scholarly journals Plastin 1 Binds to Keratin and Is Required for Terminal Web Assembly in the Intestinal Epithelium

2009 ◽  
Vol 20 (10) ◽  
pp. 2549-2562 ◽  
Author(s):  
Eva-Maria S. Grimm-Günter ◽  
Céline Revenu ◽  
Sonia Ramos ◽  
Ilse Hurbain ◽  
Neil Smyth ◽  
...  
Keyword(s):  
Brush Border ◽  
Actin Filaments ◽  
Keratin 19 ◽  
Terminal Web ◽  
Intestinal Brush Border ◽  
Important Regulator ◽  
Increased Sensitivity ◽  
Filament Network ◽  
Deficient Mice

Plastin 1 (I-plastin, fimbrin) along with villin and espin is a prominent actin-bundling protein of the intestinal brush border microvilli. We demonstrate here that plastin 1 accumulates in the terminal web and interacts with keratin 19, possibly contributing to anchoring the rootlets to the keratin network. This prompted us to investigate the importance of plastin 1 in brush border assembly. Although in vivo neither villin nor espin is required for brush border structure, plastin 1-deficient mice have conspicuous ultrastructural alterations: microvilli are shorter and constricted at their base, and, strikingly, their core actin bundles lack true rootlets. The composition of the microvilli themselves is apparently normal, whereas that of the terminal web is profoundly altered. Although the plastin 1 knockout mice do not show any overt gross phenotype and present a normal intestinal microanatomy, the alterations result in increased fragility of the epithelium. This is seen as an increased sensitivity of the brush border to biochemical manipulations, decreased transepithelial resistance, and increased sensitivity to dextran sodium sulfate-induced colitis. Plastin 1 thus emerges as an important regulator of brush border morphology and stability through a novel role in the organization of the terminal web, possibly by connecting actin filaments to the underlying intermediate filament network.

Loss of the NHE2 Na+/H+ exchanger has no apparent effect on diarrheal state of NHE3-deficient mice

2001 ◽  
Vol 281 (6) ◽  
pp. G1385-G1396 ◽  
Author(s):  
Clara Ledoussal ◽  
Alison L. Woo ◽  
Marian L. Miller ◽  
Gary E. Shull
Keyword(s):  
Absorptive Capacity ◽  
Apparent Effect ◽  
Acid Base ◽  
Intestinal Brush Border ◽  
Apparent Reduction ◽  
Acid Base Status ◽  
Deficient Mice ◽  
Additional Loss ◽  
Partial Compensation

The expression of NHE2 and NHE3 on intestinal-brush border membranes suggests that both Na+/H+ exchangers serve absorptive functions. Studies with knockout mice showed that the loss of NHE3, but not NHE2, causes diarrhea, demonstrating that NHE3 is the major absorptive exchanger and indicating that any remaining absorptive capacity contributed by NHE2 is not sufficient to compensate fully for the loss of NHE3. To test the hypothesis that NHE2 provides partial compensation for the diarrheal state of NHE3-deficient mice, we crossed doubly heterozygous mice carrying null mutations in the Nhe2and Nhe3 genes and analyzed the phenotypes of their offspring. The additional loss of NHE2 in NHE3-deficient mice caused no apparent reduction in viability, no further impairment of systemic acid-base status or increase in aldosterone levels, and no apparent worsening of the diarrheal state. These in vivo phenotypic correlates of the absorptive defect suggest that the NaCl, HCO[Formula: see text], and fluid absorption that is dependent on apical Na+/H+ exchange is due overwhelmingly to the activity of NHE3, with little contribution from NHE2.

scholarly journals Organization of the actin filament cytoskeleton in the intestinal brush border: a quantitative and qualitative immunoelectron microscope study.

