In addition, these effects were independent of p Approaches Cell culture and reagents Human STS cell lines SKLMS WFA Induces Vimentin Cleavage AKT

dynamics through cell division we generated a cell line stably expressing a C-terminal GFP tagged version of this kinase. Polo::GFP initially accumulated on centrosomes and astral microtubules throughout prophase (Figure 1, Movie S1), but as soon as the nuclear envelope started to break down, it could also be observed on kinetochores (Figure 1; -44:00 time point). Polo::GFP then accumulated inside the nucleus reaching a maximum at approximately 36 We are aware that besides the models presented here, our results may be explained by alternative models, in which the tetramer only needs one active dimer or has such a high turn over that the effects are not measurable minutes just before anaphase onset (64 minutes; n = 14), indicating that the nuclear envelope was entirely fenestrated (Figure 1; -38:00 time point). About 5 minutes later the two centrosomes had migrated to opposite poles (Figure 1; -33:00 time point). Proper chromosome congression and alignment was achieved about half an hour later when anaphase started (Figure 1; -00:30 and 00:00 time points). Polo::GFP began to localize to the spindle midzone two along with a half minutes following anaphase onset (6301 seconds; n = 14) (Figure 1; 02:30 time point) and continued to accumulate through cleavage furrow formation and ingression (Figure 1; 04:30 and 14:00 time points and Motion pictures S1 and S2). Interestingly, when the midbody formed, Polo::GFP also spread along the whole length on the central spindle microtubules (Figure 1; 14:00 time point). Because the behavior of Polo::GFP in these cells appeared indistinguishable from that of a comparable transgene in embryos or from antibody stainings of fixed preparations [22,23], we think that this distribution accurately conveys the dynamics of Polo kinase localization throughout cell division.We made use of the cell line described above to analyze Polo dynamics following depleting the kinesin Pav by RNAi. As previously described for other cell lines, Pav RNAi knockdown brought on cytokinesis failure following 48 hours of treatment and no cleavage furrow formation or ingression was observed (Figure 2; Movie S3) [24]. Unexpectedly, Polo::GFP localized usually in the spindle midzone from 2 plus a half minutes immediately after anaphase onset and continued to accumulate even inside the absence of furrow formation and ingression in all pav RNAi cells examined (n = ten) (Figure 2; pav RNAi; from the 02:30 time point onwards). This can be in contrast with earlier observations that Pav is expected for correct localization of Polo for the central spindle [6]. These reports, even so, have been based only on observations produced from fixed preparations, which might have not provided an precise examination of Polo dynamics in the course of cytokinesis. These outcomes prompted us to analyze Polo::GFP localization towards the central spindle right after RNAi knock-down of many kinesins and MAPs known to be needed for cytokinesis. Within this way, we discovered that Polo::GFP consistently failed to localize towards the spindle midzone in cells (n = 12) treated for 72 hours with dsRNA against the MAP Feo (Figure 2 and Movie S4). Two distinct dsRNAs were applied in these experiments to reduce off target effects. Polo::GFP localized ordinarily to centrosomes and kinetochores for the duration of pro-, meta- and ana-phase (Figure two; feo RNAi; time points 1:00, 00:00 and 02:30), but no fluorescence was observed at the spindle midzone immediately after anaphase onset (Figure two; feo RNAi; time point 02:30), or right after furrow formation and ingression (Figure two; feo RNAi; in the 04:30 time point onwards).