|
Article |
2 Laboratory for Structural Biochemistry, RIKEN Harima Institute at SPring-8, Hyogo 679-5148, Japan
Address correspondence to John A. Cooper, Campus Box 8228, 660 S. Euclid Ave., St. Louis, MO 63110. Tel.: (314) 362-3964. Fax: (314) 362-0098. email: jcooper{at}cellbiology.wustl.edu
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key Words: cytoskeleton; cell motility; polymerization; assembly; Saccharomyces cerevisiae
Abbreviations used in this paper: CP, capping protein; wt, wild type.
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Saccharomyces cerevisiae CP is a stable heterodimer with subunits of Mr 32 (the subunit, Cap1) and 33.7 (the ß subunit, Cap2), each encoded by one gene (Amatruda and Cooper, 1992; Amatruda et al., 1992). CP is located on cortical actin patches in vivo. In CP null mutant cells, actin patches are present, but depolarized (Amatruda and Cooper, 1992; Amatruda et al., 1992). In addition, CP null mutations are synthetic lethal with null mutations in SAC6, which encodes fimbrin, another protein of the actin patch (Adams et al., 1993). CP is necessary for the localization of twinfilin to actin patches, through a direct interaction (Palmgren et al., 2001).
The crystal structure of chicken CP inspired a "tentacle" model in which the COOH-terminal regions of each subunit are proposed to extend out to interact with barbed ends of actin filaments (Yamashita et al., 2003). In that structure, the and ß subunits have similar secondary and tertiary structures, giving the molecule a pseudo twofold rotational axis of symmetry. The overall shape resembles a mushroom, with a stalk and cap. A bundle of six anti-parallel
-helices, three from the NH2 terminus of each subunit, comprise the mushroom stalk. Next are short stretches of ß-strand and reverse turns lying to the side of the stalk and under the mushroom cap. The central region of the protein includes a single 10-stranded anti-parallel ß-sheet. Atop this ß-sheet are two long
-helices, running anti-parallel to each other. Finally, each subunit has a COOH-terminal extension proposed to function as a flexible tentacle, reaching out to interact with the barbed end of the actin filament. Each COOH-terminal region contains a short amphipathic
-helix.
The tentacle model was supported by a structure/function analysis of chicken CP, in which the actin-binding activities of recombinant mutant chicken CPs were tested (Wear et al., 2003). Here, we tested the tentacle model for yeast CP, using a similar approach. We used a structure for yeast CP prepared by homology modeling from the chicken CP structure.
More importantly, we used the resulting CP actin-binding mutants to test the functional significance of the actin-capping activity of CP in vivo. We assayed the function of the mutant CPs in vivo by testing their ability to rescue null phenotypes. We also determined the localization of the mutant CPs in vivo. Actin-capping activity in vitro correlated well with the function and localization of CP in vivo, providing insight into the mechanism of actin assembly and motility along with the function of CP.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
|
|
|
|
To test whether the Cap1 tentacle alone is sufficient to bind actin, we assayed a synthetic peptide corresponding to the COOH-terminal 30 amino acids of Cap1. The peptide inhibited actin polymerization in the seeded assembly assay. The data were fit well by a model that included both capping and monomer binding, but not by simpler models with only capping or monomer binding (Fig. 4 C). From the modeling, the capping Kd was 1.71 µM, with an on-rate constant of 0.0021 µM-1s-1 (Table I). For monomer binding, the Kd was 2.86 µM, and the on-rate constant was 0.0084 µM-1s-1. The peptide had no effect in the critical concentration assay; however, concentrations of peptide >8 µM caused the formation of gross precipitates. Thus, the proposed Cap1 tentacle sequence alone is sufficient to bind actin, and even to cap barbed ends, albeit with low affinity. Circular dichroism analyses of the free peptide showed no secondary structure (unpublished data), so the active actin-binding conformation of the tentacle may be promoted by its connection to the body of the protein.
Point mutations in the Cap1 COOH-terminal region
The proposed tentacle for Cap1 includes an amphipathic -helix (Fig. 1 A, arrowhead). The hydrophobic side includes three highly conserved residues: W251, A254, and I255. W251 is present in the CP
subunit of all species (Fig. 1 B). We replaced W251 with alanine. Purified Cap1(W251A)/Cap2 protein inhibited actin polymerization in the seeded assembly assay (Fig. 4 D). A capping model fit the data well. The Kd for capping was 91 nM, 76-fold less than that of wt CP (Table I). The off-rate constant was increased 17-fold, and the on-rate constant was decreased fourfold. Capping was also seen in the critical concentration assay, where the Kd was 250 nM (Fig. 5 D, triangle; Table I). Thus, W251 is important for capping. The residue may be important for the structure of the tentacle, or it may interact directly with actin.
The tentacle model predicts that the COOH-terminal regions, the proposed tentacles, are mobile relative to the body of the protein. The tentacle should pivot about its attachment site. R239 is a highly conserved residue at this location (Fig. 1, A and B). To test whether R239 is important for capping, we replaced it with alanine. The Cap1 (R239A)/Cap2 protein inhibited polymerization in the seeded assembly assay (Fig. 4 E). A capping model gave a good fit, and the Kd for capping was 176 nM, 150-fold higher than that of wt (Table I). The on-rate constant was decreased 7.5-fold, and the off-rate constant was increased 18-fold, relative to wt. Capping was also seen in the critical concentration assay, where the Kd was 400 nM (Fig. 5 D, circle; Table I). Thus, the R239A mutation may affect the mobility or structure of the tentacle, such that the tentacle does not properly interact with actin filaments.
