Article |
Address correspondence to Anthony Bretscher, Department of Molecular Biology and Genetics, Cornell University, 353 Biotechnology Building, Ithaca, NY. Tel.: (607) 255-5713. Fax: (607) 255-6249. E-mail: apb5{at}cornell.edu
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Abstract |
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Key Words: actin; polarity; Cdc42; PKC; MAPK
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Introduction |
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Like many formins from animals and fungi, yeast Bni1p and Bnr1p contain an NH2-terminal Rho-binding domain (RBD),* a central proline-rich formin homology (FH) domain 1 that binds profilin, and a COOH-terminal FH2 domain (Kohno et al., 1996; Evangelista et al., 1997; Imamura et al., 1997) (Fig. 1, a and d). The isolated FH2 domain of Bni1p can serve as a nucleator for actin assembly in vitro (Pruyne et al., 2002; Sagot et al., 2002b). Because the FH2 domain has the novel capacity to remain bound to the barbed end of the assembling filament, Bni1p can potentially serve as a nucleator and filament anchor during actin cable assembly at the cell cortex (Pruyne et al., 2002).
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Yeast contains six Rho-family GTPases (Cdc42p and Rho15p) that participate in multiple aspects of cell polarity, including septin organization (Holly and Blumer, 1999; Weiss et al., 2000; Gladfelter et al., 2002), the regulation of secretion (Adamo et al., 1999, 2001; Robinson et al., 1999; Guo et al., 2001; Zhang et al., 2001), and the stimulation of cell wall synthesis (Cabib et al., 1998). All six Rho proteins also have roles in regulating actin polarity. Cdc42p is required for actin polarization at bud emergence, with multiple effectors implicated in this process (Adams et al., 1990; Brown et al., 1997; Chen et al., 1997; Evangelista et al., 1997; Holly and Blumer, 1999; Bi et al., 2000; Lamson et al., 2002). Rho3p and Rho4p share a function in polarizing actin, though so far no physiologically relevant effectors have been defined (Matsui and Toh-e, 1992b; Imai et al., 1996). Rho1p functions through Pkc1p to regulate actin in a complex manner, driving MAPK-independent depolarization of the actin cytoskeleton in response to cell wall injuries and then stimulating a MAPK-dependent repolarization (Delley and Hall, 1999). Overexpressed Rho2p can substitute for the actin-polarizing activity of Rho1p (Marcoux et al., 2000), and Rho5p negatively regulates cell wall stress-induced actin depolarization (Schmitz et al., 2002), but effectors for these proteins are unknown.
The Bni1p and Bnr1p RBDs show two-hybrid interactions with multiple Rho proteins (Kohno et al., 1996; Evangelista et al., 1997; Imamura et al., 1997), but the contribution of each Rho protein to regulating formin-dependent actin cable assembly was not known. We find that multiple Rho-dependent pathways converge upon Bni1p and Bnr1p to regulate their activity, including the semiredundant Rho3p and Rho4p and the highly conserved homologues of RhoA (Rho1p) and CDC42 (Cdc42p). Thus, the formins are key targets for integrating signaling pathways in controlling actin polarity.
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Results |
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As Bni1p and Bnr1p perform an essential function in the assembly of actin cables (Evangelista et al., 2002; Sagot et al., 2002a), we generated a strain lacking both RHO3 and BNR1 to examine whether Bni1p expressed at endogenous levels also requires RHO3. Consistent with a strong dependence of actin cable assembly by Bni1p upon Rho3p, the rho3bnr1
cells grew very poorly and contained few actin cables (Fig. 3, a and b) but accumulated actin bars, a form of aggregated monomeric actin common to cytoskeletal mutants.
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Rho3p and Rho4p share an essential function in formin activation
The viability of the rho3bnr1
strain suggested that Bni1p has some residual ability to assemble cables in the absence of Rho3p. One possible explanation is that other Rho proteins play a role in this process. One likely candidate is Rho4p. Simultaneous deletion of RHO3 and RHO4 is lethal, and overexpression of Rho4p can suppress growth defects of rho3
cells (Matsui and Toh-e, 1992a), indicating that the two share some redundant function. Double conditional rho3 rho4 mutants display severe cytoskeletal defects (Matsui and Toh-e, 1992b; Imai et al., 1996), yet the effectors for these Rho isoforms in organizing the cytoskeleton are unknown. If they share an essential role in formin activation, we predicted that expression of the constitutively active Bni1p
RBD might bypass the requirement for these two Rho proteins. Indeed, expression of Bni1p
RBD from the BNI1 promoter rescued the viability of the double rho3
rho4
mutant (Fig. 3 c). An attractive explanation is that the normal function of Rho3p and Rho4p is to bind the Bni1p RBD to relieve the inhibitory interaction between the RBD and the DAD.
