Figure 4: p120 regulates RhoA activity on the equatorial cortex during cytokinesis.
(a) IF for p120 and RhoA in anaphase and telophase U2OS cells. Note the accumulation of p120 and co-localization on the equatorial cortex (arrowheads). Right panels show magnifications of representative areas denoted by the dotted squares. Scale bar, 10 μm. (b) IF for p120 and RhoA in control and dox-treated U2OS p120-iKD cells. Scale bar, 10 μm. Quantifications show the percentage of control and dox-treated U2OS p120-iKD cells with RhoA-positive membrane protrusions during anaphase. ***P<0.001. Statistical significance was determined using the χ2-test. (c) Time-lapse stills of control and dox-treated U2OS p120-iKD cells expressing the RaichuEV RhoA FRET probe. Note the focused RhoA zone in control cells (upper panels; arrows), whereas RhoA activity is non-focused and oscillates in dox-treated U2OS p120-iKD cells leading to cytokinesis failure (lower panels; arrowheads). Insets show H2B-mCherry channels. Scale bar, 20 μm. (d) Quantification of bi- and multinucleation in dox-treated U2OS p120-iKD reconstituted with empty vector (control), FL p120-1A or the RhoA-binding mutant p120-1AΔ[622–628]. Statistical significance was determined using the Student’s t-test. Shown are the data from three independent experiments. Results are expressed as mean±s.d. *P<0.05/‡P<0.05 (binucleation and multinucleation, respectively). (e) IF for p120 and RhoA in dox-treated U2OS p120-IKD cells reconstituted with FL p120-1A or p120-1AΔ[622–628]. Note the absence of co-localization of RhoA and p120 in p120-1AΔ[622–628]-expressing cells (arrowhead) compared with cells reconstituted with p120-A FL (arrow). Scale bar, 10 μm.
