scholarly journals Actin filament severing by cofilin is more important for assembly than constriction of the cytokinetic contractile ring

2011 ◽  
Vol 195 (3) ◽  
pp. 485-498 ◽  
Author(s):  
Qian Chen ◽  
Thomas D. Pollard
Keyword(s):  
Fission Yeast ◽  
Computer Simulations ◽  
Actin Filament ◽  
Actin Filaments ◽  
Actin Binding ◽  
Wild Type ◽  
Contractile Ring ◽  
Ring Constriction ◽  
Release Model ◽  
Mutant Cells

We created two new mutants of fission yeast cofilin to investigate why cytokinesis in many organisms depends on this small actin-binding protein. These mutant cofilins bound actin monomers normally, but bound and severed ADP-actin filaments much slower than wild-type cofilin. Cells depending on mutant cofilins condensed nodes, precursors of the contractile ring, into clumps rather than rings. Starting from clumped nodes, mutant cells slowly assembled rings from diverse intermediate structures including spiral strands containing actin filaments and other contractile ring proteins. This process in mutant cells depended on α-actinin. These slowly assembled contractile rings constricted at a normal rate but with more variability, indicating ring constriction is not very sensitive to defects in severing by cofilin. Computer simulations of the search-capture-pull and release model of contractile ring formation predicted that nodes clump when the release step is slow, so cofilin severing of actin filament connections between nodes likely contributes to the release step.

scholarly journals Counting actin in contractile rings reveals novel contributions of cofilin and type II myosins to fission yeast cytokinesis

2021 ◽  
Author(s):  
Qian Chen ◽  
Mamata Malla ◽  
Thomas D. Pollard
Keyword(s):  
Fission Yeast ◽  
Actin Filaments ◽  
Yeast Cells ◽  
Type Ii ◽  
Wild Type ◽  
Contractile Ring ◽  
Peak Number ◽  
Ring Constriction ◽  
Proportional Increase ◽  
Mutant Cells

Cytokinesis by animals, fungi and amoebas depends on actomyosin contractile rings, which are stabilized by continuous turnover of actin filaments. Remarkably little is known about the amount of polymerized actin in contractile rings, so we used low concentration of GFP-Lifeact to count total polymerized actin molecules in the contractile rings of live fission yeast cells. Contractile rings of wild-type cells accumulated polymerized actin molecules at 4,900/min to a peak number of ~198,000 followed by a loss of actin at 5,400/min throughout ring constriction. In adf1-M3 mutant cells with cofilin that severs actin filaments poorly, contractile rings accumulated polymerized actin at twice the normal rate and eventually had almost two-fold more actin along with a proportional increase in type II myosins Myo2, Myp2 and formin Cdc12. Although 30% of adf1-M3 mutant cells failed to constrict their rings fully, the rest lost actin from the rings at the wild-type rates. Mutations of type II myosins Myo2 and Myp2 reduced contractile ring actin filaments by half and slowed the rate of actin loss from the rings.

scholarly journals Counting actin in contractile rings reveals novel contributions of cofilin and type II myosins to fission yeast cytokinesis

2021 ◽  
Author(s):  
Mamata Malla ◽  
Thomas D. Pollard ◽  
Qian Chen
Keyword(s):  
Fission Yeast ◽  
Actin Filaments ◽  
Yeast Cells ◽  
Type Ii ◽  
Wild Type ◽  
Contractile Ring ◽  
Peak Number ◽  
Ring Constriction ◽  
Proportional Increase ◽  
Mutant Cells

Cytokinesis by animals, fungi and amoebas depends on actomyosin contractile rings, which are stabilized by continuous turnover of actin filaments. Remarkably little is known about the amount of polymerized actin in contractile rings, so we used low concentration of GFP-Lifeact to count total polymerized actin molecules in the contractile rings of live fission yeast cells. Contractile rings of wild-type cells accumulated polymerized actin molecules at 4,900/min to a peak number of ∼198,000 followed by a loss of actin at 5,400/min throughout ring constriction. In adf1-M3 mutant cells with cofilin that severs actin filaments poorly, contractile rings accumulated polymerized actin at twice the normal rate and eventually had almost two-fold more actin along with a proportional increase in type II myosins Myo2, Myp2 and formin Cdc12. Although 30% of adf1-M3 mutant cells failed to constrict their rings fully, the rest lost actin from the rings at the wild-type rates. Mutations of type II myosins Myo2 and Myp2 reduced contractile ring actin filaments by half and slowed the rate of actin loss from the rings.

scholarly journals Profilin-mediated Competition between Capping Protein and Formin Cdc12p during Cytokinesis in Fission Yeast