In mammalian cells, expression of the mDia DAD has been shown to also bypass the requirement for Rho activation of a formin by competing with the DAD of the endogenous full-length formin for binding the RBD (Alberts, 2001). To more specifically determine whether Rho3p and Rho4p play a role in regulating the interaction of the Bni1p RBD and DAD, we generated a construct containing the DAD of Bni1p (Fig. 1 c). Expression of this construct was able to rescue growth and permit actin cable assembly in rho3rho4
yeast (Fig. 3, c and d). As expected, rescue by the Bni1p DAD was dependent upon full-length formins, because Bni1pDADCOOH was unable to rescue the lethality of bni1
bnr1
(unpublished data). These results suggest that Rho3p and, to a lesser extent, Rho4p share a role in regulating the interaction between the RBD and DAD of the yeast formin Bni1p.
Rho4p is the only Rho protein that binds the Bnr1p RBD, as analyzed by two-hybrid or in vitro binding assays (Imamura et al., 1997). To ascertain whether Rho4p is the preferred Rho activator for Bnr1p, the growth rates of wild-type, rho4, bni1
, and rho4
bni1
strains were compared. No obvious growth defects were seen in any of these strains, and the actin cables of rho4
bni1
were normal, indicating that Bnr1p can function in the absence of Rho4p (Fig. 3 d).
The strong dependence of Bni1p upon Rho3p suggests that the actin cables present in rho3 yeast are dependent upon Bnr1p. Consistent with this, the growth rates of rho3
and rho3
bni1
strains were similar (unpublished data), and the actin cables of rho3
bni1
were normal (Fig. 3 d), suggesting that Bnr1p can also function in the absence of Rho3p. However, the lethality of rho3
rho4
yeast can be rescued by an activated version of Bnr1p (Bnr1p
RBD) (Fig. 3 c), suggesting that yeast lacking Rho3p and Rho4p are deficient in all formin function. Also, the deletion of RHO3 results in slow growth, but this can be rescued by the activated Bni1
RBD (unpublished data), suggesting that the slow growth of our rho3
strain results from diminished formin activity. Notably, loss of BNI1 in our strain background does not show a slow growth phenotype, suggesting that the loss of RHO3 also diminishes activation of Bnr1p. Thus, although Bnr1p-mediated filament assembly can occur in the presence of Rho3p or Rho4p, activation by Rho3p appears to be more important for normal growth. Our combined results indicate that the shared essential function of Rho3p and Rho4p is to activate the formins, specifically through disrupting the RBDDAD interaction, and that Rho3p plays a more important role in this process.
Cdc42p is required for actin cable polarization at bud emergence
Loss of Cdc42p function causes multiple defects in polarized growth (Kozminski et al., 2000). However, many conditional alleles arrest as unbudded cells that grow isotropically (Adams et al., 1990; Miller and Johnson, 1997; Kozminski et al., 2000), similar to the terminal phenotype of double conditional bni1bnr1 mutants (Evangelista et al., 2002). To determine whether Cdc42p regulates Bni1p or Bnr1p activity, we examined whether actin cables are present in a temperature-sensitive cdc42-1 strain. When cdc42-1 yeast were shifted to 35°C, polarized actin cables were lost from unbudded cells but, to a large extent, were retained in budded cells (Fig. 4, a and b). The defect in the unbudded cdc42-1 cells did not appear to be a loss of cables but an inability to properly organize them. The unbudded cdc42-1 cells retained actin cables to a similar extent as wild-type cells but in a disorganized distribution.
