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Genes to Cells (2006) 11, 893-905. doi:10.1111/j.1365-2443.2006.00987.x
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Actin-capping protein is involved in controlling organization of actin cytoskeleton together with ADF/cofilin, profilin and F-actin crosslinking proteins in fission yeast

Kentaro Nakanoa,* and Issei Mabuchi

Division of Biology, Department of Life Sciences, School of Arts and Sciences, University of Tokyo, Komaba, Meguro-ku, Tokyo 153-8902, Japan


   Abstract
Top
 Abstract
Introduction
 Results
 Discussion
 Experimental procedures
 References
 
Actin-capping protein (CP) is a heterodimeric protein which is expressed in various eukaryotic cells. CP binds to the barbed end of the actin filaments in vitro and inhibits both the association and dissociation of actin monomers at this end. However, the cellular role of CP has not been uncovered. Here we investigated the function of CP in fission yeast cells. The fission yeast CP is composed of Acp1 and Acp2. It was found that Acp2 accumulated as cortical dots at the cell ends during interphase and the mid-region of mitotic cells, which disappeared in the absence of Acp1 or F-actin. Acp1 and Acp2, when co-over-expressed, decreased F-actin structures in cells, and cytokinesis was often interrupted in these cells. On the other hand, disruption of one of the CP genes affected the distribution of F-actin patches at cell ends and decreased the rate of actin depolymerization in vivo. Moreover, genetic analysis showed that CP controls actin dynamics together with ADF/cofilin and profilin. In addition, CP is likely involved in assembling the F-actin contractile ring and F-actin patch with F-actin-crosslinking proteins.


   Introduction
Top
 Abstract
 Introduction
Results
 Discussion
 Experimental procedures
 References
 
The organization of the actin cytoskeleton is important for various cellular events including cell morphogenesis, cell migration, and cytokinesis. Eukaryotic cells express various kinds of proteins that control the architecture and behavior of the actin cytoskeleton (reviewed in Pollard & Borisy 2003). G-actin-binding proteins such as thymosin ß4 and profilin sequester G-actin and form a pool of unpolymerized actin in the cell. Actin-depolymerizing proteins sever and depolymerize F-actin. These proteins have been considered to accelerate turnover of actin in the cell. F-actin capping proteins cap the barbed end of F-actin. Ca-dependent F-actin-severing proteins such as fragmin/severin and gelsolin/villin sever F-actin in the presence of Ca ions and cap the barbed end. F-actin crosslinking proteins form the framework of the actin cytoskeleton.

The barbed end of F-actin has higher rates of association and dissociation with G-actin and a lower critical concentration for polymerization of actin than the pointed end. The capping protein (CP) is a conserved heterodimeric protein among eukaryotes (reviewed in Wear & Cooper 2005). Capping the barbed end by CP suppresses both the association and dissociation of actin monomers at that end. The capping also prevents annealing of F-actin. In addition, it can shorten the lag time for polymerization of actin in vitro by stabilizing actin nuclei although filaments are elongated from the pointed end. However, the role of CP in the cell has not been clarified. It has been demonstrated that loss of function of CP increases the amount of F-actin in Dictyostelium discoideum amoebae (Hug et al. 1995), in Drosophila melanogaster bristles (Hopmann & Miller 2003), and in the budding yeast Saccharomyces cerevisiae (Kim et al. 2004). Therefore, it functions at least to suppress excess formation of actin cytoskeleton. Animal cells often express both CP and fragmin/severin and/or gelsolin/villin family proteins whereas budding yeast cells express only CP. When platelets are stimulated with thrombin, explosive polymerization of actin is induced after both CP and gelsolin are removed from the barbed ends of F-actin (Barkalow et al. 1996). It has been suggested that phosphatidylinositol 4,5-bisphosphate (PIP2), known as a signaling molecule in the plasma membrane, mediates this process by removing CP and gelsolin from the barbed ends. Thus, the capping and uncapping of the filament end may spatially and temporally control polymerization of actin in the cell.