2005 ◽  
Vol 16 (5) ◽  
pp. 2313-2324 ◽  
Author(s):  
David R. Kovar ◽  
Jian-Qiu Wu ◽  
Thomas D. Pollard
Keyword(s):  
Fission Yeast ◽  
Actin Filament ◽  
Actin Filaments ◽  
Cell Separation ◽  
The Other ◽  
Temperature Sensitive ◽  
Contractile Ring ◽  
Capping Protein ◽  
Division Site ◽  
Ring Constriction

Fission yeast capping protein SpCP is a heterodimer of two subunits (Acp1p and Acp2p) that binds actin filament barbed ends. Neither acp1 nor acp2 is required for viability, but cells lacking either or both subunits have cytokinesis defects under stressful conditions, including elevated temperature, osmotic stress, or in combination with numerous mild mutations in genes important for cytokinesis. Defects arise as the contractile ring constricts and disassembles, resulting in delays in cell separation. Genetic and biochemical interactions show that the cytokinesis formin Cdc12p competes with capping protein for actin filament barbed ends in cells. Deletion of acp2 partly suppresses cytokinesis defects in temperature-sensitive cdc12-112 cells and mild overexpression of capping protein kills cdc12-112 cells. Biochemically, profilin has opposite effects on filaments capped with Cdc12p and capping protein. Profilin depolymerizes actin filaments capped by capping protein but allows filaments capped by Cdc12p to grow at their barbed ends. Once associated with a barbed end, either Cdc12p or capping protein prevents the other from influencing polymerization at that end. Given that capping protein arrives at the division site 20 min later than Cdc12p, capping protein may slowly replace Cdc12p on filament barbed ends in preparation for filament disassembly during ring constriction.

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 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 The number of cytokinesis nodes in mitotic fission yeast scales with cell volume

2021 ◽  
Author(s):  
Wasim A Sayyad ◽  
Thomas D Pollard
Keyword(s):  
Plasma Membrane ◽  
Fission Yeast ◽  
Large Population ◽  
Yeast Cells ◽  
Wild Type ◽  
Contractile Ring ◽  
Node Number ◽  
Nmt1 Promoter ◽  
Wide Range ◽  
Mutant Cells

Cytokinesis nodes are assemblies of stoichiometric ratios of proteins associated with the plasma membrane, which serve as precursors for the contractile ring during cytokinesis by fission yeast. The total number of nodes is uncertain, because of the limitations of the methods used previously. Here we used the ~140 nm resolution of Airyscan confocal microscopy to resolve a large population of dim, unitary cytokinesis nodes in 3D reconstructions of whole fission yeast cells. Wild-type fission yeast cells make about 200 unitary cytokinesis nodes. Most, but not all of these nodes condense into a contractile ring. The number of cytokinesis nodes scales with cell size in four strains tested, although wide rga4Δ mutant cells form somewhat fewer cytokinesis nodes than expected from the overall trend. The surface density of Pom1 kinase on the plasma membrane around the equators of cells is similar with a wide range of node numbers, so Pom1 does not control cytokinesis node number. However, varying protein concentrations with the nmt1 promoter showed that the numbers of nodes increase above a baseline of about 200 with the total cellular concentration of either Pom1 or the kinase Cdr2.

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 Formation of actin filament bundles in the ring canals of developing Drosophila follicles.

1996 ◽  
Vol 133 (1) ◽  
pp. 61-74 ◽  
Author(s):  
L G Tilney ◽  
M S Tilney ◽  
G M Guild
Keyword(s):  
Actin Filament ◽  
Actin Filaments ◽  
Actin Binding ◽  
Nurse Cells ◽  
Ring Canal ◽  
Contractile Ring ◽  
Thin Sections ◽  
Development Stages ◽  
Ring Canals ◽  
Filament Bundles

Growing the intracellular bridges that connect nurse cells with each o ther and to the developing oocyte is vital for egg development. These ring canals increase from 0.5 microns in diameter at stage 2 to 10 microns in diameter at stage 11. Thin sections cut horizontally as you would cut a bagel, show that there is a layer of circumferentially oriented actin filaments attached to the plasma membrane at the periphery of each canal. By decoration with subfragment 1 of myosin we find actin filaments of mixed polarities in the ring such as found in the "contractile ring" formed during cytokinesis. In vertical sections through the canal the actin filaments appear as dense dots. At stage 2 there are 82 actin filaments in the ring, by stage 6 there are 717 and by stage 10 there are 726. Taking into account the diameter, this indicates that there is 170 microns of actin filaments/canal at stage 2 (pi x 0.5 microns x 82), 14,000 microns at stage 9 and approximately 23,000 microns at stage 11 or one inch of actin filament! The density of actin filaments remains unchanged throughout development. What is particularly striking is that by stages 4-5, the ring of actin filaments has achieved its maximum thickness, even though the diameter has not yet increased significantly. Thereafter, the diameter increases. Throughout development, stages 2-11, the canal length also increases. Although the density (number of actin filaments/micron2) through a canal remains constant from stage 5 on, the actin filaments appear as a net of interconnected bundles. Further information on this net of bundles comes from studying mutant animals that lack kelch, a protein located in the ring canal that has homology to the actin binding protein, scruin. In this mutant, the actin filaments form normally but individual bundles that comprise the fibers of the net are not bound tightly together. Some bundles enter into the ring canal lumen but do not completely occlude the lumen. all these observations lay the groundwork for our understanding of how a noncontractile ring increases in thickness, diameter, and length during development.

