Expansion and concatenation of nonmuscle myosin IIA filaments drive cellular contractile system formation during interphase and mitosis

Fenix, Aidan M.; Taneja, Nilay; Buttler, Carmen A.; Lewis, J. E.; Engelenburg, Schuyler B. van; Ohi, Ryoma; Burnette, Dylan T.

doi:10.1091/mbc.e15-10-0725

Cited by 113 publications

(183 citation statements)

References 62 publications

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“…Recent research, using superresolution light-microscopic imaging of living cells by which individual myosin filaments could be discerned, have shown that myosin filaments are very dynamic (32)(33)(34). Myosins tagged with fluorescent fusion proteins were imaged as closely spaced "dumbbells" corresponding to the ∼300-nm-long bipolar filaments assembled in vitro.…”

Section: Discussionmentioning

confidence: 99%

“…These punctates grew brighter as they appeared to be carried away from the cell periphery by retrograde actin flow. At times, the punctates split into two parts, which then each subsequently grew brighter, presumably as more monomeric myosin added to the filaments (32,33).…”

Section: Discussionmentioning

confidence: 99%

“…Non-RLC-phosphorylated NM2A, NMB, and NM2C (260 nM) were individually polymerized overnight at 4°C in 10 mM Mops (pH 7.0), 150 mM NaCl, 0.1 mM EGTA, 1 mM DTT, 2 mM MgCl 2 , and 1 mM [γ-32 P]ATP, and then for 30 min at room temperature. The production of 32 Pi was determined as described previously (42).…”

Section: Methodsmentioning

confidence: 99%

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Effect of ATP and regulatory light-chain phosphorylation on the polymerization of mammalian nonmuscle myosin II

Liu

Billington

Shu

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

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Addition of 1 mM ATP substantially reduces the light scattering of solutions of polymerized unphosphorylated nonmuscle myosin IIs (NM2s), and this is reversed by phosphorylation of the regulatory light chain (RLC). It has been proposed that these changes result from substantial depolymerization of unphosphorylated NM2 filaments to monomers upon addition of ATP, and filament repolymerization upon RLC-phosphorylation. We now show that the differences in myosin monomer concentration of RLC-unphosphorylated and -phosphorylated recombinant mammalian NM2A, NM2B, and NM2C polymerized in the presence of ATP are much too small to explain their substantial differences in light scattering. Rather, we find that the decrease in light scattering upon addition of ATP to polymerized unphosphorylated NM2s correlates with the formation of dimers, tetramers, and hexamers, in addition to monomers, an increase in length, and decrease in width of the bare zones of RLC-unphosphorylated filaments. Both effects of ATP addition are reversed by phosphorylation of the RLC. Our data also suggest that, contrary to previous models, assembly of RLCphosphorylated NM2s at physiological ionic strength proceeds from folded monomers to folded antiparallel dimers, tetramers, and hexamers that unfold and polymerize into antiparallel filaments. This model could explain the dynamic relocalization of NM2 filaments in vivo by dephosphorylation of RLC-phosphorylated filaments, disassembly of the dephosphorylated filaments to folded monomers, dimers, and small oligomers, followed by diffusion of these species, and reassembly of filaments at the new location following rephosphorylation of the RLC.C lass II myosins, the first of 37 known myosin classes (1, 2) to be discovered and originally thought to be specific for muscle, are now known to occur widely in nonmuscle cells in numerous species. Human nonmuscle cells express three different myosin 2 paralogs: nonmuscle myosins (NM) NM2A, NM2B, and NM2C. Like all class II myosins (3, 4), NM2 monomers are hexapeptides of two identical heavy chains (HC), two essential light chains (ELC), and two regulatory light chains (RLC). The HCs of NM2A, NM2B, and NM2C are encoded by different genes, have 60-80% sequence similarity, and isoforms of each are derived from alternative splicing (4).Each HC has an N-terminal globular motor domain with an ATPase site and an actin-binding site, followed by a lever arm that binds one ELC and one RLC, and a long tail that homodimerizes to form a coiled-coil α-helix. The HC ends with a short C-terminal nonhelical tailpiece (5). Like most class II myosins, NM2 monomers assemble into bipolar filaments by antiparallel association of their elongated coiled-coil helical tails. The filaments of NM2s are appreciably smaller than filaments of muscle myosins, with lengths of about 300 nm, consisting of ∼30 monomers for filaments of NM2A and NM2B and ∼14 monomers for filaments of NM2C (5).As described originally for smooth muscle myosin based on light-scattering data (6-8), filaments of unphosp...

