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

First published online 1 September 2005
doi: 10.1242/jcs.02538

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 Google Scholar Google Scholar Articles by Lagana, A. Articles by Nabi, I. R. Search for Related Content PubMed PubMed Citation Articles by Lagana, A. Articles by Nabi, I. R. Social Bookmarking                         What's this?

pH-specific sequestration of phosphoglucose isomerase/autocrine motility factor by fibronectin and heparan sulphate

¹ Département de pathologie et biologie cellulaire, Université de Montréal, Montréal, Québec, H3C 3J7, Canada
² Department of Pharmacology, Hebrew University of Jerusalem, Jerusalem 91120, Israel
³ Department of Cellular and Physiological Sciences, University of British Columbia, Vancouver, British Columbia, V6T 1Z3, Canada

View larger version (132K): [in a new window]   Fig. 1. PGI/AMF binding to cell surface fibrils requires FN expression. FN+/– (A-C,G-I) and FN–/– (D-F,J-L) fibroblasts were plated for 2 days on glass cover slips. The cells were incubated with 25 µg ml–1 PGI/AMF conjugated to Alexa-568 (PGI/AMF-568) for 30 minutes at 37°C in medium buffered to pH 7.5 with Hepes (A-F) or to pH 5.0 with MES (G-L) and then fixed with 3% paraformaldehyde. PGI/AMF-568 labeling is shown in red (A,D,G,J) and FN labeled with mouse anti-FN mAb and Alexa-488 conjugated anti-mouse secondary antibody shown in green (B,E,H,K). Merged images show co-localization of the two in yellow (C,F,I,L). Bar, 10 µm.

View larger version (91K): [in a new window]   Fig. 2. Binding of PGI/AMF to dimeric FN at neutral and acid pH. The fluorescent signal due to binding of PGI/AMF-FITC to uncoated wells of a 96-well plate, to wells coated with 20 µg ml–1 BSA or FN, or to wells plated with NIH-3T3 cells was amplified by anti-FITC and Alexa488 anti-rabbit secondary antibodies and measured with a fluorescence plate reader (A). Absolute relative fluorescence values were normalized to maximal values and binding at pH 5 and pH 7.5 in the presence or absence of PGI/AMF-FITC was determined. To assess relative FN levels in the wells containing soluble FN or NIH-3T3 cells, parallel wells were labeled with anti-FN and Alexa488 anti-rabbit secondary antibodies (±s.e.m., n=4). Alternatively, NIH-3T3 cells were plated for 2 days on cover slips coated with 20 µg ml–1 of FN. The cells were then incubated with 25 µg ml–1 bPGI/AMF for 60 minutes at 37°C. After fixation with 3% paraformaldehyde, bPGI/AMF was revealed with Texas Red-streptavidin (B) and FN labeled with mouse anti-FN mAb and Alexa-488 conjugated anti-mouse secondary antibody (C). bPGI/AMF appears in red and FN in green and co-localization of the two appears in yellow in the merged image (D). PGI/AMF binds selectively to the fibrillar form of FN in NIH-3T3 cells plated on a FN substrate. Bar, 20 µm.

View larger version (84K): [in a new window]   Fig. 3. FRET analysis shows the direct interaction of PGI/AMF and FN at pH 5 but not at pH 7.5. NIH-3T3 cells incubated with FN-488 for 30 minutes at pH 7.5 in complete HEPES-buffered medium were then incubated with PGI/AMF-568 for 30 minutes at 37°C at either pH 7.5 in complete HEPES-buffered medium (A-D) or at pH 5 in complete MES-buffered medium (E-H), washed with PBS/CM, fixed with 3% paraformaldehyde and the cover slips mounted in Airvol for confocal photobleaching FRET analysis. Single scan images before bleaching of the acceptor PGI/AMF-568 (A,E) and donor FN-488 (C,G) were collected and then the acceptor was bleached to 60% to 90% in the zone of interest (indicated by the box). New single scan images were then acquired simultaneously for both the donor (B,F) and the acceptor (D,H). Donor images are presented in pseudocolor (see bar to right) to highlight increased donor fluorescence after acceptor photobleaching. The extent of bleaching (% bleach) and increased donor fluorescence (% FRET) in the bleached region was quantified and is presented in table form (I) for FN-488 and PGI/AMF-568 as donor/acceptor pairs at both pH 5 and 7.5. Negative controls include donor alone or acceptor alone at pH 7.5 or 5.0 and as a positive control FRET between FN-488 and FN-568 was measured, as indicated. P values were determined by ANOVA relative to the FN-488 and PGI/AMF-568 pair at pH 7.5.

View larger version (53K): [in a new window]   Fig. 4. PGI/AMF endocytosis is required for its association with cell surface FN fibrils. NIH-3T3 cells were uninfected (A,B) or infected for 48 hours with adenoviruses expressing wild-type dynamin-1 (dynWT) (C,D), the dominant negative dynamin-1 K44A mutant (dynK44A) (E,F) and the dominant negative clathrin hub (Cla-Hub) (G,H). The cells were then incubated with 25 µg ml–1 of PGI/AMF-568 for 60 minutes (A,C,E,G) and fixed with 3% paraformaldehyde. Infection rates were from 50-80% and in the selected fields, all cells were infected. Uninfected, dynWT and dynK44A infected cells were labeling with mouse anti-HA antibody and goat Alexa-488 anti-mouse secondary antibody to identify infected cells (B,D,F) and clathrin hub infected cells were identified by labeling with mouse anti-T7 antibody followed by labeling with goat Alexa-488 anti-mouse secondary antibody (H). Bar, 20 µm.

