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

2012 ◽  
Vol 23 (16) ◽  
pp. 3094-3110 ◽  
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.

scholarly journals Tropomyosin and Myosin-II Cellular Levels Promote Actomyosin Ring Assembly in Fission Yeast

2010 ◽  
Vol 21 (6) ◽  
pp. 989-1000 ◽  
Author(s):  
Benjamin C. Stark ◽  
Thomas E. Sladewski ◽  
Luther W. Pollard ◽  
Matthew Lord
Keyword(s):  
Motor Activity ◽  
Atpase Activity ◽  
Fission Yeast ◽  
Actin Filaments ◽  
Myosin Ii ◽  
Bound State ◽  
Contractile Ring ◽  
Ring Assembly

Myosin-II (Myo2p) and tropomyosin are essential for contractile ring formation and cytokinesis in fission yeast. Here we used a combination of in vivo and in vitro approaches to understand how these proteins function at contractile rings. We find that ring assembly is delayed in Myo2p motor and tropomyosin mutants, but occurs prematurely in cells engineered to express two copies of myo2. Thus, the timing of ring assembly responds to changes in Myo2p cellular levels and motor activity, and the emergence of tropomyosin-bound actin filaments. Doubling Myo2p levels suppresses defects in ring assembly associated with a tropomyosin mutant, suggesting a role for tropomyosin in maximizing Myo2p function. Correspondingly, tropomyosin increases Myo2p actin affinity and ATPase activity and promotes Myo2p-driven actin filament gliding in motility assays. Tropomyosin achieves this by favoring the strong actin-bound state of Myo2p. This mode of regulation reflects a role for tropomyosin in specifying and stabilizing actomyosin interactions, which facilitates contractile ring assembly in the fission yeast system.

scholarly journals Progress towards understanding the mechanism of cytokinesis in fission yeast

2008 ◽  
Vol 36 (3) ◽  
pp. 425-430 ◽  
Author(s):  
Thomas D. Pollard
Keyword(s):  
Fission Yeast ◽  
Actin Filaments ◽  
Biochemical Analysis ◽  
Fluorescent Proteins ◽  
Myosin Ii ◽  
Live Cells ◽  
Contractile Ring ◽  
Simple Hypothesis ◽  
A Cell ◽  
Temporal And Spatial

We use fission yeast to study the molecular mechanism of cytokinesis. We benefit from a long history in genetic analysis of the cell cycle in fission yeast, which provided the most complete inventory of cytokinesis proteins. We used fluorescence microscopy of proteins tagged with fluorescent proteins to establish the temporal and spatial pathway for the assembly and constriction of the contractile ring. We combined biochemical analysis of purified proteins (myosin-II, profilin, formin Cdc12p and cofilin), observations of fluorescent fusion proteins in live cells and mathematical modelling to formulate and test a simple hypothesis for the assembly of the contractile ring. This model involves the formation of 65 nodes containing myosin-II and formin Cdc12p around the equator of the cell. As a cell enters anaphase, actin filaments grow from formin Cdc12p in these nodes. Myosin captures actin filaments from adjacent nodes and pulls intermittently to condense the nodes into a contractile ring.

scholarly journals Roles of Formin Nodes and Myosin Motor Activity in Mid1p-dependent Contractile-Ring Assembly during Fission Yeast Cytokinesis

2009 ◽  
Vol 20 (24) ◽  
pp. 5195-5210 ◽  
Author(s):  
Valerie C. Coffman ◽  
Aaron H. Nile ◽  
I-Ju Lee ◽  
Huayang Liu ◽  
Jian-Qiu Wu
Keyword(s):  
Motor Activity ◽  
Fission Yeast ◽  
Actin Filaments ◽  
Broad Band ◽  
Strong Support ◽  
Cable Model ◽  
Contractile Ring ◽  
Myosin Motor ◽  
Ring Assembly ◽  
Myosin Motors

Two prevailing models have emerged to explain the mechanism of contractile-ring assembly during cytokinesis in the fission yeast Schizosaccharomyces pombe: the spot/leading cable model and the search, capture, pull, and release (SCPR) model. We tested some of the basic assumptions of the two models. Monte Carlo simulations of the SCPR model require that the formin Cdc12p is present in >30 nodes from which actin filaments are nucleated and captured by myosin-II in neighboring nodes. The force produced by myosin motors pulls the nodes together to form a compact contractile ring. Live microscopy of cells expressing Cdc12p fluorescent fusion proteins shows for the first time that Cdc12p localizes to a broad band of 30–50 dynamic nodes, where actin filaments are nucleated in random directions. The proposed progenitor spot, essential for the spot/leading cable model, usually disappears without nucleating actin filaments. α-Actinin ain1 deletion cells form a normal contractile ring through nodes in the absence of the spot. Myosin motor activity is required to condense the nodes into a contractile ring, based on slower or absent node condensation in myo2-E1 and UCS rng3-65 mutants. Taken together, these data provide strong support for the SCPR model of contractile-ring formation in cytokinesis.

