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Journal List > Mol Biol Cell > v.16(5); May 2005

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Mol Biol Cell. 2005 May; 16(5): 2313–2324.
doi: 10.1091/mbc.E04-09-0781.
PMCID: PMC1087237
Copyright © 2005, The American Society for Cell Biology
Profilin-mediated Competition between Capping Protein and Formin Cdc12p during Cytokinesis in Fission YeastD in Box
David R. Kovar,* Jian-Qiu Wu,* and Thomas D. Pollard*†‡
* Department of Molecular, Cellular, and Developmental Biology, Yale University, New Haven, CT 06520
† Department of Cell Biology, Yale University, New Haven, CT 06520
‡ Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT 06520
Anne Ridley, Monitoring Editor
Address correspondence to: Thomas D. Pollard (thomas.pollard/at/yale.edu).
Received September 6, 2004; Revised February 16, 2005; Accepted February 17, 2005.
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Abstract
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.
INTRODUCTION
Actin filaments in cytokinetic contractile rings have the remarkable ability to transmit the force that pinches daughter cells in two in spite of the fact that they turn over rapidly (Carter, 1967 blue right-pointing triangle; Schroeder, 1970 blue right-pointing triangle; Pelham and Chang, 2002 blue right-pointing triangle) and depolymerize naturally as the ring constricts (Schroeder, 1972 blue right-pointing triangle; Wu et al., 2003 blue right-pointing triangle). Actin binding proteins, including formins, profilin, and ADF/cofilins, are required for cytokinesis, but the mere dependence of cytokinesis on these proteins provides few insights about how they contribute to actin filament dynamics in the contractile ring.
Until recently, nothing motivated curiosity about a role for capping protein in cytokinesis. Capping proteins bind actin filament barbed ends, blocking subunit association and dissociation (Isenberg et al., 1980 blue right-pointing triangle; Cooper and Schafer, 2000 blue right-pointing triangle). No one had observed capping protein in a contractile ring, and both budding yeast (Amatruda et al., 1990 blue right-pointing triangle, 1992 blue right-pointing triangle) and fission yeast (Nakano et al., 2001 blue right-pointing triangle) survive deletion of capping protein genes without defects in cytokinesis. Capping protein is essential for viability of Drosophila (Hopmann et al., 1996 blue right-pointing triangle) and Dictyostelium (Hug et al., 1995 blue right-pointing triangle), but a link to cytokinesis was not explored. On the other hand, capping protein-yellow fluorescent protein (YFP) concentrates in the cleavage furrow of some fission yeast cells just before constriction of the contractile ring (Wu et al., 2003 blue right-pointing triangle), capping protein purifies with midbodies from Chinese hamster ovary (CHO) cells (Skop et al., 2004 blue right-pointing triangle), and capping protein transiently accumulates at the midbody of asymmetrically dividing cells of Caenorhabditis elegans embryos (Waddle et al., 1994 blue right-pointing triangle).
These observations prompted us to reconsider the participation of capping protein in cytokinesis. Our experiments on fission yeast revealed a network of genetic and biochemical interactions among capping protein, the actin monomer binding protein profilin, and the cytokinesis formin Cdc12p that contribute to cytokinesis. Because both capping protein and formin interact with actin filament barbed ends, our results focus attention of the role of actin filament barbed ends during cytokinesis.
Formins are required for cytokinesis in many cell types, including flies, yeast, and worms (Evangelista et al., 2003 blue right-pointing triangle). Formin formin homology (FH)2 domains nucleate actin filaments and interact with barbed ends (Pruyne et al., 2002 blue right-pointing triangle; Kovar et al., 2003 blue right-pointing triangle; Zigmond et al., 2003 blue right-pointing triangle; Harris et al., 2004 blue right-pointing triangle; Kovar and Pollard, 2004 blue right-pointing triangle). Some formins allow elongation of barbed ends (Zigmond et al., 2003 blue right-pointing triangle; Harris et al., 2004 blue right-pointing triangle; Kovar and Pollard, 2004 blue right-pointing triangle; Moseley et al., 2004 blue right-pointing triangle), but elongation of a barbed end associated with fission yeast Cdc12p requires gating by profilin (Kovar et al., 2003 blue right-pointing triangle; Kovar and Pollard, 2004 blue right-pointing triangle). Fission yeast has genes for three formins that are important for specific processes: For3p (interphase actin cables), Fus1p (mating), and Cdc12p (cytokinesis) (Chang et al., 1997 blue right-pointing triangle; Petersen et al., 1998b blue right-pointing triangle; Feierbach and Chang, 2001 blue right-pointing triangle; Nakano et al., 2002 blue right-pointing triangle).
Capping protein is a heterodimer of structurally related α- and β-subunits with COOH-terminal “tentacles” (Yamashita et al., 2003 blue right-pointing triangle) that attach to the barbed end of an actin filament (Wear et al., 2003 blue right-pointing triangle). Mutational analysis established that the main physiological function of capping protein in budding yeast is capping the barbed ends of filaments nucleated by Arp2/3 complex in actin patches (Kim et al., 2004 blue right-pointing triangle). Depletion of capping protein compromises the actin-based lamellae of Dictyostelium (Hug et al., 1995 blue right-pointing triangle) and Drosophila S2 cells (Kiger et al., 2003 blue right-pointing triangle; Rogers et al., 2003 blue right-pointing triangle), structures that also depend on Arp2/3 complex.
Fission yeast require the actin monomer binding protein profilin for viability and contractile ring assembly (Balasubramanian et al., 1994 blue right-pointing triangle). Viability depends on the ability of profilin to bind both actin and poly-l-proline (Lu and Pollard, 2001 blue right-pointing triangle). Actin-profilin elongates actin filament barbed ends but not pointed ends (Pollard and Cooper, 1984 blue right-pointing triangle), so the combination of capping protein and profilin inhibits elongation at both ends. On the other hand, profilin binding to the proline-rich FH1 domain of the cytokinesis formin Cdc12p (Chang et al., 1997 blue right-pointing triangle) allows filaments nucleated by Cdc12p to grow at their barbed ends while remaining attached to the formin (Kovar et al., 2003 blue right-pointing triangle; Kovar and Pollard, 2004 blue right-pointing triangle).
