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. Author manuscript; available in PMC: 2015 Mar 3.
Published in final edited form as: Curr Biol. 2014 Feb 20;24(5):579–585. doi: 10.1016/j.cub.2014.01.072

Homeostatic Actin Cytoskeleton Networks Are Regulated by Assembly Factor Competition for Monomers

Thomas A Burke 1, Jenna R Christensen 1, Elisabeth Barone 2, Cristian Suarez 1, Vladimir Sirotkin 2, David R Kovar 1,3
PMCID: PMC3979332  NIHMSID: NIHMS568306  PMID: 24560576

Summary

Controlling the quantity and size of organelles through competition for a limited supply of components is quickly emerging as an important cellular regulatory mechanism [1]. Cells assemble diverse actin filament (F-actin) networks for fundamental processes including division, motility, and polarization [2–4]. F-actin polymerization is tightly regulated by activation of assembly factors such as the Arp2/3 complex and formins at specific times and places. We directly tested an additional hypothesis that diverse F-actin networks are in homeostasis, whereby competition for actin monomers (G-actin) is critical for regulating F-actin network size. Here we show that inhibition of Arp2/3 complex in the fission yeast Schizosaccharomyces pombe not only eliminates Arp2/3 complex-mediated endocytic actin patches, but also induces a dramatic excess of formin-assembled F-actin. Conversely, disruption of formin increases the density of Arp2/3 complex-mediated patches. Furthermore, modifying actin levels significantly perturbs the fission yeast actin cytoskeleton. Increasing actin favors Arp2/3 complex-mediated actin assembly, whereas decreasing actin favors formin-mediated contractile rings. Therefore, the specific actin concentration in a cell is critical, and competition for G-actin helps regulate the proper amount of F-actin assembly for diverse processes.

Results and Discussion

To control F-actin network density, actin polymerization is tightly regulated through the activation of assembly (nucleation) factors by GTPase signaling cascades, the rate at which F-actin barbed ends are capped, the rate at which assembly factors are turned off, and F-actin disassembly factors [2, 3, 5]. The supply of unassembled G-actin is not generally considered to be limiting [6, 7]. Alternatively, it is possible that the actin cytoskeleton is homeostatic with a limited concentration of G-actin, which is competed for by assembly factors to help regulate its incorporation into diverse F-actin networks [3, 8–10]. However, this intriguing additional hypothesis has not been systematically tested.

Fission yeast forms three F-actin network structures by three different assembly factors [9]. The Arp2/3 complex assembles short-branched F-actin in endocytic actin patches, whereas the formins For3 and Cdc12 assemble long-straight F-actin in polarizing actin cables and the cytokinetic contractile ring, respectively. The amount of actin and other components incorporated into actin patches and contractile rings is remarkably consistent, varying less than 50% for each structure [11–13]. Although measuring the composition of actin cables has been technically challenging, they may be similarly consistent. Of the ~1 million actin molecules per cell, ~35 to 50% are evenly distributed between 30 to 50 actin patches, ~10% are incorporated into contractile rings, and perhaps as much as 15% are estimated to be consumed by actin cables [11–15].

To directly test the hypothesis that assembly factors compete for G-actin, we investigated the consequences of systematically disrupting individual assembly factors in fission yeast cells. Initially, we treated cells expressing the general F-actin marker Lifeact-GFP with a range of concentrations of the Arp2/3 complex inhibitor CK-666 [16], causing a dose-dependent decrease in the number of actin patches (Figures 1A and 1B and Figure S1A available online) and reduction in patch lifetime and motility (Table S1). Strikingly, actin patch depletion coincides with the dramatic formation of new ectopic cable-like F-actin (Figure 1A and Figure S1A), saturating at ~100 µM CK-666 (Figure 1B). CK-666 treatment facilitates ectopic F-actin assembly in both minimal and rich growth media, is visible with different general F-actin markers including rhodamine-phalloidin (Figures S1B–F), and is inhibited by the G-actin sequestering drug LatA (Figure S1G).

Figure 1. Pharmacological Inhibition of Arp2/3 Complex Stimulates Ectopic F-Actin Assembly.

Figure 1

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(A–D) The Arp2/3 complex inhibitor CK-666 was applied for 30 minutes to fission yeast expressing the general F-actin marker Lifeact-GFP.

(A) Fluorescent micrographs of cells treated with DMSO (control) or a range of CK-666 concentrations. Scale bar, 5 µm.

