Essential Genes for Cytokinesis in Rho-mediated Signaling
To clarify which genes are essential for cytokinesis, we treated HeLa cells with siRNAs to the genes involved in Rho-mediated signaling. These included RhoGEF (ECT2), GAP (MgcRacGAP), kinesinlike proteins (MKLP1 and CHO1), Rho GTPases (RhoA, Rac1, and Cdc42), and Rho-effector kinases (ROCK-I, ROCK-II, and Citron-kinase). We used siRNA to firefly luciferase as a control. Treatment with each siRNA yielded 90 –99% depletion of the target protein 72 h after transfection (). Because MKLP1 and CHO1 are the longer and the shorter isoforms, respectively, encoded by the same kinesin gene, siRNA to MKLP1 depleted both MKLP1 and CHO1 ().
Immunoblots showing siRNA-mediated depletion of the proteins involved in Rho-mediated signaling. HeLa cells were treated with the indicated siRNAs for 72 h. GAPDH, glyceraldehyde-3-phosphate dehydrogenase, for a loading control.
To evaluate cytokinesis failure, these siRNA-treated cells were examined by two different procedures: flow cytometry and morphological analysis. The flow cytometry profiles showed that ECT2, MgcRacGAP, MKLP1, and Citron-kinase RNAi increased the ratio of tetraploid (4N) and polyploid (8N and 16N) cells (). Morphological analysis confirmed increase in the percentage of bi/multinucleate cells in ECT2, MgcRacGAP, MKLP1, and Citron-kinase RNAi cells (). To further verify essential genes for cytokinesis, we continued siRNA treatment for 6 d. ECT2, MgcRacGAP, MKLP1, and Citron-kinase RNAi led to giant cells with multinuclei (Figure S1A) or multipolar spindles (Figure S1B), indicating multiple failures of cytokinesis. Thus, our combined flow cytometry and morphological analyses show that in HeLa cells, ECT2, MgcRacGAP, MKLP1, and Citron-kinase are required for cytokinesis, whereas CHO1, RhoA, Rac1, Cdc42, ROCK-I, and ROCK-II are not essential for cytokinesis.
Figure 2. Effects of RNAi on cytokinesis. HeLa cells were treated with the indicated siRNAs for 72 h. (A) Flow cytometry profiles; horizontal axis, DNA content; vertical axis, cell counts. Positions corresponding to 2N, 4N, 8N, and 16N are indicated. (B) Morphology (more ...)
Translocation of Active Rho Subfamily Proteins to the Equatorial Region Is Required for Contractile Ring Formation
Unexpectedly, RhoA RNAi did not inhibit cytokinesis. In the RhoA-depleted cells, the cleavage furrow was observed (), suggesting that RhoA is dispensable for cytokinesis. In mammalian cells, the Rho subfamily includes three close homologues, RhoA, RhoB, and RhoC, which might have redundant functions in cytokinesis. To confirm this, we did immunostaining and immunoblotting of RhoA-depleted cells, using RhoA-specific and pan-RhoA, B, and C antibodies (Yonemura et al., 2004
). Both antibodies stained the cleavage furrow in control luciferase RNAi cells (). In RhoA RNAi cells, RhoA-specific antibody failed to stain the cleavage furrow (), but pan-RhoA, B, and C antibody staining remained at the cleavage furrow (). Thus, besides RhoA, other Rho subfamily proteins, such as RhoB or RhoC, are localized at the cleavage furrow and may compensate for the loss of RhoA function.
Figure 3. Translocation of active Rho to the equatorial region is required for contractile ring formation. (A and B) RhoA RNAi. Cells were treated with siRNA to Luciferase or RhoA for 48 h. (A) Cells stained with RhoA-specific and pan-RhoA, B, C antibodies. Bar, (more ...)
C3 exoenzyme (C3) is an ADP-ribosyltransferase specific for Rho (but not Rac1 or Cdc42) and inactivates its functions (Fiorentini et al., 1998
). It is reported that C3-microinjected HeLa cells frequently detach from the dish or fail to initiate anaphase (O'Connell et al., 1999
). To avoid these problems, we used the scrape-loading method to deliver C3 to HeLa cells (McNeil et al., 1984
). The advantage of the scrape-loading method enabled us to analyze a large number of C3-loaded cells without decreasing cell viability. Treatment with C3 increased binucleate cells to ~80%, 24 h after loading (Figure S2, A and B). In the flow cytometry profile, C3 increased the population of tetraploid and polyploid cells (Figure S2C). Giant cells with multinuclei or multipolar spindles occurred 5 d after loading with C3 (Figure S2D). These findings support the idea that Rho plays an essential role in cytokinesis.