The CP ß (Cap2) COOH-terminal region
We tested the tentacle model for CP ß (Cap2) by removing the COOH-terminal 27 amino acids, K261L287, which comprises the entire proposed tentacle. In a previous experiment, a 21-aa truncation had normal actin-binding activity, using partially purified protein from yeast and falling ball viscometry (Sizonenko et al., 1996). Here, purified Cap1/Cap2(261287) inhibited polymerization in the seeded assembly assay, and a capping model fit the data well (Fig. 3 C). With muscle actin, the Kd for capping was 7.8 nM, increased approximately sevenfold from that of wt CP (Table I). The difference was due almost entirely to an increase in the off-rate constant. With yeast actin, the results were similar; the Kd was 17 nM, increased approximately fivefold from that of wt CP (Fig. 3 D, Table I). In the steady-state critical concentration assay, Cap1/Cap2(
261287) exhibited capping activity, with a Kd of 25 nM for both muscle and yeast actin (Fig. 5, A and B; Table I). Increased concentrations, up to 10 µM, did not change the critical concentration further. A shorter truncation of 24 residues produced similar results (Table I, Cap1/Cap2(
264287)). The proposed Cap2 tentacle alone, as a free synthetic peptide and a GST fusion protein, had no actin-binding activity (unpublished data). However, the synthetic peptide showed no evidence of a secondary structure by circular dichroism. This region includes an amphipathic
-helix in the homology model structure, so that feature may be necessary for activity and may only exist when the tentacle is attached to the protein. Overall then, the proposed tentacle of Cap2 is necessary for high affinity capping, but the proposed tentacle of Cap1 is much more important.
Function and localization of CP mutants in vivo
We assayed the ability of a number of CP mutants to function in cells, motivated by two major goals. First, actin capping is hypothesized to be a physiologically relevant function of CP. To test this hypothesis, we asked whether actin capping, as measured biochemically in vitro, correlates with the ability of CP to function normally in vivo. Second, to address the role of capping in the assembly of the actin patch, we determined the localization of these CP mutant proteins in vivo. In the sarcomere of striated muscle, CP (CapZ) appears to bind first to a periodic scaffold, independently of actin. Then, CP may capture or nucleate an actin filament (Schafer et al., 1993, 1995). On the other hand, in the dendritic nucleation model for lamellipodial protrusion in animal cells (Pollard and Borisy, 2003), CP caps older barbed ends after they have formed, which "funnels" actin polymerization to the new ends near the membrane (Carlier and Pantaloni, 1997). We asked which model applies to the yeast actin patch.
To test function in vivo, we first measured the ability of all the CP mutations to rescue two phenotypes characteristic of null mutationsdepolarization of the actin cytoskeleton and synthetic lethality with null mutations in the fimbrin gene SAC6. The assays consisted of staining cells with rhodamine-phalloidin to reveal the actin distribution and growing strains on plates after plasmid shuffle in a sac6 background (Table II). Next, CP mutant proteins were localized by anti-CP staining, using rhodamine-phalloidin as a second label to reveal actin patches (Fig. 6).
|
|
To assess the importance of the amphipathic -helical region of the Cap1 tentacle (W251S257) for function in vivo, we changed single aa residues within that region. The conserved residues Trp251, Ala254, and Ile255 are found on the hydrophobic side. W251A and W251R mutations produced large (but not complete) loss of function in both assays (Table II). W251F was similar to wt. Changing Ala254 or Ile255 to Arg resulted in wt levels of function in both assays (Table II). We also changed Gly252, Ser253, Gly256, and Ser257 to Arg, one at a time; none of the mutations showed any difference compared with wt. Thus, W251 is the only residue in the helical domain necessary for CP function in vivo. CP localization in the W251A and W251R strains showed decreased intensity of patch staining with increased diffuse cytoplasmic staining (Fig. 6).
We tested the importance of three residues near the proposed pivot point of the Cap1 tentacle: Arg239, Arg240, and Arg241 (Fig. 1 B). Residues Arg 240 and Arg 241 are located on the surface of the protein, with their side chains exposed to solvent (Fig. 1 A; only Arg240 was indicated). The side chain of Arg239 is oriented toward the body of the protein, and Arg239 makes close contacts with surrounding residues. Thus, Arg239 may anchor the tentacle and be necessary for pivoting. An R239A mutation produced severe loss of function in both assays, whereas R240A and R241A mutations had no effect (Table II). A double mutant, RR239 240AA, showed complete loss of function, as did the single change of charge mutations R239E and R240E (Table II). CP was localized normally in R240A and R241A strains. The R239A mutant showed increased diffuse staining as well as patch localization; the patch staining intensity was much less than that of wt (Fig. 6). RR239 240AA, R239E, and R240E showed diffuse CP staining (Table II).