(B) Dependence of the number of actin patches (left) and Lifeact-GFP fluorescence intensity of ectopic F-actin (right) on the concentration of CK-666. Error bars, s.d.; n = 25.

(C and D) Effects of addition and washout of CK-666 on cells in a microfluidic chamber (Movie S1).

(C)Time-lapse fluorescent micrographs after the addition of saturating CK-666 at zero minutes, and removal of CK-666 at 40 minutes. Scale bar, 5 µm.

(D) The number of actin patches (left) and ectopic F-actin (right) upon the addition and removal (dashed lines) of CK-666. Error bars, s.d.; n = 10.

(E–H) Genetic depletion of the Arp2/3 complex in fission yeast cells expressing Lifeact-GFP.

(E) Fluorescent micrographs of WT and Arp2/3 complex mutant arp3-C1 cold sensitive cells following 4 hours at 19°C. Scale bar, 5 µm.

(F) Actin patches per cell (blue) and Ectopic F-actin fluorescence (green) of cells from (E). Error bars, s.d.; n = 25.

(G) Fluorescent micrographs of WT and Arp2/3 complex shut off (SO-arp3) cells after inhibiting expression for 46 hours. Scale bar, 5 µm.

(H) Actin patches per cell (blue) and Ectopic F-actin fluorescence (green) of cells from (H). Error bars, s.d.; n = 25.

Observation of cells in a microfluidic chamber revealed that depletion of actin patches and the concomitant assembly of ectopic F-actin occurs in ~10–20 minutes after addition of saturating concentrations of CK-666 (Figure 1C and 1D and Movie S1). Ectopic F-actin rapidly disassembles upon wash out of CK-666 with a corresponding reassembly of actin patches in ~10–40 minutes (Figures 1C and 1D). Actin patch proteins ArpC5-mCherry (Arp2/3 complex component) and Acp2-GFP (actin capping protein) are released into the cytoplasm by CK-666 treatment, but do not incorporate into the ectopic F-actin (Figure S1H–J).

Genetic disruption of Arp2/3 complex also leads to ectopic F-actin assembly, albeit less prominently than with CK-666 since actin patches are not reduced completely under these conditions (Figures 1E–H). Compared to WT cells, at the restrictive temperature of 19°C Arp2/3 complex cold-sensitive mutant arp3-C1 cells [17] have approximately half the number of patches and a corresponding statistically significant 3-fold increase in ectopic F-actin (p<0.0001) (Figures 1E and 1F). Similarly, reducing Arp2/3 complex expression by shutting off Arp3 (SO-arp3) for 46 hours also halves the number of patches per cell while increasing the amount of ectopic F-actin by more than 3-fold (p<0.0001) (Figures 1G and 1H).

We next investigated whether the cable-like ectopic F-actin is spontaneously assembled, or is dependent upon remaining actin assembly factors: the formins For3 and Cdc12. 100% of single formin mutant cells (for3Δ or cdc12–112 temperature sensitive) assemble ectopic F-actin when treated with CK-666 at the restrictive temperature of 36°C, whereas double formin mutant for3Δ cdc12–112 cells do not (Figure 2A and Figures S2A–D). Disrupting the small G-actin binding protein profilin (cdc3–124), which is necessary for formin-mediated actin assembly in vivo [18, 19], also prevents CK-666 mediated ectopic F-actin assembly (Figure 2A and Figure S2E). Time-lapse imaging revealed that formin-mediated ectopic F-actin is highly dynamic, whereas smaller F-actin aggregates formed by inhibition of Arp2/3 complex in double formin mutant cells are immobile (Figure S2F). Additionally, the F-actin binding protein tropomyosin (Cdc8) [20], which associates with formin-assembled filaments [20–22], localizes to the ectopic F-actin (Figure S2G). Similarly, the formin-mediated contractile ring marker Rlc1-tdTomato localizes to robust rings in CK-666 treated cells (Figure S2H).

Figure 2. Ectopic F-Actin Assembly Requires Formin.

Figure 2

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(A–C) WT and mutant cells expressing Lifeact-GFP were grown at 36°C for 3 hours, and incubated with DMSO or 100 µM CK-666 for 30 minutes.

(A) Fluorescent micrographs of WT, single formin mutant (for3Δ and cdc12–112), double formin mutant (for3Δ cdc12–112), and profilin mutant (cdc3–124) cells. Scale bar, 5 µm.