The localization of Rho during cell division is inconclusive. It is reported that the endogenous or tagged Rho protein is localized at the cell cortex but is not concentrated at the contractile ring (Drechsel et al., 1997
; Yoshizaki et al., 2003
). Recently, Rho has been detected at the cleavage furrow, using the TCA fixation method (Yonemura et al., 2004
). We used this TCA fixation method and examined the localization of Rho in HeLa cells at various mitotic stages. Before the onset of anaphase, Rho was localized in the cytoplasm (). We discovered that after chromosome separation, Rho translocated to the equatorial region before furrow ingression (). As reported previously (Yonemura et al., 2004
), Rho accumulated at the ingressed cleavage furrow and concentrated at the midbody (). Next, we examined the localization of Rho in C3-loaded cells. We found that C3 delocalized Rho from the equatorial region (). Thus, Rho (presumably active) accumulates at the equatorial region before furrow ingression.
Rho Activity Is Required for Accumulation of Myosin II at the Equatorial Region
To clarify the relationship between Rho and contractile ring formation, we scrape-loaded cells with the Rho-specific inhibitor C3 and examined the localization of myosin II and actin. C3 inhibited accumulation of both myosin heavy chain and actin at the equatorial region (). Thus, Rho is required for acto-myosin contractile ring formation.
To directly visualize the assembly of the contractile ring in living cells, we established HeLa cells that stably express MRLC-GFP. First, we determined the distribution of MRLC-GFP in PBS-loaded control cells during cell division. MRLC-GFP accumulated at the equatorial region after chromosome separation (, +4, +6 min; and Movie S1). Then, the cleavage furrow ingressed as MRLC-GFP accumulated (, +10 to +18 min). The furrow constricted to form the midbody (, +28 min). Finally, the midbody was severed and two daughter cells were separated (, +268 min). Next, we examined the localization of MRLC in C3-loaded cells. MRLC-GFP did not accumulate at the equatorial region even after chromosome separation (, +6, +10 min; and Movie S2). The cells spread without cleavage furrow ingression (, +20 to +90 min) and binucleate cells were generated (, +90 min). We monitored the scrape-loaded cells for 24 h by time-lapse microscopy. Although mock cells completed normal cell division (>99%; n = 188), C3-loaded cells failed to form the cleavage furrow (63%; n = 90). These observations reveal that during cytokinesis, Rho regulates myosin dynamics and contractile ring formation.
Figure 4. C3 exoenzyme inhibits contractile ring formation. (A and B) HeLa cells that stably express MRLC-GFP were loaded with PBS (Mock) or C3 by the scrape-loading method. Selected images of time-lapse recording of mock cells (A; Movie S1) and C3-loaded cells (more ...)
Phosphorylation of MRLC leads to bipolar myosin filaments, resulting in the formation of acto-myosin fibers (Applegate and Pardee, 1992
). It has been reported that MRLC is phosphorylated at the cleavage furrow (Matsumura et al., 1998
; Murata-Hori et al., 1998
; Ueda et al., 2002
). We found that phosphorylated MRLC colocalized with Rho at the equatorial region before furrow ingression (, Mock). C3 suppressed accumulation of phosphorylated MRLC at the equatorial region (, C3). Because MRLC is a substrate of Citron-kinase (Yamashiro et al., 2003
) and Citron-kinase is localized at the cleavage furrow (Madaule et al., 1998
), we examined the localization of Citron-kinase. Like phosphorylated MRLC, Citron-kinase accumulated at the equatorial region before furrow ingression, and C3 inhibited this accumulation (). Thus, Rho is required for accumulation of phosphorylated MRLC and Citron-kinase at the equatorial region.
Rho Localization Is Independent of Myosin and Actin
Inhibition of myosin II ATPase activity does not prevent contractile ring formation (Straight et al., 2003
). To determine whether myosin is required for the localization of Rho, we treated cells with blebbistatin, a myosin II ATPase inhibitor. Blebbistatin blocked furrow ingression but not contractile ring formation (). Rho and the downstream signals of Rho, Citron-kinase and phosphorylated MRLC, accumulated at the equatorial region (). Thus, the motor activity of myosin II is not required for translocation of Rho to the equatorial region.
