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. 2010 Nov 15;191(4):741-9.
doi: 10.1083/jcb.201006031. Epub 2010 Nov 8.

Distinct roles of septins in cytokinesis: SEPT9 mediates midbody abscission

Mathew P Estey  1 , Caterina Di Ciano-Oliveira, Carol D Froese, Margaret T Bejide, William S Trimble
Affiliations

Distinct roles of septins in cytokinesis: SEPT9 mediates midbody abscission

Mathew P Estey et al. J Cell Biol. .

Abstract

Septins are a family of GTP-binding proteins implicated in mammalian cell division. Most studies examining the role of septins in this process have treated the family as a whole, thus neglecting the possibility that individual members may have diverse functions. To address this, we individually depleted each septin family member expressed in HeLa cells by siRNA and assayed for defects in cell division by immunofluorescence and time-lapse microscopy. Depletion of SEPT2, SEPT7, and SEPT11 causes defects in the early stages of cytokinesis, ultimately resulting in binucleation. In sharp contrast, SEPT9 is dispensable for the early stages of cell division, but is critical for the final separation of daughter cells. Rescue experiments indicate that SEPT9 isoforms containing the N-terminal region are sufficient to drive cytokinesis. We demonstrate that SEPT9 mediates the localization of the vesicle-tethering exocyst complex to the midbody, providing mechanistic insight into the role of SEPT9 during abscission.

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Figures

Figure 1.
Figure 1.
Septin distribution during cell division. HeLa cells were stained for α-tubulin (red), DNA (blue), and the indicated septin (green). Confocal microscopy was performed, and a single z slice is shown. Bars, 17 µm.
Figure 2.
Figure 2.
Roles of individual septins in cell division. (A) HeLa cells were treated with the indicated siRNA, and lysates were probed as specified. Numbers to the right of the blot represent molecular mass standards in kilodaltons. (B) Representative examples of cells treated with control siRNA, and of multinucleated cells and cells attached by persistent midbodies after SEPT9 KD (red, α-tubulin; blue, DNA). Bars, 17 µm. (C) Quantification of effect of septin KD on cytokinesis. The percentage of cells exhibiting multinucleation or midbody attachment was determined upon treatment with the indicated siRNA. Data are represented as mean ± SEM (error bars; n ≥ 300 cells from three or more independent experiments). Asterisks indicate differences between control and septin KD cells. *, P < 0.01; **, P < 0.0001.
Figure 3.
Figure 3.
SEPT9 mediates midbody abscission. (A–C) HeLa cells were transfected with siRNA, and randomly selected cells were followed through division by time-lapse microscopy. The time (in hours:minutes) since the beginning of DNA segregation is shown. Black arrows point to intact midbodies, whereas white arrows denote abscission. Bars, 32 µm. (A) Division of HeLa cells after treatment with control siRNA. (B) SEPT11 KD causes defects early in cytokinesis. (C) SEPT9 KD causes defects in midbody abscission. See Videos 1–5 for complete movies. (D and E) Quantification of 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, and 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 control KD and n = 25 cells for SEPT9 KD.
Figure 4.
Figure 4.
The SEPT9 isoforms are not all functionally equivalent. (A and B) Stable cell lines inducibly expressing siRNA-resistant Flag-SEPT9_i3 (A) or Flag-SEPT9_i4 (B) were treated with increasing amounts of doxycycline, and expression was assayed by Western blotting. (C) The parent and Flag-SEPT9_i3 cell lines were treated with control or SEPT9 siRNA in the absence of doxycycline, and SEPT9 levels were assayed by Western blotting. Note that SEPT9_i4 disappears upon treatment with SEPT9 siRNA, demonstrating efficient KD. Also note the higher SEPT9 (_i1-3) levels in the Flag-SEPT9_i3 stable line upon SEPT9 depletion, when compared with the parent line. (D) The parent and Flag-SEPT9_i4 cell lines were treated as in C. Numbers to the right of the blots represent molecular mass standards in kilodaltons. (E) The parent, Flag-SEPT9_i3, and Flag-SEPT9_i4 cell lines were treated with control or SEPT9 siRNA and assayed for defects in cytokinesis by immunofluorescence microscopy. Unresolved cytokinesis refers to cells exhibiting midbody attachment or multinucleation. Data are represented as mean ± SEM (error bars; n ≥ 300 cells from three or more independent experiments). *, P < 0.05; **, P < 0.005.
Figure 5.
Figure 5.
SEPT9 mediates exocyst complex localization to the midbody. (A) Quantification of the effect of SEPT9 KD on the localization of midbody components. The percentage of cells exhibiting enrichment of the protein of interest at the midbody was determined by immunofluorescence microscopy after control or SEPT9 KD. Data are represented as mean ± SEM (error bars; n ≥ 300 cells from three independent experiments). Asterisks indicate differences between control and SEPT9 KD cells; **, P < 0.001. (B and C) Representative example of the localization of the exocyst component Sec8 in cytokinetic cells upon control (B) or SEPT9 KD (C). Arrows point to the midbody. (D) Sec8 fluorescence at the midbody was quantified using ImageJ upon control or SEPT9 KD; **, P < 0.0001. (E) Double staining of SEPT9 and Sec8 in cytokinetic cells. Bars, 17 µm.
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