(B) Line scans of ectopic F-actin intensity along the length of five representative cells (dashed colored lines), and the average of 20 cells (solid black line).

(C) Mean fluorescence intensity of Lifeact-GFP (left) or rhodamine-phalloidin (right) in contractile rings of WT and for3Δ cells incubated with DMSO (control) or 100 µM CK-666. Error bars, s.d.; n ≥ 25.

(D) The density (red) and mean fluorescence (blue) of Lifeact-GFP labeled actin patches in WT and formin mutant cells grown at 36°C for 3 hours. Error bars, s.d.; n = 25.

These results indicate that inhibition of Arp2/3 complex amplifies formin-mediated actin assembly in fission yeast, suggesting an underlying homeostatic state whereby assembly factors compete for G-actin. Increased levels of G-actin produced by inhibiting Arp2/3 complex may allow intrinsically active formins to elongate filaments faster [23], and/or turn on inactive formin molecules [24, 25].

For3 primarily localizes to and forms actin cables from cell tips [26], whereas active Cdc12 forms contractile rings in the middle of dividing cells [18]. Consistent with these different cellular localizations, line scans of fluorescence intensity across the length of individual interphase cells treated with CK-666 revealed that single formin mutants assemble unique ectopic F-actin patterns (Figure 2B). Ectopic F-actin is present in approximately three peaks at the tips and middle of WT cells, while it localizes primarily to poles in cdc12–112 cells, and to the midzone in for3Δ cells. Ectopic F-actin in for3Δ cells is assembled by Cdc12 in the midzone rather than relocated there following its assembly elsewhere (Figure S2I and Movie S2), indicating that Cdc12 is active in the midzone during interphase when Arp2/3 complex is inhibited. The fluorescence intensities of Lifeact-GFP and rhodamine-phalloidin in Cdc12-mediated contractile rings increase in WT cells treated with CK-666 (Figure 2C). Contractile ring F-actin levels are also elevated in for3Δ cells, and even more so when for3Δ cells are treated with CK-666 (Figure 2C). Therefore, Cdc12 assembles more robust rings upon inhibition of the other assembly factors, suggesting that the formin Cdc12 competes with both Arp2/3 complex and the formin For3 for G-actin.

We next investigated whether depleting the formins enhances Arp2/3 complex-mediated actin assembly (Figures 2A and 2D, and Figures S2A–D). While formin mutant and WT cells have similar total amounts of actin (Figures S2J and S2K), single (cdc12–112 and for3Δ) and double (cdc12–112 for3Δ) formin mutant cells have increasingly higher densities of Arp2/3 complex-dependent actin patches (Figure 2D). The density of Lifeact-GFP-labeled actin patches increases from ~1.0 (per µm2 of confocal Z-projections) in WT cells to ~1.6 in cdc12–112 for3Δ cells. However, in both single and double formin mutant cells, the amount of Lifeact-GFP fluorescence, lifetime, and motility of individual patches are relatively unchanged (Figure 2D and Table S1). The assembly of an excess number of actin patches with similar dynamics suggests that the concentration of G-actin could be important for regulation of actin patch initiation by the Arp2/3 complex. Conversely, consumption of G-actin by individual patches may instead be limited by the number of activated Arp2/3 complexes, barbed end capping protein, and F-actin disassembly by cofilin [12, 27, 28].

We hypothesize that inhibition of Arp2/3 complex liberates G-actin by preventing its incorporation into new patches, thereby increasing its availability for the formins. Because the F-actin severing protein cofilin is required for actin patch disassembly, depletion of cofilin increases the density and size of actin patches and prevents formin-mediated assembly of both rings and cables (Figures 3A and 3C) [10, 27]. Given that CK-666 inhibits nucleation by Arp2/3 complex but does not disassemble pre-existing branches [29], it is therefore not surprising that treatment of cofilin mutant cells (adf1–1) with 100 µM CK-666 at the restrictive temperature of 36°C does not deplete actin patches, and consequently does not induce formin-mediated ectopic F-actin assembly (Figure 3A). Prevention of CK-666 mediated ectopic F-actin assembly appears to be specific to the cofilin adf1–1 mutant, because deletion of the F-actin bundling endocytic actin patch component fimbrin (fim1Δ) does not prevent ectopic F-actin formation (Figure 3B).