Here, we show that fission yeast capping protein SpCP is required for cytokinesis under a variety of stressful conditions, including high temperature (36°C), hyperosmotic media or mild mutations in numerous genes that participate in cell division. Capping protein competes with formin Cdc12p for actin filament barbed ends, both in biochemical experiments and in fission yeast cells, because SpCP and Cdc12p are genetically antagonistic. Without capping protein, excess actin filaments accumulate in patches, and actin cables are severely compromised.
MATERIALS AND METHODS
Strains, Growth Conditions, and Cellular Methods
Supplemental Table 5 lists the Schizosaccharomyces pombe strains used in this study (available online). We used standard growth media (YE5S complete medium and EMM5S minimal medium) and methods for genetics, electroporation, and molecular biology (http://www.bio.uva.nl/pombe/handbook; Sambrook et al., 1989 blue right-pointing triangle; Wu et al., 2001 blue right-pointing triangle). We used 100 μM latrunculin-A (Molecular Probes, Eugene, OR) to depolymerize actin (Wu et al., 2001 blue right-pointing triangle). We stained nuclei and septa with Hoechst (Sigma-Aldrich, St. Louis, MO) or with 4,6-diamidino-2-phenylindole (DAPI) (Sigma-Aldrich) and calcofluor (Sigma-Aldrich), and filamentous actin with rhodamine-phalloidin (Fluka Chemical, Ronkonkoma, NY) (Kovar et al., 2003 blue right-pointing triangle). For each experimental condition, we counted at least 400 total cells from two separate cultures.
acp1 (Nakano et al., 2001 blue right-pointing triangle), acp2 (accession AL391713), and twf1 (accession AL034490) genes were tagged and/or replaced at their chromosomal loci by PCR-based gene targeting by using kanMX6 as the selectable marker with pairs of primers that were 80–100 base pairs in length (Bahler et al., 1998 blue right-pointing triangle). A 3× GFP kanMX6 module, used as a PCR template for C-terminal integration of 3× GFP, was made by amplifying a budding yeast 3× GFP module (Lee et al., 2003 blue right-pointing triangle) with primers that introduced 5′ PacI and 3′ Asc1 restriction sites. The resulting fragment was cloned into pFA6a-kanMX6 (Bahler et al., 1998 blue right-pointing triangle), creating plasmid pFA6a-3× GFP-kanMX6. acp2, twf1, and cdc12 were tagged at their C termini with green fluorescent protein (GFP), YFP, cyan fluorescent protein (CFP), and/or 3× GFP by using forward primers that ended just upstream of the stop codon, and reverse primers that ended 1 base pair downstream of the stop codon. All tagged proteins were functional by several criteria: colony growth, generation time, morphology at different temperatures, and crosses with mutations known to give synthetic phenotypes (i.e., acp2-GFP × cdc3-6). High-level expression P3nmt1 and medium-level P41nmt1 thiamine-inducible promoters were integrated in front of acp1 and acp2 by using forward primers that end just upstream of the start codon and reverse primers that ended with the start codon. Expression under the nmt1 promoter was regulated in EMM5S medium with the presence or absence of 10.0 μg/ml thiamine (Sigma-Aldrich).
Plasmids
Plasmids pREP42-GFP-cdc4 (Balasubramanian et al., 1997 blue right-pointing triangle), pSGP573-41×-YFP-cdc8 (Wu et al., 2003 blue right-pointing triangle) and pET21-cdc12(882-1390) (Kovar and Pollard, 2004 blue right-pointing triangle) have been described previously. To make p573-81×-YFP-act1 and pREP81-cdc12(882-1375), the entire act1 coding region or appropriate cdc12 region was amplified from an S. pombe cDNA library and cloned into pSGP573-81× (Pasion and Forsburg, 1999 blue right-pointing triangle) (GFP was subsequently replaced by YFP) or pREP81 (Maundrell, 1990 blue right-pointing triangle), respectively. A bacteria expression vector for fission yeast capping protein, SpCP, was made by a published strategy (Soeno et al., 1998 blue right-pointing triangle). The entire coding regions (including start and stop) of both acp1 and acp2 were amplified from an S. pombe cDNA library, with primers that introduced 5′ NdeI and 3′ BamHI restriction sites, and cloned into plasmid pET3a (Novagen, Madison, WI). pET3a-acp2 was cut with restriction enzymes BglII and ClaI, and the resulting fragment was cloned into the EcoRV site of pET3a-acp1, creating plasmid pET3a-acp1acp2. Inserts of all new plasmids were sequenced to confirm fidelity of PCR amplification.
Microscopy of Cells
For most experiments, cells were observed by differential interference contrast (DIC) and fluorescence microscopy by using an Olympus IX-71 inverted microscope equipped with a 60× 1.4 numerical aperture Plan-apo objective and appropriate filter sets (DIC, DAPI, CFP, fluorescein isothiocyanate, and YFP) and a Hamamatsu Orca-ER cooled charge-coupled device (CCD) camera (Bridgewater, NJ). Images in Figure 2EFigure 2., Figure 5FFigure 5., and Supplemental Figure 2C were taken with an Ultraview spinning disk confocal microscope (PerkinElmer Life and Analytical Sciences, Boston, MA). Time-lapse microscopy, kymograph preparation, and data analysis have been described previously (Wu et al., 2003 blue right-pointing triangle). The amount of filamentous actin in patches was determined by measuring the integrated fluorescence intensity of pixels at the ends of rhodamine-phalloidin–stained interphase cells (no contractile ring present) by using ImageJ software (http://rsb.info.nih.gov/ij/). Twenty randomly chosen wild-type and acp1Δ acp2Δ cells from at least four different fields were measured.
Figure 2.
Figure 2.
Figure 2.
Late cytokinesis defects in the absence of capping protein. (A and B) DIC and fluorescence micrographs of wild-type (MLP198) and acp2Δ (KV301) cells expressing integrated myosin regulatory light chain-GFP (Rlc1p-GFP) from its native promoter and (more ...)
Figure 5.
Figure 5.
Figure 5.
Antagonism of capping protein and the formin Cdc12p in cells. (A) A capping protein null mutant mildly suppresses a temperature sensitive cdc12 mutant. DIC and fluorescence micrographs of acp2Δ (KV21), cdc12-112 (MBY310), and acp2Δ cdc12-112 (more ...)
