Fig. 3. Spreading of myoblasts in the presence of mutant forms of MARCKS. (A) The spreading of cells expressing the wild type, G2A-mPSD and PSD fusion proteins was monitored after the cells were plated on fibronectin (FN) or laminin (LM) for 30 minutes. G2A-mPSD and PSD cells failed to spread on fibronectin. (B) Cells were allowed to spread on laminin (open bars) or fibronectin (filled bars) for 30 minutes as in panel (A), and cell spreading was quantified by determining the percentage of cells that had spread (see Materials and Methods). Data represent the mean±SD of three independent experiments. (C) The spreading of cells expressing the G2A fusion protein compared to control and wild-type (WT) cells after 5 or 15 minutes on fibronectin. Cells expressing the G2A mutant spread faster than both other cell types. (D) Cells overexpressing MARCKS fusion proteins were treated with PMA (100 nM) and plated on fibronectin for 30 minutes. Treatment with PMA accelerated the spreading of cells expressing wild-type or G2A fusion proteins (although at this time point the difference with or without PMA is minimal because the cells have are already spread) but failed to rescue the spreading deficit of cells expressing the G2A-mPSD and PSD mutants. Bars, 10 µm.