Different contributions of nonmuscle myosin IIA and IIB to the organization of stress fiber subtypes in fibroblasts

Stress fibers (SFs) are contractile, force-generating bundled structures that can be classified into three subtypes, namely ventral SFs (vSFs), transverse arcs (TAs), and dorsal SFs. Nonmuscle myosin II (NMII) is the main component of SFs. This study examined the roles of the NMII isoforms NMIIA and NMIIB in the organization of each SF subtype in immortalized fibroblasts. Knockdown (KD) of NMIIA (a major isoform) resulted in loss of TAs from the lamella and caused the lamella to lose its flattened shape. Exogenous expression of NMIIB rescued this defect in TA formation. However, the TAs that formed on exogenous NMIIB expression in NMIIA-KD cells and the remaining TAs in NMIIB-KD cells, which mainly consisted of NMIIB and NMIIA, respectively, failed to rescue the defect in lamellar flattening. These results indicate that both isoforms are required for the proper function of TAs in lamellar flattening. KD of NMIIB resulted in loss of vSFs from the central region of the cell body, and this defect was not rescued by exogenous expression of NMIIA, indicating that NMIIA cannot replace the function of NMIIB in vSF formation. Moreover, we raised the possibility that actin filaments in vSFs are in a stretched conformation

Novel Kelch-like Protein, KLEIP, Is Involved in Actin Assembly at Cell-Cell Contact Sites of Madin-Darby Canine Kidney Cells

Dynamic rearrangements of cell-cell adhesion underlie a diverse range of physiological processes, but their precise molecular mechanisms are still obscure. Thus, identification of novel players that are involved in cell-cell adhesion would be important. We isolated a human kelch-related protein, Kelch-like ECT2 interacting protein (KLEIP), which contains the broad-complex, tramtrack, bric-a-brac (BTB)/poxvirus, zinc finger (POZ) motif and six-tandem kelch repeats. KLEIP interacted with F-actin and was concentrated at cell-cell contact sites of Madin-Darby canine kidney cells, where it colocalized with F-actin. Interestingly, this localization took place transiently during the induction of cell-cell contact and was not seen at mature junctions. KLEIP recruitment and actin assembly were induced around E-cadherin–coated beads placed on cell surfaces. The actin depolymerizing agent cytochalasin B inhibited this KLEIP recruitment around E-cadherin–coated beads. Moreover, constitutively active Rac1 enhanced the recruitment of KLEIP as well as F-actin to the adhesion sites. These observations strongly suggest that KLEIP is localized on actin filaments at the contact sites. We also found that N-terminal half of KLEIP, which lacks the actin-binding site and contains the sufficient sequence for the localization at the cell-cell contact sites, inhibited constitutively active Rac1-induced actin assembly at the contact sites. We propose that KLEIP is involved in Rac1-induced actin organization during cell-cell contact in Madin-Darby canine kidney cells

Dissecting the Role of Rho-mediated Signaling in Contractile Ring Formation

In anaphase, microtubules provide a specification signal for positioning of the contractile ring. However, the nature of the signal remains unknown. The small GTPase Rho is a potent regulator of cytokinesis, but the involvement of Rho in contractile ring formation is disputed. Here, we show that Rho serves as a microtubule-dependent signal that specifies the position of the contractile ring. We found that Rho translocates to the equatorial region before furrow ingression. The Rho-specific inhibitor C3 exoenzyme and small interfering RNA to the Rho GDP/GTP exchange factor ECT2 prevent this translocation and disrupt contractile ring formation, indicating that active Rho is required for contractile ring formation. ECT2 forms a complex with the GTPase-activating protein MgcRacGAP and the kinesinlike protein MKLP1 at the central spindle, and the localization of ECT2 at the central spindle depends on MgcRacGAP and MKLP1. In addition, we show that the bundled microtubules direct Rho-mediated signaling molecules to the furrowing site and regulate furrow formation. Our study provides strong evidence for the requirement of Rho-mediated signaling in contractile ring formation

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

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

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