Aurora B–dependent centralspindlin oligomerization contributes to spindle midzone–independent furrow ingression. (A) Scheme explaining how oligomerization of centralspindlin is induced by Aurora B–dependent phosphorylation of MKLP1. (B) HeLa cell lines with stable inducible expression of GFP::MKLP1 and GFP::MKLP1-S708E were transfected with the indicated siRNAs, treated with or without ZM447439 before anaphase, and imaged live. The number of cells showing stable furrow regression, full-furrow regression followed by furrow regression, visible but minimal furrow ingression or no furrow ingression was scored. The number of cells analyzed per condition is indicated. One representative experiment out of three is shown. **, P < 0.01; χ2 test for comparison of the indicated conditions. (C) Representative stills of HeLa cells in anaphase with stable inducible expression of GFP::MKLP1 and RACGAP1::GFP and transfected with the indicated siRNAs. Numbers indicate the number of times the depicted localization was observed/total number of cells that were imaged live. (D) Representative stills of HeLa cells in anaphase with stable inducible expression of GFP::MKLP1 and GFP::MKLP1-S708E and transfected with siPRC1 and treated with the Aurora B inhibitor ZM447439 (2 µM) before anaphase onset. (E) C. elegans embryos expressing a mCherry::PH membrane marker (pink) with a CYK-4::GFP transgene (cyan) were depleted of endogenous Aurora B (AIR-2) by RNAi. These embryos were filmed starting at metaphase in the first division cycle. Shown are montages of the equatorial region as the cell divides (n ≥ 5). The arrow indicates the appearance of cortical CYK-4::GFP under wild-type conditions. (F) Embryos expressing CYK-4::GFP were scored for the extent of recruitment of the GFP marker to the ingressing furrow tip during anaphase, relative to a cytosolic background (see Materials and methods). Error bars represent ± SD; *, P < 0.05; Student’s t test, n ≥ 5. Bars: 10 µm (C–E).