For CP ß, we truncated the entire proposed tentacle by introducing a stop site that removed 27 residues, creating Cap2261287. The mutation provided complete rescue of both null mutant phenotypes (Table II). The localization of this mutant CP was normal. A shorter truncation, Cap2
264287, produced similar results, as did the point mutation Cap2 K261A.
When a mutation produced loss of function in vivo, we asked whether the level of the mutant CP was normal, to consider loss of protein as a trivial explanation for loss of function. By immunoblots, both CP subunits were expressed at approximately normal levels in all CP mutants with any loss of function (Fig. 7).
|
First, twinfilin is located at the actin patch and binds directly to CP (Palmgren et al., 2001). In CP null mutants, twinfilin localization at patches is greatly decreased (Palmgren et al., 2001). Here, we found that twinfilin localization to actin patches in Cap1239268 was also greatly decreased (Fig. 8).
|
|
Next, we measured the levels of total actin and F-actin. By immunoblot, the level of total actin was the same in wt, CP null, and CP actin-binding mutant cells (Fig. 10 A). We measured F-actin by rhodamine-phalloidin binding assays, examining either a population of cells with a fluorometer or individual cells with a fluorescence microscope. Under both methods, the level of F-actin in both mutants was increased by similar amounts. By fluorometry, the level was 1.4 times that of wt (Fig. 10 B), and by microscopy, the level was
1.3 times that of wt (Fig. 10 C). These results differ from a previous result from our lab (Karpova et al., 1995), where the F-actin level as measured by fluorometry and rhodamine-phalloidin binding went down. As part of this paper, we repeated those experiments carefully, and we also used fluorescence microscopy as an independent approach. We do not have a good understanding of the basis for the discrepancy. Combined with the previous result that the CP mutants had increased numbers of free barbed ends, the increase in F-actin implies that the actin monomer concentration is greater than the barbed end critical concentration, which is consistent with our understanding of actin assembly in general.
|
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The tentacle model for CP binding to actin
An atomic structure for chicken CP was recently solved using x-ray crystallography (Yamashita et al., 2003). In the chicken CP structure, the COOH-terminal region of the subunit is largely exposed to solvent, but is folded down onto the surface of the protein with an apparent hydrophobic interaction between the body and the COOH-terminal region. In contrast, the COOH-terminal region of the CP ß subunit is extended, lacking any interactions with the body of the protein. These features, coupled with the symmetry of the barbed end of the actin filament, inspired a tentacle model in which the COOH-terminal regions are mobile, extend out from the body of the protein, and bind to the barbed end (Yamashita et al., 2003). The actin-binding aspect of this model was supported by experiments with chicken CP (Wear et al., 2003).
CP is highly conserved, so we constructed a homology model for yeast CP based on the chicken structure. We tested the actin-binding aspect of the tentacle model by deleting the proposed tentacles and measuring actin-capping activity in vitro. Loss of both COOH-terminal regions caused a complete loss of actin-binding activity. Loss of the COOH-terminal region caused a large decrease in actin-capping activity (8,600-fold), and loss of the ß COOH-terminal region caused a small decrease in actin-capping activity (eightfold). Single amino acid changes confirmed the primary importance of the
COOH-terminal region, and demonstrated the importance of residues on the hydrophobic side of an amphipathic helix in the region and at a potential pivot point where the proposed tentacle meets the body of the protein. The proposed
tentacle alone, as a synthetic peptide, displayed actin-capping and monomer-binding activities, although they were weak. Based on the observation that a single COOH-terminal region, either free or attached to CP, is able to cap, we suggest that the COOH-terminal regions bind to interfaces between actin subunits in the filament, as discussed previously (Wear et al., 2003).
These results test and confirm the actin-binding aspect of the tentacle model, but the experiments do not test the prediction that the COOH-terminal regions are mobile and extend away from the body of CP to bind the barbed end. Indeed, recent analyses of the interaction of S100 protein with chicken CP suggest that the COOH-terminal region of CP is not extended (Wear and Cooper, 2004).
Physiological function and localization of CP
We used these actin-binding mutants to test two hypothesesthat CP's major physiological function is to bind actin, and that CP is present at cortical actin patches because it binds actin. We found that CP actin-binding mutants had the same phenotype as CP null mutants for every phenotype assayedpolarization of the actin cytoskeleton, synthetic lethality with sac6, twinfilin localization, incorporation of rhodamine-actin into permeabilized cells, levels of F-actin and total actin, and actin patch motility. Together, these results show that CP's actin-capping activity is necessary for CP to function in vivo. CP does possess other biochemical activities, such as binding twinfilin (Palmgren et al., 2001), and those may also be important for function in vivo. The results here do not argue against that possibility, which we see as likely.
We also localized the CP actin-binding mutant proteins in vivo, performing a double localization with actin patches. The order of severity of the mutations for loss of actin binding in vitro was essentially the same as the order of severity for loss of function in vivo and for localization to actin patches (Table II). The results suggest that actin filaments with free barbed ends are formed first and are then capped by CP, which agrees with the hypothesis that actin filaments with free barbed ends are nucleated by the action of Arp2/3 complex (Machesky and Gould, 1999; Pollard and Borisy, 2003). Actin patches are still present in CP null mutants (Amatruda et al., 1990), also consistent with CP localization being "downstream" of actin assembly.
