Myosin II Recruitment during Cytokinesis Independent of Centralspindlin-mediated Phosphorylation

During cell division, the mechanisms by which myosin II is recruited to the contractile ring are not fully understood. Much recent work has focused on a model in which spatially restricted de novo filament assembly occurs at the cell equator via localized myosin II regulatory light chain (RLC) phosphorylation, stimulated by the RhoA-activating centralspindlin complex. Here, we show that a recombinant myosin IIA protein that assembles constitutively and is incapable of binding RLC still displays strong localization to the furrow in mammalian cells. Furthermore, this RLC-deficient myosin II efficiently drives cytokinesis, demonstrating that centralspindlin-based RLC phosphorylation is not necessary for myosin II localization during furrowing. Myosin II truncation analysis further reveals two distinct myosin II tail properties that contribute to furrow localization: a central tail domain mediating cortical furrow binding to heterologous binding partners and a carboxyl-terminal region mediating co-assembly with existing furrow myosin IIA or IIB filaments.Non-muscle myosin II, through its interaction with F-actin, is believed to be the dominant force-producing machinery utilized to separate daughter cells during cell division. Following anaphase onset, myosin II is recruited to the equatorial cortex where it assembles into the contractile ring. Despite much recent progress, the exact mechanism by which myosin II is recruited to and retained in the contractile ring in the proper spatio-temporal manner remains unclear.Myosin II is a member of the myosin superfamily that binds F-actin and hydrolyzes ATP to produce force. A monomer consists of two myosin heavy chains (“MHCs”),³ two essential light chains (“ELCs”), and two regulatory light chains (“RLCs”) (see Fig. 1A). The MHC consists of an amino-terminal globular head domain often referred to as the “motor” domain. It is responsible for F-actin binding and ATP binding and hydrolysis. One RLC and one ELC associate with each MHC via two IQ motifs on a neck region linking the head and tail domain. The remainder of the MHC forms a continuous α-helix that interacts with another MHC rod to create a coiled-coil-mediated dimer. At the extreme C terminus, mammalian non-muscle myosin II molecules contain an ∼30 residue “non-helical tailpiece.” Many phosphorylation sites have been identified on both the RLC and the MHC (1–4). The best characterized of these sites is Thr-18/Ser-19 on the RLC, which, when phosphorylated, has been shown to activate myosin II by increasing the affinity of MHC for F-actin, consequently increasing the ATPase activity (5, 6). Phosphorylation of these sites is also able to convert myosin II from a folded 10 S “inactive” monomer into the extended 6 S monomer that readily forms filaments (7).Open in a separate windowFIGURE 1.RLC-independent localization of myosin to furrow in HeLa and COS-7 cells. A, diagram of myosin IIA. GFP was conjugated to the amino terminus of the MHC. B, diagram of GFP-IIA constructs. GFP-IIA-ΔIQ2 removes the RLC binding site known as the IQ2 motif. C and D, at 72 h after transfection, HeLa (C) or COS-7 (D) cells expressing GFP-IIA (top row) or GFP-IIA-ΔIQ2 in early anaphase (middle row) or late anaphase (lower row) were fixed and stained with phalloidin-568 (red) for actin and DAPI (blue) for DNA. The images in the right column are merges of actin, DNA, and GFP channels.Mammalian genomes contain three genes encoding non-muscle myosin II heavy chain isoforms, mhc IIA, IIB, and IIC. MHC IIA and IIB are widely expressed in many tissues and cell lines, whereas IIC is expressed with a more limited distribution (8). In mice, gene knockouts of mhc IIA and IIB result in differing phenotypes that are only partially rescued by the other isoform, suggesting that both isoform-specific and overlapping roles exist (9). Previous reports have suggested that myosin IIA and IIB isoforms are capable of co-assembling into mixed or heterotypic filaments (10, 11). However, there is also evidence showing that myosin II isoforms have different subcellular localization in non-mitotic cells, supportinga model in which homotypic filaments are the dominant filamentous structure in live cells (12, 13). Whether myosin IIA and IIB can co-assemble in the contractile ring of dividing cells is not known.The dominant model for furrow localization of myosin II during cell division hypothesizes spatially restricted equatorial activation and filament assembly via phosphorylation of the RLC on Thr-18/Ser-19. The most prominent upstream signaling pathway implicated in this furrow recruitment model is the centralspindlin pathway. In this pathway, the kinesin-6 protein MKLP1 anchors MgcRacGAP and a RhoGEF (ECT2) to the spindle midzone (14). This in turn locally activates RhoA, leading to activation of Rho kinase and/or citron kinase, both of which have been shown capable of phosphorylating RLC (15–18). Centralspindlin-based signals clearly contribute to myosin II-cytokinesis functions. However, whether these signals contribute to initial myosin II binding/recruitment, to myosin II contractile activation, or to both, is unresolved.Another recent study reported that GFP-tagged RLC constructs with alanine substitutions at the activating Thr-18/Ser-19 sites were still able to localize to the furrow of dividing HeLa cells, suggesting that RLC phosphorylation is not required for myosin equatorial localization (19). However, in that study, it was not clear how much endogenous wild type RLC was present; thus this result may represent a tracer amount of the T18A/S19A mutant RLC passively co-assembling with a larger pool of endogenous RLC-phosphorylated myosin II.Another proposed model for myosin recruitment to the equatorial region of a dividing cell is cortical flow. In this model, supported by observations in both Dictyostelium and mammalian cells, myosin filaments move along the cortex and into the furrow in a motor-dependent manner (20–22). However, recent studies using total internal reflection fluorescence imaging in normal rat kidney cells revealed no detectable myosin II cortical flow (23), raising uncertainty as to whether cortical flow is an important mechanism for myosin recruitment in mammalian cells.In this study, we provide the first evidence that mammalian myosin II can localize to the furrow of dividing cells independent of the regulatory light chain. These studies demonstrate that both robust myosin II recruitment to the furrow and efficient cell division can be achieved without spatially localized centralspindlin-mediated RLC phosphorylation. We conclude that other mechanisms such as cortical flow (22, 24) and/or equatorial myosin II binding partners (25, 26) must be sufficient for myosin II recruitment and cell furrowing in mammalian cells. Furthermore, we show that a headless myosin construct can localize to the contractile ring, supporting a model in which actin binding and ATPase activity are not required for myosin II recruitment. We also provide novel evidence that MHC isoforms are capable of co-assembling in the contractile ring.