The fission yeast Schizosaccharomyces pombe, like the budding yeast S. cerevisiae, serves as an excellent model for studying the molecular mechanisms of organization of the actin cytoskeleton (Bähler & Peter 2000). Actin is organized into cortical F-actin patches at growing ends and longitudinal F-actin cables during interphase in this organism (Marks & Hyams 1985; Arai et al. 1998). F-actin patches are considered to be required for maintaining the cylindrical shape of the cell enveloped with a cell wall (Kobori et al. 1989) while F-actin cables are considered to support transport of materials necessary for polarized growth (Kamasaki et al. 2005). During mitosis, an F-actin ring is assembled at the mid region of the cell and shrinks to divide the cell into two. Thus it is thought to correspond to the contractile ring in animal cells. Several actin-modulating proteins have been revealed in fission yeast including Cdc3 (profilin; Balasubramanian et al. 1994), Cdc8 (tropomyosin; Balasubramanian et al. 1992), Fim1 (fimbrin; Nakano et al. 2001; Wu et al. 2001), Ain1 ({alpha}-actinin; Wu et al. 2001), Rng2 (IQGAP; Eng et al. 1998), Stg1 (SM22/transgelin; Nakano et al. 2005) and Adf1 (ADF/cofilin; Nakano & Mabuchi 2006). An {alpha}-subunit of CP (Acp1) has also been reported to be involved in the organization of the actin cytoskeleton in this organism (Nakano et al. 2001). It has been reported that a complex of Acp1 and Acp2, ß-subunit of CP, possesses a capping activity for the barbed end of F-actin and that the complex functions competitively with the formin Cdc12 in formation of the F-actin ring (Kovar et al. 2005). Here, we report that CP controls turnover of actin together with Cdc3 and Adf1, and organization of the actin cytoskeleton together with F-actin-crosslinking proteins Fim1, Ain1 and Rng2 in S. pombe.


   Results
Top
 Abstract
 Introduction
 Results
Discussion
 Experimental procedures
 References
 
Acp1 and Acp2 form a complex in fission yeast cells

SPAC631.01c encodes a possible candidate for a ß-subunit of CP in S. pombe (Fig. 1A). This gene product, Acp2, exhibited 48.1% similarity to the ß1-subunit of chicken CapZ (Schafer et al. 1994), 47.4% to Drosophila melanogaster CPB (Hopmann et al. 1996), and 38.1% to the budding yeast Cap2p (Amatruda et al. 1990).


Figure 1
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  		Figure 1  Acp1 and Acp2 form a capping protein complex in S. pombe. (A) Primary structure of the ß-subunit of the capping protein. Amino acids which are conserved among all, or more than three, proteins are reversed or enclosed, respectively. Gg, Gallus gallus. Dm, Drosophila melanogaster. Sc, Saccharomyces cerevisiae. Sp, Schizosaccharomyces pombe. (B) Acp2 forms a complex with Acp1 in vivo. GST or GST-Acp2 were pulled down from extracts of acp2 cells expressing each protein with glutathione-Sepharose beads and subjected to immunoblotting using the antibodies indicated. An arrow or arrowhead indicates signal derived from GST or GST-Acp2, respectively.

 
To elucidate whether Acp2 forms a complex with Acp1 in vivo, a pull-down assay was performed. A cytoplasmic extract was obtained from cells expressing GST-Acp2 or GST alone, and the extract was incubated with glutathione beads. Bound proteins were subjected to SDS-PAGE followed by immunoblotting with anti-Acp1 antiserum. Acp1 was pulled down with GST-Acp2 but not with GST alone (Fig. 1B). Therefore, Acp2 is likely to be a ß-subunit of CP in S. pombe.

CP may be a component of F-actin patches

We examined localization of Acp2 by expressing an N-terminal YFP-fused protein in the cell, since structural analysis data suggested that the C-termini of both subunits of CP play a critical role in capping the barbed end of F-actin (Yamashita et al. 2003). YFP-Acp2 seemed to be functional since its expression could compensate for a defect caused by deletion of acp2+ (data not shown). Deconvolved 3-D images clearly revealed that YFP-Acp2 accumulated as cortical dots at the cell ends during interphase and at the mid-region during mitosis in {Delta}acp2-cells expressing YFP-Acp2 (Fig. 2A). Next, we examined the role of Acp1 and F-actin in the localization of Acp2. No cortical localization of Acp2 was seen in acp1-null cells or cells treated with a G-actin-sequestering substance, Latrunculin-A (Lat-A) (Fig. 2B,C). Thus, Acp2 may interact with F-actin in the cortical dots as a complex with Acp1. Although we tried to examine co-localization of Acp2 with F-actin by immunofluorescence microscopy, this was not successful since YFP-Acp2 dispersed immediately during the fixing of cells with formaldehyde (data not shown). On the other hand, YFP-Acp2 was often accumulated in the nucleus although its intensity varied among the cells (Fig. 2). The nuclear localization of Acp2 is probably not related to the function of CP since YFP-Acp2 remained in the nucleus in the absence of Acp1 or F-actin.