One interesting aspect of these results was how much of a defect in CP's actin-capping activity was compatible with apparently normal function and localizationat least 10-fold. One interpretation of this result is that the cell contains a 10-fold excess of CP, so that actin filaments are still capped in adequate numbers and with adequate speed for viability and growth in these CP mutants.
Comparison of yeast and chicken CP results
A recent analysis of actin-binding mutations for chicken CP had results qualitatively similar to those here for yeast CP (Wear et al., 2003). Loss of both COOH-terminal regions produced a complete loss of actin binding, and the COOH terminus was more important than the ß COOH terminus. Previous experiments, performed without the benefit of the CP crystal structure or bacterial expression system, suggested that yeast and chicken CP might bind actin differently (Hug et al., 1992; Sizonenko et al., 1996). Our current analysis shows that this is not the case. The high degree of sequence conservation among CP subunits from various eukaryotes suggests that this model for binding actin may apply to other CPs. Indeed, nematode CP can function in yeast, rescuing null mutant phenotypes (Waddle et al., 1993).
Implications of the biochemical results for actin assembly in vivo
One interesting and potentially important difference between yeast and chicken CP is that yeast CP binds actin less well than does chicken CP, by a factor of 10 in Kd. This difference holds for actin from yeast as well as from muscle. Moreover, the off-rate constant for the dissociation of yeast CP from the actin filament is higher than that of chicken CP by a factor of 10 or more. Therefore, the half-life of a capped actin filament in yeast should be 22 s, compared with 30 min in vertebrates (Schafer et al., 1996).
One can also calculate the expected half-life of a free barbed end, and how much that barbed end should grow before being capped. We confirmed previous values for cellular content of actin and CP in yeast (Amatruda and Cooper, 1992; Nefsky and Bretscher, 1992; Karpova et al., 1995). The total concentration of actin in the cytoplasm is 5.3 µM, and the total concentration of CP is 1.3 µM (see Materials and methods). To estimate the half-life of a new free barbed end until the time when it is bound by CP, one must assume a value for the concentration of free cytoplasmic CP. Most CP appears to be localized at patches in images of wt cells (Fig. 6; Amatruda and Cooper, 1992), and the localization results here indicate that CP found at patches is indeed bound to actin. Thus, one might estimate the free CP cytoplasmic concentration at 10% of the total, or 0.13 µM. With the on-rate constant of 9 µM-1 s-1 for yeast CP and yeast actin (Table I), the rate of capping is 9 µM-1 s-1 x 0.13 µM = 1.17 s-1, which converts to a half-life of 0.4 s. The total actin concentration is 5.3 µM, and a conservative estimate of the actin monomer concentration might be 0.5 µM, near the critical concentration for the pointed end. At an actin monomer concentration of 0.5 µM, a free barbed end would grow by two subunits, given the actin elongation rate constants and the half-life of 0.4 s. This value of two subunits is probably a lower limit. The actin monomer concentration may be higher if pointed ends are capped by Arp2/3 complex. Also, the effective actin monomer concentration in the patch may be higher if proteins that bind monomer in the patch are able to "deliver" that monomer to a free barbed end. Even so, this analysis suggests that actin filaments of patches are short, which agrees with the size of patches (Mulholland et al., 1994). Actin cables of yeast presumably contain longer actin filaments, bundled together, which were nucleated by formins. Formins remain with the barbed end of the nucleated filament and protect it from capping by capping protein (Zigmond et al., 2003), which may account for the increased filament length in cables versus patches.
The results here also address the question of how the dendritic nucleation model (Pollard and Borisy, 2003) applies to actin patch assembly and motility, especially the funneling model for CP's role in dendritic nucleation (Carlier and Pantaloni, 1997). Previous papers showed that Arp2/3 complex and cofilin activity are important for actin patch assembly and motility (Lappalainen and Drubin, 1997; Winter et al., 1997; Idrissi et al., 2002), and that actin polymerization is important for patch movement (Carlsson et al., 2002). These findings are in accord with the dendritic nucleation model. Here, we found that the loss of CP's actin-capping activity led to an increase in free barbed ends per patch and per small bud, based on addition of rhodamine-actin to permeabilized cells. This result is also predicted by the dendritic nucleation model.
The funneling model for CP's role in dendritic nucleation predicts that the absence of CP should cause decreased assembly and motility at the locations of Arp2/3-induced nucleation, secondary to increased actin assembly at other locations of barbed ends in the cell. Here, CP actin-binding and null mutants showed an increase in the level of F-actin per cell and per bud, based on rhodamine-phalloidin binding, in agreement with the funneling model. However, CP actin-binding and null mutants showed no change in actin patch motility, based on measurements of individual patch speed, which runs counter to the model. One potential explanation for this result is that yeast cells may not have a substantial pool of barbed ends in the cytoplasm in general, outside of patches and perhaps cable ends, and thus do not need CP to cap those ends to keep the actin monomer concentration high enough to support actin assembly at patches. Another possibility is that some other factor caps barbed ends. Aip1 has been reported to cap barbed ends as a complex with cofilin (Cof1) in Xenopus and Saccharomyces (Okada et al., 2002; Balcer et al., 2003); however, an aip1 cap2 double-null mutant shows only a minimal synthetic effect in terms of growth (Rodal et al., 1999). Biochemical and sequence analyses have not revealed any other types of barbed end capping proteins, such as gelsolins, in Saccharomyces.
