Actin turnover maintains actin filament homeostasis during cytokinetic ring contraction

Cytokinesis in many eukaryotes involves a tension-generating actomyosin-based contractile ring. Many components of actomyosin rings turn over during contraction, although the significance of this turnover has remained enigmatic. Here, using Schizosaccharomyces japonicus, we investigate the role of turnover of actin and myosin II in its contraction. Actomyosin ring components self-organize into ∼1-µm-spaced clusters instead of undergoing full-ring contraction in the absence of continuous actin polymerization. This effect is reversed when actin filaments are stabilized. We tested the idea that the function of turnover is to ensure actin filament homeostasis in a synthetic system, in which we abolished turnover by fixing rings in cell ghosts with formaldehyde. We found that these rings contracted fully upon exogenous addition of a vertebrate myosin. We conclude that actin turnover is required to maintain actin filament homeostasis during ring contraction and that the requirement for turnover can be bypassed if homeostasis is achieved artificially.

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Role of Tropomyosin in Formin-mediated Contractile Ring Assembly in Fission Yeast

Molecular Biology of the Cell ◽

10.1091/mbc.e08-12-1201 ◽

2009 ◽

Vol 20 (8) ◽

pp. 2160-2173 ◽

Cited By ~ 57

Author(s):

Colleen T. Skau ◽

Erin M. Neidt ◽

David R. Kovar

Keyword(s):

Fission Yeast ◽

Actin Filament ◽

Actin Filaments ◽

Actin Polymerization ◽

Actin Assembly ◽

Animal Cells ◽

Contractile Ring ◽

Direct Role ◽

Ring Assembly

Like animal cells, fission yeast divides by assembling actin filaments into a contractile ring. In addition to formin Cdc12p and profilin, the single tropomyosin isoform SpTm is required for contractile ring assembly. Cdc12p nucleates actin filaments and remains processively associated with the elongating barbed end while driving the addition of profilin-actin. SpTm is thought to stabilize mature filaments, but it is not known how SpTm localizes to the contractile ring and whether SpTm plays a direct role in Cdc12p-mediated actin polymerization. Using “bulk” and single actin filament assays, we discovered that Cdc12p can recruit SpTm to actin filaments and that SpTm has diverse effects on Cdc12p-mediated actin assembly. On its own, SpTm inhibits actin filament elongation and depolymerization. However, Cdc12p completely overcomes the combined inhibition of actin nucleation and barbed end elongation by profilin and SpTm. Furthermore, SpTm increases the length of Cdc12p-nucleated actin filaments by enhancing the elongation rate twofold and by allowing them to anneal end to end. In contrast, SpTm ultimately turns off Cdc12p-mediated elongation by “trapping” Cdc12p within annealed filaments or by dissociating Cdc12p from the barbed end. Therefore, SpTm makes multiple contributions to contractile ring assembly during and after actin polymerization.

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Myosin-II activity generates a dynamic steady state with continuous actin turnover in a minimal actin cortex

10.1101/312512 ◽

2018 ◽

Author(s):

Sonal ◽

Kristina A. Ganzinger ◽

Sven K. Vogel ◽

Jonas Mücksch ◽

Philipp Blumhardt ◽

...

Keyword(s):

Steady State ◽

Actin Polymerization ◽

Myosin Ii ◽

Fine Tuning ◽

Cellular Functions ◽

Actin Cortex ◽

Actin Turnover ◽

Cytoskeletal Dynamics

ABSTRACTDynamic reorganization of the actomyosin cytoskeleton allows a fine-tuning of cell shape that is vital to many cellular functions. It is well established that myosin-II motors generate the forces required for remodeling the cell surface by imparting contractility to actin networks. An additional, less understood, role of myosin-II in cytoskeletal dynamics is believed to be in the regulation of actin turnover; it has been proposed that myosin activity increases actin turnover in various cellular contexts, presumably by contributing to disassembly. In vitro reconstitution of actomyosin networks has confirmed the role of myosin in actin network disassembly, but factors such as diffusional constraints and the use of stabilized filaments have thus far limited the observation of myosin-assisted actin turnover in these networks. Here, we present the reconstitution of a minimal dynamic actin cortex where actin polymerization is catalyzed on the membrane in the presence of myosin-II activity. We demonstrate that myosin activity leads to disassembly and redistribution in this simplified cortex. Consequently, a new dynamic steady state emerges in which actin filaments undergo constant turnover. Our findings suggest a multi-faceted role of myosin-II in fast remodeling of the eukaryotic actin cortex.