Figure 5. Translocation of Rho to the equatorial region is independent of actin and myosin. HeLa cells were treated with DMSO (Control), blebbistatin, or latrunculin B (Lat B). Cells were fixed and stained with anti-Rho A, B, and C, anti-α-tubulin, anti-Citron-kinase (more ...)
It has been reported that in yeast, accumulation of myosin at the cleavage furrow does not depend on F-actin (Bi et al., 1998
; Motegi et al., 2000
). To test whether actin is required for Rho localization, we disrupted F-actin structure, using latrunculin B (). Rho accumulated at the equatorial region (), indicating that F-actin is not required for Rho localization. Also, Citron-kinase and phosphorylated MRLC accumulated at the equatorial region (). Myosin heavy chain accumulated at the equatorial region but the distribution of myosin heavy chain was abnormal (, granular structures), suggesting the requirement of actin for proper distribution of myosin II.
ECT2, MgcRacGAP, and MKLP1 Regulate Translocation of Rho to the Equatorial Region
Overexpression of oncogenic Dbl GEF has been shown to translocate Rho to the plasma membrane in interphase cells (Michaelson et al., 2001
). We examined whether ECT2 Rho-GEF is responsible for translocation of Rho to the equatorial region in anaphase. Depletion of ECT2 by RNAi diminished both RhoA and RhoA, B, and C staining at the equatorial region (). In ECT2-depleted anaphase cells, RhoA was delocalized from the equatorial region or exhibited aberrant localization (79 and 16%, respectively; ). Thus, ECT2 regulates translocation of Rho to the equatorial region.
Figure 6. ECT2, MgcRacGAP, and MKLP1-dependent translocation of Rho to the equatorial region. HeLa cells were treated with siRNA to luciferase, ECT2, MgcRacGAP, or MKLP1 for 36 h. The cells were fixed with 10% TCA. Bars, 10 μm. (A) Cells stained with RhoA-specific (more ...)
MgcRacGAP and MKLP1 form a complex at the central spindle in anaphase and are required for completion of cytokinesis (Mishima et al., 2002
). To test whether MgcRac-GAP and MKLP1 regulate contractile ring formation, we examined the localization of Rho in MgcRacGAP- or MKLP1-depleted cells. As in ECT2 RNAi cells, Rho was delocalized or exhibited abnormal localization in MgcRac-GAP or MKLP1 RNAi cells (). Thus, MgcRac-GAP and MKLP1 are also responsible for translocation of Rho to the equatorial region.
Because Citron-kinase and phosphorylated MRLC are the downstream signals of Rho, we examined the localization of these proteins in the siRNA-treated cells. Like C3 treatment, ECT2, MgcRacGAP, or MKLP1 RNAi inhibited accumulation of phosphorylated MRLC and Citron-kinase at the equatorial region (). Thus, ECT2, MgcRacGAP, and MKLP1 regulate the downstream signals of Rho for contractile ring formation.
ECT2, MgcRacGAP, and MKLP1 Regulate Contractile Ring Formation
The requirement of ECT2, MgcRacGAP, and MKLP1 for completion of cytokinesis (severing of the intercellular bridge or abscission) has been reported (Tatsumoto et al., 1999
; Jantsch-Plunger et al., 2000
; Kuriyama et al., 2002
; Matuliene and Kuriyama, 2002
; Minoshima et al., 2003
). To confirm which step of cytokinesis is regulated by these genes, we treated HeLa cells with siRNAs and recorded time-lapse images. ECT2, MgcRacGAP, or MKLP1 RNAi caused a variety of defective cytokinesis phenotypes, including no cleavage furrow formation, incomplete furrowing, and regression (Figure S3, A and B). These observations suggest that ECT2, MgcRacGAP, or MKLP1 regulates multiple steps of cytokinesis, including contractile ring formation, furrow ingression, and completion of cytokinesis. Alternatively, they may only regulate the initial step of cytokinesis (contractile ring formation), which affects later cytokinetic events.
Oceguera-Yanez et al
)reported that ECT2 and MgcRacGAP regulate bipolar spindle assembly in prometa- to metaphase. However, by our time-lapse microscopy, we were not able to detect major detects in mitosis, such as prolonged prometaphase or chromosome mis-separation, in ECT2 or MgcRacGAP RNAi cells.
