Figure 6.

PIPKIγ is functionally related with AP1 and E-cadherin trafficking in vivo. (A) Endogenous PIPKIγ and AP1 colocalize at vesicular compartments. PIPKIγ (FITC, green) and AP1 (Texas red, red) were visualized in MDCK cells by indirect inmmunofluorescence. (B) PIPKIγ and AP1 colocalize with the internalized E-cadherin. Parental MDCK cells were plated on coverslips and allowed to form AJs. They were then treated with 2 mM EGTA for 30 min. Cells were fixed and indirect immunofluorescence staining was performed using anti-PIPKIγ and anti–γ-adaptin, followed by Cy5-conjugated anti–rabbit IgG antibody and Texas red–conjugated anti–mouse IgG antibodies. Cells were then incubated with 0.5 μg/ml of normal mouse IgG at 37°C for 30 min and then with FITC-conjugated anti– E-cadherin antibodies. Arrows indicate the colocalization of AP1, internalized ECD, and PIPKIγ. (C) PIPKIγ661 recruits AP1 to functional sites of E-cadherin recycling. Parental or PIPKIγ isoform–overexpressing MDCK cells were treated with 2 mM EGTA for 30 min followed by complete medium for 10 min at 37°C to induce the recycling of E-cadherin back to the PM. Immunofluorescence staining was then performed to visualize AP1 (γ-adaptin), E-cadherin, and PIPKIγ. Both horizontal and vertical sections were collected to show precise subcellular localizations. Bars, 10 μm.