![]() |
Materials and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Oligonucleotide primers are listed in Table III, CP mutant plasmids in Table IV, and yeast strains in Table V. Oligonucleotide-mediated site-directed mutagenesis was performed using the QuikChange® Site-Directed Mutagenesis Kit (Stratagene). Mutations were verified by DNA sequencing.
|
|
|
Bacterial expression of CP
A tandem bacterial expression plasmid to simultaneously express Cap1 and Cap2 was constructed in pET-3d (Novagen) (Studier et al., 1990) following the strategy developed by Obinata and colleagues (Soeno et al., 1998). The coding regions of CAP1 and CAP2 were amplified by PCR using wt yeast genomic DNA as a template. Oligonucleotides MAW-33 and MAW-34 were used for CAP1, and MAW-48 and MAW-36 were used for CAP2 (Table III). The amplified PCR products were gel purified and subcloned into separate pET-3d vectors; the resulting constructs were verified by DNA sequencing in both directions. The CAP1 PCR product was digested with AlfIII and BamHI, and then ligated into pET-3d digested with NcoI and BamHI, to make pBJ 1353. The CAP2 PCR product was digested with BspHI and BamHI, and then ligated into pET-3d digested with NcoI and BamHI, to make pBJ 1354. The tandem expression vector pBJ 1355, allowing simultaneous expression of Cap1p and Cap2p, was then constructed as follows: the DNA sequences corresponding to the T7 promoter region, the ribosome-binding site, the coding sequence of CAP2, and the T7 terminator region were amplified by PCR using the forward primer MAW-47 and the reverse primer MAW-46 with pBJ 1354 as template. The amplified product was gel purified, digested with HindIII, and subcloned into pBJ 1353 digested with HindIII. The orientation and sequence of the CAP2 insert was verified by restriction digest and DNA sequencing of the coding region of CAP2.
CP mutants were expressed in bacteria and purified to homogeneity as described previously (Wear et al., 2003), with minor modifications. Yeast actin was purified from strain YJC 0094 (Table V) as described previously (Goode, 2002). Muscle actin was purified and labeled with pyrene as described in Wear et al. (2003). Actin polymerization assays, both kinetic and steady state, were performed as described in Wear et al. (2003). Kinetic rate constants were determined from the time course of seeded actin assembly using Berkeley Madonna, as described previously (Wear et al., 2003).
Yeast CP expression mutants: mutagenesis and strain construction
Plasmids expressing mutant forms of Cap1 or Cap2 from their endogenous promoters (Table IV) were introduced into cap1 and cap2
strains (YJC 0390 and YJC 0171, respectively) and into cap1
sac6
CEN::CAP1 and cap2
sac6
CEN::CAP2 strains (YJC 0596 and YJC 0922, respectively).
Mutations of CAP1
The template for mutagenesis was a CAP1 expression plasmid (pBJ 217) containing CAP1 and its flanking sequences in the HIS3 CEN plasmid pRS 313 (Sikorski and Hieter, 1989). Cap1258268: deletion of Cap1 residues Y258K268. Bases 19701971 were changed from AT to GA with oligonucleotides KKT-07 and KKT-08. Cap1
251268: base 1950 was changed from G to A with oligonucleotides KKT-09 and KKT-10. Cap1-
239268: base 1912 was changed from A to T with oligonucleotides KKT-11 and KKT-12. Cap1 R239A: bases 19121914 were changed from AGA to GCT with primers KKT-01 and KKT-02. Cap1 R240A: bases 19151917 were changed from AGA to GCT with oligonucleotides KKT-03 and KKT-04. Cap1 R240E: bases 19151916 were changed from AG to GA with oligonucleotides KKT-13 and KKT-14. Cap1 RR239, 240AA: bases 19121917 were changed from AGAAGA to GCTGCT with oligonucleotides KKT-05 and KKT-06. Cap1 W251R: bases 19481950 were changed from TGG to CGT with oligonucleotides KKT-17 and KKT-18. Cap1 W251A: bases 19481950 were changed from TGG to GCT with oligonucleotides KKT-77 and KKT-78. Cap1 W251F: bases 19491950 were changed from GG to TC with oligonucleotides KKT-89 and KKT-90. Cap1 G252R: base 1951 was changed from G to C with oligonucleotides KKT-91 and KKT-92. Cap1 S253R: base 1954 was changed from A to C with oligonucleotides KKT-93 and KKT-94. Cap1 A254R: bases 19571959 were changed from GCG to CGT with oligonucleotides KKT-19 and KKT-20. Cap1 I255R: bases 19601961 were changed from AT to CG with oligonucleotides KKT-21 and KKT-22. Cap1 G256R: bases 19631965 were changed from GGC to ACG with oligonucleotides KKT-95 and KKT-96. Cap1 S257R: base 1966 was changed from A to C with oligonucleotides KKT-97 and KKT-98.