Figure 2
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  		Figure 2  Three-dimensional localization of YFP-Acp2. {Delta}acp2-cells expressing YFP-Acp2 under a conventional fluorescence microscope (A). Arrowheads and a large arrow indicate accumulation of YFP-Acp2 at cell ends and the division site, respectively. YFP-Acp2 dots disappeared upon treatment with 1 µM Lat-A for 5 min (B) or in the absence of Acp1 (C). Small arrows indicate nuclear localization of YFP-Acp2. Bar 10 µm.

 
CP is able to inhibit actin polymerization in vivo

To study the function of CP in fission yeast cells, we examined the effect of over-expression of Acp1 and/or Acp2 from nmt1+ promoter in the wild-type cells. Expression of these genes was induced by removing thiamine from the medium. The over-expression of both proteins moderately inhibited cell growth while that of each protein alone did not as compared with the control (Fig. 3A). The F-actin organization in these cells were observed at 20 h after removal of thiamine at 30 °C. The control cells and the cells over-expressing either Acp1 or Acp2 had normally organized F-actin structures; F-actin patches at the cell ends, longitudinal F-actin cables and the F-actin ring in the dividing cells were all properly formed (Fig. 3Ba–c). In contrast, almost all the F-actin structures disappeared in the cells over-expressing both Acp1 and Acp2 (Fig. 3Bd). This result indicates that CP suppressed actin polymerization in vivo. Moreover, cells defective in cytokinesis were often observed, probably due to loss of the F-actin ring.


Figure 3
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  		Figure 3  F-actin disappears in the cells co-over-expressing Acp1 and Acp2. (A) Co-over-expression of Acp1 and Acp2 affects cell growth. Wild-type cells carrying pREP1acp1 and/or pREP2acp2 were incubated on EMM plates with or without 4 µM thiamine at 30 °C for 3 days. Control cells contain each empty vector. (B) Effect of over-expression of Acp1 and Acp2 on in vivo F-actin structures. The wild-type cells carrying pREP1acp1 and/or pREP2acp2 were fixed and stained with Bodipy-phallacidin and DAPI after incubation at 30 °C for 20 h in EMM. An arrowhead indicates a telophase cell having no F-actin ring. Bar 10 µm.

 
CP is required for proper distribution of F-actin during interphase

Gene disruption analyses have revealed that neither acp1+ nor acp2+ was essential for cell viability (Nakano et al. 2001; Kovar et al. 2005), although a moderate defect in localization of F-actin patches has been seen in {Delta}acp1-cells (Nakano et al. 2001). Here we investigated the appearance of actin cytoskeleton in {Delta}acp2-cells and {Delta}acp1 {Delta}acp2-cells. It was found that F-actin patches were moderately delocalized from the ends of these cells during interphase as in {Delta}acp1-cells (Fig. 4A). F-actin cables were only faintly seen in all of these mutant cells, as reported by Kovar et al. 2005. On the other hand, the F-actin ring was formed normally during mitosis in these cells. These phenotypes were observed at 20–37 °C. Our result was a little different from that reported by Kovar et al. (2005): they have noted that disorganization of the F-actin ring is seen in ~15% of the cells. This difference may be due to the media; we used the rich medium YES while they used the minimum medium EMM under which the cells were somewhat stressed.


Figure 4
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  		Figure 4  Acp1 and Acp2 are involved in controlling the organization of actin cytoskeleton. (A) F-actin patches were partially delocalized in the absence of Acp1 and/or Acp2. Cells grown at 30 °C in YES were fixed and stained with Bodipy-phallacidin (top) and DAPI (bottom). Arrows and arrowheads indicate the F-actin ring and F-actin cables, respectively. Bar represents 5 µm. (B) Deletion of Acp1 or Acp2 cause a decrease in the rate of F-actin depolymerization in vivo. The wild-type cells, {Delta}acp1-cells and {Delta}acp2-cells grown at 25 °C were fixed and stained with Bodipy-phallacidin after incubation with 1 µM Lat-A for the period indicated. Percentages of cells containing F-actin after addition of Lat-A are shown.