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Overexpression of Cdc42p is reported to bypass the requirement for Rho3p and Rho4p (Matsui and Toh-e, 1992b). To examine the ability of the various Rho isoforms to replace Rho3p and Rho4p function, we introduced CDC42, RHO1, RHO2, or RHO5 on high-copy plasmids into rho3rho4
yeast grown in the presence of pRS316-RHO3 and then tested each strain for its ability to lose the RHO3 plasmid. Consistent with Matsui and Toh-e (1992b), CDC42 was able to rescue the lethality of rho3
rho4
, and the rescued cells displayed normal actin cables (Fig. 5, a and b), indicating that at least one yeast formin was active under these conditions. However, none of the other three Rho proteins was able to rescue the lethality of rho3
rho4
(unpublished data). Cdc42p was also able to bypass the synthetic sickness and actin cable defects of rho3
bnr1
yeast (unpublished data), indicating that when overexpressed, Cdc42p can relieve the dependence of Bni1p activation upon Rho3p.
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Although PCK1 is essential, a deletion can be rescued by a dominant active allele of the MAPK kinase kinase BCK1 (BCK1-20) (Lee and Levin, 1992; Levin and Bartlett-Heubusch, 1992). To determine whether Pkc1p is required for Rho1p-dependent activation of the formins, pkc1 BCK1-20 cells were examined at several temperatures. At room temperature, actin cables were present (unpublished data), but when shifted to 37°C, actin cables disassembled in the pck1
BCK1-20 cells within 15 min, just like the rho1-2 mutants (Fig. 8, d and e). A temperature-sensitive PKC1 allele (pkc1-2ts) (Lee and Levin, 1992) yielded similar results (unpublished data), whereas wild-type PKC1 or PKC1 BCK1-20 controls showed no loss of actin cables. Thus, Rho1p works through its effector Pkc1p to activate the formins in a MAPK-independent manner, though this signaling appears to be required only at elevated temperatures.
To determine whether Pkc1p signaling can bypass the requirement for Rho3p-dependent activation of Bni1p, Pkc1p* was expressed in rho3bnr1
yeast. The presence of the activated kinase was unable to rescue the slow growth of this strain (Fig. 8 f) or restore actin cables and eliminate actin bars (Fig. 8 g), suggesting that Pkc1p cannot bypass the requirement for Rho3p to activate Bni1p. To determine whether Rho3p can bypass the need for Rho1p/Pkc1p signaling in activating Bni1p, we coexpressed full-length Bni1p with an activated GTPase-deficient allele of RHO3 (RHO3-V25) (Adamo et al., 1999) from a CEN plasmid in rho1-2 yeast. The presence of activated Rho3p was unable to bypass the requirement of Bni1p-stimulated filament assembly for Rho1p (unpublished data), indicating that both Rho1p/Pkc1p and Rho3p are required for efficient activation of Bni1p at 37°C. Similarly, overexpression of CDC42 was unable to suppress the rho1-2 mutant at 37°, either with respect to growth or the actin cable defect. Based on the loss of all cables in rho1-2 yeast, Rho1p/Pkc1p is likely to also be required for the activation of Bnr1p.
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Discussion |
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Rho3p and Rho4p were previously known to share an essential role in yeast growth (Matsui and Toh-e, 1992a), and we find here that this function appears to be regulation of the inhibitory interaction between the RBD and DAD of the yeast formins. Thus, these two Rho proteins become dispensable in cells expressing Bni1p or Bnr1p from which the RBD has been deleted, or when the RBDDAD interaction is disrupted by overexpression of exogenous DAD sequence. By several criteria, Rho3p appears to be the more important of the two GTPases. Loss of Rho3p almost completely eliminates Bni1p-dependent filament assembly. Furthermore, rho3 yeast grow slowly, but this slow growth can be bypassed by an activated Bni1p, suggesting that the growth defect is related to a loss of signaling to the formins. As bni1
does not cause a similar decrease in growth rate in our strain background, we suggest that the loss of Rho3p eliminates a significant component of Bnr1p activation.
An attractive model would have Rho3p and Rho4p activate the formins directly. GTP-bound forms of multiple Rho proteins show two-hybrid interactions with the RBD of the formins, although the reported interactions do not completely coincide with our in vivo analysis; Bni1p RBD interacts with GTP-Cdc42p, -Rho1p, -Rho3p, and -Rho4p (Kohno et al., 1996; Evangelista et al., 1997), but Bnr1p interacts only with GTP-Rho4p (Imamura et al., 1997). Our results suggest that the most physiologically important interaction for Bni1p is with Rho3p. However, with no reported interaction between Bnr1p and Rho3p, it is possible that signaling between these two molecules is indirect.