Protein Purification
Recombinant fission yeast capping protein SpCP (Acp1p and Acp2p) was purified from bacteria. The pET3a-acp1acp2 construct was transformed into Escherichia coli strain BL21-DE3 pLysS (Stratagene, La Jolla, CA) and grown overnight at 37°C. After subculturing into fresh media, cells were grown at 37°C for ~2 h and then induced for 6 h with the addition of 0.5 mM isopropyl β-d-thiogalactopyranoside. Cells harvested by centrifugation were frozen, resuspended in extraction buffer (50 mM Tris, pH 8.0, 1 mM EDTA, 1 mM dithiothreitol [DTT], and 50 mM NaCl) supplemented with a complete protease inhibitor tablet (Roche Diagnostics, Indianapolis, IN), and sonicated. The sonicate was clarified at 30,000 and 50,000 × g for 25 min each. A 45–65% ammonium sulfate cut was taken, and the pellet was resuspended in (15 ml) and dialyzed against 2 liters (for 2 h) of DE52 buffer (10 mM Tris, pH 8.0, 100 mM NaCl, 1 mM EDTA, 1 mM DTT, and 0.01% NaN3). The dialyzed protein was separated on a 30-ml bed volume DE52 (Whatman, Clifton, NJ) column eluted with a 400-ml 100–400 mM NaCl gradient. SpCP-containing fractions were dialyzed overnight against 2 liters of HA buffer (500 mM KCl, 10 mM KH2PO4, 1 mM DTT, and 0.01% NaN3) and separated on a 15-ml Hydroxyapatite (Bio-Rad, Hercules, CA) column eluted with a 400-ml 10–250 mM KH2PO4 gradient. SpCP-containing fractions were dialyzed overnight against 2 liters of SQ(7.5) buffer (10 mM Tris, pH 7.5, 100 mM NaCl, 1 mM DTT, and 0.01% NaN3) and separated on a 1-ml Source 15Q (Amersham Biosciences, Piscataway, NJ) column eluted with a 100-ml 100–300 mM NaCl gradient. SpCP-containing fractions were dialyzed overnight against 2 liters of SQ(6.0) buffer [10 mM 2-(N-morpholino)ethanesulfonic acid, pH 6.0, 50 mM NaCl, 1 mM DTT, and 0.01% NaCl] and separated on a 1-ml Source 15Q (Amersham Biosciences) column eluted with a 100-ml 50–300 mM NaCl gradient. Pure SpCP was dialyzed against CP buffer (10 mM Tris, pH 7.5, 40 mM KCl, 0.5 mM DTT, 50% glycerol, and 0.01% NaN3), flash frozen in liquid nitrogen, and stored at –80°C.
Mouse capping protein MmCP (Palmgren et al., 2001 blue right-pointing triangle), Cdc12(FH1FH2)p residues 882-1390 (Kovar and Pollard, 2004 blue right-pointing triangle), and S. pombe profilin (Lu and Pollard, 2001 blue right-pointing triangle) were purified from bacteria. Ca-ATP actin was purified from rabbit skeletal muscle (Kovar et al., 2003 blue right-pointing triangle). Gel filtered actin was labeled on Cys-374 with pyrenyliodoacetamide or Oregon green 488 iodoacetamide (Molecular Probes) (Kovar et al., 2003 blue right-pointing triangle). Just before each experiment, Ca-ATP actin was converted to Mg-ATP actin by adding 0.2 volume of 5 mM EGTA and 0.5 mM MgCl2 for 5 min at 25°C.
Protein concentrations were calculated with the following extinction coefficients: actin, A290 = 26,600 M–1 cm–1 (Houk and Ue, 1974 blue right-pointing triangle); profilin, A280 = 1.63 OD mg–1 ml–1 (Lu and Pollard, 2001 blue right-pointing triangle); MmCP, A280 = 76.3 mM–1 cm–1 (Palmgren et al., 2001 blue right-pointing triangle); Cdc12(882-1390)p, A280 = 49,860 M–1 cm–1 (Kovar and Pollard, 2004 blue right-pointing triangle); and SpCP, A280 = 72,000 M–1 cm–1 (estimated based on amino acid composition by ProtParam; http://us.expasy.org/tools/).
Fluorescence Spectroscopy
Actin assembly was measured from the fluorescence of a trace of pyrene-actin (excitation at 365 nm and emission at 407 nm) with a PTI Alphascan spectroflourimeter (Photon Technology International, Monmouth Junction, NJ) (Higgs et al., 1999 blue right-pointing triangle; Kovar et al., 2003 blue right-pointing triangle). Final protein concentrations are indicated in the figure legends.
For spontaneous assembly assays (Higgs et al., 1999 blue right-pointing triangle; Kovar et al., 2003 blue right-pointing triangle), separate drops of a 20 μM mixture of pyrene-labeled and unlabeled Mg-ATP-actin, other proteins, and 10× KMEI (500 mM KCl, 10 mM MgCl2, and 100 mM imidazole, pH 7.0) were placed on the side of a plastic Eppendorf tube, and the reaction was started by mixing with Mg-buffer G (2 mM Tris, pH 8.0, 0.2 mM ATP, 0.1 mM MgCl2, and 0.5 mM DTT).
For assembly from F-actin seeds, 6 μM unlabeled preassembled actin filaments was incubated in a drop for 5 min with a range of concentrations of SpCP or Cdc12(FH1FH2)p. To start the reaction, a separate drop with 15 μM (5% pyrene-labeled) Mg-ATP-actin and 75 μM profilin was washed into the F-actin seeds with F buffer (G buffer supplemented with 1× KMEI). In some cases, indicated in the Figure 6Figure 6. legend, the seeds were incubated with either Cdc12p or capping protein (CP) for 5 min and then either CP or Cdc12p was added at the start of the reaction.
Figure 6.
Figure 6.
Figure 6.
Biochemical competition between SpCP and formin Cdc12p for actin filament barbed ends. The conditions were as follows: 10 mM imidazole, pH 7.0, 50 mM KCl, 1 mM MgCl2, 1 mM EGTA, 0.5 mM DTT, 0.2 mM ATP, 90 μM CaCl2, and 0.25% glycerol at 25°C. (more ...)
The critical concentration for actin assembly was determined two ways (Kovar et al., 2003 blue right-pointing triangle). First, a range of concentrations of Mg-ATP actin (5% pyrene labeled) was polymerized alone or with the indicated concentrations of either SpCP, Cdc12(882-1390)p, and/or profilin in F buffer for 16 h in the dark at 25°C. Second, a 5 μM stock of 10% pyrene F-actin was diluted to 1.0 μM with F buffer, in the presence of a range of concentrations of SpCP, Cdc12(882-1390)p, and/or profilin (Caldwell et al., 1989 blue right-pointing triangle), and incubated for 16 h in the dark at 25°C.