Mutations of CAP2
The template for mutagenesis was a CAP2 expression plasmid (pBJ 119) containing CAP2 and its flanking sequences in the LEU2 CEN plasmid pRS 315 (Sikorski and Hieter, 1989). Cap2264287: deletion of Cap2 residues A264 through L287. Bases 26502652 were changed from AAG to TGA with oligonucleotides KKT-27 and KKT-28. Cap2
261287: bases 26412643 were changed from AAG to TGA with oligonucleotides KKT-113 and KKT-114. Cap2 K261A: bases 26412643 were changed from AAG to GCT with oligonucleotides KKT-29 and KKT-30.
Electrophoresis and immunoblotting
Whole-yeast cell extracts were subjected to 10% SDS-PAGE and transferred to nitrocellulose. The membrane was blocked with 3% BSA in PBS. Affinity-purified rabbit polyclonal antibodies against Cap1 (R13) and Cap2 (R12) were incubated for 1 h at RT. HRP-conjugated mouse antirabbit IgG (Biosource International) was used at 1:40,000. To detect actin, affinity-purified goat anti-yeast actin pAbs (G2) (Karpova et al., 1993) were used, followed by HRP-rabbit antigoat IgG. An ECL detection kit (Amersham Biosciences) was used. To quantitate total cell content of CP and actin, known quantities of purified yeast CP and actin were loaded on the gel as standards, and the blot was scanned and analyzed by densitometry. The number of cells loaded on the gel was calculated, and mean cell volume was calculated from the major and minor axes of cells from the culture by light microscopy. In two independent experiments, the total actin concentration in the cytoplasm was determined to be 4.4 and 6.1 µM, and the total CP concentration was 0.98 and 1.5 µM.
Fluorescence microscopy
CP and actin were localized with anti-CP antibodies and rhodamine-phalloidin as described previously (Amatruda and Cooper, 1992) in the same strains used to test actin patch polarization. Twinfilin was localized with anti-twinfilin antibodies (Palmgren et al., 2001), provided by Pekka Lappalainen (University of Helsinki, Helsinki, Finland). Digital images were collected on an inverted microscope (model IX70; Olympus) with a PlanApo 100x oil (NA 1.4) objective lens and a cooled CCD camera (Dage-MTI), using NIH Image software on a PowerMac G4.
Assays for null mutant phenotypes
For actin patch polarization, mutated CP genes on plasmids were transfected into strains carrying cap1 or cap2 null mutations YJC 0390 or YJC 0171, respectively. Cultures were fixed and stained with rhodamine-phalloidin as described previously (Sizonenko et al., 1996). As a quantitative measure of actin cytoskeleton polarization, we counted the number of actin patches in the mother portion of small-budded cells and defined polarized cells as having <4 patches in the mother. For sac6 synthetic lethality, mutated CP genes on URA3 plasmids were shuffled as described previously (Sizonenko et al., 1996). Serial 10-fold dilutions of multiple independent transformants were grown on selective media and were replica plated onto 5-fluoro-orotic acid.
Rhodamine-actin and permeabilized cells
Addition of 0.5 µM rhodamine-labeled actin to permeabilized cells was performed as described previously (Li et al., 1995) with minor modifications. Three strains, YJC 2954 (wt), YJC 2953 (Cap1), and YJC 2967 (Cap1
239268) were used (Table V). Fluorescence images were collected on an inverted microscope (model IX70; Olympus) using NIH Image software. The fluorescence intensity in specific regions (i.e., whole bud or single patch in bud) was measured using NIH image software. For each dataset, 20 small-budded cells (bud size <1/3 of the mother) from 10 different fields were chosen at random and were measured. Background intensity was measured from an equal-sized area of each field that did not have cells, and was subtracted from the measured intensity of the buds or patches. The average, SD, and SEM were calculated from the 20 measurements. In some experiments, purified yeast CP was added at 500 nM before the addition of rhodamine-actin. The CP concentration was maintained during the incubation with rhodamine-actin.
F-actin by fluorometry
To determine F-actin levels in wt (YJC 2954), Cap1 (YJC 2953), and Cap1
239268 (YJC 2967) strains, a rhodamine-phalloidin binding assay was performed as described previously (Lillie and Brown, 1994; Karpova et al., 1995). In brief, the total protein content of cell aliquots was measured by Bradford assay. Before staining, fixed cells were divided into three equal aliquots. Cells were suspended in 1x PBS containing 0.2% Triton X-100, and were stained with 0.33 µM rhodamine-phalloidin (Molecular Probes, Inc.). In a control aliquot, 300 µM unlabeled phalloidin was also added (Molecular Probes, Inc.). The cells were washed twice with 1x PBS. The bound rhodamine-phalloidin was extracted by incubation in 500 µl methanol. Fluorescence intensity was measured on a spectrofluorometer (QuantaMasterTM; Photon Technology International), with excitation at 550 nm and emission at 580 nm. The values obtained for unlabeled phalloidin-containing control samples were subtracted from those obtained for the corresponding samples to correct for nonspecific binding of labeled phalloidin.
