Figure 7. PIPKIγi2 Integrates Exocyst Complex and Talin with Integrin, and is Required for Integrin Trafficking.
(A) HeLa cells cotransfected with Flag-tagged exocyst complex components (Sec6 or Sec8 or Exo70) and HA-tagged PIPKIγi2 were allowed to adhere on FN-coated coverslips for 1-2 hours before immunostaining with anti-Flag (green) and anti-HA (red) antibodies.
(B) PIPKIγi2, Exo70 and α5-GFP integrin colocalize at cell membrane and intracellular compartments. HeLa cells (bottom) or HeLa cells stably expressing HA-tagged PIPKIγi2 (top) were co-transfected with Flag-tagged Exo70 and α5 integrin-GFP or GFP. Cells were fixed and immunostained using anti-HA (blue) or anti-Flag (red) antibodies.
(C) Exo70 colocalizes with talin at focal adhesions and intracellular sites. HeLa cells transiently transfected with Flag-tagged Exo70 alone or cotransfected with HA-tagged PIPKIγi2 were allowed to adhere on FN-coated coverslips for 1-2 hours before immunostaining with anti-HA (blue), anti-Flag (red) and anti-talin (green) antibodies.
(D) PIPKIγi2 integrates talin, β1-integrin and exocyst complex in the same complex. HeLa cells were transfected with PIPKIγi1 or PIPKIγi2. Talin were immunoprecipitated 24 hours post-transfection followed by immunoblotting for exocyst complex and β1-integrin.
(E) PIPKIγi2 were immunoprecipitated from HeLa cells stably expressing PIPKIγi2 or PIPKIγi2KD at different time points following wounding to induce migration. Immunocomplexes were examined for presence of integrins and/or talin by immunoblotting.
(F) Model depicting the role of PIPKIγi2 in integrin trafficking in directionally migrating cells. Cell migration induces the integration of PIPKIγi2, talin, β1-integrin into the complex either in plasma membrane or in intracellular recycling compartments. Further, PIP2 generation by PIPKIγi2 into the complex facilitates the assembly of the exocyst complex. Thus, coordinated activity of PIPKIγi2 and the exocyst complex in concert with talin promotes the polarized recruitment and trafficking of integrin molecules to migrating cell fronts. Loss of PIPKIγi2 or the exocyst complex or talin compromises the polarized recruitment/trafficking of integrin impairing cell polarization and directional cell migration. See also Figure S6