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Requirements for Hirano Body Formation

Eukaryotic Cell ◽

10.1128/ec.00044-14 ◽

2014 ◽

Vol 13 (5) ◽

pp. 625-634 ◽

Cited By ~ 4

Author(s):

Paul Griffin ◽

Ruth Furukawa ◽

Cleveland Piggott ◽

Andrew Maselli ◽

Marcus Fechheimer

Keyword(s):

Actin Filament ◽

Actin Filaments ◽

Myosin Ii ◽

Actin Binding ◽

Hirano Bodies ◽

Latrunculin B ◽

Content Type ◽

Inhibitory Domain

ABSTRACT Hirano bodies are paracrystalline F-actin-rich structures associated with diverse conditions, including neurodegeneration and aging. Generation of model Hirano bodies using altered forms of Dictyostelium 34-kDa actin-bundling protein allows studies of their physiological function and mechanism of formation. We describe a novel 34-kDa protein mutant, E60K, with a point mutation within the inhibitory domain of the 34-kDa protein. Expression of E60K in Dictyostelium induces the formation of model Hirano bodies. The E60K protein has activated actin binding and is calcium regulated, unlike other forms of the 34-kDa protein that induce Hirano bodies and that have activated actin binding but lack calcium regulation. Actin filaments in the presence of E60K in vitro show enhanced resistance to disassembly induced by latrunculin B. Actin filaments in model Hirano bodies are also protected from latrunculin-induced depolymerization. We used nocodazole and blebbistatin to probe the role of the microtubules and myosin II, respectively, in the formation of model Hirano bodies. In the presence of these inhibitors, model Hirano bodies can form but are smaller than controls at early times of formation. The ultrastructure of model Hirano bodies did not reveal any major difference in structure and organization in the presence of inhibitors. In summary, these results support the conclusion that formation of model Hirano bodies is promoted by gain-of-function actin filament bundling, which enhances actin filament stabilization. Microtubules and myosin II contribute to but are not required for formation of model Hirano bodies.

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Actin-depolymerizing Protein Adf1 Is Required for Formation and Maintenance of the Contractile Ring during Cytokinesis in Fission Yeast

Molecular Biology of the Cell ◽

10.1091/mbc.e05-09-0900 ◽

2006 ◽

Vol 17 (4) ◽

pp. 1933-1945 ◽

Cited By ~ 69

Author(s):

Kentaro Nakano ◽

Issei Mabuchi

Keyword(s):

Actin Cytoskeleton ◽

Fission Yeast ◽

Actin Filaments ◽

Myosin Ii ◽

Yeast Cells ◽

Contractile Ring ◽

Capping Protein ◽

Division Site ◽

Family Protein

The role of the actin-depolymerizing factor (ADF)/cofilin-family protein Adf1 in cytokinesis of fission yeast cells was studied. Adf1 was required for accumulation of actin at the division site by depolymerizing actin at the cell ends, assembly of the contractile ring through severing actin filaments, and maintenance of the contractile ring once formed. Genetic and cytological analyses suggested that it collaborates with profilin and capping protein in the mitotic reorganization of the actin cytoskeleton. Furthermore, it was unexpectedly found that Adf1 and myosin-II also collaborate in assembling the contractile ring. Tropomyosin was shown to antagonize the function of Adf1 in the contractile ring. We propose that formation and maintenance of the contractile ring are achieved by a balanced collaboration of these proteins.

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Specification of Actin Filament Function and Molecular Composition by Tropomyosin Isoforms

Molecular Biology of the Cell ◽

10.1091/mbc.e02-04-0244 ◽

2003 ◽

Vol 14 (3) ◽

pp. 1002-1016 ◽

Cited By ~ 180

Author(s):

Nicole S. Bryce ◽

Galina Schevzov ◽

Vicki Ferguson ◽

Justin M. Percival ◽

Jim J.-C. Lin ◽

...

Keyword(s):

Cell Size ◽

Functional Properties ◽

Actin Filament ◽

Actin Filaments ◽

Myosin Ii ◽

Molecular Composition ◽

Cellular Migration ◽

Stress Fibers ◽

Human Homologue ◽

Tropomyosin Isoforms

The specific functions of greater than 40 vertebrate nonmuscle tropomyosins (Tms) are poorly understood. In this article we have tested the ability of two Tm isoforms, TmBr3 and the human homologue of Tm5 (hTM5NM1), to regulate actin filament function. We found that these Tms can differentially alter actin filament organization, cell size, and shape. hTm5NM1was able to recruit myosin II into stress fibers, which resulted in decreased lamellipodia and cellular migration. In contrast, TmBr3 transfection induced lamellipodial formation, increased cellular migration, and reduced stress fibers. Based on coimmunoprecipitation and colocalization studies, TmBr3 appeared to be associated with actin-depolymerizing factor/cofilin (ADF)-bound actin filaments. Additionally, the Tms can specifically regulate the incorporation of other Tms into actin filaments, suggesting that selective dimerization may also be involved in the control of actin filament organization. We conclude that Tm isoforms can be used to specify the functional properties and molecular composition of actin filaments and that spatial segregation of isoforms may lead to localized specialization of actin filament function.