F-actin by fluorescence microscopy
Cells were stained with rhodamine-phalloidin as described previously (Sizonenko et al., 1996). Fluorescence microscopy was performed as described above, and intensities of individual cells were measured using NIH Image software. For each dataset, 10 cells in 23 different fields chosen at random were measured. Background intensity was measured from an area of each field that did not have cells, and was subtracted.
Actin patch motility
Cortical actin patches were tracked as described previously (Carlsson et al., 2002), using Sac6-GFP. Strain YJC 3481(SAC6-GFP-HIS3 cap1) was transfected with a plasmid that expressed nothing (pBJ 82), wt CAP1 (pBJ 108), or Cap1
239268 (pBJ 1461). We confirmed the functionality of the genes by the polarization of the actin patches. Movies of actin patches were collected from a single focal plane near the upper or lower surface of the cell with a microscope system described previously (Castillon et al., 2003). Patch movement was tracked and analyzed with custom software (Carlsson et al., 2002). The software was adapted to Mac OS X by Jill Jeanblanc and Kevin Schmidt in our lab, and is available from the authors.
![]() |
Acknowledgments |
---|
This work was supported by grants to J.A. Cooper from the National Institutes of Health (GM47337) and to Y. Maeda from the Special Coordination Funds for Promoting Science and Technology from the Ministry of Education, Culture, Sports, Science and Technology of the Japanese Government. K. Kim was supported by a postdoctoral fellowship from the American Heart Association.
Submitted: 12 August 2003
Accepted: 12 January 2004
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Adams, A.E.M., J.A. Cooper, and D.G. Drubin. 1993. Unexpected combinations of null mutations in genes encoding the actin cytoskeleton are lethal in yeast. Mol. Biol. Cell. 4:459468.[Abstract]
Amatruda, J.F., J.F. Cannon, K. Tatchell, C. Hug, and J.A. Cooper. 1990. Disruption of the actin cytoskeleton in yeast capping protein mutants. Nature. 344:352354.[CrossRef][Medline]
Amatruda, J.F., and J.A. Cooper. 1992. Purification, characterization and immunofluorescence localization of Saccharomyces cerevisiae capping protein. J. Cell Biol. 117:10671076.[Abstract]
Amatruda, J.F., D.J. Gattermeir, T.S. Karpova, and J.A. Cooper. 1992. Effects of null mutations and overexpression of capping protein on morphogenesis, actin distribution and polarized secretion in yeast. J. Cell Biol. 119:11511162.[Abstract]
Balcer, H.I., A.L. Goodman, A.A. Rodal, E. Smith, J. Kugler, J.E. Heuser, and B.L. Goode. 2003. Coordinated regulation of actin filament turnover by a high-molecular-weight Srv2/CAP complex, cofilin, profilin, and Aip1. Curr. Biol. 13:21592169.[Medline]
Carlier, M.F., and D. Pantaloni. 1997. Control of actin dynamics in cell motility. J. Mol. Biol. 269:459467.[CrossRef][Medline]
Carlsson, A.E., A.D. Shah, D. Elking, T.S. Karpova, and J.A. Cooper. 2002. Quantitative analysis of actin patch movement in yeast. Biophys. J. 82:23332343.
Castillon, G.A., N.R. Adames, C.H. Rosello, H.S. Seidel, M.S. Longtine, J.A. Cooper, and R.A. Heil-Chapdelaine. 2003. Septins have a dual role in controlling mitotic exit in budding yeast. Curr. Biol. 13:654658.[CrossRef][Medline]
Cooper, J.A., M.C. Hart, T.S. Karpova, and D.A. Schafer. 1999. Capping protein. Guidebook to the Cytoskeletal and Motor Proteins. T. Kreis and R. Vale, editors. Oxford University Press, New York. 6264.
Goode, B.L. 2002. Purification of yeast actin and actin-associated proteins. Methods Enzymol. 351:433441.[Medline]
Guex, N., and M.C. Peitsch. 1997. SWISS-MODEL and the Swiss-PdbViewer: An environment for comparative protein modeling. Electrophoresis. 18:27142723.[Medline]
Hug, C., T.M. Miller, M.A. Torres, J.F. Casella, and J.A. Cooper. 1992. Identification and characterization of an actin-binding site of CapZ. J. Cell Biol. 116:923931.[Abstract]
Idrissi, F.Z., B.L. Wolf, and M.I. Geli. 2002. Cofilin, but not profilin, is required for myosin-I-induced actin polymerization and the endocytic uptake in yeast. Mol. Biol. Cell. 13:40744087.
Karpova, T.S., M.M. Lepetit, and J.A. Cooper. 1993. Mutations that enhance the cap2 null mutant phenotype in Saccharomyces cerevisiae affect the actin cytoskeleton, morphogenesis and pattern of growth. Genetics. 135:693709.
