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 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 UCS protein Rng3p activates actin filament gliding by fission yeast myosin-II

2004 ◽  
Vol 167 (2) ◽  
pp. 315-325 ◽  
Author(s):  
Matthew Lord ◽  
Thomas D. Pollard
Keyword(s):  
Atpase Activity ◽  
Heavy Chain ◽  
Actin Filaments ◽  
Myosin Ii ◽  
Gliding Motility ◽  
Light Chains ◽  
Temperature Sensitive ◽  
Contractile Ring ◽  
In Vitro Motility

We purified native Myo2p/Cdc4p/Rlc1p (Myo2), the myosin-II motor required for cytokinesis by Schizosaccharomyces pombe. The Myo2p heavy chain associates with two light chains, Cdc4p and Rlc1p. Although crude Myo2 supported gliding motility of actin filaments in vitro, purified Myo2 lacked this activity in spite of retaining full Ca-ATPase activity and partial actin-activated Mg-ATPase activity. Unc45-/Cro1p-/She4p-related (UCS) protein Rng3p restored the full motility and actin-activated Mg-ATPase activity of purified Myo2. The COOH-terminal UCS domain of Rng3p alone restored motility to pure Myo2. Thus, Rng3p contributes directly to the motility activity of native Myo2. Consistent with a role in Myo2 activation, Rng3p colocalizes with Myo2p in the cytokinetic contractile ring. The absence of Rlc1p or mutations in the Myo2p head or Rng3p compromise the in vitro motility of Myo2 and explain the defects in cytokinesis associated with some of these mutations. In contrast, Myo2 with certain temperature-sensitive forms of Cdc4p has normal motility, so these mutations compromise other functions of Cdc4p required for cytokinesis.

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 The novel proteins Rng8 and Rng9 regulate the myosin-V Myo51 during fission yeast cytokinesis

2014 ◽  
Vol 205 (3) ◽  
pp. 357-375 ◽  
Author(s):  
Ning Wang ◽  
Libera Lo Presti ◽  
Yi-Hua Zhu ◽  
Minhee Kang ◽  
Zhengrong Wu ◽  
...  
Keyword(s):  
Fission Yeast ◽  
Molecular Motors ◽  
Coiled Coil ◽  
Myosin V ◽  
Contractile Ring ◽  
Novel Proteins ◽  
Ring Assembly ◽  
Actin Cables ◽  
Order Complexes

The myosin-V family of molecular motors is known to be under sophisticated regulation, but our knowledge of the roles and regulation of myosin-Vs in cytokinesis is limited. Here, we report that the myosin-V Myo51 affects contractile ring assembly and stability during fission yeast cytokinesis, and is regulated by two novel coiled-coil proteins, Rng8 and Rng9. Both rng8Δ and rng9Δ cells display similar defects as myo51Δ in cytokinesis. Rng8 and Rng9 are required for Myo51’s localizations to cytoplasmic puncta, actin cables, and the contractile ring. Myo51 puncta contain multiple Myo51 molecules and walk continuously on actin filaments in rng8+ cells, whereas Myo51 forms speckles containing only one dimer and does not move efficiently on actin tracks in rng8Δ. Consistently, Myo51 transports artificial cargos efficiently in vivo, and this activity is regulated by Rng8. Purified Rng8 and Rng9 form stable higher-order complexes. Collectively, we propose that Rng8 and Rng9 form oligomers and cluster multiple Myo51 dimers to regulate Myo51 localization and functions.

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

2016 ◽  
Vol 27 (11) ◽  
pp. 1821-1833 ◽  
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.

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 Fission yeast tropomyosin specifies directed transport of myosin-V along actin cables

2014 ◽  
Vol 25 (1) ◽  
pp. 66-75 ◽  
Author(s):  
Joseph E. Clayton ◽  
Luther W. Pollard ◽  
Maria Sckolnick ◽  
Carol S. Bookwalter ◽  
Alex R. Hodges ◽  
...  
Keyword(s):  
Fission Yeast ◽  
Actin Filaments ◽  
Total Internal Reflection ◽  
Single Molecules ◽  
Myosin V ◽  
Class V ◽  
Physiological Relevance ◽  
Actin Cables

A hallmark of class-V myosins is their processivity—the ability to take multiple steps along actin filaments without dissociating. Our previous work suggested, however, that the fission yeast myosin-V (Myo52p) is a nonprocessive motor whose activity is enhanced by tropomyosin (Cdc8p). Here we investigate the molecular mechanism and physiological relevance of tropomyosin-mediated regulation of Myo52p transport, using a combination of in vitro and in vivo approaches. Single molecules of Myo52p, visualized by total internal reflection fluorescence microscopy, moved processively only when Cdc8p was present on actin filaments. Small ensembles of Myo52p bound to a quantum dot, mimicking the number of motors bound to physiological cargo, also required Cdc8p for continuous motion. Although a truncated form of Myo52p that lacked a cargo-binding domain failed to support function in vivo, it still underwent actin-dependent movement to polarized growth sites. This result suggests that truncated Myo52p lacking cargo, or single molecules of wild-type Myo52p with small cargoes, can undergo processive movement along actin-Cdc8p cables in vivo. Our findings outline a mechanism by which tropomyosin facilitates sorting of transport to specific actin tracks within the cell by switching on myosin processivity.

scholarly journals Cytokinesis Depends on the Motor Domains of Myosin-II in Fission Yeast but Not in Budding Yeast

2005 ◽  
Vol 16 (11) ◽  
pp. 5346-5355 ◽  
Author(s):  
Matthew Lord ◽  
Ellen Laves ◽  
Thomas D. Pollard
Keyword(s):  
Atpase Activity ◽  
Fission Yeast ◽  
Mechanism Of Action ◽  
Budding Yeast ◽  
Myosin Ii ◽  
Cellular Function ◽  
Motor Domain ◽  
Actin Binding ◽  
Contractile Ring ◽  
Head Domain

Budding yeast possesses one myosin-II, Myo1p, whereas fission yeast has two, Myo2p and Myp2p, all of which contribute to cytokinesis. We find that chimeras consisting of Myo2p or Myp2p motor domains fused to the tail of Myo1p are fully functional in supporting budding yeast cytokinesis. Remarkably, the tail alone of budding yeast Myo1p localizes to the contractile ring, supporting both its constriction and cytokinesis. In contrast, fission yeast Myo2p and Myp2p require both the catalytic head domain as well as tail domains for function, with the tails providing distinct functions ( Bezanilla and Pollard, 2000 ). Myo1p is the first example of a myosin whose cellular function does not require a catalytic motor domain revealing a novel mechanism of action for budding yeast myosin-II independent of actin binding and ATPase activity.

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 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.

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