Ptins coexist in complexes, they may have distinct functions in the course of cell division. To address this, we followed septin-depleted cells via cell division by Tubastatin-A site time-lapse microscopy. Separation of the DNA, ingression with the cleavage furrow, and formation with the midbody appeared normal upon handle KD (Fig. three A and Video 1). However, SEPT2 (Video 2)- and SEPT11 (Fig. three B and Video three)depleted cells exhibited abnormal cleavage furrow contraction at the early stages of cytokinesis. Though cleavage furrow ingression started normally, the size in the nascent daughter cells then fluctuated considerably. In most instances, the cleavage furrow did sooner or later totally ingress; on the other hand, it did so to the side ofboth daughter nuclei, hence creating a multinucleated cell and an enucleated cell. Within a few situations, cleavage furrow regression was observed, as described previously (Spiliotis et al., 2005). Depletion of all septins with SEPT7 siRNA had exactly the same effects. A related phenotype has been observed upon depletion of the septin-binding protein anillin (Straight et al., 2005), raising the possibility that SEPT2 and SEPT11 might mediate anillin function during cytokinesis. Regardless of the enhance in multinucleated cells observed upon SEPT9 KD (Fig. 2 C), these cells showed no defects in DNA separation, cleavage furrow constriction, or midbody formation (Fig. 3 C and Video 4). Rather, SEPT9-depleted cells exhibited dramatic defects in midbody abscission (Fig. three C and Video five). The midbody of control KD cells abscised an average of three.9 h after the onset of cytokinesis, and all of these cells effectively completed abscission (Fig. 3 D). In contrast, only 70 of SEPT9 KD cells successfully abscised, together with the imply abscission time getting 9 h just after the initiation of cytokinesis (Fig. three D;SEPT9 mediates midbody abscission Estey et al.Figure three. SEPT9 mediates midbody abscission. (A ) HeLa cells have been transfected with siRNA, and randomly selected cells have been followed by way of division by time-lapse microscopy. The time (in hours:minutes) since the starting of DNA segregation is shown. Black arrows point to intact midbodies, whereas white arrows denote abscission. Bars, 32 . (A) Division of HeLa cells just after remedy with control siRNA. (B) SEPT11 KD causes defects early in cytokinesis. (C) SEPT9 KD causes defects in midbody abscission. See Videos 1 for comprehensive films. (D and E) Quantification in the effect of SEPT9 KD on midbody abscission in HeLa (D) and ARPE-19 cells (E). The time from DNA segregation to midbody abscission was determined, plus the cumulative percentage of cells that were abscised is plotted as a function of time. For D, n = 45 cells for control KD and n = 30 cells for SEPT9 KD; for E, n = 34 cells for handle KD and n = 25 cells for SEPT9 KD.P 0.0001). Remarkably, in 20 of SEPT9 KD cells, the midbody either persisted until the next division (Fig. three, C and D) or had not broken 40 h just after the onset of cytokinesis. In some circumstances, the midbody broke when each daughter cells reentered mitosis, whereas in other instances it persisted even right after a second round of division. The remaining ten of SEPT9 KD cells exhibited one of two phenotypes: either the midbody regressed soon after failing to abscise, giving rise to a multinucleated cell (hence accounting for our fixed-cell benefits), or one or both daughter cells underwent apoptosis just after getting unable to complete744 JCB VOLUME 191 Number 4 abscission. Impaired midbody abscission upon SEPT9 KD was also observed in noncancer.