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 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 Inhibition of acanthamoeba actomyosin-II ATPase activity and mechanochemical function by specific monoclonal antibodies.

1984 ◽  
Vol 99 (3) ◽  
pp. 1024-1033 ◽  
Author(s):  
D P Kiehart ◽  
T D Pollard
Keyword(s):  
Monoclonal Antibodies ◽  
Atpase Activity ◽  
Conformational Changes ◽  
Binding Sites ◽  
Polyclonal Antibodies ◽  
Myosin Ii ◽  
Antigenic Site ◽  
Actin Binding ◽  
Filamentous Form ◽  
Antibody Effects

Monoclonal and polyclonal antibodies that bind to myosin-II were tested for their ability to inhibit myosin ATPase activity, actomyosin ATPase activity, and contraction of cytoplasmic extracts. Numerous antibodies specifically inhibit the actin activated Mg++-ATPase activity of myosin-II in a dose-dependent fashion, but none blocked the ATPase activity of myosin alone. Control antibodies that do not bind to myosin-II and several specific antibodies that do bind have no effect on the actomyosin-II ATPase activity. In most cases, the saturation of a single antigenic site on the myosin-II heavy chain is sufficient for maximal inhibition of function. Numerous monoclonal antibodies also block the contraction of gelled extracts of Acanthamoeba cytoplasm. No polyclonal antibodies tested inhibited ATPase activity or gel contraction. As expected, most antibodies that block actin-activated ATPase activity also block gel contraction. Exceptions were three antibodies M2.2, -15, and -17, that appear to uncouple the ATPase activity from gel contraction: they block gel contraction without influencing ATPase activity. The mechanisms of inhibition of myosin function depends on the location of the antibody-binding sites. Those inhibitory antibodies that bind to the myosin-II heads presumably block actin binding or essential conformational changes in the myosin heads. A subset of the antibodies that bind to the proximal end of the myosin-II tail inhibit actomyosin-II ATPase activity and gel contraction. Although this part of the molecule is presumably some distance from the ATP and actin-binding sites, these antibody effects suggest that structural domains in this region are directly involved with or coupled to catalysis and energy transduction. A subset of the antibodies that bind to the tip of the myosin-II tail appear to inhibit ATPase activity and contraction through their inhibition of filament formation. They provide strong evidence for a substantial enhancement of the ATPase activity of myosin molecules in filamentous form and suggest that the myosin filaments may be required for cell motility.

scholarly journals Myosins generate contractile force and maintain organization in the cytokinetic contractile ring

2021 ◽  
Author(s):  
Zachary A. McDargh ◽  
Shuyuan Wang ◽  
Harvey F. Chin ◽  
Sathish Thiyagarajan ◽  
Erdem Karatekin ◽  
...  
Keyword(s):  
Fission Yeast ◽  
Striated Muscle ◽  
Protein Complexes ◽  
Myosin Ii ◽  
Force Production ◽  
Super Resolution ◽  
Building Blocks ◽  
Contractile Ring ◽  
Ring Model ◽  
Self Assembling

During cytokinesis, cells assemble an actomyosin contractile ring whose tension constricts and divides cells, but the ring tension was rarely measured. Actomyosin force generation is well understood for the regular sarcomeric architecture of striated muscle, but recent super-resolution studies of fission yeast contractile rings revealed organizational building blocks that are not sarcomeres but irregularly positioned plasma membrane-anchored protein complexes called nodes. Here, we measured contractile ring tensions in fission yeast protoplast cells. The myosin II isoforms Myo2 and Myp2 generated the tension, with a ~2-fold greater contribution from Myo2. Simulations of a molecularly detailed ring model revealed a sliding node mechanism for tension, where nodes hosting tense actin filaments were pulled bidirectionally around the ring. Myo2 and Myp2 chaperoned self-assembling components into the ring organization, and anchored the ring against bridging instabilities. Thus, beyond force production, Myo2 and Myp2 are the principal organizers, bundlers and anchors of the contractile ring.

scholarly journals Fission yeast IQGAP maintains F-actin-independent localization of myosin-II in the contractile ring

2013 ◽  
Vol 19 (2) ◽  
pp. 161-176 ◽  
Author(s):  
Masak Takaine ◽  
Osamu Numata ◽  
Kentaro Nakano
Keyword(s):  
Fission Yeast ◽  
Myosin Ii ◽  
Contractile Ring

scholarly journals Cooperation between tropomyosin and α-actinin inhibits fimbrin association with actin filament networks in fission yeast

eLife ◽  
2019 ◽  
Vol 8 ◽  
Author(s):  
Jenna R Christensen ◽  
Kaitlin E Homa ◽  
Alisha N Morganthaler ◽  
Rachel R Brown ◽  
Cristian Suarez ◽  
...  
Keyword(s):  
Fission Yeast ◽  
Cellular Networks ◽  
Residence Time ◽  
Actin Filament ◽  
Binding Proteins ◽  
Actin Binding ◽  
Contractile Ring ◽  
Actin Networks ◽  
Filament Networks ◽  
Actin Patch