Calculation of the Concentration of Apparent Ends, Initial Polymerization Rates, and Depolymerization Rates
The concentration of apparent ends was calculated from elongation rates by using the equation [Endsapp] = elongation rate/(k+[actin monomers]), where k+ = 11.6 μM–1 s–1 at pH 7.0 (Higgs et al., 1999 blue right-pointing triangle), where 25% (1 μM) of the actin had polymerized. Barbed end polymerization rates from preassembled F-actin seeds were measured from the slope of a linear fit of the first 300 s. The affinity of capping proteins for actin filament barbed ends was determined by fitting a plot of the dependence of the initial assembly rate on the concentration of capping protein with the equation Vi = Vif + (Vib – Vif) ((Kd + [ends] + [CP] – square root((Kd + [ends] + [CP])2 – 4[ends][CP])/2[ends])), where Vi is the observed elongation rate, Vif is the elongation rate when barbed ends are free, Vib is the elongation rate when barbed ends are capped, [ends] is barbed end concentration, and [CP] is capping protein concentration (Huang et al., 2003 blue right-pointing triangle). The rate of depolymerization was calculated by fitting the data from 300 to 1000 s with a single exponential curve. Depolymerization rates were expressed as a percent normalized to the rate of actin alone.
Total Internal Reflection Fluorescence (TIRF) Microscopy
Images of Oregon green-labeled fluorescent actin filaments excited by total internal reflection were collected at 15-s intervals with a cooled CCD camera (Orca-ER) as described previously (Kovar et al., 2003 blue right-pointing triangle; Kovar and Pollard, 2004 blue right-pointing triangle). Initially, a mixture of unlabeled Mg-ATP actin and 20% Mg-ATP Oregon green actin was mixed with 2× TIRF buffer (1×: 10 mM imdazole, pH 7.0, 50 mM KCl, 1 mM MgCl2, 1 mM EGTA, 50 mM DTT, 0.2 mM ATP, 50 μM CaCl2, 15 mM glucose, 20 μg/ml catalase, 100 μg/ml glucose oxidase, and 0.5% [4000 centipoise] methylcellulose) and water and transferred to a flow cell for imaging. Subsequently, a second sample with 40% labeled actin in the absence or presence of the indicated concentrations of capping protein replaced the initial sample during continuous imaging.
RESULTS
Biochemical Comparison of Fission Yeast and Mouse Capping Proteins
SpCP is a heterodimer of α-subunits (Acp1p; Nakano et al., 2001 blue right-pointing triangle) and β-subunits (Acp2p) (Figure 1AFigure 1.) with sequences similar to budding yeast capping protein (Amatruda et al., 1990 blue right-pointing triangle, 1992 blue right-pointing triangle). Like mouse capping protein MmCP, purified recombinant fission yeast capping protein SpCP increased the critical concentration for actin assembly (Figure 1BFigure 1.), but this required 10-fold more SpCP than MmCP (Figure 1CFigure 1.). Direct observation of actin filament elongation in real time by evanescent wave fluorescence microscopy (Figure 1DFigure 1.) confirmed that MmCP (Figure 1EFigure 1.) and SpCP (Figure 1FFigure 1.) stopped subunit addition at barbed ends but not pointed ends. Inhibition of elongation (Figure 1GFigure 1.) suggested barbed end affinities of 16 nM for SpCP and 0.8 nM for MmCP (Figure 1HFigure 1.). Similarly, 20-fold more SpCP than MmCP was required to inhibit the depolymerization of actin filament barbed ends (Figure 1, I and JFigure 1.). MmCP stimulated spontaneous assembly of Mg-actin monomers (Cooper and Pollard, 1985 blue right-pointing triangle; Caldwell et al., 1989 blue right-pointing triangle; Kovar et al., 2003 blue right-pointing triangle), but SpCP inhibited spontaneous assembly (Figure 1KFigure 1.). Inhibition of nucleation (the apparent concentration of ends produced) was biphasic, with maximal inhibition at 100 nM SpCP and less inhibition at higher concentrations (Figure 1LFigure 1.). Inhibition of end-to-end annealing of actin filaments required 10-fold more SpCP than MmCP (Figure 1MFigure 1.).
Figure 1.
Figure 1.
Figure 1.
Biochemical comparison of purified SpCP and MmCP. The conditions were as follows: 10 mM imidazole, pH 7.0, 50 mM KCl, 1 mM MgCl2, 1 mM EGTA, 0.5 mM DTT, 0.2 mM ATP, 90 μM CaCl2, and 0.25% glycerol at 25°C. (A) A Coomassie Blue-stained (more ...)
Background Information on Fission Yeast Capping Protein
This section summarizes the basic cellular properties of fission yeast capping protein SpCP, which are documented by supplemental materials (available online), because SpCP is similar to orthologues in other cells. GFP-tagged SpCP concentrates with actin filaments in patches at the ends of interphase cells (Supplemental Figure 1, A–G). These patches form, move, and disappear on a time scale of seconds (Supplemental Figure 1, E and F). Localization of Acp2p-GFP to patches depends on Acp1p (Supplemental Figure 1A) and actin filaments (Supplemental Figure 1B). Acp2p-CFP patches overlap with some but not all patches of PCH protein Cdc15p-YFP (Supplemental Figure 1D). As in budding yeast (Goode et al., 1998 blue right-pointing triangle; Palmgren et al., 2001 blue right-pointing triangle), SpCP is required for twinfilin (Twf1p-GFP) to concentrate in actin patches (Supplemental Figure 1G), but deletion of twinfilin (twf1Δ) has no effect on cellular morphology or localization of Acp2p-GFP to patches (Supplemental Figure 1G) under any condition that we tested. We confirmed that cells lacking Acp1p are viable with normal morphology in complete media at 25°C (Nakano et al., 2001 blue right-pointing triangle) and found the same to be true for cells lacking Acp2p or both Acp1p and Acp2p (Supplemental Figure 2 and Supplemental Table 1).
During cell division, patches marked by Acp2p-GFP accumulate lateral to the cleavage site (Supplemental Figure 1, A–C and F), and Acp2p-CFP concentrates along with actin filaments in ~2% of contractile rings (Figure 2FFigure 2.). The contractile ring signal bleaches rapidly and is obscured by the bright fluorescence of patches clustered near the cleavage furrow, so 2% may be an underestimate. Because cells lacking Acp1p are viable without cytokinesis defects (Nakano et al., 2001 blue right-pointing triangle), SpCP seemed unlikely to have a role in cytokinesis. Nonetheless, the following sections document evidence that SpCP does function in cytokinesis in a role that is antagonistic to the formin Cdc12p.
