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

First published online 5 August 2003
doi: 10.1242/jcs.00694

This Article Summary Full Text Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend 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 Levchenko, T. Articles by Holmgren, L. Search for Related Content PubMed PubMed Citation Articles by Levchenko, T. Articles by Holmgren, L.

Loss of responsiveness to chemotactic factors by deletion of the C-terminal protein interaction site of angiomotin

Department of Oncology-Pathology, Cancer Center Karolinska Institutet, S-17176 Stockholm, Sweden

View larger version (29K): [in a new window]   Fig. 1. The C terminus of angiomotin contains a conserved putative PDZ-binding motif. (A) Eight amino acids of the C terminus of human and mouse angiomotin are aligned with glycophorin C, a protein with a characterized PDZ binding motif. The last four amino acids that determine the binding specificity to PDZ-containing proteins are boxed. (B) Coomassie staining of SDS-PAGE showing specific binding of a 90 kDa protein isolated by peptide affinity chromatography using the last 20 amino acids of angiomotin (lane 1). The protein did not bind the angiomotin peptide that lacked the C-terminal amino acids YLI (lane 2). (C) Generation of 3' deletion mutations of the angiomotin gene. A series of 3' deletions were generated by PCR amplifications from the angiomotin cDNA as described in the Materials and Methods. The deletions ranged from 20-3 amino acids and were cloned into the pBabe retroviral vector. (D) Western blot analysis showing expression levels of angiomotin and the 1-4 mutants after transfection into MAE cells.

View larger version (13K): [in a new window]   Fig. 2. Defective chemotactic response by cells transfected with the C-terminal deletion mutants. Mouse aortic endothelial cells (MAE) transfected with vector (Vec), angiomotin (Amot) or deletion mutants (1-4) were stimulated by addition of (A) 20 ng/ml bFGF, (B) 10 ng/ml VEGF or (C) 200 ng/ml LPA. The differences between angiomotin and the deletion mutants were statistically significant (asterisks denotes P<0.01, error bars=s.d.). Cell migration was stimulated by the addition the chemo-attractant to the lower chamber in a modified Boyden chamber. The number of cells migrating through a collagen 1-coated filter was estimated after 5 hours. All assays were performed in quadruplicate.

View larger version (67K): [in a new window]   Fig. 3. Deletion mutants of angiomotin inhibit endothelial tube formation. Tube formation of vector, angiomotin and 1 and 4 transfected MAE cells was analyzed by in the matrigel assay. (A) Images show tube formation 16 hours after the start of the experiment. Scale bar: 300 µm. (B) Total tube length formed by vector, angiomotin and 1 and 4 transfected MAE cells. All analyses were performed in triplicate. The differences between vector and the 1 and 4 mutants were statistically significant (asterisks denotes P<0.001, error bars=s.d.).

View larger version (89K): [in a new window]   Fig. 4. Similar localization of angiomotin and 4-angiomotin in MAE cells. Immunofluorescent staining with a polyclonal antibody against angiomotin shows that both angiomotin (top panel) and 4 (middle panel) localizes to the central area of the cell and at the leading front of lamellipodia. Arrows and phalloidin stainings indicate location of the lamellipodia. Vector control cells (bottom panel) show some background staining in the nucleus. Scale bar: 10 µm.

View larger version (40K): [in a new window]   Fig. 5. Expression of wild-type and 4-angiomotin in endothelial cells of transgenic mice. (A) TIE-angiomotin and TIE-4 angiomotin constructs. (B) Southern blot showing integration of TIE constructs in angiomotin (TIE-amot) and 4-angiomotin (TIE-4) transgenic embryos (E9.5) and adult mice, and expression of the transgenes, analyzed by RT-PCR, showing angiomotin expression in angiomotin (TIE-ang) and 4-angiomotin (TIE-4) in transgenic embryos (E9.5) and adult mice (hAmot, human angiomotin). (C-H) Phenotype of TIE-angiomotin and TIE-4 angiomotin embryos at E9.5. TIE-angiomotin embryos (D) are phenotypically indistinguishable from their wild-type littermates (C). (E) Wild-type littermate of the TIE-4 angiomotin embryos. (F) Cranial hemorrhage in TIE-4 angiomotin-expressing embryo. (G,H) Embryonic malformations in TIE-4 angiomotin embryos.

View larger version (97K): [in a new window]   Fig. 6. PECAM whole-mount staining of TIE-angiomotin (A) and TIE-4 angiomotin embryos (B). PECAM whole-mount staining shows aberrant cranial vascularization in the TIE-4 angiomotin embryo.

View larger version (115K): [in a new window]   Fig. 7. Transverse histological sections of wild-type and TIE-4 angiomotin E9.5 brains. Whole-mount PECAM-stained embryos were sectioned and stained with Hematoxylin and Eosin. (A) In wild-type brain, blood vessels align the neuroepithelium (arrows). (B) In contrast, the TIE-4 angiomotin brain has dilated blood vessels within the cephalic mesenchyme (arrows). This panel is representative of embryos 1-9 in Table 1. (C) Magnification of boxed area in A, showing capillaries residing and entering the neuroepithelium (arrowheads). (D) Magnification of boxed area in B, showing the presence of dilated blood vessels (open arrow) within the mesenchyme and with only a few capillaries entering the neuroectoderm (arrowhead). Note also the ruptured vessel (open arrow) with nucleated fetal blood cells leaking into the mesenchyme. (E) A more severe phenotype of a TIE-4-angiomotin mutant embryo displaying sack-like structures adjacent to the neuroepithelium (arrows), which has collapsed into the brain vesicle (asterisk). (F) Magnification of the boxed area in E, showing the dilated blood vessels. No vessel enters the neuroepithelium. cm, cephalic mesenchyme; ne, neuroepithelium. Scale bars: 125 µm.