1988 ◽  
Vol 107 (3) ◽  
pp. 1037-1048 ◽  
Author(s):  
D Drenckhahn ◽  
R Dermietzel
Keyword(s):  
Brush Border ◽  
Actin Filaments ◽  
Close Association ◽  
Zonula Occludens ◽  
Terminal Web ◽  
Ultrathin Sections ◽  
Microfilament System ◽  
Human Intestinal Epithelium ◽  
Insight Into ◽  
Specific Label

In the present study we have used immunogold labeling of ultrathin sections of the intact chicken and human intestinal epithelium to obtain further insight into the molecular structure of the brush-border cytoskeleton. Actin, villin, and fimbrin were found within the entire microvillus filament bundle, from the tip to the basal end of the rootlets, but were virtually absent from the space between the rootlets. This suggests that the bulk of actin in the brush border is kept in a polymerized and cross-linked state and that horizontally deployed actin filaments are virtually absent. About 70% of the label specific for the 110-kD protein that links the microvillus core bundle to the lipid bilayer was found overlying the microvilli. The remaining label was associated with rootlets and the interrootlet space, where some label was regularly observed in association with vesicles. Since the terminal web did not contain any significant amounts of tubulin and microtubules, the present findings would support a recently proposed hypothesis that the 110-kD protein (which displays properties of an actin-activated, myosin-like ATPase) might also be involved in the transport of vesicles through the terminal web. Label specific for myosin and alpha-actinin was confined to the interrootlet space and was absent from the rootlets. About 10-15% of the myosin label and 70-80% of the alpha-actinin label was observed within the circumferential band of actin filaments at the zonula adherens, where myosin and alpha-actinin displayed a clustered, interrupted pattern that resembles the spacing of these proteins observed in other contractile systems. This circular filament ring did not contain villin, fimbrin, or the 110-kD protein. Finally, actin-specific label was observed in close association with the cytoplasmic aspect of the zonula occludens, suggesting that tight junctions are structurally connected to the microfilament system.

Turnover of intestinal brush-border proteins during postnatal development in rat

1980 ◽  
Vol 239 (6) ◽  
pp. G524-G531
Author(s):  
B. Seetharam ◽  
K. Y. Yeh ◽  
D. H. Alpers
Keyword(s):  
Postnatal Development ◽  
Brush Border ◽  
Cathepsin B ◽  
Leucine Incorporation ◽  
Turnover Rates ◽  
Distal Intestine ◽  
Rapid Turnover ◽  
Intestinal Brush Border ◽  
Proximal Intestine

Protein turnover in brush-border membranes of rats during postnatal development has been studied by the double isotope technique. Unlike adult animals where only large proteins (mol wt > 140,000) show relatively rapid turnover, most brush-border proteins in 12-day-old rats show high 3H-to-14C ratios of leucine incorporation, consistent with rapid turnover. Lysosomal proteases, including cathepsin B, are partly responsible for this rapid turnover. This conclusion is based on the following findings: 1) In vivo treatment of 12-day-old animals with leupeptin, an inhibitor of cathepsin B, alters relative turnover rates, enzyme activity, and content of many brush-border proteins. Activities of maltase and trehalase rise while lactase falls. 2) Cathepsin B activity falls rapidly in intestine after the animals are 16 days of age, at a time when luminal pancreatic proteases are rising. Moreover, cathepsin B activity shows less latency in distal intestine at 12 and 16 days than at later ages or in proximal intestine. It is suggested that during postnatal development lysosomal enzymes, e.g., cathepsin B, play an important role in the turnover of intestinal brush-border proteins.

scholarly journals Increased Severity of Local and Systemic Anaphylactic Reactions in Gp49b1-Deficient Mice