Cytokinesis Depends on Capping Protein under a Variety of Stressful Conditions
Cells lacking one or both capping protein genes have conditional defects in cytokinesis that are evident at elevated temperatures or in 1 M ethylene glycol at 25°C. At 36°C in minimal EMM5S media ~20% of acp1Δ, acp2Δ, and acp1Δ acp2Δ cells have cytokinesis defects (Figure 2AFigure 2., Supplemental Figure 2, A and B, and Supplemental Table 1) with disorganized contractile rings (Figure 2, A and EFigure 2. and Supplemental Figure 2B) and improper deposition of septal material (Figure 2AFigure 2. and Supplemental Figure 2A). At 25°C in 1 M ethylene glycol, ~15% of capping protein null cells have cytokinesis defects in minimal EMM5S media (Figure 2BFigure 2. and Supplemental Table 1).
Contractile rings form in acp2Δ cells with the same time course as wild-type cells, but delays emerge late in the process as the contractile ring disassembles and daughter cells separate (Figure 2, C and DFigure 2.). We followed these events by time-lapse microscopy of cells expressing integrated myosin regulatory light chain-GFP (Rlc1p-GFP) (Wu et al., 2003 blue right-pointing triangle). In the presence of 1 M ethylene glycol in minimal media at 25°C, these defects are more severe and extend to include a delay in contractile ring constriction. With the exception of cell separation, wild-type cells in both the absence and presence of 1 M ethylene glycol faithfully follow the time course of cytokinesis with remarkably little variation (Figure 2DFigure 2.; Wu et al., 2003 blue right-pointing triangle). On the other hand, the timing of all late cytokinesis events was highly variable in acp2Δ cells as reflected in larger standard deviations (Figure 2DFigure 2.).
Excess capping protein also caused cytokinesis defects. Cells overexpressing both α- and β-subunits, but neither alone, grew abnormally long (Figure 3AFigure 3. and Supplemental Table 1). Nuclei divided normally but contractile rings failed to form (Figure 3CFigure 3.), and septa were incomplete (Figure 3BFigure 3.). Cells accumulated up to eight nuclei before lysing. Because actin patches and actin cables looked normal (Figure 3CFigure 3.), SpCP overexpression specifically affected contractile ring assembly. The formin Cdc12p concentrated at the division site but failed to form a contractile ring between separated nuclei in cells overexpressing SpCP (Figure 3DFigure 3.). Capping protein activity (measured by the effect on the low shear viscosity of pure actin filaments; Isenberg et al., 1980 blue right-pointing triangle) was 10 times higher in extracts from strains overexpressing SpCP than control extracts.
Figure 3.
Figure 3.
Figure 3.
Overexpression of capping protein causes a specific lethal defect in cytokinesis. (A–D) Wild-type, capping protein α-subunit overexpressing (P3nmt1-acp1; KV37), capping protein β-subunit overexpressing (P3nmt1-acp2; KV40), and (more ...)
Genetic crosses revealed synthetic interactions between the capping protein null mutation acp2Δ and mutations in other genes required for cytokinesis. We tested the ability of the double mutants to form colonies on complete YE5S media agar plates at a range of temperatures (Supplemental Table 2) where acp2Δ single mutant cells formed normal colonies. We stained selected single and double mutants with Hoechst or DAPI and calcofluor to visualize and count nuclei and septa (Figure 4Figure 4. and Supplemental Table 3).
Figure 4.
Figure 4.
Figure 4.
Genetic interactions of capping protein null mutants with other mutations. Fluorescent micrographs of single and double mutant cells grown at the indicated temperature in complete YE5S liquid media and stained with Hoechst to visualize DNA and septa. (more ...)
Double mutants that formed colonies less well than the single non-acp2Δ mutant parent had more severe defects in septal organization and contained more multinucleated cells. Whereas 1% of tubulin cold-sensitive nda3-KM311 cells had abnormal septa, 14% of nda3-KM311 acp2Δ double mutant cells had abnormal septa at 20°C (Figure 4BFigure 4.). Compared with α-actinin null ain1-Δ1 cells at 25°C, 26% more ain1-Δ1 acp2Δ double mutant cells had abnormal septa (Figure 4CFigure 4.). Compared with myosin essential light chain temperature-sensitive (ts) cdc4-8 cells at 25°C, 47% more cdc4-8 acp2Δ double mutant cells had abnormal septa (Figure 4DFigure 4.). Compared with IQGAP ts rng2-D5 cells at 25°C, 41% more rng2-D5 acp2Δ double mutant cells had abnormal septa (Figure 4EFigure 4.). Compared with anillin-like mid1-ΔF null cells at 25°C, 49% more mid1-ΔF acp2Δ double mutant cells had abnormal septa (Figure 4FFigure 4.). Compared with PCH protein cdc15-127 ts cells at 32°C, 16% more cdc15-127 acp2Δ double mutant cells had abnormal septa (Figure 4GFigure 4.).
On the other hand, some double mutants were indistinguishable morphologically from the non-acp2Δ parent mutant. For example, tropomyosin ts cdc8-27 and cdc8-27 acp2Δ double mutant cells both formed normal septa and had similar numbers of binucleate cells at 25°C (Figure 4AFigure 4.).
Cellular and Biochemical Competition between SpCP and Formin Cdc12p
Genetic crosses revealed suppressor interactions between the capping protein null mutation acp2Δ and temperaturesensitive mutations in genes for two proteins required for cytokinesis: the formin cdc12 and type-II myosin myo2. Compared with myo2-E1 ts cells at 28°C, myo2-E1 acp2Δ double mutant cells had 16% fewer abnormal septa and 26% less were multinucleate (Figure 4HFigure 4.). We studied the formin interaction in detail.
No genetic interaction was found between acp2Δ and for3Δ, a formin required for actin cables (our unpublished data). However, deleting acp2 partly suppresses the severe defects of ts cdc12-112 cells grown at 33°C in complete YE5S liquid media (Figure 5AFigure 5. and Supplemental Table 4). Compared with formin cdc12-112 ts cells at 33°C, 25% fewer cdc12-112 acp2Δ double mutant cells had abnormal septa and 11% fewer were multinucleate (Figure 5AFigure 5.). In 87% of cdc12-112 cells the septa failed to span the entire cell width, whereas only 25% of cdc12-112 acp2Δ double mutant cells had this “partial” septa defect.