To analyze the relationship between the depletion level of the ECT2, MgcRacGAP, or MKLP1, and defects in contractile ring formation, we treated MRLC-GFP-expressing cells with siRNA to luciferase, ECT2, MgcRacGAP, or MKLP1. When ECT2, MgcRacGAP, or MKLP1 were almost completely depleted, MRLC-GFP did not accumulate at the equatorial region and furrow ingression was prevented (Figure S4F). In contrast, residual amounts of ECT2, MgcRacGAP, and MKLP1 led to accumulation of MRLC-GFP at the equatorial region and ingression of the furrow (Figure S4, C–E). Therefore, it seems that the severity of abnormal cytokinesis phenotypes correlate with the depletion level of each protein.
To show failure of contractile ring formation directly, we treated MRLC-GFP-expressing cells with siRNAs and recorded time-lapse images. Luciferase RNAi cells exhibited normal furrow ingression with accumulation of MRLC at the equatorial region ( and Movie S3). In contrast, ECT2 RNAi inhibited accumulation of MRLC at the equatorial region, indicating that contractile ring formation was disrupted (, 0 to +20 min; and Movie S4). These cells spread without dividing, resulting in binucleate cells (, +25 to +72). MgcRacGAP or MKLP1 RNAi caused similar defects in accumulation of MRLC without forming the cleavage furrow (, and Movies S5 and S6, respectively). Thus, ECT2, MgcRacGAP, and MKLP1 all regulate myosin dynamics and contractile ring formation during cytokinesis.
Figure 7. Formation of the contractile ring depends on ECT2, MgcRacGAP, and MKLP1. (A–D) Distribution of MRLC during cell division. Selected images from time-lapse recording. HeLa cells that stably express MRLC-GFP were treated with siRNA to Luciferase (more ...)
ECT2 Forms a Complex with MgcRacGAP and MKLP1 at the Central Spindle
, PBL RhoGEF forms a complex with RacGAP50C and PAV kinesin. RacGAP50C and PAV are colocalized at the central spindle but PBL is localized at the equatorial cell cortex (Somers and Saint, 2003
). We examined the localization of mammalian orthologues ECT2, MgcRac-GAP, and MKLP1 during cell division. In metaphase, they were localized in the cytoplasm. (, Metaphase). After the onset of anaphase, MgcRacGAP and MKLP1 accumulated at the central spindle (, Anaphase). Although, in Drosophila
, PBL RhoGEF does not colocalize with RacGAP50C and PAV at the central spindle (Somers and Saint, 2003
), we found that in mammalian cells, ECT2 colocalized with MgcRacGAP and MKLP1 at the interdigitating portion of the central spindle (, Anaphase).
Figure 8. ECT2 forms complex with MgcRacGAP and MKLP1 at the central spindle. (A) Subcellular localization of ECT2, MgcRacGAP, and MKLP1 in metaphase and anaphase. HeLa cells were stained with indicated antibodies and anti-α-tubulin. Bar, 10 μm. (more ...)
To examine whether ECT2 forms a complex with MgcRac-GAP and MKLP1, we coimmunoprecipitated these proteins, using an extract from HeLa cells synchronized in anaphase to cytokinesis. MgcRacGAP was coimmunoprecipitated with MKLP1 (, lane 5) and MKLP1 was coimmunoprecipitated with MgcRacGAP (, lane 4), confirming formation of an equimolar complex known as centralspindlin (Mishima et al., 2002
). We found that ECT2 was coimmunoprecipitated with MgcRacGAP and MKLP1 (, lane 4 and lane 5, respectively) and vice versa (, lane 3). Thus, ECT2 RhoGEF forms a complex with MgcRacGAP and MKLP1.
To identify which component of centralspindlin (MgcRac-GAP or MKLP1) directly interacts with ECT2, we did a yeast two-hybrid analysis (). As expected, an interaction between MgcRacGAP and MKLP1 was detected. We found that ECT2 interacted with MgcRacGAP but not MKLP1.
We tested whether this complex formation determines the localization of ECT2, MgcRacGAP, and MKLP1 at the central spindle. First, we examined the localization of ECT2 in MgcRacGAP or MKLP1-depleted cells. Luciferase RNAi did not affect the localization of ECT2 at the central spindle (). In contrast, MgcRacGAP or MKLP1 RNAi delocalized ECT2 from the central spindle (). Thus, the localization of ECT2 at the central spindle depends on both MgcRacGAP and MKLP1. Next, we examined the localization of MgcRacGAP and MKLP1. ECT2 RNAi did not affect the localization of these proteins (). However, MgcRacGAP RNAi delocalized MKLP1 () and MKLP1 RNAi delocalized MgcRacGAP () from the central spindle. Thus, MgcRacGAP localization depends on MKLP1 and MKLP1 localization depends on MgcRacGAP.
