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. 2013 Jan 1;126(1):312–326. doi: 10.1242/jcs.116244

Fig. 5.

Fig. 5.

PtdIns(4,5)P2 regulates villin self-association and villin-induced filopodial assembly. (A) Co-localization of SEYPF–villin and CFP–PH-domain in MDCK Tet-Off cells. Scale bar: 10 µm. (B) Recombinant villin was incubated with increasing concentrations of PtdIns(4,5)P2 (PIP2; 0–80 µM) followed by cross-linking with a sub-optimal concentration (10-fold molar excess) of DFDNB for 1 hour at room temperature. The cross-linked proteins were separated by 8% SDS-PAGE and villin dimers were identified by western analysis. (C) The optimal concentration of adenovirus required to express HA-tagged PIP5-kinase I (PIP5KI) in MDCK Tet-Off cells was determined by infecting cells with different aliquots of recombinant adenovirus for 16 h followed by incubation for 24 h for protein expression. PIP5-kinase expression was detected by western analysis using an HA antibody. (D) Schematic diagram shows the dimerization [amino acids (a.a.) 21–67 and 112–119] and the two PtdIns(4,5)P2 binding (PB2: a.a. 138–146; PB5: a.a. 816–824) sites in human villin protein. (E) Filopodial assembly in MDCK Tet-Off cells (VIL/NULL), MDCK Tet-Off cells expressing SEYFP-tagged villin (VIL/WT) or mutant villin proteins (VIL/Δ21–67/112–119 or VIL/ΔPB2 or VIL/ΔPB5) were infected with adenovirus to express EGFP or PIP5-kinase I. Scale bar: 10 µm. (F) Quantification of filopodia in MDCK Tet-Off VIL/NULL and VIL/WT cells expressing EGFP (−) or PIP5-kinase (+). There was a significant increase in filopodial assembly in cells expressing VIL/WT compared to VIL/NULL cells (#P<0.001, n = 20) and a more significant increase in VIL/WT cells compared to VIL/NULL cells when they expressed higher levels of intracellular PtdIns(4,5)P2 (*P<0.001). Overexpression of PIP5-kinase had no effect on filopodial assembly in the mutant villin cell lines. Values are means ± s.e.m. (G) MDCK Tet-Off cells expressing VIL/WT and the PtdIns(4,5)P2 phosphatase SigD show a significant decrease (P<0.001, n = 30) in the average number of filopodia assembled per cell compared to cells overexpressing the phosphatase-deficient mutant SigD (C462S). (H) MDCK Tet-Off cells expressing VIL/WT treated with the PLC-γ1 inhibitor U73122 show a significant increase (P<0.001, n = 30) in the average number of filopodia assembled per cell compared to cells treated with the negative control (U73343).