View larger version (113K): [in a new window]   Fig. 5. PGI/AMF and FN do not co-internalize and interact at the cell surface. NIH-3T3 cells were co-incubated with 25 µg ml–1 bPGI/AMF and 10 µg ml–1 FN-FITC for 10 (A-C) or 60 (D-F) minutes at 37°C. The cells were then fixed with 3% paraformaldehyde and bPGI/AMF was revealed with Texas Red-streptavidin (A,D) and FN with anti-FN mAb and Alexa-488 conjugated secondary antibodies (B,E). Colocalization of the two appears in yellow in the merged images (C,F). Bar, 10 µm.

View larger version (92K): [in a new window]   Fig. 6. Endocytosed bPGI/AMF can undergo more than one cycle of endocytosis. NIH-3T3 cells were incubated with 25 µg ml–1 bPGI/AMF in complete medium (A-F) or with complete medium in the absence of bPGI/AMF (G-I) for 30 minutes at 37°C. The cells were then washed three times with complete medium and then incubated 30 minutes with Texas Red-streptavidin (SA-TR) at 37°C (A-C, G-I) or at 4°C (D-F). Cells were fixed with 3% paraformaldehyde, permeabilized with 0.1% Triton X-100, and labeled with FITC-streptavidin (SA-FITC) to detect endocytosed bPGI/AMF that had not been bound by Texas Red-streptavidin (B,E,H). Texas Red (red) and FITC-streptavidin (green) confocal images were merged and confocal co-localization appears in yellow (C,F,I). When added at 37°C (A-C) but not at 4°C (D-F), Texas Red-streptavidin can be seen to label PGI/AMF positive MVBs and was therefore captured for a second round of endocytosis by recycling bPGI/AMF. Bar, 10 µm.

View larger version (67K): [in a new window]   Fig. 7. Heparan sulphate stimulates FN-independent binding of PGI/AMF at pH 5. (A) NIH-3T3 cells were left untreated or treated with 1, 10, 30 and 100 µg ml–1 HS for 30 minutes at 37°C and then incubated for 30 minutes at 37°C with 25 µg ml–1 PGI/AMF-568 in HEPES adjusted medium at pH 7.5 or 5 µg ml–1 PGI/AMF-568 in MES adjusted medium at pH 5. Representative images show PGI/AMF-568 labeling in red and FN labeling in green in NIH-3T3 cells at pH 7.5 (B) or pH 5 (C) and PGI/AMF-568 labeling in FN–/– cells (D) in control cells and cells treated with 10 µg ml–1 HS. Bar, 20 µm. The bar graphs in B, C and D present the quantification of the fluorescent intensity of total cell-associated (Cell) and FN fibril-associated (FN) PGI/AMF-568 in the absence (CTL; white bars) or following pretreatment with 10 µg ml–1 HS (+HS; green bars). In NIH-3T3 cells at pH 5 (C), PGI/AMF-568 binding was competed for by incubating the cells with 100 µg ml–1 of unlabelled PGI/AMF before addition of 5 µg ml–1 PGI/AMF-568 in complete medium adjusted to pH 5 in the absence (CTL comp; grey bars) or presence of 10 µg ml–1 HS (HS comp; dark green). PGI/AMF-568 fluorescent intensity was quantified from 10 random images per condition and the data normalized to the condition presenting the maximum intensity (±s.e.m.; n=3; P<0.01).

View larger version (22K): [in a new window]   Fig. 8. Acid-dependent sequestration of PGI/AMF by FN stimulates cell motility. Confluent monolayers of FN+/– (blue bars) and FN–/– (green bars) cells were grown for 1 day in regular medium, wounded by scraping and cell migration from the wound measured after 14 hours. The scraped monolayers were treated with 25 µg ml–1 PGI/AMF for the complete 14 hour period or treated for 30 minutes with the same concentration of PGI/AMF at pH 5 or 7.5 and then rinsed and incubated in regular medium for the remainder of the 14 hour period. Alternatively, cells were pre-incubated with 10 µg ml–1 HS for 60 minutes and then incubated with HS or HS plus PGI/AMF for 30 minutes at pH 5 prior to rinsing. The data was normalized to the migration of FN+/–cells in the absence of PGI/AMF or HS (±s.e.m.; n=3).

View larger version (16K): [in a new window]   Fig. 9. The complex biology of PGI/AMF and its receptor. At neutral pH, PGI/AMF binds its receptor AMFR at the cell surface and can be endocytosed via two different pathways: caveolae/raft-dependent endocytosis to the smooth ER (1) or clathrin-dependent endocytosis to multivesicular bodies (MVBs) (2). Internalized PGI/AMF can recycle from MVBs to the plasma membrane (3) where it can undergo further rounds of endocytosis and recycling (4). Recycling receptor-ligand complexes can also be sequestered via stable association with FN fibrils (5). PGI/AMF can also interact directly with IGFBP-3 (6), an interaction that reduces its cellular binding. At acid pH, endocytosis is inhibited and PGI/AMF binds directly to FN fibrils (7) or to heparan sulphate (HS) (8).

CiteULike

Complore

Connotea

Del.icio.us

Digg

Reddit

Technorati

Twitter