surface of mouse monocytes. No H-2 antigens could be detected at the apical surface of dissociated or undissociated epithelial cells. It is unlikely that the fuzzy coat masked H-2 antigens at the apical surface because it was essentially absent from the apical membranes of dissociated cells. These observations extend our knowledge of the cellular distribution of transplantation antigens, and provide further evidence of a discontinuity in the expression of membrane components at the junctional complex of epithelial cells.

scholarly journals Regulation of microvillus structure: calcium-dependent solation and cross-linking of actin filaments in the microvilli of intestinal epithelial cells.

1980 ◽  
Vol 87 (3) ◽  
pp. 809-822 ◽  
Author(s):  
M S Mooseker ◽  
T A Graves ◽  
K A Wharton ◽  
N Falco ◽  
C L Howe
Keyword(s):  
Epithelial Cells ◽  
Actin Filaments ◽  
High Speed ◽  
Core Protein ◽  
Intestinal Epithelial Cells ◽  
Gel Filtration ◽  
Intestinal Epithelial ◽  
Oligomeric State ◽  
Core Proteins ◽  
Presence And Absence

The bundle of filaments within microvilli of intestinal epithelial cells contains five major proteins including actin, calmodulin, and subunits of 105-, 95-, and 70-kdaltons. It has been previously shown (Howe, C. L., M. S. Mooseker, and T. A. Graves. 1980. Brush-border calmodulin: a major component of the isolated microvillus core. J. Cell Biol. 85: 916-923) that the addition of Ca++ (> 10(-6) M) to microvillus cores causes a rapid, drastic, but at least partially reversible disruption of this actin filament bundle. High-speed centrifugation of microvillus cores treated with Ca++ indicates that several core proteins are solubilized, including 30-50% of the actin and calmodulin, along with much of the 95- and 70-kdalton subunits. Gel filtration of such Ca++ extracts in the presence and absence of Ca++ indicates that microvillar actin "solated" by Ca++ is in an oligomeric state probably complexed with the 95-kdalton subunit. Removal of Ca++ results in the reassembly of F-actin, probably still complexed with 95-kdalton subunit, as determined by gel filtration, cosedimentation, viscometry, and electron microscopy. The 95-kdalton subunit (95K) was purified from Ca++ extracts by DEAE-Sephadex chromatography and its interaction with actin characterized by viscometry, cosedimentation, and EM in the presence and absence of Ca++. In the presence, but not absence, of Ca++, 95K inhibits actin assembly (50% inhibition at 1:50-60 95K to actin) and also reduces the viscosity of F-actin solutions. Similarly, sedimentation of actin is inhibited by 95K, but a small, presumably oligomeric actin- 95K complex formed in the presence of Ca++ is pelletable after long-term centrifugation. In the absence of Ca++, 95K cosediments with F-actin. EM of 95K-actin mixtures reveals that 95K "breaks" actin into small, filamentous fragments in the presence of Ca++. Reassembly of filaments occurs once Ca++ is removed. In the absence of Ca++, 95K has no effect on filament structure and, at relatively high ratios (1:2-6) of 95K to actin, this core protein will aggregate actin filaments into bundles.

scholarly journals Organization of actin, myosin, and intermediate filaments in the brush border of intestinal epithelial cells.

1982 ◽  
Vol 94 (2) ◽  
pp. 425-443 ◽  
Author(s):  
N Hirokawa ◽  
L G Tilney ◽  
K Fujiwara ◽  
J E Heuser
Keyword(s):  
Epithelial Cells ◽  
Intermediate Filaments ◽  
Actin Filaments ◽  
Intestinal Epithelial Cells ◽  
Intestinal Cell ◽  
Intestinal Epithelial ◽  
Replication Method ◽  
Terminal Web ◽  
Filament Bundles ◽  
Lateral Margins

Terminal webs prepared from mouse intestinal epithelial cells were examined by the quick-freeze, deep-etch, and rotary-replication method. The microvilli of these cells contain actin filaments that extend into the terminal web in compact bundles. Within the terminal web these bundles remain compact; few filaments are separated from the bundles and fewer still bend towards the lateral margins of the cell. Decoration with subfragment 1 (S1) of myosin confirmed that relatively few actin filaments travel horizontally in the web. Instead, between actin bundles there are complicated networks of the fibrils. Here we present two lines of evidence which suggest that myosin is one of the major cross-linkers in the terminal web. First, when brush borders are exposed to 1 mM ATP in 0.3 M KCl, they lose their normal ability to bind antimyosin antibodies as judged by immunofluorescence, and they lose the thin fibrils normally found in deep-etch replicas. Correspondingly, myosin is released into the supernatant as judged by SDS gel electrophoresis. Second, electron microscope immunocytochemistry with antimyosin antibodies followed by ferritin-conjugated second antibodies leads to ferritin deposition mainly on the fibrils at the basal part of rootlets. Deep-etching also reveals that the actin filament bundles are connected to intermediate filaments by another population of cross-linkers that are not extracted by ATP in 0.3 M KCl. From these results we conclude that myosin in the intestinal cell may not only be involved in a short range sliding-filament type of motility, but may also play a purely structural role as a long range cross-linker between microvillar rootlets.

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