Figure 9. Localization of ECT2 at the central spindle depends on MgcRacGAP and MKLP1. Localization of ECT2, MgcRacGAP, and MKLP1 in cells depleted of these protein by RNAi. Bar, 10 μm. In control cells, ECT2 (A), MgcRacGAP (E), and MKLP1 (I) were localized (more ...)
Bundled Microtubules Direct the Furrowing Site via Rho-mediated Signaling
Because anaphase microtubules specify the position of the contractile ring, we examined the relationship between microtubules and Rho. The microtubule inhibitor nocodazole inhibits polymerization of microtubules and arrests cells in prometaphase. Therefore, nocodazole prevented the onset of anaphase and translocation of Rho to the equatorial region (, Nocodazole). Inactivation of Cdk1/cyclin B regulates the timing of mitotic exit and cytokinesis as well as the mitotic spindle array (Wheatley et al., 1997
; Echard and O'Farrell, 2003
; Mishima et al., 2004
). Recently, it has been reported that the Cdk1 inhibitors BM-1026 and purvanalol A promote premature mitotic exit and furrow ingression (Niiya et al., 2005
). To test whether the Cdk1 inhibitor roscovitine affects initiation of cytokinesis, we treated synchronized prometaphase cells with DMSO (control) or roscovitine (). Control cells entered anaphase 1–3 h after nocodazole release, and 88% (n = 65) of the cells completed normal mitosis and cytokinesis within 4 h (; and Movie S7). Like other Cdk1 inhibitors (Niiya et al., 2005
), roscovitine promoted premature exit from mitosis (Movie S8). The cells formed ectopic furrows (, arrows) and exhibited cortical contraction (81%; n = 115). The cortical contraction started ~20 min after nocodazole release and lasted ~1 h. Finally, the cells spread out again without dividing.
Figure 10. Bundled microtubules direct the Rho-signaling molecules to the furrowing site. Note that a, b, and c in C–E correspond to the experimental diagram shown in A. (A) Schematic diagram showing roscovitine treatment. Cells were treated with nocodazole (more ...)
To investigate whether ectopic furrow formation, like normal cleavage furrow formation, depends on Rho-mediated signaling, we examined the localization of Rho in roscovitine-treated cells. Rho accumulated at the ectopic furrows 30 min after nocodazole release (, arrows). Adjacent to the ectopic furrows, bundled microtubules were observed. Citron-kinase and phosphorylated MRLC, the downstream signals of Rho, also accumulated at the ectopic furrows (, arrows). Most cells completed cortical contraction 90 min after nocodazole release, when microtubule bundles and Rho accumulation disappeared (). These results suggest that ectopic furrow formation depends on Rho-mediated signaling.
To test whether the timing of Cdk1 inhibition affects microtubule organization and furrow formation, we pretreated cells with roscovitine 15 min before nocodazole washout (). Roscovitine pretreatment suppressed cortical contraction to 11% (n = 82; ), and cells spread without dividing (; and Movie S9). Microtubule bundling was not observed either in the presence of nocodazole (, Nocodazole + Roscovitine) or after washout of nocodazole (). Ectopic furrow formation was suppressed, and Rho (), Citron-kinase or phosphorylated MRLC () did not accumulate. These results suggest that proper temporal regulation of Cdk1 inhibition is required for microtubule bundling, and bundled microtubules spatially direct the localization of the Rho-mediated signaling molecules.
To confirm whether ECT2 RhoGEF regulates translocation of Rho and formation of the furrows, luciferase or ECT2 RNAi cells were treated with roscovitine. Luciferase RNAi cells exhibited cortical contraction (92%; n = 109; Movie S10). In contrast, as in the case of BMI-1026 treatment (Niiya et al., 2005
), roscovitine-induced cortical contraction was suppressed by ECT2 RNAi (3%; n = 93; Movie S11). In luciferase RNAi cells, ECT2 accumulated at the bundled microtubules () and Rho accumulated at the ectopic furrows (). Although microtubules were bundled by roscovitine treatment, ECT2 RNAi suppressed ectopic furrow formation () and accumulation of Rho (). These results show that the ECT2-Rho pathway is the link between microtubules and furrow formation.