Fig. 4. Adhesion and spreading on different types of extracellular matrix. B16-F1 populations expressing different GFP-vinculin mutants were analysed on laminin, fibronectin and collagen. Identical numbers of cells were seeded onto 12-well plates coated with the matrix molecules. (A) Incubation times of 10 minutes on laminin and fibronectin, and 90 minutes on collagen, were chosen to obtain comparable cell adhesion. After washing and fixation, cells were counted and adhesion was quantified as a proportion of adherent cells (mean±s.d.; n=4-6) compared with adherent cells expressing the GFP-vinculin wild type. (B) To analyse spreading, cells were allowed to attach for 15 minutes on laminin or fibronectin and, after washing and fixation, stained with TRITC-phalloidin. Cells were classified into three groups: spread (S), non-spread (N) and ambiguous (A), as seen in cells expressing vinculin-wt or vinculin-LD. Depicted cells were scored as spread or non-spread, respectively, unless indicated otherwise. Spreading was quantified as a proportion of spread cells (mean±s.e.; n=3) compared with the total number of adherent cells.