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

See 1 more Smart Citation

Effect of ATP and regulatory light-chain phosphorylation on the polymerization of mammalian nonmuscle myosin II

Liu

Billington

Shu

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

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“…This ability to bundle F-actin is likely important for structuring the apical actomyosin bundles at the AJC. Moreover, recent work has shown that Myosin IIA filaments can form large lateral stacks of motor groups, which may be important for organizing actin structure at junctions (Fenix et al, 2016). Interestingly, these stacks of Myosin IIA filaments can be assembled, although to a lesser extent, from mutants of Myosin IIA that have reduced motor activity (Fenix et al, 2016).…”

Section: Junctional Roles For Myosin IImentioning

confidence: 99%

Rho GTPases and actomyosin: Partners in regulating epithelial cell-cell junction structure and function

Arnold

Stephenson

Miller

2017

Experimental Cell Research

116

111

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Epithelial tissues are defined by polarized epithelial cells that are integrated into tissues and exhibit barrier function in order to regulate what is allowed to pass between cells. Cell-cell junctions must be stable enough to promote barrier function and tissue integrity, yet plastic enough to remodel when necessary. This remarkable ability to dynamically sense and respond to changes in cell shape and tissue tension allows cell-cell junctions to remain functional during events that disrupt epithelial homeostasis including morphogenesis, wound healing, and cell division. In order to achieve this plasticity, both tight junctions and adherens junctions are coupled to the underlying actomyosin cytoskeleton. Here, we discuss the importance of the junctional linkage to actomyosin and how a localized zone of active RhoA along with other Rho GTPases work together to orchestrate junctional actomyosin dynamics. We focus on how scaffold proteins help coordinate Rho GTPases, their upstream regulators, and their downstream effectors for efficient, localized Rho GTPase signaling output. Additionally, we highlight important roles junctional actin-binding proteins play in addition to their traditional roles in organizing actin. Together, Rho GTPases, their regulators, and effectors form compartmentalized signaling modules that regulate actomyosin structure and contractility to achieve proper cell-cell adhesion and tissue barriers.

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“…This pathway could be mobilized in dividing cells by mitotic signaling, via BAG3 phosphorylation, to facilitate accurate turnover of non-muscle contractile structures that are submitted to high tensile forces. It has been hypothesized that the organization of actin and myosin II in the cytokinetic AR is similar to that seen in muscle sarcomere; recent evidence supports a sliding filament-based purse string contraction mechanism for AR constriction (Fenix et al 2016;Pollard and Wu 2010). Although it has been established that the AR needs to be disassembled to allow for the final abscission of daughter cells, the mechanisms remain poorly understood.…”

Section: Discussionmentioning

confidence: 99%

Fine-tuning of actin dynamics by the HSPB8-BAG3 chaperone complex facilitates cytokinesis and contributes to its impact on cell division

Varlet

Fuchs

Luthold

et al. 2017

Cell Stress and Chaperones

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The small heat shock protein HSPB8 and its cochaperone BAG3 are proposed to regulate cytoskeletal proteostasis in response to mechanical signaling in muscle cells. Here, we show that in dividing cells, the HSPB8-BAG3 complex is instrumental to the accurate disassembly of the actin-based contractile ring during cytokinesis, a process required to allow abscission of daughter cells. Silencing of HSPB8 markedly decreased the mitotic levels of BAG3 in HeLa cells, supporting its crucial role in BAG3 mitotic functions. Cells depleted of HSPB8 were delayed in cytokinesis, remained connected via a disorganized intercellular bridge, and exhibited increased incidence of nuclear abnormalities that result from failed cytokinesis (i.e., bi-and multi-nucleation). Such phenotypes were associated with abnormal accumulation of F-actin at the intercellular bridge of daughter cells at telophase. Remarkably, the actin sequestering drug latrunculin A, like the inhibitor of branched actin polymerization CK666, normalized F-actin during cytokinesis and restored proper cell division in HSPB8-depleted cells, implicating deregulated actin dynamics as a cause of abscission failure. Moreover, this HSPB8-dependent phenotype could be corrected by rapamycin, an autophagy-promoting drug, whereas it was mimicked by drugs impairing lysosomal function. Together, the results further support a role for the HSPB8-BAG3 chaperone complex in quality control of actin-based structure dynamics that are put under high tension, notably during cell cytokinesis. They expand a so-far underappreciated connection between selective autophagy and cellular morphodynamics that guide cell division.

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Expansion and concatenation of nonmuscle myosin IIA filaments drive cellular contractile system formation during interphase and mitosis

Cited by 113 publications

References 62 publications

Effect of ATP and regulatory light-chain phosphorylation on the polymerization of mammalian nonmuscle myosin II

Effect of ATP and regulatory light-chain phosphorylation on the polymerization of mammalian nonmuscle myosin II

Rho GTPases and actomyosin: Partners in regulating epithelial cell-cell junction structure and function

Fine-tuning of actin dynamics by the HSPB8-BAG3 chaperone complex facilitates cytokinesis and contributes to its impact on cell division

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