Figure 3. Depletion of ADF/Cofilin Prevents CK-666 Mediated Ectopic F-Actin Assembly.

Figure 3

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(A–C) Mutant cells expressing Lifeact-GFP were grown at 25 or 36°C for 2 hours, and incubated with DMSO or 100 µM CK-666 for 30 minutes

(A) Fluorescent micrographs of cofilin mutant adf1–1 cells at 25 and 36°C. Scale bar, 5 µm.

(B) Fluorescent micrographs of fimbrin mutant fim1Δ cells at 25°C. Scale bar, 5 µm.

(C) Actin patches per cell (red) and mean patch fluorescence (blue) of cofilin mutant adf1–1 cells from (A). Error bars, s.d.; n = 25.

Because the disruption of actin assembly factors and their associated structures leads to extraneous F-actin assembly by competing factors, we hypothesized that the specific cellular actin concentration is critical for proper F-actin network formation. We replaced the endogenous actin (act1) promoter with the thiamine-repressible Pnmt1 promoter, which in the presence or the absence of thiamine for 22 hours results in either a ~5-fold under- or ~5-fold over-expression of actin, respectively (Figure S3A–C). Fluorescent images of Lifeact-GFP revealed that under- and over-expressing actin has contrasting effects; under-expression (U.E.) favors formin-mediated contractile rings whereas over-expression (O.E.) favors Arp2/3 complex-mediated actin patches (Figure 4A). Because actin cables are difficult to image, we focused our quantitative analysis on actin patches and contractile rings. There are ~3-fold fewer actin patches per cell when actin is under-expressed (Figure 4B), but individual patch behaviors (lifetime, mean Lifeact-GFP fluorescence, and motility) are only affected slightly (Figures 4C–E and Movie S3 and Table S1). Conversely, actin over-expression cells contain at least 2-fold more actin patches per cell (Figure 4B), an underestimate as individual patches are extremely difficult to discern within broad swaths of patch-like material (Figures 4A–D) that colocalizes with the actin patch component fimbrin Fim1-mCherry (Figure S3D). The lifetime of distinguishable individual patches in actin over-expression cells is ~2-fold longer, and they travel an ~3.5-fold shorter distance from the cortex during internalization (Figures 4C–E and Movie S3 and Table S1). Interestingly, actin patch dynamics are significantly altered in actin over-expression cells where actin is increased by ~500%, whereas patch dynamics are not altered in formin mutant cells where the available actin may be increased by only ~20% (Table S1).

Figure 4. Varying Actin Concentrations Disrupts Formin- and Arp2/3 Complex-Mediated F-Actin Assembly.

Figure 4

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(A–J) Comparison of Lifeact-GFP labeled F-actin networks in WT cells, and in cells under-expressing (U.E.) or over-expressing (O.E.) actin for 22 hours.

(A) Representative fluorescent micrographs. Scale bar, 5 µm.

(B) Actin patches per cell. Error bars, s.d.; n = 25. Actin patches per O.E. cell is underrepresented due to patch aggregation (#).

(C) Time-lapse (seconds) fluorescent micrographs of actin patches (arrows) (Movie S3). Scale bar, 5.0 µm.

(D) Kymographs (Time = 0 to 45 seconds) of the cell tips shown in (C), revealing actin patch (red triangles) dynamics. Scale bar, 5 µm.

(E) Plots of the position of two representative patches (red and black) over time (0.75 seconds per point).

(F) Percent of cells with contractile rings.

(G) Lifeact-GFP fluorescence intensity in rings. Error bars, s.d.; n = 25. P = 0.0098.

(H) Percent of cells with 1, 2, or >2 nuclei.

(I) Fluorescent micrographs of cells incubated with DMSO (control) or a range of CK-666 concentrations for 30 minutes. Scale bar, 5 µm.

(J) Dependence of Lifeact-GFP fluorescence in ectopic F-actin structures per cell on the concentration of CK-666. Error bars, s.d.; n = 25.

