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First published online 2 March 2004
doi: 10.1242/jcs.01003

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Laminin 5 deposition regulates keratinocyte polarization and persistent migration

Fred Hutchinson Cancer Research Center, 1100 Fairview Avenue North, Seattle, Washington 98109 and the Department of Pathobiology, University of Washington, Seattle, Washington 98195, USA

View larger version (108K): [in a new window]   Fig. 1. Activated keratinocytes migrated processively on laminin 5. (A) Immunofluorescence of precursor laminin 5 deposited by a keratinocyte migrating on exogenous laminin 5 for 3 hours. Arrow, site of initial adhesion. Anti-laminin 5 mAb D2-1 (green), phalloidin (red). Scale bar: 20 µm. (B) mAb D2-1 stains the initial site of adhesion (arrow, A) and the band of deposits that was under the symmetrically spread cell. The cell is no longer in this field. The deposit pattern reflects the change in cell morphology as it polarized and elongated perpendicular to the direction of migration (arrowheads, P) and began linear migration (large arrow). Scale bar: 10 µm. (C) Phase image of a keratinocyte with polarized fan cell morphology (see also Movie 1, http://dev.biologists.org.supplemental/). Large arrow denotes direction of migration. Cell characteristics r, retracted rear, n, nucleus, b, cell body-lamellipodium transition region, L, lamellipodium, f, filopodia. Scale bar: 5 µm. (D) Tracks from 2-hour migration on exogenous laminin 5, overlay of phase image of cells at the end (see also Movie 2, http://dev.biologists.org.supplemental/). Scale bar: 20 µm.

View larger version (13K): [in a new window]   Fig. 2. Polarization is affected by exogenous ligand and requires growth factors, PI 3-kinase and protein translation. Keratinocytes were plated for 2 hours on laminin 5 (black bars) or collagen surfaces (white bars) with either no treatment (nt), absence of growth factors (–GF), presence of 5 µM LY294002 (LY29) or presence of 10 µg/ml cycloheximide (CHX). Values represent the average and standard deviation of at least three independent experiments.

View larger version (51K): [in a new window]   Fig. 3. Polarization on collagen requires interactions of integrin 3ß1 with precursor laminin 5 deposits. Cells were plated onto exogenous laminin 5 (A,B) or collagen (C-F) in the absence of antibody (C) or the presence of anti-2 mAb P1H5 (A), anti-3 mAb P1B5 (D), anti-6 goH3 (G) or anti-laminin 5 mAb C2-9 (E) for 5 hours then the percentage of polarized cells were determined (see Materials and Methods). Alternatively, for those antibodies that would block initial adhesion, cells were instead plated for 1 hour to allow for adhesion and spreading prior to the addition of anti-3 mAb P1B5 (B) or anti-2 P1H5 (F) for 3 hours before cells were quantified. (G) Summary of data obtained from polarization experiments. Values represent the average and standard deviation of at least three independent experiments.

View larger version (64K): [in a new window]   Fig. 4. Activated keratinocytes on laminin 5 assembled small, punctate focal complexes during processive migration. Cells were plated onto exogenous laminin 5 (A-F) or collagen (G-L) for 2 hours, then fixed and stained for immunofluorescence. Deconvoluted optical sections of the basal cell surface are shown. Phospho-FAK-Y397 (red) (A,D,G,J), ß1 integrin mAb 9EG7 (B) and 3 integrin mAb P1F2 (E,K) and 2 integrin mAb P1H5 (H) (green). Scale bar: 10 µm.

View larger version (39K): [in a new window]   Fig. 6. Processive migration required deposition of precursor laminin 5. (A) Time-lapse analysis from 2-hour recordings as described in Fig. 5 were analyzed to determine the percentage of cells that fell into the region defining processive migration (shaded region, Fig. 5) on laminin 5 (black bars) or collagen (white bars). Cells that do not synthesize laminin 5 (JEB-G) showed decreased processive migration. Treatment of cells with 1 µM nocodazole (noc), 1 µM taxol (tax) or 6 µM rottlerin (rot) blocked processive migration. Values are averages and standard deviations from at least three independent experiments. (B) Rottlerin and nocodazole caused an increase in detergent-insoluble integrin 6ß4. Triton-insoluble integrin 6 was quantified by ELISA using anti-6 antibody goH3. Cells pretreated for 3 hours with 1 µM nocodazole or 6 µM rottlerin were plated on recombinant laminin 5 adsorbed to Petri plates for 5 hours then extracted and processed for ELISA. Representative data from one of three experiments is shown. (C) Triton-insoluble integrin ß4 was quantified by western blot analysis using mAb 3D5. Log phase cells plated on recombinant laminin 5 adsorbed to Petri plates were either not treated (–) or treated (+) with 6 µM rottlerin for 7 hours, extracted with 1% Triton (sol) followed by 0.5% SDS (insol) and immunoblotted with anti-ß4 antibodies. Equal amounts of protein from Triton X-100 extracts and equal amounts for SDS samples were loaded. Gels were quantified using ImageQuant software (Materials and Methods). (D) Integrin 6 stained with goH3 antibody is absent from basal contacts in polarized cells (indicated by arrows) migrating on laminin 5. Cells were fixed and processed for immunofluroescence as described in Fig. 4 using deconvoluted microscopy. (E,F) Cells were plated on laminin 5 for 5 hours then extracted with 1% Triton X-100, fixed and processed for standard epifluorescence. (E) Processively migrating cells on laminin 5, indicated by arrows, lack detergent-insoluble integrin 6ß4. (F) Cells pretreated with 6 µM rottlerin for 3 hours then plated on laminin 5 had increased 6ß4 that was resistant to Triton X-100 extraction.