In addition to formins, Rho3p and Rho4p can bind to several proteins involved in polarized secretion, including the exocyst proteins Sec3p and Exo70p and (for Rho3p) the myosin-V Myo2p (Adamo et al., 1999; Robinson et al., 1999; Guo et al., 2001). Furthermore, mutants for RHO3 show defects in exocytosis (Adamo et al., 1999), but the contributions of the Rho proteins to secretion and to actin organization seem to be distinct. Many suppressors of rho3 growth defects encode proteins involved in the secretory pathway, but these are not able to rescue rho3
rho4
lethality (Matsui and Toh-e, 1992b; Imai et al., 1996; Kagami et al., 1997). Also, defects in the function of Sec3p or Myo2p do not directly affect the organization of actin cables (Haarer et al., 1996; Schott et al., 1999). Finally, rho3 alleles specifically defective in secretion retain a normal actin cytoskeleton (Adamo et al., 1999), suggesting that they are not defective in activating the formins. Despite the importance of Rho3p for exocytosis, the growth defects of our rho3
mutants appear to be limited to formin activation, as they can be rescued by an activated allele of Bni1p. This discrepancy might reflect a difference in strain background or a difference in growth conditions, such that the requirement for Rho3p in exocytosis does not impact the growth rate under the conditions used in this study.
A previous screen identified two high-copy suppressors of rho3rho4
lethality, the highly conserved Rho family member Cdc42p and the Cdc42p-binding scaffold protein Bem1p (Matsui and Toh-e, 1992b). We found that overexpressed Cdc42p could activate the formins to generate actin cables in rho3
rho4
yeast and could specifically activate Bni1p in rho3
bnr1
yeast. The ability of Cdc42p to associate with the Bni1p RBD (Evangelista et al., 1997; Imamura et al., 1997) supports the possibility that Cdc42p might directly activate Bni1p.
The need to overexpress Cdc42p to rescue the rho3rho4
lethality suggests that the normal role of Cdc42p is more restricted. Previous work demonstrated that Cdc42p plays an important role in organizing actin during bud emergence (Adams et al., 1990), and we found that the loss of Cdc42p function in six conditional cdc42 alleles specifically compromised actin cable organization in unbudded cells, but cables appeared to be unperturbed in budded cells. Notably, the levels of actin cables did not appear to be diminished in the unbudded cells, suggesting that the formins were still activated, presumably by Rho3p and Rho4p. This suggests that Cdc42p plays a role in properly recruiting the active formins to the nascent bud site. In fact, previous results have demonstrated that Bni1p is unable to localize in the absence of Cdc42p function (Jaquenoud and Peter, 2000; Ozaki-Kuroda et al., 2001). The inability of cdc42-ts strains to organize actin cables in unbudded cells can account for two other phenotypes of the arrested cells, isotropic growth and depolarized cortical patches, because both of these phenotypes can also arise as a secondary consequence of loss of transport along actin cables (Pruyne et al., 1998; Schott et al., 1999; Evangelista et al., 2002; Sagot et al., 2002a). The ability of Cdc42p to replace Rho3p and Rho4p when overexpressed may reflect a subtle role for Cdc42p in regulating formin activity for cable polarization under normal conditions.
The conserved RhoA homologue, Rho1p, makes an additional, independent contribution to the activation of the formins. Loss of Rho1p function at 37°C in rho1-2 yeast eliminates both actin cable assembly by endogenous formins and cable-like filament accumulation stimulated by exogenous Bni1p. The expression of the activated Bni1pRBD construct restored cables to rho1-2 yeast at the restrictive temperature, though the viability of the rho1-2 yeast was not rescued. Despite the ability of Rho1p to bind the Bni1p RBD in two-hybrid assays (Kohno et al., 1996), our evidence suggests that Rho1p does not directly regulate the formin RBDDAD interaction but acts indirectly through the Rho1p effector, Pkc1p. Yeast without functional Pkc1p also lost actin cables at 37°C, whereas expression of an activated kinase Pkc1p* restored cables to rho1-2 yeast and permitted overexpressed Bni1p to generate ectopic filaments in rho1-2 yeast. The Pkc1p/Rho1p dependence of formin activation was only observable at elevated temperatures, suggesting that some other change in the cell status under these conditions impinges on the formins such that a Pkc1p-dependent signal is required to maintain their activity.
