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

First published online 20 July 2004
doi: 10.1242/jcs.01251

This Article Summary Full Text Full Text (PDF) Movies 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 Choma, D. P. Articles by DiPersio, C. M. Search for Related Content PubMed PubMed Citation Articles by Choma, D. P. Articles by DiPersio, C. M.

Integrin 3ß1 directs the stabilization of a polarized lamellipodium in epithelial cells through activation of Rac1

Center for Cell Biology and Cancer Research, Albany Medical College, MC-165, 47 New Scotland Avenue, Albany, NY 12208, USA

View larger version (98K): [in a new window]   Fig. 1. Genetic ablation of 3ß1 in keratinocytes causes loss of wound edge integrity. (A-F) MK+/+ cells (A,D,G,J), MK–/– cells (B,E,H,K) and MK3 cells (C,F,I,L) were grown to confluence on either collagen-coated surfaces (A-F) or LN-5 ECM (G-L). Monolayers were then scrape wounded and wound-edge morphology and migration were observed for 8 hours by time-lapse video microscopy. Results are representative of at least three experiments for each substrate. Bar, 100 µm.

View larger version (102K): [in a new window]   Fig. 2. Cells that lack 3ß1 do not extend lamellipodia at a wound edge. (A) MK+/+, MK–/– or MK3 cells were grown to confluence on collagen-coated glass coverslips, scrape wounded and fixed either immediately after wounding (0 hours) or 2 hours after wounding. Cells were stained for F-actin with TRITC-conjugated phalloidin. (B) MK3 cells were stained for ß-actin 2 hours after wounding. The corresponding phase image is shown for each field. Arrowheads point to edges of lamellipodia; arrows point to actin purse strings. Bar, 10 µm.

View larger version (64K): [in a new window]   Fig. 3. Integrin 3ß1 is localized at leading lamellipodia and its adhesion to LN-5 is required for lamellipodia formation by wound-edge cells. (A,B) MK3 cells were grown to confluence on collagen-coated glass coverslips, wounded, fixed 2 hours after wounding and then stained with P1B5 for 3ß1 integrin (B); the corresponding phase-contrast image is shown in (A). Arrowheads point to lamellipodium edge. Bar, 10 µm. (C) P1B5 staining of the MK3 monolayer away from the scrape wound shows the expected cell-cell localization of 3ß1. (D) Confluent MK–/– cells show a lack of staining with P1B5. (E) MK3 cells on collagen-coated surfaces were scrape wounded and photographed immediately after wounding (0 hours) or 2 hours after wounding in the presence of a control IgG, a function-blocking antibody against integrin 3ß1 (P1B5) or a function-blocking antibody against integrin 6ß4 (GoH3), as indicated. Bar, 100 µm. (E') Panels show close-up images of wound-edge cells from IgG-treated or P1B5-treated scrape wounds, as indicated. (F) The proportions of leading-edge cells displaying lamellipodia were determined for three separate wound edges, 60 cells for each wound edge. Error bars represent s.e.m.

View larger version (69K): [in a new window]   Fig. 4. MK cells display integrin 3ß1-dependent fan formation. (A) MK3 cells were plated sparsely onto LN-5 ECM and stained for F-actin and the 3 integrin subunit. Arrowheads indicate leading lamellipodium. Bar, 10 µm. (B) MK–/– cells were plated sparsely onto LN-5 ECM and stained for F-actin. Arrows indicate filopodia. Corresponding phase-contrast images are shown in (A,B). (C) MK3 cells were plated sparsely in the presence of a control IgG, a function-blocking antibody against integrin 3ß1 (P1B5) or a function-blocking antibody against integrin 6ß4 (GoH3), as indicated, and cell morphology was observed by time-lapse video microscopy. (D) The proportion of MK cells that assumed a fan shape in either serum-free (open bars) or serum-containing (filled bars) conditions was determined. Data represent the average of three separate experiments, with 100 cells being scored per experiment. Error bars represent s.e.m.

View larger version (72K): [in a new window]   Fig. 5. Integrin 3ß1-deficient cells fail to polarize and migrate in a persistent and directional fashion. MK+/+ cells, MK–/– cells or MK3 cells were plated sparsely onto LN-5 ECM and observed for 2 hours by time-lapse video microscopy. Intervals are indicated in minutes above each column. Arrows mark the position of a short-lived protrusion from an MK–/– cell.

View larger version (32K): [in a new window]   Fig. 6. Rac1 activation in keratinocytes is dependent on integrin 3ß1. (A) Cell lysates from MK+/+ cells, MK–/– cells and MK3 cells were immunoblotted for Rac1, keratin 14 or 3 integrin. (B) Representative immunoblot of GST-PAK pull-down assay to detect levels of active Rac1 in MK cells after adhesion to LN-5 ECM for 30 minutes in the absence of exogenous growth factors. (C) Quantitation of Rac1 activity for three separate experiments. Error bars represent s.e.m.

View larger version (76K): [in a new window]   Fig. 7. Rac1 activity is required for assumption of a fan shape by 3ß1-expressing MK cells. MK+/+ cells (A-F) or MK3 cells (G-L) expressing either dominant negative Rac1N17 (A-C,G-I) or GFP (D-F,J-L), as indicated. F-actin staining was performed using TRITC-conjugated phalloidin. Infected cells were visualized by GFP fluorescence. Corresponding phase-contrast images are shown for each field. Bar, 10 µm.

View larger version (37K): [in a new window]   Fig. 8. Activated Rac1 does not induce stable lamellipodia in the absence of 3ß1. MK–/– cells were infected with adenovirus encoding either Rac1L61 (A,B,E-G) or a GFP control (C,D). Infected cells were plated on LN-5 ECM and stained for either F-actin with TRITC-conjugated phalloidin (A,C) or the Myc epitope (F). Infected cells were visualized by GFP fluorescence. Bar, 10 µm. (H) Cell lysates from Rac1L61-infected or control-infected cells were immunoblotted for Myc epitope or GFP.

View larger version (46K): [in a new window]   Fig. 9. Model for role of 3ß1 in the establishment of polarity by migrating wound-edge keratinocytes. (A) In quiescent epidermis, 6ß4-containing hemidesmosomes mediate stable adhesion of basal keratinocytes to the basement membrane, whereas 3ß1 is localized primarily to cell-cell borders. (B) Upon wounding, growth factors and cytokines in the wound bed stimulate actin polymerization and the formation of an initial protrusion. These initial protrusions contain 3ß1 that interacts with newly deposited LN-5 (C), stabilizing the protrusion and subsequently stimulating Rac1 activity. Rac1 activation leads to growth of a stable leading lamellipodium.