Abstract
The small GTPase Rac1 has been implicated in regulation of cell migration and cell-cell adhesion in epithelial cells. Little is known, however, about the spatial and temporal coordination of Rac1 activity required to balance these competing processes. We fractionated endogenous Rac1-containing protein complexes from membranes of Madin-Darby canine kidney cells and identified three major complexes comprising a Rac1.PAK (p21-activated kinase) complex, and 11 S and 16 S Rac1 complexes. Significantly, Rac1 shifts from the 11 S to a 16 S particle during initiation of cell-cell adhesion. This shift may reflect a diffusion trapping mechanism by which these Rac1 complexes are localized to cadherin-mediated cell-cell contacts through an interaction with annexin II.
Highlights
From an 11 S to a 16 S particle in response to cadherin-based adhesion
We sought to define endogenous Rac1 complexes in membranes isolated from whole MDCK cells and examine whether these Rac1 complexes are dynamically altered during cell-cell contact assembly
Membranes isolated from a mixed population of migratory and cell-cell adherent MDCK cells were extracted in octyl glucoside-containing buffer to solubilize membrane-bound Rac1; note that Ͻ5% of endogenous Rac1 is tightly membrane-bound, but can be quantitatively removed by extraction with detergents [18]
Summary
Cell Culture—MDCK cells were cultured in Dulbecco’s modified Eagle’s medium ϩ 10% fetal bovine serum. Cells were replated at confluent density (10 ϫ 106 cells/7.5-cm dish) in low calcium medium ϩ 10% dialyzed fetal bovine serum and cultured for 3 h. To initiate adhesion synchronously [13], Dulbecco’s modified Eagle’s medium ϩ 10% fetal calf serum was perfused into the culture and the cells incubated for the appropriate time. LE cells [16] were cultured in Dulbecco’s modified Eagle’s medium ϩ 5% fetal bovine serum. Resulting extract was loaded onto 4-ml 10 – 40% (w/v) sucrose gradients and subjected to centrifugation at 48,000 rpm for 9 h at 4 °C in an SW 60 rotor.
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