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Tropomyosin inhibits ADF/cofilin-dependent actin filament dynamics

The Journal of Cell Biology ◽

10.1083/jcb.200110013 ◽

2002 ◽

Vol 156 (6) ◽

pp. 1065-1076 ◽

Cited By ~ 180

Author(s):

Shoichiro Ono ◽

Kanako Ono

Keyword(s):

Actin Filament ◽

Actin Filaments ◽

Actin Polymerization ◽

Biological Properties ◽

Actin Dynamics ◽

Background Suppression ◽

Thin Filaments ◽

Actin Organization ◽

C Elegans ◽

Filament Dynamics

Tropomyosin binds to actin filaments and is implicated in stabilization of actin cytoskeleton. We examined biochemical and cell biological properties of Caenorhabditis elegans tropomyosin (CeTM) and obtained evidence that CeTM is antagonistic to ADF/cofilin-dependent actin filament dynamics. We purified CeTM, actin, and UNC-60B (a muscle-specific ADF/cofilin isoform), all of which are derived from C. elegans, and showed that CeTM and UNC-60B bound to F-actin in a mutually exclusive manner. CeTM inhibited UNC-60B–induced actin depolymerization and enhancement of actin polymerization. Within isolated native thin filaments, actin and CeTM were detected as major components, whereas UNC-60B was present at a trace amount. Purified UNC-60B was unable to interact with the native thin filaments unless CeTM and other associated proteins were removed by high-salt extraction. Purified CeTM was sufficient to restore the resistance of the salt-extracted filaments from UNC-60B. In muscle cells, CeTM and UNC-60B were localized in different patterns. Suppression of CeTM by RNA interference resulted in disorganized actin filaments and paralyzed worms in wild-type background. However, in an ADF/cofilin mutant background, suppression of CeTM did not worsen actin organization and worm motility. These results suggest that tropomyosin is a physiological inhibitor of ADF/cofilin-dependent actin dynamics.

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Dynamics of Tpm1.8 domains on actin filaments with single molecule resolution

10.1101/2020.06.15.152033 ◽

2020 ◽

Cited By ~ 1

Author(s):

Ilina Bareja ◽

Hugo Wioland ◽

Miro Janco ◽

Philip R. Nicovich ◽

Antoine Jégou ◽

...

Keyword(s):

Actin Filament ◽

Single Molecule ◽

Actin Filaments ◽

High Probability ◽

Actin Binding ◽

Single Molecule Level ◽

Filament Dynamics ◽

Kinetics Of ◽

Insight Into

ABSTRACTTropomyosins regulate dynamics and functions of the actin cytoskeleton by forming long chains along the two strands of actin filaments that act as gatekeepers for the binding of other actin-binding proteins. The fundamental molecular interactions underlying the binding of tropomyosin to actin are still poorly understood. Using microfluidics and fluorescence microscopy, we observed the binding of fluorescently labelled tropomyosin isoform Tpm1.8 to unlabelled actin filaments in real time. This approach in conjunction with mathematical modeling enabled us to quantify the nucleation, assembly and disassembly kinetics of Tpm1.8 on single filaments and at the single molecule level. Our analysis suggests that Tpm1.8 decorates the two strands of the actin filament independently. Nucleation of a growing tropomyosin domain proceeds with high probability as soon as the first Tpm1.8 molecule is stabilised by the addition of a second molecule, ultimately leading to full decoration of the actin filament. In addition, Tpm1.8 domains are asymmetrical, with enhanced dynamics at the edge oriented towards the barbed end of the actin filament. The complete description of Tpm1.8 kinetics on actin filaments presented here provides molecular insight into actin-tropomyosin filament formation and the role of tropomyosins in regulating actin filament dynamics.

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Anchoring of actin to the plasma membrane enables tension production in the fission yeast cytokinetic ring

Molecular Biology of the Cell ◽

10.1091/mbc.e19-03-0173 ◽

2019 ◽

Vol 30 (16) ◽

pp. 2053-2064 ◽

Cited By ~ 3

Author(s):

Shuyuan Wang ◽

Ben O’Shaughnessy

Keyword(s):

