Phosphorylation of myosin II regulatory light chain controls its accumulation, not that of actin, at the contractile ring in HeLa cells

During cytokinesis in eukaryotic cells, an actomyosin-based contractile ring (CR) is assembled along the equator of the cell. Myosin II ATPase activity is stimulated by the phosphorylation of the myosin II regulatory light chain (MRLC) in vitro, and phosphorylated MRLC localizes at the CR in various types of cells. Previous studies have determined that phosphorylated MRLC plays an important role in CR furrowing. However, the role of phosphorylated MRLC in CR assembly remains unknown. Here, we have used confocal microscopy to observe dividing HeLa cells expressing fluorescent protein-tagged MRLC mutants and actin during CR assembly near the cortex. Di-phosphomimic MRLC accumulated at the cell equator earlier than non-phosphorylatable MRLC and actin. Interestingly, perturbation of myosin II activity by non-phosphorylatable MRLC expression or treatment with blebbistatin, a myosin II inhibitor, did not alter the time of actin accumulation at the cell equator. Furthermore, inhibition of actin polymerization by treatment with latrunculin A had no effect on MRLC accumulation at the cell equator. Taken together, these data suggest that phosphorylated MRLC temporally controls its own accumulation, but not that of actin, in cultured mammalian cells

Aurora B but Not Rho/MLCK Signaling Is Required for Localization of Diphosphorylated Myosin II Regulatory Light Chain to the Midzone in Cytokinesis

<div><p>Non-muscle myosin II is stimulated by monophosphorylation of its regulatory light chain (MRLC) at Ser19 (1P-MRLC). MRLC diphosphorylation at Thr18/Ser19 (2P-MRLC) further enhances the ATPase activity of myosin II. Phosphorylated MRLCs localize to the contractile ring and regulate cytokinesis as subunits of activated myosin II. Recently, we reported that 2P-MRLC, but not 1P-MRLC, localizes to the midzone independently of myosin II heavy chain during cytokinesis in cultured mammalian cells. However, the mechanism underlying the distinct localization of 1P- and 2P-MRLC during cytokinesis is unknown. Here, we showed that depletion of the Rho signaling proteins MKLP1, MgcRacGAP, or ECT2 inhibited the localization of 1P-MRLC to the contractile ring but not the localization of 2P-MRLC to the midzone. In contrast, depleting or inhibiting a midzone-localizing kinase, Aurora B, perturbed the localization of 2P-MRLC to the midzone but not the localization of 1P-MRLC to the contractile ring. We did not observe any change in the localization of phosphorylated MRLC in myosin light-chain kinase (MLCK)-inhibited cells. Furrow regression was observed in Aurora B- and 2P-MRLC-inhibited cells but not in 1P-MRLC-perturbed dividing cells. Furthermore, Aurora B bound to 2P-MRLC <i>in vitro</i> and <i>in vivo</i>. These results suggest that Aurora B, but not Rho/MLCK signaling, is essential for the localization of 2P-MRLC to the midzone in dividing HeLa cells.</p></div

Rho signaling is not required for the localization of 2P-MRLC to the midzone.

<p>(A) Western blots for the detection of MKLP1, MgcRacGAP, and ECT2. HeLa cells were transfected with siRNA targeting luciferase, MKLP1, MgcRacGAP, or ECT2, respectively. Loading control: α-tubulin. (B) HeLa cells treated with luciferase, MKLP1, MgcRacGAP, or ECT2 siRNAs were fixed and immunostained with pLC1. Bar, 10 µm. (C) Quantitative analysis of 1P-MRLC fluorescence at the contractile ring in cells depleted of luciferase, MKLP1, MgcRacGAP, or ECT2. The averages of at least 2 independent experiments are shown. Error bars indicate the standard error of the mean (±SEM). *p<0.05, **p<0.01 (<i>t</i>-test). <i>n</i> ≥15. (D) HeLa cells treated with luciferase, MKLP1, MgcRacGAP, or ECT2 siRNAs were fixed and immunostained as indicated. (E) Quantitative analysis of 2P-MRLC fluorescence at the midzone in cells depleted of luciferase, MKLP1, MgcRacGAP, or ECT2. Error bars indicate ±SEM. (F) HeLa cells treated with 10 µM Y-27632 or 10 µM ML-7 for 30 min were fixed and immunostained with CS1P pAb or 4F12. Bar, 10 µm. (G) Quantitative analysis of 1P-MRLC fluorescence at the contractile ring in cells treated as indicated. Representative data from 2 independent experiments are shown. **p<0.01 (<i>t</i>-test). <i>n</i> ≥11. (H) Quantitative analysis of 2P-MRLC fluorescence at the midzone (white bar) or contractile ring (black bar) in cells treated as indicated. Representative data from 2 independent experiments are shown. **p<0.01 (<i>t</i>-test). <i>n</i> ≥17.</p

Aurora B and Rho signaling proteins are independent regulators of the localization of F-actin to the contractile ring.