 
CP accelerates depolymerization of actin in vivo

To examine the rate of turnover of F-actin in cells lacking CP-activity, we performed an actin-depolymerization assay using Lat-A (Lappalainen & Drubin 1997). The disappearance of F-actin from cells treated with Lat-A is expected to reflect the rate at which subunits dissociate from filaments in vivo since Lat-A sequesters G-actin but does not directly depolymerize F-actin (Ayscough et al. 1997). The addition of 1 µM Lat-A disrupted F-actin structures in the wild-type cells within 2 min (Fig. 4B). On the other hand, the rate of depolymerization of F-actin was reduced in {Delta}acp1- or {Delta}acp2-cells as compared with the wild-type cells. It was unlikely that the uptake of Lat-A was altered in either mutant, since there was no significant difference in the minimal concentration of Lat-A that inhibited cell growth between the wild-type strain and either the {Delta}acp1- or the {Delta}acp2-cells (data not shown). This result suggested that CP is involved in accelerating depolymerization of actin in vivo.

CP may function cooperatively with ADF/cofilin and profilin in vivo

We studied genetic interactions between CP and other actin-modulating proteins listed in Table 1. Tetrad analyses revealed that cells containing a mutation in adf1+ or cdc3+ were not viable at 25 °C without acp1+ or acp2+ while each of the single mutant cells grew normally. We then examined the phenotype of {Delta}acp2 adf1- and {Delta}acp2 cdc3-cells containing pREP41acp2 with or without thiamine. These cells grew normally and had a properly organized actin cytoskeleton as compared to each single mutant in the absence of thiamine since Acp2 was expressed from pREP41acp2. On the other hand, the cells ceased to grow and showed severe defects in the organization of F-actin 13 h after the expression of Acp2 was repressed by adding thiamine to the medium.


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  		Table 1 Genetic interaction between CP and actin-modulating proteins
 
The phenotype of {Delta}acp2 adf1-cells was observed in detail (Fig. 5A). Remarkably, F-actin patches were abundant at the ends of these cells during mitosis and formation of the F-actin ring was suppressed after incubation for 13 h. As a result, these cells were frequently unable to accomplish cytokinesis. Numerous F-actin patches were seen all over the cell cortex after prolonged incubation for 18 h (Fig. 5A). Moreover, longitudinal F-actin cables were not seen in these cells: cables were short and random. Then, we examined the rate of actin depolymerization in {Delta}acp2 adf1-cells (Fig. 5B). Treatment with Lat-A induced disappearance of F-actin from cells incubated without thiamine although a small amount of F-actin still remained since Adf1-activity was partially reduced in the adf1-1 mutant. On the other hand, almost all of the F-actin remained in the {Delta}acp2 adf1-cells with thiamine. This result suggested that CP plays a cooperative role in the depolymerization of actin induced by Adf1.


Figure 5
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  		Figure 5  CP collaborates with ADF/cofilin Adf1 in the organization of actin cytoskeleton. (A) Depletion of Acp2 in adf1-cells. {Delta}acp2 adf1-cells containing pREP41acp2 grown in EMM were incubated at 25 °C with thiamine for the period indicated and stained with Bodipy-phallacidin (top) and DAPI (bottom). Arrow indicates a cell assembling an abnormal F-actin ring. Note that many F-actin patches remain at the ends of this cell as shown by arrowheads. (B) Effect of Lat-A on the {Delta}acp2 adf1-cells. {Delta}acp2 adf1-cells containing pREP41acp2 were grown in EMM with or without thiamine for 13 h at 25 °C and stained with Bodipy-phallacidin after incubation with 1 µM Lat-A for 10 min. Bars 5 µm.

 
Next, the terminal phenotype of {Delta}acp2 cdc3-cells was examined. The actin cytoskeleton was organized almost normally in cdc3-single mutant cells at 25 °C although F-actin cables were poor so that they were hard to be recognized in a micrograph (Fig. 6). On the other hand, {Delta}acp2 cdc3-cells containing pREP41acp2 showed severe defects in the organization of actin at 13 h after thiamine was added to the medium. F-actin patches were delocalized in the cell cortex throughout the cell cycle and formation of the F-actin ring was strongly interfered with in the mitotic cells. Moreover, no F-actin cable was seen in these cells. This phenotype was similar to that of {Delta}acp2 adf1-cells at 18 h. Thus, CP may control the organization of actin in cooperation with Adf1 and Cdc3.


Figure 6
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  		Figure 6  Depletion of Acp2 in profilin-mutant cells. cdc3- or {Delta}acp2 cdc3-cells containing pREP41acp2 were incubated in EMM with thiamine for 13 h at 25 °C and stained with Bodipy-phallacidin (top) and DAPI (bottom). Bar 5 µm.