Plasma Membrane ◽

Fission Yeast ◽

Actin Filaments ◽

Tensile Force ◽

Myosin Ii ◽

Quantitative Evidence ◽

Explicit Simulation ◽

Adjacent Segments ◽

Develop Tension

The cytokinetic ring generates tensile force that drives cell division, but how tension emerges from the relatively disordered ring organization remains unclear. Long ago, a musclelike sliding filament mechanism was proposed, but evidence for sarcomeric order is lacking. Here we present quantitative evidence that in fission yeast, ring tension originates from barbed-end anchoring of actin filaments to the plasma membrane, providing resistance to myosin forces that enables filaments to develop tension. The role of anchoring was highlighted by experiments on isolated fission yeast rings, where sections of ring became unanchored from the membrane and shortened ∼30-fold faster than normal. The dramatically elevated constriction rates are unexplained. Here we present a molecularly explicit simulation of constricting partially anchored rings as studied in these experiments. Simulations accurately reproduced the experimental constriction rates and showed that following anchor release, a segment becomes tensionless and shortens via a novel noncontractile reeling-in mechanism at about the velocity of load-free myosin II. The ends are reeled in by barbed end–anchored actin filaments in adjacent segments. Other actin anchoring schemes failed to constrict rings. Our results quantitatively support a specific organization and anchoring scheme that generate tension in the cytokinetic ring.

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The F-actin bundler α-actinin Ain1 is tailored for ring assembly and constriction during cytokinesis in fission yeast

Molecular Biology of the Cell ◽

10.1091/mbc.e16-01-0010 ◽

2016 ◽

Vol 27 (11) ◽

pp. 1821-1833 ◽

Cited By ~ 35

Author(s):

Yujie Li ◽

Jenna R. Christensen ◽

Kaitlin E. Homa ◽

Glen M. Hocky ◽

Alice Fok ◽

...

Keyword(s):

Fission Yeast ◽

Actin Filament ◽

Actin Filaments ◽

Biochemical Properties ◽

Ring Formation ◽

Cross Linking ◽

Contractile Ring ◽

Mixed Polarity ◽

Dividing Cells

The actomyosin contractile ring is a network of cross-linked actin filaments that facilitates cytokinesis in dividing cells. Contractile ring formation has been well characterized in Schizosaccharomyces pombe, in which the cross-linking protein α-actinin SpAin1 bundles the actin filament network. However, the specific biochemical properties of SpAin1 and whether they are tailored for cytokinesis are not known. Therefore we purified SpAin1 and quantified its ability to dynamically bind and bundle actin filaments in vitro using a combination of bulk sedimentation assays and direct visualization by two-color total internal reflection fluorescence microscopy. We found that, while SpAin1 bundles actin filaments of mixed polarity like other α-actinins, SpAin1 has lower bundling activity and is more dynamic than human α-actinin HsACTN4. To determine whether dynamic bundling is important for cytokinesis in fission yeast, we created the less dynamic bundling mutant SpAin1(R216E). We found that dynamic bundling is critical for cytokinesis, as cells expressing SpAin1(R216E) display disorganized ring material and delays in both ring formation and constriction. Furthermore, computer simulations of initial actin filament elongation and alignment revealed that an intermediate level of cross-linking best facilitates filament alignment. Together our results demonstrate that dynamic bundling by SpAin1 is important for proper contractile ring formation and constriction.

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α-Actinin and fimbrin cooperate with myosin II to organize actomyosin bundles during contractile-ring assembly

Molecular Biology of the Cell ◽

10.1091/mbc.e12-02-0123 ◽

2012 ◽

Vol 23 (16) ◽

pp. 3094-3110 ◽

Cited By ~ 61

Author(s):

Damien Laporte ◽

Nikola Ojkic ◽

Dimitrios Vavylonis ◽

Jian-Qiu Wu

Keyword(s):

Actin Filaments ◽

Myosin Ii ◽

Broad Band ◽

Live Cells ◽

Condensation Process ◽

Contractile Ring ◽

Ring Assembly ◽

Cooperative Process ◽

Normal Ring ◽

Work Supports

The actomyosin contractile ring assembles through the condensation of a broad band of nodes that forms at the cell equator in fission yeast cytokinesis. The condensation process depends on actin filaments that interconnect nodes. By mutating or titrating actin cross-linkers α-actinin Ain1 and fimbrin Fim1 in live cells, we reveal that both proteins are involved in node condensation. Ain1 and Fim1 stabilize the actin cytoskeleton and modulate node movement, which prevents nodes and linear structures from aggregating into clumps and allows normal ring formation. Our computer simulations modeling actin filaments as semiflexible polymers reproduce the experimental observations and provide a model of how actin cross-linkers work with other proteins to regulate actin-filament orientations inside actin bundles and organize the actin network. As predicted by the simulations, doubling myosin II Myo2 level rescues the node condensation defects caused by Ain1 overexpression. Taken together, our work supports a cooperative process of ring self-organization driven by the interaction between actin filaments and myosin II, which is progressively stabilized by the cross-linking proteins.

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