QUICK SEARCH:   [advanced] Author: Keyword(s):
Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents

First published online 22 January 2008
doi: 10.1242/jcs.020800

This Article Summary Full Text Full Text (PDF) Supplementary Material Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Related articles in JCS Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Delacour, D. Articles by Jacob, R. Search for Related Content PubMed PubMed Citation Articles by Delacour, D. Articles by Jacob, R. Social Bookmarking                         What's this?

Loss of galectin-3 impairs membrane polarisation of mouse enterocytes in vivo

Delphine Delacour^1,*, Annett Koch^1,*, Waltraud Ackermann¹, Isabelle Eude-Le Parco², Hans-Peter Elsässer¹, Francoise Poirier² and Ralf Jacob^1,

¹ Department of Cell Biology and Cell Pathology, Philipps University, D-35037 Marburg, Germany
² Department of Development, Institut Jacques Monod, CNRS UMR 7592, Universités Paris 6 and Paris 7, Cedex 05 Paris, France

View larger version (60K): [in this window] [in a new window]   Fig. 1. Galectin-3 distribution in the mouse small intestine. The distribution of galectin-3 and villin in murine small intestine sections was analysed by immunohistochemistry with polyclonal antibodies directed against galectin-3 (Alexa Fluor 546, red) and monoclonal anti-villin antibodies (Alexa Fluor 488, green). (A) Overview, showing the gradual increase in galectin-3 expression along the crypt-villus axis, whereas villin distributes uniformly at the brush border membrane. Arrowheads indicate goblet cells. Enlarged view of villus tip (B) and crypt area (C). Nuclear counterstaining with Hoechst 33342 is blue. Scale bars: 40 µm (A) and 20 µm (B,C).

View larger version (101K): [in this window] [in a new window]   Fig. 2. Distribution of brush border hydrolases in wt and gal3–/– mice. Transverse sections of small intestines of wt (gal3+/+) and gal3-null mutant (gal3–/–) mice were stained with antibodies against LPH, DPPIV, SI (Alexa Fluor 488, green) and APN (Alexa Fluor 546, red). Cross sections were taken from the upper third of the villi. Higher magnifications of two adjacent brush border domains are shown in the bottom half of each panel. Hoechst 33342 nuclear counterstaining is blue. Scale bars: 20 µm.

View larger version (45K): [in this window] [in a new window]   Fig. 3. Interaction of brush border hydrolases with galectin-3 and their association with detergent-resistant membranes. (A) Binding of galectin-3 to hydrolases was assessed by coimmunoprecipitation from small intestinal lysates, using the corresponding antibodies for LPH, DPPIV, SI and APN. Precipitates were subjected to SDS-PAGE and immunoblot analysis with a galectin-3 antibody. When indicated, the coimmunoprecipitation was performed in the presence of galactose (0.3 M) or lactose (0.1 M). (B) Crude membrane pellets of small intestine were extracted with 1% Triton X-100 and separated on a Nycodenz density gradient. Fractions were collected from the top of the gradient (1-12) and subsequently analysed by immunoblot using antibodies against LPH, DPPIV, SI and APN. The distribution of floating flotillin-1 indicates detergent-resistant membrane (DRM)-containing fractions.

View larger version (107K): [in this window] [in a new window]   Fig. 4. Distribution of giantin, Lamp1, Rab8 and Rab11 in enterocytes from wt and gal3–/– mice. Transverse sections of small intestines of wt (gal3+/+) and gal3-null mutant (gal3–/–) mice were stained with antibodies against giantin (Alexa Fluor 546; red), Lamp1 (Alexa Fluor 488; green), Rab8 (Alexa Fluor 488; green) and Rab11 (Alexa Fluor 546; red). Cross sections were taken from the upper third of the villi. Higher magnifications of two adjacent brush border domains are shown in the bottom half of each panel. Hoechst 33342 nuclear counterstaining is blue. Scale bars: 20 µm.

View larger version (179K): [in this window] [in a new window]   Fig. 5. Morphological changes in gal3–/– enterocytes. Electron microscopy of enterocytes from wt (gal3+/+) and galectin-3 null mutant (gal3–/–) mice. Numerous intracellular vesicles and vacuoles are visible in some enterocytes of gal3–/– mice (B,E). Higher magnifications of the brush border and tight junction area (D,E,F) show no differences between wt and gal3–/– mice. Lateral membranes of enterocytes from wt mice depict typical interdigitations (G,H), whereas in gal3–/– mice, numerous infoldings and protrusions (I-L, arrows) are visible. The intracellular space between two adjacent cells is indicated by arrowheads. In M-O the focus is on the basal part of the basolateral membrane. Infoldings (arrows) are seen in the enterocytes of gal3–/– mice. Scale bars: 2 µm.

View larger version (172K): [in this window] [in a new window]   Fig. 6. Localisation of LPH in wt and gal3–/– mouse enterocytes. Fixation of the tissues samples was performed as described by Elsasser et al. (Elsasser et al., 1993). Antibody binding was visualised by incubation with a monoclonal anti-LPH antibody and 10 nm immunogold-conjugated goat anti-mouse IgG antibody, as indicated by arrowheads. (A) In the absence of anti-LPH, no staining was observed. (B,C) Antibody staining of brush borders from wt mice. Enterocytes from gal3–/– mice revealed intense LPH labelling of accumulated intracellular vesicles as well as brush border staining (B). This is also visible in the higher magnifications of intracellular vesicles (G,H), whereas minor intracellular LPH staining could be detected in enterocytes from wt mice (D,E). Scale bars: 2 µm.

View larger version (107K): [in this window] [in a new window]   Fig. 7. Distribution of basolateral and apical markers. Cross-sections of small intestine villi of wt (gal3+/+) and gal3-null mutant (gal3–/–) mice stained with antibodies against apical markers villin (Alexa Fluor 488, green), β-actin and ezrin (Alexa Fluor 546, red), and with the basolateral marker E-cadherin. Cross sections were taken from the upper third of the villi. Higher magnifications are shown in the bottom half of each panel. Hoechst 33342 nuclear counterstaining is blue. Scale bars: 20 µm.

View larger version (57K): [in this window] [in a new window]   Fig. 8. Distribution of actin and villin in 3D reconstructed enterocytes from wt and gal3–/– mice. Transverse sections of small intestine of wt (A, supplementary material Movie 1) (gal3+/+) and gal3-null mutant mice (B, supplementary material Movie 2) (gal3–/–) were stained with antibodies against β-actin (Alexa Fluor 546, red) and villin (Alexa Fluor 488, green). For 3D reconstruction, 35 layers of each sample were recorded and processed by deconvolution and 3D rendering (see also supplementary material Movies 1 and 2). In A, co-staining of actin and villin in the apical brush border is indicated by arrowheads. Arrows in B indicate actin- and villin-positive structures in the lateral membranes (B). Hoechst 33342 nuclear counterstaining is blue. Scale bars: 20 µm.

CiteULike

Complore

Connotea

Del.icio.us

Digg

Reddit

Technorati

Twitter