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

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.

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

eLife ◽

10.7554/elife.47279 ◽

2019 ◽

Vol 8 ◽

Cited By ~ 7

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.

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Competition between Tropomyosin, Fimbrin, and ADF/Cofilin drives their sorting to distinct actin filament networks

eLife ◽

10.7554/elife.23152 ◽

2017 ◽

Vol 6 ◽

Cited By ~ 44

Author(s):

Jenna R Christensen ◽

Glen M Hocky ◽

Kaitlin E Homa ◽

Alisha N Morganthaler ◽

Sarah E Hitchcock-DeGregori ◽

...

Keyword(s):

Actin Filaments ◽

Actin Binding ◽

Actin Network ◽

Actin Networks ◽

Cooperative Interactions ◽

Filament Networks ◽

Collective Interactions ◽

Functionally Diverse ◽

Cooperative Association

The fission yeast actin cytoskeleton is an ideal, simplified system to investigate fundamental mechanisms behind cellular self-organization. By focusing on the stabilizing protein tropomyosin Cdc8, bundling protein fimbrin Fim1, and severing protein coffin Adf1, we examined how their pairwise and collective interactions with actin filaments regulate their activity and segregation to functionally diverse F-actin networks. Utilizing multi-color TIRF microscopy of in vitro reconstituted F-actin networks, we observed and characterized two distinct Cdc8 cables loading and spreading cooperatively on individual actin filaments. Furthermore, Cdc8, Fim1, and Adf1 all compete for association with F-actin by different mechanisms, and their cooperative association with actin filaments affects their ability to compete. Finally, competition between Fim1 and Adf1 for F-actin synergizes their activities, promoting rapid displacement of Cdc8 from a dense F-actin network. Our findings reveal that competitive and cooperative interactions between actin binding proteins help define their associations with different F-actin networks.

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MICAL2 acts through Arp3B isoform-specific Arp2/3 complexes to destabilize branched actin networks

10.1101/2020.09.21.306522 ◽

2020 ◽

Author(s):

Chiara Galloni ◽

Davide Carra ◽

Jasmine V. G. Abella ◽

Svend Kjær ◽

Pavithra Singaravelu ◽

...

Keyword(s):

Functional Properties ◽

Actin Filament ◽

Actin Assembly ◽

Actin Network ◽

Network Stability ◽

Actin Networks ◽

Cellular Processes ◽

Actin Turnover ◽

Filament Networks

AbstractThe Arp2/3 complex (Arp2, Arp3 and ARPC1-5) is essential to generate branched actin filament networks for many cellular processes. Human Arp3, ARPC1 and ARPC5 exist as two isoforms but the functional properties of Arp2/3 iso-complexes is largely unexplored. Here we show that Arp3B, but not Arp3 is subject to regulation by the methionine monooxygenase MICAL2, which is recruited to branched actin networks by coronin-1C. Although Arp3 and Arp3B iso-complexes promote actin assembly equally efficiently in vitro, they have different cellular properties. Arp3B turns over significantly faster than Arp3 within the network and upon its depletion actin turnover decreases. Substitution of Arp3B Met293 by Thr, the corresponding residue in Arp3 increases actin network stability, and conversely, replacing Arp3 Thr293 with Gln to mimic Met oxidation promotes network disassembly. Thus, MICAL2 regulates a subset of Arp2/3 complexes to control branched actin network disassembly.

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Moesin and cortactin control actin-dependent multivesicular endosome biogenesis

Molecular Biology of the Cell ◽

10.1091/mbc.e15-12-0853 ◽

2016 ◽

Vol 27 (21) ◽

pp. 3305-3316 ◽

Cited By ~ 12

Author(s):

Olivia Muriel ◽

Alejandra Tomas ◽

Cameron C. Scott ◽

Jean Gruenberg

Keyword(s):

Network Formation ◽

Late Endosome ◽

Actin Binding ◽

Membrane Remodeling ◽

Actin Network ◽

Degradative Pathway ◽

Actin Networks ◽

Early Endosomes

We used in vivo and in vitro strategies to study the mechanisms of multivesicular endosome biogenesis. We found that, whereas annexinA2 and ARP2/3 mediate F-actin nucleation and branching, respectively, the ERM protein moesin supports the formation of F-actin networks on early endosomes. We also found that moesin plays no role during endocytosis and recycling to the plasma membrane but is absolutely required, much like actin, for early-to-late-endosome transport and multivesicular endosome formation. Both actin network formation in vitro and early-to-late endosome transport in vivo also depend on the F-actin–binding protein cortactin. Our data thus show that moesin and cortactin are necessary for formation of F-actin networks that mediate endosome biogenesis or maturation and transport through the degradative pathway. We propose that the primary function of endosomal F-actin is to control the membrane remodeling that accompanies endosome biogenesis. We also speculate that this mechanism helps segregate tubular and multivesicular membranes along the recycling and degradation pathways, respectively.