Formin-mediated contractile rings are not detected in cells over-expressing actin (Figure 4F), and >80% of these cells are multi-nucleate with malformed septa (Figure 4H and Figures S3E and S3F). The type II myosin regulatory light chain Rlc1-GFP contractile ring marker confirmed that multi-nucleate cells over-expressing actin fail to form normal contractile rings (Figure S3G). Conversely, ~2-fold more cells under-expressing actin have contractile rings compared to WT cells (Figure 4F), and those rings have significantly more Lifeact-GFP fluorescence (actin) (Figure 4G), resulting in >2-fold more bi-nucleate cells with deformed septa than WT cells (Figure 4H and Figure S3F). Thus, reduced actin concentrations appear to decrease Arp2/3 complex-mediated actin patches and favor formin-mediated contractile rings, whereas elevated actin concentrations favors actin patches over contractile rings. Increased actin concentration may stimulate excessive actin patch initiation by Arp2/3 complex, which subsequently consumes the majority of actin at the expense of formins. Conversely, reduced actin concentration may increase the ratio of profilin to G-actin, which is critical for formin Cdc12 function [19].

Lastly, treatment of cells under- or over-expressing actin with a range of concentrations of CK-666 supports our hypothesis that competition for a common pool of G-actin helps regulate the extent of F-actin network assembly (Figures 4I and 4J). Cells under-expressing actin require a lower concentration of CK-666 to fully disassemble the fewer number of actin patches, and form less formin-mediated ectopic F-actin than WT cells. On the other hand, cells over-expressing actin require ~2-fold more CK-666 to fully disassemble the excess of actin patches, and form >3-fold more ectopic F-actin.

We discovered that depletion of either Arp2/3 complex- or formin-dependent F-actin networks leads to enhanced F-actin assembly by the remaining actin assembly factors. Furthermore, raising actin levels favors Arp2/3 complex actin patches, whereas lowering actin levels favors formin contractile rings. These results suggest an important regulatory mechanism whereby the actin cytoskeleton is in homeostasis, characterized by an intrinsic competition for a common pool of G-actin that helps set the number and size of diverse F-actin structures in fission yeast. Consumption of G-actin by one F-actin network is critical to limit the amount of Gactin available for other networks. This competition could explain why Arp2/3 complex mutations suppress profilin mutations in fission yeast [17, 30].

Although competition for G-actin had not been systematically tested before, and has only occasionally been suggested as a possible actin cytoskeleton regulatory mechanism [3, 8–10, 31, 32], multiple Arp2/3 complex inhibition experiments from diverse cell types can be interpreted similarly. For example, depletion of actin patches by either over-expressing the Arp2/3 complex inhibitor Gmf1 or depleting the Arp2/3 complex activator Dip1 both lead to ectopic F-actin formation in fission yeast [31, 33]. Budding yeast Arp2/3 complex mutant cells have excessive formin-like F-actin cables [34]. Furthermore, in insect and diverse animal cell types the inhibition of Arp2/3 complex leads to depletion of lamellipodia with a simultaneous increase in the number of long, straight, bundled F-actin networks such as formin- and Ena/VASP-dependent filopodia-like structures [32, 35–40]. Therefore, considerable care is required to interpret experiments that perturb actin assembly factors or actin levels.

Supplementary Material

01
02. Movie S1.

Effect of microfluidic flow of CK-666 into and out of cells. Related to Figures 1C and 1D. Time-lapse of the maximum fluorescent intensity Z-projections of cells expressing Lifeact-GFP, showing F-actin organization upon the addition of EMM5S + DMSO (−20 to 0 min), EMM5S + 2 mM CK-666 (0 to 40 min), and EMM5S + DMSO (40 to 100 min). Boxed region is shown in Figure 1C. Scale bar, 5 µm.

Download video file (5.7MB, mov)
03. Movie S2.

Effect of microfluidic flow of CK-666 into for3Δ cells. Related to Figure S2I. Time-lapse of maximum intensity Z-projections of Lifeact-GFP in for3Δ cells, showing F-actin organization upon the addition of EMM5S + DMSO (−15 to 0 min), and EMM5S + 2 mM CK-666 (0 to 30 min). Scale bar, 5 µm.

Download video file (232.5KB, mov)
04. Movie S3.

Varying actin concentrations disrupts actin patches. Related to Figure 4. Time-lapse of Lifeact-GFP labeled actin patches in a single Z-plane for WT, actin under-expression (U.E.), and actin over-expression (O.E.) cells at 25°C. White arrows indicate individual patches. Scale bar, 5 µm.

Download video file (4.1MB, mov)

Highlights.

  • -

    Inhibition of Arp2/3 complex leads to excessive formin F-actin networks.

  • -

    Inhibition of formin leads to excessive Arp2/3 complex F-actin networks.

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    Altering actin levels affects Arp2/3 complex and formin F-actin networks differently.