On the other hand, mild overexpression of SpCP (α- and β-subunits expressed from the P41nmt1 promoter) was lethal in formin ts cdc12-112 and profilin ts cdc3-124 cells (Figure 5BFigure 5.), but not wild-type, tropomyosin ts cdc8-27, IQ-GAP ts rng2-D5, myosin essential light chain ts cdc4-8, or type-II myosin ts myo2-E1 cells at semipermissive temperature (30°C) (Figure 5, B and CFigure 5., and Supplemental Table 4). Mild overexpression of SpCP was not lethal in cells with deletion of for3, the formin required for interphase actin cables (our unpublished data), suggesting a specific antagonistic relationship between SpCP and the cytokinesis formin Cdc12p. P41nmt1-acp1 P41nmt1acp2 cdc12-112 cells continued cycles of nuclear division but could not assemble contractile rings, resulting in partial septa (Figure 5CFigure 5.), like wild-type cells strongly overexpressing SpCP (Figure 3Figure 3.).
Mild overexpression of Cdc12(FH1FH2)p from the P81nmt1 promoter on a plasmid was lethal in acp2Δ cells, but not in a variety of other ts genetic backgrounds (Figure 5, D–FFigure 5., and Supplemental Table 4). These pREP81-cdc12(FH1FH2) acp2Δ cells had aberrant thick actin cables in aster-like accumulations (Figure 5FFigure 5.) and suffered a variety of defects in morphology and cytokinesis (Figure 5EFigure 5.).
Biochemical experiments showed competition between SpCP and formin Cdc12p for actin filament barbed ends (Figure 6Figure 6.). We used an active fragment of Cdc12p consisting of the FH1 and 2 domains, Cdc12(FH1FH2)p (Kovar et al., 2003 blue right-pointing triangle; Kovar and Pollard, 2004 blue right-pointing triangle). We consider first the individual proteins.
At steady state, both SpCP and Cdc12(FH1FH2)p cap actin filament barbed ends and increase the critical concentration for assembly to the critical concentration of the pointed end (Figure 6, A and BFigure 6.). Profilin has opposite effects on these capped filaments, depolymerizing filaments capped by SpCP (Figure 6, A and BFigure 6.) (like other capping proteins; Cooper and Schafer, 2000 blue right-pointing triangle) but overcoming barbed end capping by Cdc12(FH1FH2)p (Figure 6, A and BFigure 6.; Kovar et al., 2003 blue right-pointing triangle). Both effects depend on the concentration of profilin. Similarly, profilin allowed Cdc12(FH1FH2)p capped filaments (Kovar et al., 2003 blue right-pointing triangle), but not SpCP capped filaments, to elongate their barbed ends (Figure 6EFigure 6.). Both SpCP and Cdc12(FH1FH2)p stimulate spontaneous assembly of Mg-actin monomers by nucleating filaments that grow at their pointed ends. Profilin has opposite effects on these reactions, inhibiting spontaneous assembly in the presence of SpCP but accelerating spontaneous assembly by Cdc12(FH1FH2)p (Figure 6CFigure 6.).
Three different experiments showed that SpCP and Cdc12(FH1FH2)p exclude each other from actin filament barbed ends. The effects of the two proteins are additive in the absence of profilin, because both cap tightly. Profilin allows barbed ends capped by Cdc12(FH1FH2)p but not SpCP to elongate. With both proteins, the outcome depends entirely on which protein binds the barbed end first.
  • When SpCP, profilin and Cdc12(FH1FH2)p are added to Mg-actin monomers simultaneously, Cdc12(FH1FH2)p overcomes the inhibition of spontaneous assembly of Mg-actin by SpCP and profilin, strongly stimulating polymerization in a concentration dependent manner, even at concentrations 10-fold lower than SpCP (Figure 6DFigure 6.).
  • Both SpCP and Cdc12(FH1FH2)p inhibit actin filament elongation, but if the two proteins are added to filaments simultaneously in the presence of profilin, substoichiometric concentrations of Cdc12(FH1FH2)p overcome capping by SpCP (Figure 6EFigure 6.). The rate of elongation depends on the concentration of Cdc12(FH1FH2)p (Figure 6FFigure 6.).
  • Preincubation of filaments with SpCP or MmCP prevents elongation of the barbed ends by actin monomers, Cdc12(FH1FH2)p, and profilin. Conversely, preincubation of filaments with Cdc12(FH1FH2)p allows actin monomers and profilin to elongate the barbed ends even with excess SpCP or MmCP (Figure 6, G and HFigure 6.). This is explained by persistent association of Cdc12(FH1FH2)p (Kovar and Pollard, 2004 blue right-pointing triangle) and the capping proteins with barbed ends, such that neither a formin nor a capping protein can displace the other from a barbed end.
Other Functions of Fission Yeast Capping Protein
Actin Filament Cables and Patches. Cells lacking either capping protein subunit had fewer actin cables but ~35% more actin filaments in actin patches judging from rhodamine-phalloidin fluorescence (Supplemental Figure 2C). The actin cable deficiency resulted in mislocalization of the type V myosin Myo52p (Supplemental Figure 2D). In wild-type cells, Myo52p-GFP localized to patches primarily at the ends during interphase and the middle during division. Spots of Myo52p-GFP were dispersed throughout acp2Δ cells, similar to cells lacking for3 (Feierbach and Chang, 2001 blue right-pointing triangle; Nakano et al., 2002 blue right-pointing triangle), the formin required for actin filament cables (Supplemental Figure 2D; Feierbach and Chang, 2001 blue right-pointing triangle).
Sexual Cycle. Like actin and fimbrin (Petersen et al., 1998a blue right-pointing triangle; Wu et al., 2001 blue right-pointing triangle), Acp2p-GFP concentrates in patches at the tips of mating cells, in patches in the cortex of developing spores and in patches at the tips of germinating spores (Supplemental Figure 3A). Crosses of two wild-type strains yielded four spores in 99% of the asci after 48 h (Supplemental Figure 3B). However, crosses of two acp1Δ strains, two acp2Δ strains, and two fim1-1Δ strains yielded four spores in only 58, 61, and 7% of asci, respectively (Supplemental Figure 3B). Few additional spores formed with longer incubation.