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Fission yeast myosin Myo2 is down-regulated in actin affinity by light chain phosphorylation

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1703161114 ◽

2017 ◽

Vol 114 (35) ◽

pp. E7236-E7244 ◽

Cited By ~ 12

Author(s):

Luther W. Pollard ◽

Carol S. Bookwalter ◽

Qing Tang ◽

Elena B. Krementsova ◽

Kathleen M. Trybus ◽

...

Keyword(s):

Fission Yeast ◽

Light Chain ◽

Force Production ◽

Class Ii ◽

Cell System ◽

Cell Cortex ◽

Animal Cells ◽

Contractile Ring ◽

Biophysical Studies

Studies in fission yeast Schizosaccharomyces pombe have provided the basis for the most advanced models of the dynamics of the cytokinetic contractile ring. Myo2, a class-II myosin, is the major source of tension in the contractile ring, but how Myo2 is anchored and regulated to produce force is poorly understood. To enable more detailed biochemical/biophysical studies, Myo2 was expressed in the baculovirus/Sf9 insect cell system with its two native light chains, Rlc1 and Cdc4. Milligram yields of soluble, unphosphorylated Myo2 were obtained that exhibited high actin-activated ATPase activity and in vitro actin filament motility. The fission yeast specific chaperone Rng3 was thus not required for expression or activity. In contrast to nonmuscle myosins from animal cells that require phosphorylation of the regulatory light chain for activation, phosphorylation of Rlc1 markedly reduced the affinity of Myo2 for actin. Another unusual feature of Myo2 was that, unlike class-II myosins, which generally form bipolar filamentous structures, Myo2 showed no inclination to self-assemble at approximately physiological salt concentrations, as analyzed by sedimentation velocity ultracentrifugation. This lack of assembly supports the hypothesis that clusters of Myo2 depend on interactions at the cell cortex in structural units called nodes for force production during cytokinesis.

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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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In vitro reactivation of the cytokinetic contractile ring of fission yeast cells

Methods in Cell Biology - Cytokinesis ◽

10.1016/bs.mcb.2016.03.037 ◽

2017 ◽

pp. 387-394 ◽

Cited By ~ 1

Author(s):

I. Mabuchi ◽

J. Kashiwazaki ◽

M. Mishra

Keyword(s):

Fission Yeast ◽

Yeast Cells ◽

Contractile Ring

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

Molecular Biology of the Cell ◽

10.1091/mbc.e09-10-0852 ◽

2010 ◽

Vol 21 (6) ◽

pp. 989-1000 ◽

Cited By ~ 55

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.

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Coronin 1C harbours a second actin-binding site that confers co-operative binding to F-actin

Biochemical Journal ◽

10.1042/bj20120209 ◽

2012 ◽

Vol 444 (1) ◽

pp. 89-96 ◽

Cited By ~ 20

Author(s):

Keefe T. Chan ◽

David W. Roadcap ◽

Nicholas Holoweckyj ◽

James E. Bear

Keyword(s):

Binding Site ◽

Leading Edge ◽

Actin Binding ◽

Filamentous Actin ◽

Unique Region ◽

Equivalent Residue ◽

Filament Networks ◽

Actin Binding Site

Dynamic rearrangement of actin filament networks is critical for cell motility, phagocytosis and endocytosis. Coronins facilitate these processes, in part, by their ability to bind F-actin (filamentous actin). We previously identified a conserved surface-exposed arginine (Arg30) in the β-propeller of Coronin 1B required for F-actin binding in vitro and in vivo. However, whether this finding translates to other coronins has not been well defined. Using quantitative actin-binding assays, we show that mutating the equivalent residue abolishes F-actin binding in Coronin 1A, but not Coronin 1C. By mutagenesis and biochemical competition, we have identified a second actin-binding site in the unique region of Coronin 1C. Interestingly, leading-edge localization of Coronin 1C in fibroblasts requires the conserved site in the β-propeller, but not the site in the unique region. Furthermore, in contrast with Coronin 1A and Coronin 1B, Coronin 1C displays highly co-operative binding to actin filaments. In the present study, we highlight a novel mode of coronin regulation, which has implications for how coronins orchestrate cytoskeletal dynamics.

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