scholarly journals αE-catenin actin-binding domain alters actin filament conformation and regulates binding of nucleation and disassembly factors

2013 ◽  
Vol 24 (23) ◽  
pp. 3710-3720 ◽  
Author(s):  
Scott D. Hansen ◽  
Adam V. Kwiatkowski ◽  
Chung-Yueh Ouyang ◽  
HongJun Liu ◽  
Sabine Pokutta ◽  
...  
Keyword(s):  
Actin Filament ◽  
Actin Filaments ◽  
Binding Domain ◽  
Actin Binding ◽  
Filamentous Actin ◽  
Filament Structure ◽  
Actin Binding Domain ◽  
Filament Bundles ◽  
Underlying Mechanisms ◽  
Cell Cell

The actin-binding protein αE-catenin may contribute to transitions between cell migration and cell–cell adhesion that depend on remodeling the actin cytoskeleton, but the underlying mechanisms are unknown. We show that the αE-catenin actin-binding domain (ABD) binds cooperatively to individual actin filaments and that binding is accompanied by a conformational change in the actin protomer that affects filament structure. αE-catenin ABD binding limits barbed-end growth, especially in actin filament bundles. αE-catenin ABD inhibits actin filament branching by the Arp2/3 complex and severing by cofilin, both of which contact regions of the actin protomer that are structurally altered by αE-catenin ABD binding. In epithelial cells, there is little correlation between the distribution of αE-catenin and the Arp2/3 complex at developing cell–cell contacts. Our results indicate that αE-catenin binding to filamentous actin favors assembly of unbranched filament bundles that are protected from severing over more dynamic, branched filament arrays.

Actin filaments regulate epithelial Na+ channel activity

1991 ◽  
Vol 261 (5) ◽  
pp. C882-C888 ◽  
Author(s):  
H. F. Cantiello ◽  
J. L. Stow ◽  
A. G. Prat ◽  
D. A. Ausiello
Keyword(s):  
Actin Filament ◽  
Actin Filaments ◽  
Functional Role ◽  
Cytochalasin D ◽  
Actin Binding ◽  
Na Channel ◽  
Na Channels ◽  
Channel Activity ◽  
Cortical Actin ◽  
Excised Patches

The functional role of the cytoskeleton in the control of ion channel activity is unknown. In the present study, immunocolocalization of Na+ channels with specific antibodies and fluorescein isothiocyanate-phalloidin to stain the cortical cytoskeleton indicates that actin is always present in close proximity to apical Na+ channels in A6 cells. The patch-clamp technique was used to assess the effect of cortical actin networks on apical Na+ channels in these A6 epithelial cells. The actin filament disrupter, cytochalasin D (5 micrograms/ml), induced Na+ channel activity in cell-attached patches within 5 min of addition. Cytochalasin D also induced and/or increased Na+ channel activity in 90% of excised patches tested within 2 min. Addition of short actin filaments (greater than 5 microM) to excised patches also induced channel activity. This effect was enhanced by addition of ATP and/or cytochalasin D. The effect of actin on Na+ channel activity was reversed by addition of the G actin-binding protein DNase I or completely prevented by treatment of the excised patches with this enzyme. Addition of the actin-binding protein, filamin, reversibly inhibited both spontaneous and actin-induced Na+ channels. Thus actin filament networks, achieved by either depolymerizing endogenous actin filaments by treatment with cytochalasin D, the addition of exogenous short actin filaments plus ATP, or actin plus cytochalasin D, regulate apical Na+ channel activity. This conclusion was supported by the observation that the addition of short actin filaments in the form of actin-gelsolin complexes in molar ratios less than 8:1 was also effective in activating Na+ channels. We have thus demonstrated a functional role for the cortical actin network in the regulation of epithelial Na+ channels that may complement a structural role for membrane protein targetting and assembly.

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