DISCUSSION
Capping protein SpCP and formin Cdc12p bind to actin filament barbed ends. In the absence of profilin, both simply prevent subunit association and dissociation. The presence of profilin reveals an important difference: ends capped by Cdc12p elongate, but ends capped by SpCP do not. Because both SpCP and Cdc12p dissociate slowly from barbed ends, whichever protein is bound will dominate the behavior of the associated end persistently. Thus, excess capping protein cannot prevent barbed ends capped by Cdc12p from elongating in the presence of profilin. Interactions of profilin, SpCP, and Cdc12p contribute to contractile ring function as detailed below.
Competition between Capping Protein and Formin in Dividing Cells
Fission yeast capping protein contributes to the fidelity of cytokinesis, but it is not absolutely required for assembly or constriction of the contractile ring. Under nonstress conditions (25°C), most cells lacking capping protein (acp1Δ, acp2Δ, and acp1Δ acp2Δ) completed cytokinesis normally, although the fraction of cells with two nuclei was ~5% higher than wild-type cells (Supplemental Table 1). Under stressful conditions (36°C or 1 M ethylene glycol), ~20% of cells lacking capping protein had aberrant septa resulting from disorganized contractile rings. Consistent with the arrival of capping protein at the division site well after the contractile ring forms (Wu et al., 2003 blue right-pointing triangle), defects arise only as the contractile ring constricts and disassembles.
Biochemical and genetic evidence support the hypothesis that fission yeast cytokinesis is influenced by profilin-mediated competition between capping protein and the formin Cdc12p for actin filament barbed ends at the division site. Fission yeast depend on Cdc12p and profilin to assemble contractile ring actin filaments de novo (Balasubramanian et al., 1994 blue right-pointing triangle; Chang et al., 1997 blue right-pointing triangle; Pelham and Chang, 2002 blue right-pointing triangle). SpCP competes with Cdc12p in vivo, because they are genetically antagonistic (Figure 5Figure 5.): the absence of capping protein partly suppresses the septation defect of temperature sensitive cdc12-112 cells (acp2Δ cdc12-112) at a semipermissive temperature; and mild overexpression of SpCP kills cdc12-112 cells as does mild overexpression of Cdc12(FH1FH2)p in capping protein null cells (acp2Δ). Furthermore, temperature-sensitive mutations in the gene for fission yeast profilin (cdc3) are synthetically lethal with both capping protein null acp2Δ and cdc12-112 temperature-sensitive mutations (Chang et al., 1997 blue right-pointing triangle). On the contrary, mutations in Drosophila profilin (chickadee) suppress bristle abnormalities in capping protein (cpb) mutants (Hopmann and Miller, 2003 blue right-pointing triangle).
Profilin is key to the competition between SpCP and formin Cdc12p. Mg-ATP-actin bound to profilin elongates free barbed ends but not pointed ends (Pollard and Cooper, 1984 blue right-pointing triangle; Kang et al., 1999 blue right-pointing triangle; Blanchoin and Pollard, 2002 blue right-pointing triangle). Therefore, profilin-Mg-ATP-actin elongates neither end of filaments capped by SpCP. Instead, subunits dissociate from pointed ends and result in depolymerization in proportion to the concentration of profilin (Pantaloni and Carlier, 1993 blue right-pointing triangle). On the other hand, by interacting with both an actin monomer and the FH1 domain of Cdc12p (Chang et al., 1997 blue right-pointing triangle), profilin allows barbed ends to elongate at near full-speed while continuously associated with Cdc12(FH1FH2)p (Kovar et al., 2003 blue right-pointing triangle; Kovar and Pollard, 2004 blue right-pointing triangle). Once Cdc12(FH1HF2)p is bound, this profilin-gated, barbed end elongation persists even with saturating concentrations of SpCP (Figure 6Figure 6.). Other formins such as budding yeast Bni1p (Pruyne et al., 2002 blue right-pointing triangle; Sagot et al., 2002 blue right-pointing triangle) and mDiaI (Li and Higgs, 2003 blue right-pointing triangle; Harris et al., 2004 blue right-pointing triangle) are less dependent on profilin to gate barbed end elongation. However, Bni1p absolutely requires profilin to assemble actin filaments in cells (Evangelista et al., 2002 blue right-pointing triangle; Sagot et al., 2002 blue right-pointing triangle), and Bni1p and mDia1 compete with capping protein for barbed ends (Zigmond et al., 2003 blue right-pointing triangle; Harris et al., 2004 blue right-pointing triangle; Moseley et al., 2004 blue right-pointing triangle; Romero et al., 2004 blue right-pointing triangle).
The evidence suggests that Cdc12p and SpCP interact sequentially with actin filament barbed ends at two distinct stages of contractile ring function. Cdc12p (Cdc12p-3XGFP) arrives early at the division site and cooperates with profilin to trigger the polymerization of the actin filaments required to assemble a compact contractile ring. SpCP arrives 20 min later (Wu et al., 2003 blue right-pointing triangle) and may slowly replace Cdc12p on some of the barbed ends in preparation for disassembly (of pointed ends) during constriction of the ring (Wu et al., 2003 blue right-pointing triangle). Regulation of the competition between formins and capping protein during cytokinesis is far from understood. Profilin regulates Cdc12p, Rho family GTPases overcome autoinhibition of some formins (Wallar and Alberts, 2003 blue right-pointing triangle), and polyphosphoinositides inhibit capping proteins in vitro (Cooper and Schafer, 2000 blue right-pointing triangle), but none of these simple binary interactions begins to explain the biology.
An alternative mechanism also should be considered. Because the contractile ring assembles de novo (Marks and Hyams, 1985 blue right-pointing triangle; Pelham and Chang, 2002 blue right-pointing triangle), the loss of capping protein may compromise the contractile ring indirectly, as postulated for interphase cables. However, some evidence is consistent with direct participation: 1) a small fraction of Acp2p-CFP concentrates in the contractile ring; 2) capping protein null mutations interact synthetically with mutations in some (i.e., anillin-like mid1 and IQGAP rng2) but not all (i.e., myosin-II myp2 and regulatory light chain rlc1) genes contributing to cytokinesis; 3) capping protein deletion (acp2Δ) mildly suppresses mutations in both the myosin-II (myo2) and formin (cdc12) required for cytokinesis; and 4) the timing of the arrival of Rlc1p-GFP to the division site, ring assembly, and initiation of constriction seem normal in acp2Δ cells (only late cytokinetic events are aberrant). Additionally, capping protein null mutations do not interact with mutations in tropomyosin cdc8 and the formin for3, which are required for actin cables, although cables are affected in capping protein null cells.
