Myosin II Recruitment during Cytokinesis Independent of Centralspindlin-mediated Phosphorylation

Abstract

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 ␣-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 ϳ30 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).Mammalian genomes contain three genes encoding nonmuscle 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 ...

BRCA2 Phosphorylated by PLK1 Moves to the Midbody to Regulate Cytokinesis Mediated by Nonmuscle Myosin IIC

“…Attachment of a contractile ring, consisting of a network of actin filaments (actomyosin) and NM-II, to the cytoplasmic membrane induces the formation of a cleavage furrow. Mammalian cells use not IIC but IIA or IIB during this process (10). NM-IIA exhibited a diffuse distribution throughout the entire midbody as observed by immunofluorescence microscopy ( Fig.…”

“…BRCA2 also contributes to the regulation of cytokinesis (6,7). During anaphase, constriction of the actomyosin ring leads to formation of a cleavage furrow (8)(9)(10). Continued furrowing results in the formation of a narrow intercellular bridge, which contains the midbody, consisting of a bundled microtubule and a ringlike structure called the Flemming body within its central portion (11).…”

RUNX1-induced silencing of non-muscle myosin heavy chain IIB contributes to megakaryocyte polyploidization

“…This experiment indicates that there still exists some myosin II activity at the contractile ring at the end of MK telophase, even with a reduced myosin heavy chain accumulation. MLC2 phosphorylation regulates myosin II activity without affecting the recruitment of myosin II and F-actin in the contractile ring 13,17,18 and controls the actin dynamics (assembly and disassembly) necessary for completion of cytokinesis 19 . To study actin turnover, CD34 + cells were transfected with a GFP-β-actin construct and induced to differentiate into hematopoietic cells by a cocktail of growth factors.…”

Role for the actomyosin complex in regulated exocytosis revealed by intravital microscopy

“…The role of F-actin in facilitating the collapse of the SCGs may be linked to the assembly of an actomyosin complex, as described for the contractile ring during cytokinesis (39,40). Indeed, nonmuscle myosin IIa has been previously implicated in the regulation of the opening and the closing of the fusion pore during regulated secretion (14,15,41).…”