 
Functional interaction of CP with F-actin-crosslinking protein

Cells lacking both CP and the F-actin-crosslinking protein Fim1 or Ain1 grew more slowly than control cells (Table 1). However, to date, no synthetic effect has been detected in cdc3 {Delta}fim1-, cdc3 {Delta}ain1-, adf1 {Delta}fim1- and adf1 {Delta}ain1-cells compared with each single mutant (our unpublished observation). Thus, it is possible that CP may be involved in controlling the actin cytoskeleton together with the F-actin-crosslinking proteins independent of Adf1 and Cdc3.

The phenotype of {Delta}acp1 {Delta}fim1-cells has already been reported (Nakano et al. 2001). Here, we investigated the phenotype of cells lacking Acp2 and Ain1. {Delta}acp2 {Delta}ain1-cells were dumpy and showed defects in the organization of the actin cytoskeleton (Fig. 7): some F-actin patches seemed to be tightly packed at cell ends while the others were scattered in the cell during interphase. Moreover, formation of the F-actin ring was frequently impaired in mitotic cells, which has also been reported by Kovar et al. (2005). These phenotypes were identical to that of {Delta}acp1 {Delta}ain1-cells (data not shown) and very similar to the phenotype of {Delta}acp1 {Delta}fim1-cells as reported (Nakano et al. 2001). Thus, CP has a strong relationship with F-actin-crosslinking proteins in the organization of F-actin throughout the cell cycle.


Figure 7
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  		Figure 7  CP has a genetic interaction with {alpha}-actinin Ain1. {Delta}ain1- or {Delta}acp2 {Delta}ain1-cells grown in YES at 25 °C were stained with Bodipy-phallacidin (upper panels) and DAPI (lower panels). Arrows indicate abnormal F-actin rings formed in mitotic cells. Arrowheads indicate F-actin patches packed at cell ends. Bar represents 5 µm.

 
CP may be involved in assembling the F-actin ring together with IQGAP Rng2

The cells containing a mutation in myosin II essential light chain Cdc4 were lethal even at a permissive temperature when CP was removed (Table 1). To explore the terminal phenotype of cdc4 {Delta}acp2-cells, we performed a shut-off experiment of Acp2 in this strain. It was found that formation of the F-actin ring was specifically impaired in cdc4 {Delta}acp2-cells while cdc4-single mutant cells properly formed the ring (Fig. 8A). This defect was restricted to the ring since the organization of F-actin patches and F-actin cables seemed to be unaffected in the double mutant cells. On the other hand, we found no genetic interaction between CP and either of the type II myosin heavy chains Myo2 and Myp2/Myo3 (Table 1, Fig. 8B). Instead, we found a defect in formation of the F-actin ring and cytokinesis in {Delta}acp2 rng2-cells while each single mutant cell formed the ring and divided normally under the same conditions (Fig. 8B,C). This result is consistent with the suggestion that Cdc4 may function in formation of the F-actin ring by interacting with IQGAP Rng2 (Eng et al. 1998; D'souza et al. 2001). Thus, CP may function in assembling the F-actin ring with Rng2 and Cdc4.


Figure 8
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  		Figure 8  Acp2 is involved in assembling the F-actin ring during cytokinesis. (A) Formation of the F-actin ring is interfered with in {Delta}acp2 cdc4-double mutant cells. cdc4- or {Delta}acp2 cdc4-cells containing pREP41acp2 were grown in EMM with thiamine for 14 h at 25 °C and stained with Bodipy-phallacidin (top, green in bottom), anti-tubulin antiserum (red) and DAPI (blue). Arrowheads indicate abnormal F-actin cables accumulated in the medial cortex in mitotic cells. (B) Acp2 has a genetic interaction with IQGAP-like protein Rng2 but not with type II myosin heavy chain Myo2. Cells grown in YES at 25 °C were fixed and stained with DAPI (blue). (C) Formation of the F-actin ring is interfered with in {Delta}acp2 rng2-double mutant cells. {Delta}acp2-, rng2- or {Delta}acp2 rng2-cells were incubated in YES at 25 °C and stained with Bodipy-phallacidin (left, green in right), anti-tubulin antiserum (red) and DAPI (blue). Arrowheads indicate abnormal accumulation of F-actin in the middle cortex in a mitotic cell. Bars represent 10 µm.