scholarly journals Molecular organization of cytokinesis node predicts the constriction rate of the contractile ring

2021 ◽  
Vol 220 (3) ◽  
Author(s):  
Kimberly Bellingham-Johnstun ◽  
Erica Casey Anders ◽  
John Ravi ◽  
Christina Bruinsma ◽  
Caroline Laplante
Keyword(s):  
Single Molecule ◽  
Actin Filaments ◽  
Molecular Organization ◽  
Yeast Cells ◽  
Live Cells ◽  
Contractile Ring ◽  
Ring Assembly ◽  
Assembly Mechanism ◽  
Excess Number ◽  
Single Molecule Localization Microscopy

The molecular organization of cytokinesis proteins governs contractile ring function. We used single molecule localization microscopy in live cells to elucidate the molecular organization of cytokinesis proteins and relate it to the constriction rate of the contractile ring. Wild-type fission yeast cells assemble contractile rings by the coalescence of cortical proteins complexes called nodes whereas cells without Anillin/Mid1p (Δmid1) lack visible nodes yet assemble contractile rings competent for constriction from the looping of strands. We leveraged the Δmid1 contractile ring assembly mechanism to determine how two distinct molecular organizations, nodes versus strands, can yield functional contractile rings. Contrary to previous interpretations, nodes assemble in Δmid1 cells. Our results suggest that Myo2p heads condense upon interaction with actin filaments and an excess number of Myo2p heads bound to actin filaments hinders constriction thus reducing the constriction rate. Our work establishes a predictive correlation between the molecular organization of nodes and the behavior of the contractile ring.

scholarly journals Assembly and architecture of precursor nodes during fission yeast cytokinesis

2011 ◽  
Vol 192 (6) ◽  
pp. 1005-1021 ◽  
Author(s):  
Damien Laporte ◽  
Valerie C. Coffman ◽  
I-Ju Lee ◽  
Jian-Qiu Wu
Keyword(s):  
Plasma Membrane ◽  
Fission Yeast ◽  
Actin Filaments ◽  
Myosin Ii ◽  
Stable Node ◽  
Animal Cells ◽  
Contractile Ring ◽  
Cell Interior ◽  
Ring Assembly ◽  
Positional Marker

The contractile ring is essential for cytokinesis in most fungal and animal cells. In fission yeast, cytokinesis nodes are precursors of the contractile ring and mark the future cleavage site. However, their assembly and architecture have not been well described. We found that nodes are assembled stoichiometrically in a hierarchical order with two modules linked by the positional marker anillin Mid1. Mid1 first recruits Cdc4 and IQGAP Rng2 to form module I. Rng2 subsequently recruits the myosin-II subunits Myo2 and Rlc1. Mid1 then independently recruits the F-BAR protein Cdc15 to form module II. Mid1, Rng2, Cdc4, and Cdc15 are stable node components that accumulate close to the plasma membrane. Both modules recruit the formin Cdc12 to nucleate actin filaments. Myo2 heads point into the cell interior, where they efficiently capture actin filaments to condense nodes into the contractile ring. Collectively, our work characterizing the assembly and architecture of precursor nodes defines important steps and molecular players for contractile ring assembly.

scholarly journals A novel coordinated function of Myosin II with GOLPH3 controls centralspindlin localization during cytokinesis in Drosophila

2020 ◽  
Vol 133 (21) ◽  
pp. jcs252965
Author(s):  
Stefano Sechi ◽  
Anna Frappaolo ◽  
Angela Karimpour-Ghahnavieh ◽  
Roberta Fraschini ◽  
Maria Grazia Giansanti
Keyword(s):  
Plasma Membrane ◽  
Heavy Chain ◽  
Light Chain ◽  
Animal Cell ◽  
Myosin Ii ◽  
Contractile Ring ◽  
Ring Assembly ◽  
Missense Allele ◽  
Phosphatidylinositol 4 ◽  
Muscle Myosin

ABSTRACTIn animal cell cytokinesis, interaction of non-muscle myosin II (NMII) with F-actin provides the dominant force for pinching the mother cell into two daughters. Here we demonstrate that celibe (cbe) is a missense allele of zipper, which encodes the Drosophila Myosin heavy chain. Mutation of cbe impairs binding of Zipper protein to the regulatory light chain Spaghetti squash (Sqh). In dividing spermatocytes from cbe males, Sqh fails to concentrate at the equatorial cortex, resulting in thin actomyosin rings that are unable to constrict. We show that cbe mutation impairs localization of the phosphatidylinositol 4-phosphate [PI(4)P]-binding protein Golgi phosphoprotein 3 (GOLPH3, also known as Sauron) and maintenance of centralspindlin at the cell equator of telophase cells. Our results further demonstrate that GOLPH3 protein associates with Sqh and directly binds the centralspindlin subunit Pavarotti. We propose that during cytokinesis, the reciprocal dependence between Myosin and PI(4)P–GOLPH3 regulates centralspindlin stabilization at the invaginating plasma membrane and contractile ring assembly.