2001 ◽  
Vol 194 (2) ◽  
pp. 227-234 ◽  
Author(s):  
Massoud Daheshia ◽  
Daniel S. Friend ◽  
Michael J. Grusby ◽  
K. Frank Austen ◽  
Howard R. Katz
Keyword(s):  
Mast Cell ◽  
Cell Activation ◽  
Mast Cell Activation ◽  
Critical Question ◽  
Increased Sensitivity ◽  
Antibody Levels ◽  
Deficient Mice

gp49B1 is an immunoglobulin (Ig) superfamily member that inhibits FcεRI-induced mast cell activation when the two receptors are coligated with antibodies in vitro. The critical question of in vivo function of gp49B1 is now addressed in gene-disrupted mice. gp49B1-deficient mice exhibited a significantly increased sensitivity to IgE-dependent passive cutaneous anaphylaxis as assessed by greater tissue swelling and mast cell degranulation in situ. Importantly, by the same criteria, the absence of gp49B1 also resulted in a lower threshold for antigen challenge in active cutaneous anaphylaxis, in which the antigen-specific antibody levels were comparable in gp49B1-deficient and sufficient mice. Moreover, the absence of gp49B1 resulted in a significantly greater and faster death rate in active systemic anaphylaxis. These results indicate that gp49B1 innately dampens adaptive immediate hypersensitivity responses by suppressing mast cell activation in vivo. In addition, this study provides a new concept and target for regulation of allergic disease susceptibility and severity.

scholarly journals Reduced DOCK4 expression leads to erythroid dysplasia in myelodysplastic syndromes

2015 ◽  
Vol 112 (46) ◽  
pp. E6359-E6368 ◽  
Author(s):  
Sriram Sundaravel ◽  
Ryan Duggan ◽  
Tushar Bhagat ◽  
David L. Ebenezer ◽  
Hui Liu ◽  
...  
Keyword(s):  
Myelodysplastic Syndromes ◽  
Actin Filaments ◽  
Poor Prognosis ◽  
Its Gene ◽  
Rac1 Gtpase ◽  
Gtpase Activation ◽  
Filament Network ◽  
Gene Maps

Anemia is the predominant clinical manifestation of myelodysplastic syndromes (MDS). Loss or deletion of chromosome 7 is commonly seen in MDS and leads to a poor prognosis. However, the identity of functionally relevant, dysplasia-causing, genes on 7q remains unclear. Dedicator of cytokinesis 4 (DOCK4) is a GTPase exchange factor, and its gene maps to the commonly deleted 7q region. We demonstrate that DOCK4 is underexpressed in MDS bone marrow samples and that the reduced expression is associated with decreased overall survival in patients. We show that depletion of DOCK4 levels leads to erythroid cells with dysplastic morphology both in vivo and in vitro. We established a novel single-cell assay to quantify disrupted F-actin filament network in erythroblasts and demonstrate that reduced expression of DOCK4 leads to disruption of the actin filaments, resulting in erythroid dysplasia that phenocopies the red blood cell (RBC) defects seen in samples from MDS patients. Reexpression of DOCK4 in −7q MDS patient erythroblasts resulted in significant erythropoietic improvements. Mechanisms underlying F-actin disruption revealed that DOCK4 knockdown reduces ras-related C3 botulinum toxin substrate 1 (RAC1) GTPase activation, leading to increased phosphorylation of the actin-stabilizing protein ADDUCIN in MDS samples. These data identify DOCK4 as a putative 7q gene whose reduced expression can lead to erythroid dysplasia.

Characterization and modulation of rat small intestinal brush-border membrane transbilayer fluidity

1991 ◽  
Vol 260 (4) ◽  
pp. G586-G594
Author(s):  
P. K. Dudeja ◽  
R. K. Wali ◽  
J. M. Harig ◽  
T. A. Brasitus
Keyword(s):  
Brush Border ◽  
Brush Border Membrane ◽  
Leucine Aminopeptidase ◽  
Aminopeptidase Activity ◽  
Intestinal Brush Border Membrane ◽  
Intestinal Brush Border ◽  
Leucine Transport ◽  
Small Intestinal

