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. 2012 Sep 3;198(5):865–880. doi: 10.1083/jcb.201204107

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© 2012 Bastos et al.

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Figure 8. — Increased Rac1 activity in the cleavage furrow of CYK4 GAP-defective cells. (A–C) HeLa cells expressing EGFP-PAK1 CRIB domain and inducible mCherry-CYK4 (A) or GAP activity–defective CYK4^R385A (B and C) were transfected with CYK4 3′-UTR siRNA duplexes and then used for time-lapse imaging. A bright-field (BF) image is shown at the time points indicated together with the bottom section where the cells touch the glass growth surface and a maximum intensity projection along the z axis of all sections. Timings are relative to the onset of anaphase. Cells expressing only GAP activity–defective CYK4^R385A showed either early (B) or late (C) stage failure of cytokinesis, and examples of both outcomes are therefore shown. CYK4 wild-type cells underwent abscission after 171.1 ± 29.8 min, whereas CYK4^R385A cells showed early failure after 81.0 ± 21.8 min or late failure after 293.1 ± 53.6 min (n = 29). Bars, 10 µm. (D) Rac1 activity at the cell equator (red) and cell poles (green) was determined by measuring the intensity of the PAK1 CRIB domain probe every minute as cells exited mitosis using the Volocity 5 volume and quantitation tools. This was performed on cells expressing wild-type and R385A GAP mutant CYK4. Mean values are plotted in the graphs, and error bars indicate the standard deviation from the means (n = 5). WT, wild type.