We previously discovered that competition between fission yeast actin binding proteins (ABPs) for binding F-actin facilitates their sorting to different cellular networks. Specifically, competition between endocytic actin patch ABPs fimbrin Fim1 and cofilin Adf1 enhances their activities, and prevents tropomyosin Cdc8’s association with actin patches. However, these interactions do not explain how Fim1 is prevented from associating strongly with other F-actin networks such as the contractile ring. Here, we identified α-actinin Ain1, a contractile ring ABP, as another Fim1 competitor. Fim1 competes with Ain1 for association with F-actin, which is dependent upon their F-actin residence time. While Fim1 outcompetes both Ain1 and Cdc8 individually, Cdc8 enhances the F-actin bundling activity of Ain1, allowing Ain1 to generate F-actin bundles that Cdc8 can bind in the presence of Fim1. Therefore, the combination of contractile ring ABPs Ain1 and Cdc8 is capable of inhibiting Fim1’s association with F-actin networks.

scholarly journals Tropomyosin and α-actinin cooperation inhibits fimbrin association with actin filament networks in fission yeast

2017 ◽  
Author(s):  
Jenna R. Christensen ◽  
Kaitlin E. Homa ◽  
Meghan E. O’Connell ◽  
David R. Kovar
Keyword(s):  
Fission Yeast ◽  
Fluorescent Imaging ◽  
Actin Binding ◽  
Yeast Genetics ◽  
Actin Network ◽  
Contractile Ring ◽  
Actin Networks ◽  
Filament Networks ◽  
Actin Patch

ABSTRACTWe previously discovered that competition between fission yeast actin binding proteins (ABPs) for association with F-actin helps facilitate their sorting to different F-actin networks. Specifically, competition between actin patch ABPs fimbrin Fim1 and cofilin Adf1 enhances each other’s activities, and rapidly displaces tropomyosin Cdc8 from the F-actin network. However, these interactions don’t explain how Fim1, a robust competitor, is prevented from associating equally well with other F-actin networks. Here, with a combination of fission yeast genetics, live cell fluorescent imaging, and in vitro TIRF microscopy, we identified the contractile ring ABP α-actinin Ain1 as a key sorting factor. Fim1 competes with Ain1 for association with F-actin, which is dependent upon their residence time on F-actin. Remarkably, although Fim1 outcompetes both contractile ring ABPs Ain1 and Cdc8 individually, Cdc8 enhances the bundling activity of Ain1 10-fold, allowing the combination of Ain1 and Cdc8 to inhibit Fim1 association with contractile ring F-actin.

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 Assembly of the cytokinetic contractile ring from a broad band of nodes in fission yeast

2006 ◽  
Vol 174 (3) ◽  
pp. 391-402 ◽  
Author(s):  
Jian-Qiu Wu ◽  
Vladimir Sirotkin ◽  
David R. Kovar ◽  
Matthew Lord ◽  
Christopher C. Beltzner ◽  
...  
Keyword(s):  
Fission Yeast ◽  
Fusion Proteins ◽  
Cell Separation ◽  
Actin Polymerization ◽  
Myosin Ii ◽  
Broad Band ◽  
Compact Ring ◽  
Contractile Ring ◽  
Single Spot ◽  
Anaphase B

We observed live fission yeast expressing pairs of functional fluorescent fusion proteins to test the popular model that the cytokinetic contractile ring assembles from a single myosin II progenitor or a Cdc12p-Cdc15p spot. Under our conditions, the anillin-like protein Mid1p establishes a broad band of small dots or nodes in the cortex near the nucleus. These nodes mature by the addition of conventional myosin II (Myo2p, Cdc4p, and Rlc1p), IQGAP (Rng2p), pombe Cdc15 homology protein (Cdc15p), and formin (Cdc12p). The nodes coalesce laterally into a compact ring when Cdc12p and profilin Cdc3p stimulate actin polymerization. We did not observe assembly of contractile rings by extension of a leading cable from a single spot or progenitor. Arp2/3 complex and its activators accumulate in patches near the contractile ring early in anaphase B, but are not concentrated in the contractile ring and are not required for assembly of the contractile ring. Their absence delays late steps in cytokinesis, including septum formation and cell separation.

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