Karpova, T.S., K. Tatchell, and J.A. Cooper. 1995. Actin filaments in yeast are unstable in the absence of capping protein or fimbrin. J. Cell Biol. 131:14831493.[Abstract]
Lappalainen, P., and D.G. Drubin. 1997. Cofilin promotes rapid actin filament turnover in vivo. Nature. 388:7882.[CrossRef][Medline]
Li, R., Y. Zheng, and D.G. Drubin. 1995. Regulation of cortical actin cytoskeleton assembly during polarized cell growth in budding yeast. J. Cell Biol. 128:599615.[Abstract]
Lillie, S.H., and S.S. Brown. 1994. Immunofluorescence localization of the unconventional myosin, Myo2p, and the putative kinesin-related protein, Smy1p, to the same regions of polarized growth in Saccharomyces cerevisiae. J. Cell Biol. 125:825842.[Abstract]
Loisel, T.P., R. Boujemaa, D. Pantaloni, and M.F. Carlier. 1999. Reconstitution of actin-based motility of Listeria and Shigella using pure proteins. Nature. 401:613616.[CrossRef][Medline]
Machesky, L.M., and K.L. Gould. 1999. The Arp2/3 complex: A multifunctional actin organizer. Curr. Opin. Cell Biol. 11:117121.[CrossRef][Medline]
Mulholland, J., D. Preuss, A. Moon, A. Wong, D. Drubin, and D. Botstein. 1994. Ultrastructure of the yeast actin cytoskeleton and its association with the plasma membrane. J. Cell Biol. 125:381391.[Abstract]
Nefsky, B., and A. Bretscher. 1992. Yeast actin is relatively well behaved. Eur. J. Biochem. 206:949955.[Abstract]
Okada, K., L. Blanchoin, H. Abe, H. Chen, T.D. Pollard, and J.R. Bamburg. 2002. Xenopus actin-interacting protein 1 (XAip1) enhances cofilin fragmentation of filaments by capping filament ends. J. Biol. Chem. 277:4301143016.
Palmgren, S., P.J. Ojala, M.A. Wear, J.A. Cooper, and P. Lappalainen. 2001. Interactions with PIP2, ADP-actin monomers, and capping protein regulate the activity and localization of yeast twinfilin. J. Cell Biol. 155:251260.
Pollard, T.D., and G.G. Borisy. 2003. Cellular motility driven by assembly and disassembly of actin filaments. Cell. 112:453465.[Medline]
Rodal, A.A., J.W. Tetreault, P. Lappalainen, D.G. Drubin, and D.C. Amberg. 1999. Aip1p interacts with cofilin to disassemble actin filaments. J. Cell Biol. 145:12511264.
Schafer, D.A., J.A. Waddle, and J.A. Cooper. 1993. Localization of CapZ during myofibrillogenesis in cultured chicken muscle. Cell Motil. Cytoskeleton. 25:317335.[Medline]
Schafer, D.A., C. Hug, and J.A. Cooper. 1995. Inhibition of CapZ during myofibrillogenesis alters assembly of actin filaments. J. Cell Biol. 128:6170.[Abstract]
Schafer, D.A., P.B. Jennings, and J.A. Cooper. 1996. Dynamics of capping protein and actin assembly in vitro: Uncapping barbed ends by polyphosphoinositides. J. Cell Biol. 135:169179.[Abstract]
Sikorski, R.S., and P. Hieter. 1989. A system of shuttle vectors and yeast host strains designed for efficient manipulation of DNA in Saccharomyces cerevisiae. Genetics. 122:1927.
Sizonenko, G.I., T.S. Karpova, D.J. Gattermeir, and J.A. Cooper. 1996. Mutational analysis of capping protein function in Saccharomyces cerevisiae. Mol. Biol. Cell. 7:115.[Abstract]
Soeno, Y., H. Abe, S. Kimura, K. Maruyama, and T. Obinata. 1998. Generation of functional ß-actinin (CapZ) in an E. coli expression system. J. Muscle Res. Cell Motil. 19:639646.[CrossRef][Medline]
Studier, F.W., A.H. Rosenberg, J.J. Dunn, and J.W. Dubendorff. 1990. Use of T7 RNA polymerase to direct expression of cloned genes. Methods Enzymol. 185:6089.[Medline]
Waddle, J.A., J.A. Cooper, and R.H. Waterston. 1993. The alpha and beta subunits of nematode actin capping protein function in yeast. Mol. Biol. Cell. 4:907917.[Abstract]
Wear, M.A., and J.A. Cooper. 2004. Capping protein binding to S100B: implications for the "tentacle" model for capping the actin filament barbed end. J. Biol. Chem. In press.
Wear, M.A., A. Yamashita, K. Kim, Y. Maéda, and J.A. Cooper. 2003. How capping protein binds the barbed end of the actin filament. Curr. Biol. 13:15311537.[CrossRef][Medline]
Winter, D., A.V. Podtelejnikov, M. Mann, and R. Li. 1997. The complex containing actin-related proteins Arp2 and Arp3 is required for the motility and integrity of yeast actin patches. Curr. Biol. 7:519529.[Medline]
Yamashita, A., K. Maéda, and Y. Maéda. 2003. Crystal structure of CapZ: Structural basis for actin filament barbed end capping. EMBO J. 22:15291538.
Zigmond, S.H., M. Evangelista, C. Boone, C. Yang, A.C. Dar, F. Sicheri, J. Forkey, and M. Pring. 2003. Formin leaky cap allows elongation in the presence of tight capping proteins. Curr. Biol. 13:18201823.[CrossRef][Medline]