 

   Discussion
Top
 Abstract
 Introduction
 Results
 Discussion
Experimental procedures
 References
 
We characterized the fission yeast Acp2 which was the only ortholog of the ß-subunit of CP of budding yeast. Acp2 formed a complex with Acp1 in the fission yeast cells. {Delta}acp2-cells showed somewhat depolarized F-actin patches and faint F-actin cables while the F-actin ring was normally formed during cytokinesis. This phenotype was indistinguishable from that of {Delta}acp1-cells (Nakano et al. 2001). No additive effect was observed in {Delta}acp1 {Delta}acp2-cells as compared with each of the single deletion mutants (Kovar et al. 2005; this study). Thus, each of those proteins seemed to require the other to function. Moreover, Kovar et al. (2005) expressed Acp1 and Acp2 simultaneously in bacteria and purified a complex of these proteins. Therefore, Acp1 and Acp2 function as a CP complex and are responsible for organizing the actin cytoskeleton in fission yeast cells.

It has been shown that Acp2 is localized to the F-actin patches (Kovar et al. 2005). What role does CP play in the F-actin patches? We obtained results which suggested that CP controls actin dynamics in the patches. First, over-expression of the CP subunits in the cell lead to disappearance of the F-actin structures. Thus, CP is able to inhibit actin polymerization in F-actin patches, F-actin cables and F-actin ring. It has been reported that over-expression of CP in a chromosome-integrated strain suppressed formation of F-actin ring although formation of F-actin patches looked normal (Kovar et al. 2005). Their experimental conditions for over-expression of CP might have been milder than that of ours. Second, it was found that the depolymerization of actin occurred more slowly in the absence of Acp1 or Acp2 than in the wild-type cells. Third, CP showed a strong genetic interaction with Adf1 (ADF/cofilin), and the rate of depolymerization of actin was severely decreased in {Delta}acp2 adf1-cells. Thus, CP is likely to accelerate the depolymerization of actin together with Adf1 in the cell. A considerable number of F-actin patches remained at the ends of {Delta}acp2 adf1-cells even after the cells had entered mitosis, and these cells were frequently unable to form an F-actin ring. This may be explained by the idea that depolymerization of F-actin induced by these proteins at the cell ends is required for assembly of the F-actin ring when the cell enter mitosis. Moreover, a large number of F-actin patches accumulated in the cortex of {Delta}acp2 adf1-cells after prolonged repression of the expression of Acp2. Thus, CP and Adf1 may control the cellular level of F-actin. We propose a model for function of these proteins in maintaining the structure of the F-actin patch in Fig. 9. ADF/cofilin increases the number of ends of F-actin by severing it and induces monomer dissociation at the pointed end (reviewed by Chen et al. 2000). Following the severing of F-actin by Adf1, CP binds to the barbed end to inhibit reannealing of F-actin and suppress addition of G-actin at the barbed ends and thereby maintains increased number of pointed ends. These processes would accelerate depolymerization of actin at the pointed ends induced by Adf1.


Figure 9
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  		Figure 9  Proposed role of CP in actin dynamics in the F-actin patch. Adf1 produces new ends of F-actin by severing it, and induces depolymerization of actin. De novo polymerization of actin by the Arp2/3 complex also produces new barbed ends (not shown). Capping the barbed end of F-actin by CP may prevent G-actin addition to this end and annealing of filaments with each other. Accordingly, CP is capable of accelerating the depolymerization of actin induced by Adf1. On the other hand, Cdc3 may make a G-actin pool by substituting for G-actin-bound Adf1 and facilitate the polymerization of actin by accelerating the exchange of bound nucleotides. Regulation of removal of CP from the barbed end or competition of binding to this end by CP and G-actin is important for controlling the polymerization of actin in the patch.

 
F-actin patches were increased in {Delta}acp2 cdc3-cells at a permissive temperature for the cdc3-mutant. This may be explained as follows. ADF/cofilin and profilin cooperatively stimulate the turnover of actin (Didry et al. 1998; Wolven et al. 2000), probably because profilin is involved in recycling of Adf1 by replacing it on the Adf1-G-actin complex. Profilin also sequesters G-actin, which leads to depolymerization of actin. Actually, Cdc3 inhibits spontaneous nucleation of actin by sequestering G-actin in fission yeast cells (Lu & Pollard 2001). Our result indicated that CP also accelerated depolymerization of actin by Adf1 as described above. Thus, it appears that both CP and Cdc3 facilitate depolymerization of F-actin. The F-actin patches may thus be increased in number in {Delta}acp2 cdc3-cells. On the other hand, it has been considered that profilin stimulates in vitro polymerization of actin at the barbed end (Pantaloni & Carlier 1993). This activity of Cdc3 may not be strong in fission yeast, since in cdc3-mutant cells numerous fine F-actin patches are distributed widely in the cortex (Balasubramanian et al. 1994). Similar case has been found in Dictyostelium discoideum amoeba in which depletion of profilin isoforms increased the amount of F-actin (Haugwitz et al. 1994).

