Fig. 6.

Cellular PtdIns(4,5)P₂ levels regulate dimerization of villin and filopodial assembly in Caco-2 BBe1 cells. (A) The optimal concentration of adenovirus required to express HA-tagged PIP5-kinase I in Caco-2 BBe1 cells was determined by infecting cells with different amounts of recombinant adenovirus for 16 h, followed by incubation for 24–72 h for protein expression. PIP5-kinase expression was detected by western blot analysis using an HA antibody. (B) Quantification of filopodia in Caco-2 cells expressing vector alone (control) or PIP5-kinase I shows significant increase in the number of cells expressing filopodia compared to control cells (*P<0.001 n = 20). Values are means ± s.e.m. (C) The effect of increased intracellular PtdIns(4,5)P₂ (PIP₂) levels on villin dimerization was determined in Caco-2 BBe1 cells expressing either control Ad-EGFP or HA-tagged PIP5-kinase Iβ and Iγ635 for 48 h at 37°C. Villin protein was cross-linked by incubating cells with 2.5 mM DFDNB for 1 h at 37°C. Villin monomers and dimers were identified by western blot analysis. This is a representative of four experiments with similar results. (D) Binding of PtdIns(4,5)P₂ to villin results in the exposure of dimerization site. Vesicles containing 100% PC were incubated with either buffer or with villin (VIL/WT) for 30 min at room temperature. Similarly, vesicles containing 99% PC, 1% PtdIns(4,5)P₂ were incubated with buffer, 2 µM wild-type villin (VIL/WT) or 2 µM mutant protein VIL/Δ21–67/112–119. The vesicles were made fluorescent by the incorporation of NBD-PC. Scale bar: 10 µm.