View larger version (23K): [in a new window]   Fig. 5. Laminin 5 provided a better substratum for processive migration than did collagen. Cells were plated onto exogenous laminin 5 or collagen for 1-2 hours before migration was recorded at 37°C by time-lapse imaging every 2 minutes for a total of 2 hours. (A) Plots of cells on exogenous laminin 5 (a) or collagen (b) showing 2 minute interval positions for all cells in one 10x field oriented so that cell origins are at x(0), y(0). Distance migrated in microns is indicated on the plots. (B) Processive index for each cell in A is shown as a function of the total distance traveled for cells on laminin 5 (a) or collagen (b). Shaded regions indicate processively migrating cells with a total migration >40 µm and PI>0.7.

View larger version (18K): [in a new window]   Fig. 7. Laminin 5 deposits and integrin 3ß1 mediate adhesion to BSA. Activated keratinocytes were plated onto a BSA blocked surface for 4 hours and the percentage adhered cells relative to nontreated control (nt=1.0) was determined. mAb C2-9 against laminin 5 (lm5) blocked adhesion, and JEB-gravis cells, which lack laminin 5, failed to adhere. Nocodazole and taxol at 1 µM and rotterlin at 6 µM completely blocked adhesion. Inhibitory antibodies against integrin ß1 (P5D2) and integrin 3 (P1B5) inhibited adhesion while antibodies against integrin 6 (goH3) did not. JEB-PA cells lacking integrin ß4 were able to adhere to BSA. Cytochalasin D at 5 µM did not block adhesion. Data shown is the average and standard deviation of three independent experiments. *Significantly different from nt value, P<0.01; Student's t-test.

View larger version (35K): [in a new window]   Fig. 8. The deposition of laminin 5 is regulated independently of secretion. (A) ELISA assay for laminin 5 deposits confirmed loss of deposition. Cells were culture for 4 hours on laminin 5 then removed with 5 mM EDTA in PBS for 30 minutes at 37°C and new laminin 5 deposits were quantified by ELISA. Signals were normalized relative to D2-1, DMSO=1.0. No cells (–), DMSO treated cells (nt), 1 µM nocodazole (noc), 1 µM taxol (tax) or 6 µM rottlerin (rot). Presented are the average and standard deviation of three wells from a representative experiment. (B) Secretion of laminin 5 into the culture medium was not blocked by nocodazole. Keratinocytes labeled with [35S]methionine were adhered to exogenous laminin 5 for 3 hours in the presence of 0, 0.1, 0.5 or 1.0 µM nocodazole (lanes 1-4 and lanes 5-8) and the conditioned media were collected. Immunoprecipitation of secreted laminin 5 from cell culture media was performed in RIPA buffer using mAb C2-5 against the laminin 3 chain (lanes 1-4) or mAb B4-6 against the laminin 2 chain (lanes 5-8). Molecular masses (in kDa) of the three chains of the laminin 5 trimer are indicated.

View larger version (119K): [in a new window]   Fig. 9. Deposition of laminin 5 is restricted to the rear of the cell and colocalizes with sites of collagen removal. (A-C) Removal of collagen colocalized with sites of laminin 5 deposition. Migrating keratinocyte on Rhodamine-conjugated human placental type-I collagen (A), stained by immuofluorescence with mAb D2-1 for precursor laminin 5 (B). Images overlayed in C. (D-I) Deconvoluted optical sections at the substratum interface of migrating cells on collagen. mAb D2-1 was added for 15 minutes prior to cross-linking for 10 minutes with 0.4 mM BS3. The cells were then extracted with 1% Triton X-100 for 10 minutes, fixed with formaldehyde and processed for immunofluorescent staining for laminin 5 (D2-1; D,G), integrin 3 (E) and 2 (H). The nucleus is visible in E,H as a gray area. F,I merged images of D,E and G,H, respectively. Scale bar, 10 µm.

View larger version (22K): [in a new window]   Fig. 10. Laminin 5 deposits direct processive migration on collagen. Step 1. Deposition contacts: rapid switch of adhesion from exogenous collagen (blue) and integrin 2 (open ovals) to laminin 5 deposits (green ovals) and integrin 3ß1 (red bars). Deposition of laminin 5 down onto the substratum is regulated differently than apical secretion of laminin 5, fibronectin, laminin 1 and thrombospondin into the culture medium. Step 2. Polarization: integrin 3-laminin 5 interactions mediate polarization. Focal complexes form under the lamellipodia with exogenous ligand while laminin 5 deposits are restricted to the rear of the cell. Step 3. Processive migration removes exogenous collagen at precise sites of laminin 5 deposits that are restricted to the rear of the cell. Persistence of linear migration is not in response to a chemotactic gradient.