In the present experiments, selective quenching by trinitrophenyl groups as well as steady-state fluorescence polarization and differential polarized phase fluorescence techniques, using three different lipid soluble fluorophores, were used to directly examine the fluidity of the exofacial and cytofacial leaflets of rat small intestinal brush-border membranes. These studies revealed that the fluidity of the exofacial hemileaflet was greater than the cytofacial hemileaflet. Differences in the distribution of phosphatidylcholine and phosphatidylethanolamine, as assessed by phospholipase A2 treatment and trinitrophenylation of aminophospholipids, were, at least partially, responsible for the asymmetrical fluidity of the hemileaflets. Moreover, in vitro addition of benzyl alcohol (final concn 25 mM) preferentially fluidized the exofacial leaflet and concomitantly decreased leucine aminopeptidase activity but did not affect the activities of maltase, sucrase, alkaline phosphatase, or gamma-glutamyltranspeptidase. In vivo addition of the membrane-mobility agent 2-(2-methoxyethoxy)ethyl 8-(cis-2-n-octylcyclopropyl)octanate] (A2C) (final concn 7.5 microM) preferentially fluidized the cytofacial leaflet and increased Na(+)-gradient-dependent D-glucose transport but not Na(+)-gradient-dependent L-leucine transport.

scholarly journals Localization of actin and microfilament-associated proteins in the microvilli and terminal web of the intestinal brush border by immunofluorescence microscopy.

1978 ◽  
Vol 79 (3) ◽  
pp. 839-845 ◽  
Author(s):  
A Bretscher ◽  
K Weber
Keyword(s):  
Epithelial Cells ◽  
Indirect Immunofluorescence ◽  
Brush Border ◽  
Immunofluorescence Microscopy ◽  
Intestinal Epithelial Cells ◽  
Intestinal Epithelial ◽  
Indirect Immunofluorescence Microscopy ◽  
Terminal Web ◽  
Intestinal Brush Border ◽  
Associated Proteins

Indirect immunofluorescence microscopy was used to localize microfilament-associated proteins in the brush border of mouse intestinal epithelial cells. As expected, antibodies to actin decorated the microfilaments of the microvilli, giving rise to a very intense fluorescence. By contrast, antibodies to myosin, tropomyosin, filamin, and alpha-actinin did not decorate the microvilli. All these antibodies, however, decorated the terminal web region of the brush border. Myosin, tropomyosin, and alpha-actinin, although present throughout the terminal web, were found to be preferentially located around the periphery of the organelle. Therefore, two classes of microfilamentous structures can be documented in the brush border. First, the highly ordered microfilaments which make up the cores of the microvilli apparently lack the associated proteins. Second, seemingly less-ordered microfilaments are found in the terminal web, in which region the myosin, tropomyosin, filamin and alpha-actinin are located.

Regional specificity of iron uptake by small intestinal brush-border membranes from normal and iron-deficient mice

1985 ◽  
Vol 248 (3) ◽  
pp. G376-G379 ◽  
Author(s):  
A. Muir ◽  
U. Hopfer
Keyword(s):  
Brush Border ◽  
Iron Uptake ◽  
Iron Absorption ◽  
Membrane Vesicles ◽  
Soluble Form ◽  
Brush Border Membranes ◽  
Intestinal Brush Border ◽  
Iron Deficient ◽  
Small Intestinal ◽  
Deficient Mice

Fe(II)-ascorbate uptake by purified small intestinal brush-border membrane vesicles prepared from proximal and distal segments was studied in normal and iron-deficient mice. Iron was maintained in a reduced, soluble form by a 20-fold excess of ascorbate at a physiological pH of 7.2-7.4. In normal mice, iron uptake by proximal membrane vesicles was three- to fourfold greater (approximately 1,700 pmol/mg prot) than from distal segments (approximately 500 pmol/mg prot). In iron-deficient mice, uptake of Fe(II) was also greater in proximal membranes (approximately 3,200 pmol/mg prot) than uptake from distal segments (approximately 350 pmol/mg prot), and the regional difference was almost 10-fold, without any change in distal segmental iron uptake. These results are consistent with the pattern of intestinal iron absorption in iron-replete and iron-deficient animals and indicate that regulatory changes in proximal intestinal brush-border membranes may account for the increased iron absorption known to occur in iron deficiency.

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