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    The actin cytoskeleton is homeostatic, whereby assembly factors compete for G-actin.

Acknowledgments

This work was supported by NIH R01 GM079265 and ACS RSG-11-126-01-CSM (to D.R.K.), NIH MCB Training Grant T32 GM0071832 (to T.A.B. and J.R.C.), and AHA 11SDG5470024 (to V.S.). We thank members of the Kovar lab for helpful comments, Mohan Balasubramanian for fluorescent Lifeact strains and Jian-qiu Wu for double formin mutant strains before they were published, and Michael James for technical assistance.

Footnotes

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Supplemental Information

Supplemental Information includes two tables, three figures, three movies, and Supplemental Experimental Procedures and can be found with this article online.

References

  • 1.Goehring NW, Hyman AA. Organelle growth control through limiting pools of cytoplasmic components. Curr Biol. 2012;22:R330–R339. doi: 10.1016/j.cub.2012.03.046. [DOI] [PubMed] [Google Scholar]
  • 2.Campellone KG, Welch MD. A nucleator arms race: cellular control of actin assembly. Nat Rev Mol Cell Biol. 2010;11:237–251. doi: 10.1038/nrm2867. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 3.Chesarone MA, Goode BL. Actin nucleation and elongation factors: mechanisms and interplay. Curr Opin Cell Biol. 2009;21:28–37. doi: 10.1016/j.ceb.2008.12.001. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 4.Chhabra ES, Higgs HN. The many faces of actin: matching assembly factors with cellular structures. Nat Cell Biol. 2007;9:1110–1121. doi: 10.1038/ncb1007-1110. [DOI] [PubMed] [Google Scholar]
  • 5.Michelot A, Drubin DG. Building distinct actin filament networks in a common cytoplasm. Curr Biol. 2011;21:R560–R569. doi: 10.1016/j.cub.2011.06.019. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 6.Mullins RD, Pollard TD. Rho-family GTPases require the Arp2/3 complex to stimulate actin polymerization in Acanthamoeba extracts. Curr Biol. 1999;9:405–415. doi: 10.1016/s0960-9822(99)80187-0. [DOI] [PubMed] [Google Scholar]
  • 7.Zigmond SH, Joyce M, Yang C, Brown K, Huang M, Pring M. Mechanism of Cdc42-induced actin polymerization in neutrophil extracts. J Cell Biol. 1998;142:1001–1012. doi: 10.1083/jcb.142.4.1001. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 8.Gao L, Bretscher A. Analysis of unregulated formin activity reveals how yeast can balance F-actin assembly between different microfilament-based organizations. Mol Biol Cell. 2008;19:1474–1484. doi: 10.1091/mbc.E07-05-0520. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 9.Kovar DR, Sirotkin V, Lord M. Three's company: the fission yeast actin cytoskeleton. Trends Cell Biol. 2011 doi: 10.1016/j.tcb.2010.11.001. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 10.Nakano K, Mabuchi I. Actin-depolymerizing protein Adf1 is required for formation and maintenance of the contractile ring during cytokinesis in fission yeast. Mol Biol Cell. 2006;17:1933–1945. doi: 10.1091/mbc.E05-09-0900. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 11.Laporte D, Coffman VC, Lee IJ, Wu JQ. Assembly and architecture of precursor nodes during fission yeast cytokinesis. J Cell Biol. 2011;192:1005–1021. doi: 10.1083/jcb.201008171. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 12.Sirotkin V, Berro J, Macmillan K, Zhao L, Pollard TD. Quantitative analysis of the mechanism of endocytic actin patch assembly and disassembly in fission yeast. Mol Biol Cell. 2010;21:2894–2904. doi: 10.1091/mbc.E10-02-0157. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 13.Wu JQ, Pollard TD. Counting cytokinesis proteins globally and locally in fission yeast. Science. 2005;310:310–314. doi: 10.1126/science.1113230. [DOI] [PubMed] [Google Scholar]
  • 14.Martin SG, Chang F. Dynamics of the formin for3p in actin cable assembly. Curr Biol. 2006;16:1161–1170. doi: 10.1016/j.cub.2006.04.040. [DOI] [PubMed] [Google Scholar]
  • 15.Wang H, Vavylonis D. Model of For3p-mediated actin cable assembly in fission yeast. PLoS One. 2008;3:e4078. doi: 10.1371/journal.pone.0004078. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 16.Nolen BJ, Tomasevic N, Russell A, Pierce DW, Jia Z, McCormick CD, Hartman J, Sakowicz R, Pollard TD. Characterization of two classes of small molecule inhibitors of Arp2/3 complex. Nature. 2009;460:1031–1035. doi: 10.1038/nature08231. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 17.McCollum D, Feoktistova A, Morphew M, Balasubramanian M, Gould KL. The Schizosaccharomyces pombe actin-related protein, Arp3, is a component of the cortical actin cytoskeleton and interacts with profilin. Embo J. 1996;15:6438–6446. [PMC free article] [PubMed] [Google Scholar]
  • 18.Chang F, Drubin D, Nurse P. cdc12p, a protein required for cytokinesis in fission yeast, is a component of the cell division ring and interacts with profilin. J Cell Biol. 1997;137:169–182. doi: 10.1083/jcb.137.1.169. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 19.Kovar DR, Kuhn JR, Tichy AL, Pollard TD. The fission yeast cytokinesis formin Cdc12p is a barbed end actin filament capping protein gated by profilin. J Cell Biol. 2003;161:875–887. doi: 10.1083/jcb.200211078. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 20.Balasubramanian MK, Helfman DM, Hemmingsen SM. A new tropomyosin essential for cytokinesis in the fission yeast S. pombe. Nature. 1992;360:84–87. doi: 10.1038/360084a0. [DOI] [PubMed] [Google Scholar]