Participation of capping protein in cytokinesis is less clear in other eukaryotes. Capping protein purifies with midbodies from CHO cells (Skop et al., 2004 blue right-pointing triangle) and accumulates transiently in the midbodies of C. elegans embryos (Waddle et al., 1994 blue right-pointing triangle). On the other hand, depletion of capping protein from either Dictyostelium (Hug et al., 1995 blue right-pointing triangle) and cultured Drosophila cells (Kiger et al., 2003 blue right-pointing triangle; Rogers et al., 2003 blue right-pointing triangle) compromises the leading lamellae with little impact on cytokinesis, although the ploidy of Dictyostelium increased slightly. Deletion of the Drosophila capping protein β-subunit is lethal (Hopmann et al., 1996 blue right-pointing triangle), but the mechanism was not investigated. Budding yeast accomplish cytokinesis without capping protein (Amatruda et al., 1990 blue right-pointing triangle, 1992 blue right-pointing triangle), perhaps because only limited constriction of the contractile ring is required (Watts et al., 1987 blue right-pointing triangle; Bi et al., 1998 blue right-pointing triangle; Tolliday et al., 2003 blue right-pointing triangle).
Biochemical Properties of SpCP
In each biochemical assay, SpCP was ~10- to 20-fold less active than mouse capping protein. The affinity of SpCP for muscle actin barbed ends seems similar to plant and budding yeast capping proteins (Amatruda and Cooper, 1992 blue right-pointing triangle; Huang et al., 2003 blue right-pointing triangle; Kim et al., 2004 blue right-pointing triangle) but lower than vertebrate capping protein (Wear et al., 2003 blue right-pointing triangle). SpCP may bind differently to fission yeast actin, although budding yeast capping protein has the same affinity for muscle actin and yeast actin barbed ends (Kim et al., 2004 blue right-pointing triangle). Most capping proteins stimulate spontaneous assembly of actin monomers by nucleating filaments (capturing actin monomers and dimers) that grow slowly at their pointed ends (Cooper and Pollard, 1985 blue right-pointing triangle; Caldwell et al., 1989 blue right-pointing triangle; Kovar et al., 2003 blue right-pointing triangle), but concentrations of SpCP up to 100 nM inhibit spontaneous assembly of actin. Higher concentrations of SpCP stimulate spontaneous assembly like other capping proteins (Figure 1LFigure 1.). The biphasic dependence of actin assembly on the concentration of SpCP indicates that SpCP has a much higher affinity for filament barbed ends than for monomers and/or dimers. Nucleation of actin filaments by capping proteins may not be relevant in cells (Hug et al., 1995 blue right-pointing triangle), because these filaments cannot grow at either end in the presence of profilin, and dimers bound by capping protein may not be accessible to formin.
Capping Protein Functions during Interphase
Although fission yeast lacking either or both capping protein subunits had normal morphology at 25°C, interphase actin patches stained more intensely with rhodamine-phalloidin, and interphase actin cables were diminished compared with wild-type cells. Actin filaments are also more abundant in patches of budding yeast lacking capping protein (Li et al., 1995 blue right-pointing triangle; Kim et al., 2004 blue right-pointing triangle). Kim et al. (2004 blue right-pointing triangle) proposed a reasonable explanation that may apply to fission yeast: filaments nucleated in patches by Arp2/3 complex grow unchecked without capping such that each nucleation event produces a longer actin filament until most monomers are consumed. Budding yeast actin patches contain a network of branched actin filaments (Young et al., 2004 blue right-pointing triangle), similar to the leading edge of motile cells (Pollard and Borisy, 2003 blue right-pointing triangle). Surprisingly, actin filaments in latrunculin treated patches isolated from budding yeast cells lacking capping protein are not longer than filaments from wild-type patches (Young et al., 2004 blue right-pointing triangle).
The reason for compromised actin cables in capping protein null cells is less clear. Cable assembly and stability depend on formins, For3p in the case of fission yeast (Feierbach and Chang, 2001 blue right-pointing triangle; Nakano et al., 2002 blue right-pointing triangle), which remain associated with growing barbed ends (Higashida et al., 2004 blue right-pointing triangle; Kovar et al., 2004; Romero et al., 2004 blue right-pointing triangle) and prevent capping protein from inhibiting barbed end growth (Zigmond et al., 2003 blue right-pointing triangle; Harris et al., 2004 blue right-pointing triangle; Moseley et al., 2004 blue right-pointing triangle; Romero et al., 2004 blue right-pointing triangle; this report). The persistent association of formins with barbed ends protects them from capping protein and accounts for the absence of capping protein in cables.
Because neither Acp2p-GFP, GFP-Acp2p, nor budding yeast capping protein (Amatruda and Cooper, 1992 blue right-pointing triangle) concentrates in cables, the loss of capping protein may compromise cables indirectly. Unregulated incorporation of actin into filaments in patches may deplete the cytoplasmic pool of actin monomers, compromising cable growth. This indirect mechanism requires that nucleation by formins be more sensitive to the actin monomer concentration than nucleation by Arp2/3 complex. Consistent with this idea, yeast formins are inefficient nucleators at actin concentrations below 1 μM in vitro (Pruyne et al., 2002 blue right-pointing triangle; Kovar et al., 2003 blue right-pointing triangle), whereas Arp2/3 complex efficiently nucleates assembly until all monomers are consumed (Higgs et al., 1999 blue right-pointing triangle).
Acknowledgments
We thank Fred Chang (Columbia University, New York, NY) for helpful suggestions and strains; Greg Law (Perkin Elmer-Cetus Life Sciences) for help with the spinning disk confocal microscope; Matt Lord for Rlc1p-GFP strains; and members of our laboratory for discussions, reagents, and technical expertise. This work was supported by National Institutes of Health research grants GM-26338 and GM-26132 (to T.D.P.), a National Institutes of Health postdoctoral fellowship (to D.R.K.), and an Anna Fuller Fund postdoctoral fellowship (to J.-Q.W.).
Notes
This article was published online ahead of print in MBC in Press (http://www.molbiolcell.org/cgi/doi/10.1091/mbc.E04–09–0781) on March 2, 2005.
D in BoxThe online version of this article contains supplemental material at MBC Online (http://www.molbiolcell.org).
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