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

2016 ◽  
Vol 27 (9) ◽  
pp. 1465-1478 ◽  
Author(s):  
Aidan M. Fenix ◽  
Nilay Taneja ◽  
Carmen A. Buttler ◽  
John Lewis ◽  
Schuyler B. Van Engelenburg ◽  
...  
Keyword(s):  
Actin Filaments ◽  
Molecular Motor ◽  
Myosin Ii ◽  
Cell Movement ◽  
Nonmuscle Myosin ◽  
Contractile Ring ◽  
Contractile System ◽  
Nonmuscle Myosin Ii ◽  
Dividing Cells ◽  
Contractile Forces

Cell movement and cytokinesis are facilitated by contractile forces generated by the molecular motor, nonmuscle myosin II (NMII). NMII molecules form a filament (NMII-F) through interactions of their C-terminal rod domains, positioning groups of N-terminal motor domains on opposite sides. The NMII motors then bind and pull actin filaments toward the NMII-F, thus driving contraction. Inside of crawling cells, NMIIA-Fs form large macromolecular ensembles (i.e., NMIIA-F stacks), but how this occurs is unknown. Here we show NMIIA-F stacks are formed through two non–mutually exclusive mechanisms: expansion and concatenation. During expansion, NMIIA molecules within the NMIIA-F spread out concurrent with addition of new NMIIA molecules. Concatenation occurs when multiple NMIIA-Fs/NMIIA-F stacks move together and align. We found that NMIIA-F stack formation was regulated by both motor activity and the availability of surrounding actin filaments. Furthermore, our data showed expansion and concatenation also formed the contractile ring in dividing cells. Thus interphase and mitotic cells share similar mechanisms for creating large contractile units, and these are likely to underlie how other myosin II–based contractile systems are assembled.

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

2009 ◽  
Vol 20 (8) ◽  
pp. 2160-2173 ◽  
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.

scholarly journals Actin turnover maintains actin filament homeostasis during cytokinetic ring contraction

2017 ◽  
Vol 216 (9) ◽  
pp. 2657-2667 ◽  
Author(s):  
Ting Gang Chew ◽  
Junqi Huang ◽  
Saravanan Palani ◽  
Ruth Sommese ◽  
Anton Kamnev ◽  
...  
Keyword(s):  
Actin Filament ◽  
Actin Filaments ◽  
Actin Polymerization ◽  
Myosin Ii ◽  
Contractile Ring ◽  
Ring Contraction ◽  
Actin Turnover ◽  
Exogenous Addition ◽  
Turn Over

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.

scholarly journals The fission yeast cytokinesis formin Cdc12p is a barbed end actin filament capping protein gated by profilin

2003 ◽  
Vol 161 (5) ◽  
pp. 875-887 ◽  
Author(s):  
David R. Kovar ◽  
Jeffrey R. Kuhn ◽  
Andrea L. Tichy ◽  
Thomas D. Pollard
Keyword(s):  
Fission Yeast ◽  
Actin Filaments ◽  
Maximum Yield ◽  
Restrictive Temperature ◽  
Permissive Temperature ◽  
Contractile Ring ◽  
Capping Protein ◽  
Ring Assembly ◽  
End Capping ◽  
Actin Cables

Cytokinesis in most eukaryotes requires the assembly and contraction of a ring of actin filaments and myosin II. The fission yeast Schizosaccharomyces pombe requires the formin Cdc12p and profilin (Cdc3p) early in the assembly of the contractile ring. The proline-rich formin homology (FH) 1 domain binds profilin, and the FH2 domain binds actin. Expression of a construct consisting of the Cdc12 FH1 and FH2 domains complements a conditional mutant of Cdc12 at the restrictive temperature, but arrests cells at the permissive temperature. Cells overexpressing Cdc12(FH1FH2)p stop growing with excessive actin cables but no contractile rings. Like capping protein, purified Cdc12(FH1FH2)p caps the barbed end of actin filaments, preventing subunit addition and dissociation, inhibits end to end annealing of filaments, and nucleates filaments that grow exclusively from their pointed ends. The maximum yield is one filament pointed end per six formin polypeptides. Profilins that bind both actin and poly-l-proline inhibit nucleation by Cdc12(FH1FH2)p, but polymerization of monomeric actin is faster, because the filaments grow from their barbed ends at the same rate as uncapped filaments. On the other hand, Cdc12(FH1FH2)p blocks annealing even in the presence of profilin. Thus, formins are profilin-gated barbed end capping proteins with the ability to initiate actin filaments from actin monomers bound to profilin. These properties explain why contractile ring assembly requires both formin and profilin and why viability depends on the ability of profilin to bind both actin and poly-l-proline.

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