  • 21.Skau CT, Kovar DR. Fimbrin and tropomyosin competition regulates endocytosis and cytokinesis kinetics in fission yeast. Curr Biol. 2010;20:1415–1422. doi: 10.1016/j.cub.2010.06.020. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 22.Skoumpla K, Coulton AT, Lehman W, Geeves MA, Mulvihill DP. Acetylation regulates tropomyosin function in the fission yeast Schizosaccharomyces pombe. J Cell Sci. 2007;120:1635–1645. doi: 10.1242/jcs.001115. [DOI] [PubMed] [Google Scholar]
  • 23.Kovar DR, Harris ES, Mahaffy R, Higgs HN, Pollard TD. Control of the assembly of ATP- and ADP-actin by formins and profilin. Cell. 2006;124:423–435. doi: 10.1016/j.cell.2005.11.038. [DOI] [PubMed] [Google Scholar]
  • 24.Higashida C, Suetsugu S, Tsuji T, Monypenny J, Narumiya S, Watanabe N. G-actin regulates rapid induction of actin nucleation by mDia1 to restore cellular actin polymers. J Cell Sci. 2008;121:3403–3412. doi: 10.1242/jcs.030940. [DOI] [PubMed] [Google Scholar]
  • 25.Ramabhadran V, Hatch AL, Higgs HN. Actin monomers activate inverted formin 2 by competing with its autoinhibitory interaction. J Biol Chem. 2013;288:26847–26855. doi: 10.1074/jbc.M113.472415. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 26.Feierbach B, Chang F. Roles of the fission yeast formin for3p in cell polarity, actin cable formation and symmetric cell division. Curr Biol. 2001;11:1656–1665. doi: 10.1016/s0960-9822(01)00525-5. [DOI] [PubMed] [Google Scholar]
  • 27.Chen Q, Pollard TD. Actin filament severing by cofilin dismantles actin patches and produces mother filaments for new patches. Curr Biol. 2013;23:1154–1162. doi: 10.1016/j.cub.2013.05.005. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 28.Berro J, Sirotkin V, Pollard TD. Mathematical modeling of endocytic actin patch kinetics in fission yeast: disassembly requires release of actin filament fragments. Mol Biol Cell. 2010;21:2905–2915. doi: 10.1091/mbc.E10-06-0494. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 29.Hetrick B, Han MS, Helgeson LA, Nolen BJ. Small molecules CK-666 and CK-869 inhibit actin-related protein 2/3 complex by blocking an activating conformational change. Chem Biol. 2013;20:701–712. doi: 10.1016/j.chembiol.2013.03.019. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 30.Balasubramanian MK, Feoktistova A, McCollum D, Gould KL. Fission yeast Sop2p: a novel and evolutionarily conserved protein that interacts with Arp3p and modulates profilin function. Embo J. 1996;15:6426–6437. [PMC free article] [PubMed] [Google Scholar]
  • 31.Basu R, Chang F. Characterization of dip1p reveals a switch in Arp2/3-dependent actin assembly for fission yeast endocytosis. Curr Biol. 2011;21:905–916. doi: 10.1016/j.cub.2011.04.047. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 32.Hotulainen P, Lappalainen P. Stress fibers are generated by two distinct actin assembly mechanisms in motile cells. J Cell Biol. 2006;173:383–394. doi: 10.1083/jcb.200511093. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 33.Nakano K, Kuwayama H, Kawasaki M, Numata O, Takaine M. GMF is an evolutionarily developed Adf/cofilin-super family protein involved in the Arp2/3 complex-mediated organization of the actin cytoskeleton. Cytoskeleton (Hoboken) 2010;67:373–382. doi: 10.1002/cm.20451. [DOI] [PubMed] [Google Scholar]
  • 34.Winter D, Podtelejnikov AV, Mann M, Li R. The complex containing actin-related proteins Arp2 and Arp3 is required for the motility and integrity of yeast actin patches. Curr Biol. 1997;7:519–529. doi: 10.1016/s0960-9822(06)00223-5. [DOI] [PubMed] [Google Scholar]
  • 35.Rogers SL, Wiedemann U, Stuurman N, Vale RD. Molecular requirements for actin-based lamella formation in Drosophila S2 cells. J Cell Biol. 2003;162:1079–1088. doi: 10.1083/jcb.200303023. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 36.Sarmiento C, Wang W, Dovas A, Yamaguchi H, Sidani M, El-Sabai M, DesMarais V, Holman HA, Kitchen S, Backer JM, et al. WASP family members and formin proteins coordinate regulation of cell protrusions incarcinoma cells. J Cell Biol. 2008;180:1245–1260. doi: 10.1083/jcb.200708123. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 37.Steffen A, Faix J, Resch GP, Linkner J, Wehland J, Small JV, Rottner K, Stradal TE. Filopodia formation in the absence of functional WAVE- and Arp2/3-complexes. Mol Biol Cell. 2006;17:2581–2591. doi: 10.1091/mbc.E05-11-1088. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 38.Suraneni P, Rubinstein B, Unruh JR, Durnin M, Hanein D, Li R. The Arp2/3 complex is required for lamellipodia extension and directional fibroblast cell migration. J Cell Biol. 2012;197:239–251. doi: 10.1083/jcb.201112113. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 39.Wu C, Asokan SB, Berginski ME, Haynes EM, Sharpless NE, Griffith JD, Gomez SM, Bear JE. Arp2/3 is critical for lamellipodia and response to extracellular matrix cues but is dispensable for chemotaxis. Cell. 2012;148:973–987. doi: 10.1016/j.cell.2011.12.034. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 40.Yang Q, Zhang XF, Pollard TD, Forscher P. Arp2/3 complex-dependent actin networks constrain myosin II function in driving retrograde actin flow. J Cell Biol. 2012;197:939–956. doi: 10.1083/jcb.201111052. [DOI] [PMC free article] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