It is interesting that the F-actin patches delocalized in {Delta}acp2 cdc3-cells and in {Delta}acp2 adf1-cells. It seems that F-actin cables have some role in translocation of F-actin patches: attachment of F-actin patches to the cables (Arai et al. 1998) and movements of F-actin patches along the cables (Pelham & Chang 2001) have been reported. In cdc8-mutant cells which have no cables F-actin patches are scattered throughout the cortex (Balasubramanian et al. 1992; Arai et al. 1998). We could not detect any cables in {Delta}acp2 cdc3-cells and could detect only short and random cables in {Delta}acp2 adf1-cells. Although it is not clear at present why these mutant cells did not have proper longitudinal cables, it is plausible that the patches could not accumulate at the ends of these cells in the absence of such cables.

It has been reported that F-actin patches depolarize in cells lacking F-actin-crosslinking proteins such as Fim1 and Ain1 (Nakano et al. 2001; Wu et al. 2001). The disruption of the CP genes in these cells enhanced the depolarization of the patches or induced abnormal packing of the patches at the ends (Nakano et al. 2001; this study). Therefore, CP is likely to be involved in proper distribution of F-actin patches cooperatively with F-actin-crosslinking proteins. Since no genetic interaction between Fim1 or Ain1 and Cdc3 or Adf1 has been identified yet (Nakano et al. 2001; Wu et al. 2001; our unpublished observations), mode of cooperation between CP and the F-actin-crosslinking proteins would be different from that between CP and Adf1 or Cdc3. CP and the F-actin-crosslinking proteins may contribute to form structure of F-actin patches; CP may control length of filaments by capping the barbed end, while Fim1 and Ain1 may construct the three dimensional structure of the patch. Proper structure of the F-actin patch may be needed for their localization.

In {Delta}acp2 cdc4-cells, assembly of the F-actin ring was disrupted although the localization of both F-actin patches and F-actin cables was relatively normal. Cdc4 has been identified as an essential light chain of type II myosins Myo2 and Myp2/Myo3 in fission yeast (McCollum et al. 1994; Naqvi et al. 1999; Motegi et al. 2000). It has also been shown to bind to IQGAP Rng2 which plays an essential role in formation of the F-actin ring (Eng et al. 1998; D'souza et al. 2001) although the significance of the interaction of these proteins has not been uncovered yet. The positive genetic interaction between Acp2 and Rng2 and the negative one between Acp2 and heavy chains of type II myosin supported the possibility that CP functions with Rng2 rather than the type II myosins in cytokinesis. It has been shown that sea urchin egg IQGAP is localized to the cleavage furrow (Nishimura & Mabuchi 2003) and that mammalian IQGAP1 directly crosslinks F-actin (Bashour et al. 1997). In addition, genetic studies have revealed that Rng2 seems to have a close relationship with Fim1 and Ain1 (Nakano et al. 2001; Wu et al. 2001). Moreover, we observed that the F-actin ring was abnormally formed in {Delta}fim1- (Nakano et al. 2001) or {Delta}ain1- (Kavor et al. 2005; this study) cells in the absence of capping activity. Thus, CP may be involved in formation of the F-actin ring together with Fim1, Ain1 and probably Rng2. However, at this moment, it is not clear whether or not CP functions directly in assembling the F-actin ring, since it has not obviously been detected as a component of the F-actin ring (Kovar et al. 2005; this study). Further study such as observations of actin dynamics in living cells lacking both CP and F-actin-crosslinking proteins may help to solve this problem.


   Experimental procedures
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 Abstract
 Introduction
 Results
 Discussion
 Experimental procedures
References
 
Genetic techniques and DNA manipulation

S. pombe strains used in this study are listed in Table 2. The media used have been previously described (Moreno et al. 1991). Complete medium YES and minimum medium EMM were used for growing the S. pombe strains. MEA was used for induction of conjugation and sporulation. All plates contained 2% agar. Standard procedures for S. pombe genetics were followed according to Alfa et al. (1993) and Moreno et al. (1991). Standard methods were used for DNA manipulations (Sambrook et al. 1989).


