Figure 5.

Loss of cell polarity on deletion of Cdc42. (A) MA9 cells were stained with anti-tubulin primary and Rhodamine secondary antibodies (Tubulin) and 4′,6-diamidino-2-phenylindole (DAPI) nuclear stain. Confocal images were analyzed using NIS Elements software (Nikon) to measure fluorescence intensity along a bisecting vector through individual cells. Scale bar, 10 μm. (A) Cells were considered polar when clear asymmetric distribution of tubulin was seen or (B) apolar in the absence of tubulin asymmetry, as previously described.³ Data are plotted as the percentage of the total number of cells scored per sample. (C) A loss of polarity and increase in apolar cell fraction was observed following Cdc42 deletion from MA9 cells. This change became more pronounced as Cdc42KO-MA9 cells were passaged in culture, as shown at 3 weeks postdeletion of Cdc42. Functional polarity was assayed by MA9 cell adhesion and migration. (D) Cre⁺;Cdc42FL-MA9 cells treated with TAM to induce deletion of Cdc42 had decreased adhesion to fibronectin compared with vehicle (EtOH)–treated controls whereas no difference was seen in Cre null (–) MA9 control cells. (E) Similarly, Cre⁺;Cdc42FL-MA9 cells treated with TAM had a sustained decrease in migration to SDF-1α compared with controls.