01
NIHMS568306-supplement-01.pdf (3MB, pdf)
02. Movie S1.

Effect of microfluidic flow of CK-666 into and out of cells. Related to Figures 1C and 1D. Time-lapse of the maximum fluorescent intensity Z-projections of cells expressing Lifeact-GFP, showing F-actin organization upon the addition of EMM5S + DMSO (−20 to 0 min), EMM5S + 2 mM CK-666 (0 to 40 min), and EMM5S + DMSO (40 to 100 min). Boxed region is shown in Figure 1C. Scale bar, 5 µm.

Download video file (5.7MB, mov)
03. Movie S2.

Effect of microfluidic flow of CK-666 into for3Δ cells. Related to Figure S2I. Time-lapse of maximum intensity Z-projections of Lifeact-GFP in for3Δ cells, showing F-actin organization upon the addition of EMM5S + DMSO (−15 to 0 min), and EMM5S + 2 mM CK-666 (0 to 30 min). Scale bar, 5 µm.

Download video file (232.5KB, mov)
04. Movie S3.

Varying actin concentrations disrupts actin patches. Related to Figure 4. Time-lapse of Lifeact-GFP labeled actin patches in a single Z-plane for WT, actin under-expression (U.E.), and actin over-expression (O.E.) cells at 25°C. White arrows indicate individual patches. Scale bar, 5 µm.

Download video file (4.1MB, mov)

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