Enhancement of myosin II/actin turnover at the contractile ring induces slower furrowing in dividing HeLa cells

Abstract

Myosin II ATPase activity is enhanced by the phosphorylation of MRLC (myosin II regulatory light chain) in non-muscle cells. It is well known that pMRLC (phosphorylated MRLC) co-localizes with F-actin (filamentous actin) in the CR (contractile ring) of dividing cells. Recently, we reported that HeLa cells expressing non-phosphorylatable MRLC show a delay in the speed of furrow ingression, suggesting that pMRLC plays an important role in the control of furrow ingression. However, it is still unclear how pMRLC regulates myosin II and F-actin at the CR to control furrow ingression during cytokinesis. In the present study, to clarify the roles of pMRLC, we measured the turnover of myosin II and actin at the CR in dividing HeLa cells expressing fluorescent-tagged MRLCs and actin by FRAP (fluorescence recovery after photobleaching). A myosin II inhibitor, blebbistatin, caused an enhancement of the turnover of MRLC and actin at the CR, which induced a delay in furrow ingression. Furthermore, only non-phosphorylatable MRLC and a Rho-kinase inhibitor, Y-27632, accelerated the turnover of MRLC and actin at the CR. Interestingly, the effect of Y-27632 was cancelled in the cell expressing phosphomimic MRLCs. Taken together, these results reveal that pMRLC reduces the turnover of myosin II and also actin at the CR. In conclusion, we show that the enhancement of myosin II and actin turnover at the CR induced slower furrowing in dividing HeLa cells.

Cytokinesis in Animal Cells

“…Photobleaching data in C. elegans embryos and Drosophila spermatocytes suggest that myosin II, anillin, and the septins progressively disassemble, but exchange only slowly, if at all, with cytoplasmic and cortical pools as the ring constricts (Carvalho et al 2009, Goldbach et al 2010. By contrast, rapid exchange of myosin II was observed in the furrow region of HeLa cells (Kondo et al 2011). In Drosophila S2 cells, some rapid turnover of myosin II was observed in the cleavage furrow, but both the rate and extent of exchange decreased dramatically as cytokinesis Contractile ring assembly in Schizosaccharomyces pombe versus in metazoans.…”

Actin Flows Mediate a Universal Coupling between Cell Speed and Cell Persistence

“…Non-uniform actin flow profiles, as observed for example in (Wilson et al, 2010;Ofer et al, 2011) would change the exponential shape of concentration profiles, but would leave the dependence on the actin flow speed qualitatively unchanged. To check this general prediction, we measured on motile mBMDCs the concentration profiles along the polarity axis for various molecules with different affinity for actin ( Figure 2E; Movie S3): Tamra (that unspecifically labels cytoplasmic proteins), Lifeact-GFP (low affinity) (Riedl et al, 2008), MLC-GFP (high affinity) (Kondo et al, 2011), and Utrophin-GFP (high affinity) (Burkel et al, 2007). As expected from the model, we observed that increasing the actin retrograde flow could significantly increase the slope of the concentration profile of strong actin binders (MLC, Utrophin), whereas the profiles of molecules with low (Lifeact) or no (Tamra) affinity to actin remained unchanged ( Figures 2E and 2F).…”

Signaling and cytoskeletal requirements in erythroblast enucleation

“…Nonmuscle myosin IIB was recently shown to participate in enucleation. 34 The exact mechanism of actin-myosin contraction, mediated either by myosin's head, 17,35,36 or by a tail-hinge apparatus, 37 but in any way regulated by MRLC phosphorylated at Ser19, a target of Rhokinase (ROCK) or MLCK, 38 remains to be elucidated. Using our fast in vitro enucleation assays, we evaluated the action of cytochalasin-D and ML7, inhibitors of actin polymerization and MLCK, respectively.…”