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  		Table 2 List of strains used in this study
 
Gene disruption of acp2+

To disrupt acp2+, the gene was isolated from S. pombe genomic DNA and amplified by PCR using oligonucleotides capKpn (5'-gggggtaccgttaacaaaacaaattaaatgttc-3') and capSac (5'-tttgagctcagtataccagtattcccttacc-3'). The 2.5 kb PCR product containing a full-length genomic acp2+ was digested with KpnI and SacI and inserted into pBluescript II SK–. Then, we replaced a EcoRV-EcoRV region in this vector, pgacp2, with ura4+. This vector, pgacp2::ura4+, was digested with HpaI and SnaBI, and used to transform a diploid constructed by mating JY741 and JY746. Correct integration was verified by Southern blotting. We also disrupted acp2+ using another marker gene ADE2 by the same strategy.

Gene expression in fission yeast

Fission yeast expression vectors, pREP1, pREP2, and pREP41 (Maundrell 1993) were used in this study. pREP1 has a stronger nmt1 promoter than pREP41 (Basi et al. 1993). pREP2 contains a selectable marker ura4+ instead of LEU2 in pREP1. Expression of the exogenous genes is induced by removing thiamine from the medium.

The full-length acp2+ cDNA was amplified using the oligonucleotides acp2EN (5'-cccgaattccatatgaattctgaagacgctgcattagatt-3') and acp2BS (5'-ggcgtcgactggatctctaaatggaaagatcgtttaaaactg-3'). This PCR product was digested with NdeI and SalI, and a 0.8 kb fragment was ligated at the NdeI- and SalI-sites of the above vectors.

Pull down assay

pESP-2 (Stratagene) was used to express GST-Acp2 in {Delta}acp2-cells. Soluble proteins were extracted from cells expressing GST alone or GST-Acp2 in the cell lysis buffer (Moreno et al. 1991) and incubated with glutathione-Sepharose beads (Amersham Biosciences) at 4 °C for 2 h. After the beads were washed 5 times, bound proteins were subjected to SDS-PAGE and transferred on to a PVDF membrane. Immunoblotting was perfomed using anti-Acp1 antibodies produced in a rabbit.

Microscopy

Staining of the cells with DAPI, anti-tubulin antiserum (TAT-1) or Bodipy-phallacidin (Molecular Probes) was performed as described (Alfa et al. 1993; Arai et al. 1998). Stained cells were viewed by two approaches. Conventional images were captured using a Zeiss Axioskop fluorescence microscope equipped with a Plan Apochromat x63 lens and a CCD camera SPOTTM (Diagnostic Institute Inc.) and were analyzed with IPLab software (Solution Systems Co. Ltd). Three-dimensional (3-D) reconstitution was performed using a Delta Vision system (Applied Precision Inc.) attached to an Olympus IX-70-SIF fluorescence microscope equipped with a UplanApo x100 lens (Motegi et al. 2000).

Expression and observation of YFP fusion protein

The pYFP gene (Clontech Laboratory) was inserted into the NdeI site of pREP41acp2. {Delta}acp2- or {Delta}acp1-cells were transformed with this plasmid. Each transformant was grown in EMM with 4 µM thiamine at 30 °C, and examined by fluorescence microscopy at the exponentially growing stage.


   Acknowledgements
 
We thank Dr K. Gull of University of Manchester, Drs J. Wu and J. R. Pringle of the University of North Carolina, Dr M. K. Balasubramanian of the National University of Singapore, and Dr K. Maundrell of the Glaxo Institute for Molecular Biology in Switzerland for supplying us with TAT-1, {Delta}ain1 strain, rng2 mutant strain, and pREP vectors, respectively. This study was supported by a Grant-in-aid for Young Scientists and a Grant-in-aid for Scientific Research on Priority Areas for K. N. from the Ministry of Education, Culture, Sports, Science and Technology (MEXT) (nos. 15770122, 16044205, and 17049005) and Grants-in aid for Scientific Research to I. M. from the Japan Society for the Promotion of Science (no. 15207013) and from the MEXT (no. 15024213).


   Footnotes
 
Communicated by: Yasunori Machida

aPresent address: Doctoral Program in Structural Biosciences, Graduate School of Life and Environmental Sciences, University of Tsukuba, Tennohdai, Tsukuba, Ibaraki 305-8572, Japan. Back

* Correspondence: E-mail: knakano{at}biol.tsukuba.ac.jp


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Received: 4 January 2006
Accepted: 28 April 2006





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