The Pkc1p-dependent signaling did not appear to act through the downstream MAPK cascade, as cells lacking MAPK components retained actin cables even at 37°C, and overexpression of the MAPK Slt2p was unable to restore cables in rho1-2 yeast or restore Bni1p function in rho1-2 yeast. As further confirmation that formin function and MAPK signaling are distinct events, defects in formin function (e.g., bni1) and loss of MAPK signaling (e.g., slt2
) have additive deleterious effects (Fujiwara et al., 1998). While this paper was in preparation, it was reported that Rho1p and formins are necessary for the assembly of the contractile ring at elevated temperatures (Tolliday et al., 2002). These findings are consistent with our results and suggest that the role of Rho1p in that process might also be indirect through Pkc1p.
Thus, we find that three distinct Rho-dependent signals regulate the proper function of the formins. Rho3p and Rho4p share a critical role in activating the formins. This activity cannot be replaced by Rho1p/Pkc1p signaling, either through Rho1p overexpression or through activation of Pkc1p. Cdc42p can replace Rho3p/Rho4p when overexpressed, but its role appears to normally be restricted to organizing the formins for bud emergence. Similarly, the requirements for Rho1p and Pkc1p appear to be restricted to conditions that trigger a cell wall stress response (i.e., 37°C). Thus, regulation of the formins may require multiple events, for example, recruitment of the formin, a specific phosphorylation of the RBD, and the binding of a Rho protein to the RBD. A similar phenomenon is seen with Pkc1p, where binding of Rho1 and phosphorylation by the redundant kinases Pkh1/2p contribute to Pkc1p activation (Inagaki et al., 1999). It will be interesting to determine whether the animal Rho1p homologue, RhoA, regulates formins in a similarly indirect manner through its Pkc1p-related kinase effectors, the PRKs (Amano et al., 1996; Watanabe et al., 1996; Vincent and Settleman, 1997).
The multiple inputs to Bni1p and Bnr1p activation link formin-mediated actin assembly into multiple essential regulatory pathways. Undoubtedly, other inputs contribute to the regulation of formin activity. For example, osmotic stabilization can also rescue viability of rho3rho4
(Matsui and Toh-e, 1992b), suggesting that high osmolarity activates an alternative signaling pathway to bypass the requirement for Rho3p/Rho4p. The conservation of formins and their roles in regulating cytoskeletal organization suggests that these principles of multiple activating inputs are likely to be conserved across the eukaryotes.
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Materials and methods |
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Immunofluorescence microscopy
Cells were prepared, fixed, and stained using antibodies to actin, tropomyosin, and the myc epitope as previously described (Pruyne et al., 1998; Evangelista et al., 2002). Cells were categorized as unbudded, small budded (bud lengths <1/3 the length of the mother cell), medium budded (bud lengths <2/3 the length of the mother cell but ≥1/3 the length of the mother cell), or large budded (bud length ≥2/3 the length of the mother cell). For each assay, 100 cells of the indicated categories from asynchronous cultures were scored. When scoring for the presence of actin cables, cells of all categories were scored positive if cables were visible by eye, and negative if not. When scoring for the presence of polarized actin cables, cells of all categories were scored as positive if the majority of cables present in a cell were aligned along the axis of the cell or emanated from a nascent bud site. Cells were scored as negative for polarized actin cables if no cables were detectable or if half or more of the cables present in a cell were not aligned along the growth axis or associated with a bud site. When scoring for the accumulation of cable-like filaments in the bud, small- and medium-budded cells were scored positive if the stain in the bud showed a clearly visible increase in fluorescence beyond that seen in wild-type cells. Note that for the images shown in this paper, the intensity has been digitally reduced to allow dimmer portions of the displayed cells to be visible. The increased intensity of stain due to formin-stimulated accumulation of filaments is clearly discernible by eye when compared with controls. For the galactose induction experiments, midlog phase cultures grown in defined raffinose medium were induced by the addition of 2% galactose for 2 h.
Western blotting
Cells were grown up to midlog phase in defined raffinose medium and induced by the addition of 2% galactose for 2 h. Samples were equalized based on OD600, and extracts were isolated as previously described (Horvath and Riezman, 1994) and resolved by SDS-PAGE. Blots were probed with 9E10 (anti-myc) or B28 (antiyeast actin).
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Acknowledgments |
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This work was supported by the National Institutes of Health (GM39066) to A. Bretscher.
Submitted: 4 December 2002
Revised: 1 May 2003
Accepted: 7 May 2003
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References |
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