<p>HeLa cells treated with siRNAs targeting luciferase, Aurora B (A), MKLP1 (B), MgcRacGAP (C), or ECT2 (D) were fixed and immunostained as indicated. Bar, 10 µm. (E) Quantitative analysis of F-actin fluorescence at the contractile ring in cells treated as indicated. All values are presented as a percentage of the control. Error bars indicate ± SEM. *, p<0.05 (<i>t</i>-test). <i>n</i> ≥20.</p

Aurora B is not involved in the localization of 1P-MRLC to the contractile ring.

<p>(A and B) HeLa cells treated with either luciferase or Aurora B siRNAs were fixed and immunostained as indicated. Bar, 10 µm. (C) Quantitative analysis of 1P-MRLC fluorescence at the contractile ring in luciferase- or Aurora B-depleted cells. Error bars indicate ±SEM. <i>n</i> ≥19.</p

Aurora B activity, but not INCENP or survivin, is crucial for the localization of 2P-MRLC at the midbody.

<p>(A) Representative time-lapse imaging of HeLa cells treated with DMSO or 0.1 µM hesperadin for 3 h. Normal: a dividing cell; Regression: a furrow ingression (white arrowheads) but not in cells undergoing abscission; No furrowing: a cell showing no furrow ingression and no abscission; Round: a cell appearing round for at least 3 h. Bar, 10 µm. (B) Quantitative analysis of cells like those shown in (A). <i>n</i> ≥63. (C–E) HeLa cells treated with DMSO or 0.1 µM hesperadin for 30 min were fixed and immunostained as indicated. (F) Western blot detection of total MRLC and 2P-MRLC in mitotic HeLa cells treated with siRNA targeting luciferase or Aurora B. (G) Western blot detection of Aurora B in immunoprecipitates of endogenous 2P-MRLC from mitotic HeLa cell lysates. Endogenous 2P-MRLC was immunoprecipitated by 4F12. IP, immunoprecipitates; IgG, normal mouse IgG for a negative control. (H) Western blot detection of Aurora B in immunoprecipitates of endogenous 1P-MRLC from mitotic HeLa cell lysates. Endogenous 1P-MRLC was immunoprecipitated by CS1P mAb. IP, immunoprecipitates; IgG, normal mouse IgG for a negative control. (I) GST-Aurora B and GST were incubated with 2, 6, 18, or 54 µg 2P-MRLC. The upper panel indicates GST-Aurora B (66 kDa) or GST (26 kDa) on a Coomassie Brilliant Blue (CBB)-stained membrane, and the bottom panel shows MRLC detected by western blotting.</p

Distinct roles of 1P- and 2P-MRLC during cytokinesis.

<p>At the midzone (upper), the localization of 2P-MRLC is controlled by Aurora B and is essential for cell abscission. Moreover, at the contractile ring (lower), Rho signaling proteins, including MKLP1, MgcRacGAP, ECT2, Rho, and Rho kinase, but not MLCK and Aurora B, regulate the localization of 1P-MRLC, 2P-MRLC, and F-actin during cytokinesis, and they are essential for constriction of the contractile ring. The localization of MLCK and Rho kinase was described according to the literature (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0070965#pone.0070965-Poperechnaya1" target="_blank">[40]</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0070965#pone.0070965-Kosako2" target="_blank">[68]</a>, respectively). The Rho-dependent regulation of F-actin by mDia/formin was performed as previously described <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0070965#pone.0070965-Watanabe1" target="_blank">[69]</a>.</p

Aurora B is required for the localization of 2P-MRLC to the midzone.

<p>(A) Western blots for Aurora B detection. HeLa cells were transfected with siRNAs targeting luciferase or Aurora B. Loading control: α-tubulin. (B) HeLa cells treated with luciferase or Aurora B siRNAs were fixed and immunostained as indicated. Bar, 10 µm. (C) Quantitative analysis of 2P-MRLC fluorescence at the contractile ring (left) or midzone (right) in cells depleted of luciferase or Aurora B. Error bars indicate ±SEM. **p<0.01 (<i>t</i>-test). <i>n</i> ≥21.</p