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.