Article |
Address correspondence to Francis C. Luca, University of Pennsylvania, School of Veterinary Medicine, 3800 Spruce St., Philadelphia, PA 19104. Tel.: (215) 573-5664. Fax: (215) 573-5188. E-mail: fluca{at}vet.upenn.edu
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key Words: cell cycle; polarized growth; cell separation; mitotic exit network; nuclear export
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Mitotic exit in the budding yeast Saccharomyces cerevisiae is analogous to that of animal cells and involves close coupling of chromosome segregation with actomyosin ring contraction and septum deposition at the motherbud junction. In S. cerevisiae, a regulatory cascade called the mitotic exit network (MEN) appears to play a critical role in coordinating several processes associated with mitotic exit. The MEN is comprised of highly conserved proteins that include the protein kinases Cdc5p, Cdc15p, Dbf2p, and Dbf20p, the phosphatase Cdc14p, the GTPase Tem1p and its GEF Lte1p, and Mob1p, a novel protein that binds Dbf2p and Dbf20p (McCollum and Gould, 2001). This regulatory network inactivates mitotic CDK by turning on a cyclin destruction pathway and activating the CDK inhibitor Sic1p, and promoting the action of Swi5p, a transcription factor necessary for G1 transcription (Visintin et al., 1998; Jaspersen et al., 1999). The MEN also controls the onset of cytokinesis: actomyosin ring contraction and septum deposition do not occur when MEN function is abrogated, even when progress from mitosis to G1 is allowed by artificial suppression of mitotic CDK activity (Lippincott et al., 2001; Luca et al., 2001).
The association of Mob1p with Dbf2p is critical for MEN function and likely permits Dbf2p activation by Cdc15p (Komarnitsky et al., 1998; Lee et al., 2001; Mah et al., 2001). Mob1p and Dbf2p are necessary for late mitotic changes in Cdc14p localization that are critical for its regulatory action (Visintin et al., 1998; Shou et al., 1999; Stegmeier et al., 2002). Other Mob1p- and Dbf2p-related proteins are present in S. cerevisiae, raising the possibility that related or parallel pathways control important aspects of cell division. Mob1p is closely related to the nonessential protein Mob2p (Luca and Winey, 1998), and Dbf2p is very similar to the Cbk1p protein kinase. Intriguingly, two-hybrid analysis suggests that Mob2p associates with Cbk1p (Racki et al., 2000). Cbk1p is important for polarized cell growth and required for final separation of mother and daughter cells, an event that follows cytokinesis and involves degradation of the chitin-rich septum deposited between mother and daughter cells (Racki et al., 2000; Bidlingmaier et al., 2001). Cbk1p was shown to localize to the periphery of the growing bud and to the bud neck during late mitosis. Moreover, Cbk1p was demonstrated to be essential for M/G1 transcription of genes that are regulated by the Swi5-related transcription factor Ace2p, which encode proteins that are required for septum degradation and cell separation, such as Cts1p and Scw11p (Dohrmann et al., 1992; Racki et al., 2000; Bidlingmaier et al., 2001; Doolin et al., 2001). It is therefore possible that Mob2p and Cbk1p function analogously to Mob1p and Dbf2p and are part of a MEN-like signaling cascade that controls morphogenesis and transcription at the end of mitosis.
The transcription factor Ace2p is a likely downstream target for Cbk1p. Ace2p drives the expression of cell separation genes at the end of mitosis, and thus its activity must be coordinated with mitotic exit. Ace2p nuclear import is inhibited by CDK phosphorylation (O'Conallain et al., 1999), as is the related Swi5p transcription factor (Moll et al., 1991); its nuclear export is mediated by Xpo1p, a Crm1-like nuclear exportin (Jensen et al., 2000). Recently, and in parallel to work presented here, Ace2p was reported to exhibit Mob2- and Cbk1p-dependent localization to the daughter cell nucleus at the end of mitosis and to control daughter cellspecific transcription of genes that are necessary for septum degradation (Colman-Lerner et al., 2001). The mechanism by which Ace2p is restricted to the daughter cell nucleus is unknown. It is also not known how the nuclear localization or function of Ace2p is coordinated with mitotic exit. Here, we present findings that implicate complexed Mob2pCbk1p in the coordination of cell morphogenesis, mitotic exit, and nuclear exportmediated daughter cell localization of active Ace2p.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
Yeast cells exposed to cognate mating pheromone arrest in G1 and form mating projections. Mating projection formation requires sustained polarized growth and localized wall remodeling, with secretion and F-actin focused to a small region of the cell cortex (Pruyne and Bretscher, 2000). Chitin is deposited in the cell wall at the base of the mating projection. We found wild-type and ace2 cells capable of producing obvious mating projections, with F-actin focused to a region of polarized growth flanked by chitin deposition (Fig. 2 C). In contrast, neither cbk1
nor mob2
cells sustained actin polarization or formed pronounced mating projections when treated with pheromone (Fig. 2 C). Small chitin-rich cell wall deformations were evident in many cbk1
and mob2
cells, but these generally did not correspond with F-actin polarization (Fig. 2 C,
). Polarized F-actin organization was not completely absent in pheromone-treated cbk1
and mob2
cultures: after 1.5 h of treatment,
30% of mob2
and
35% of cbk1
cells exhibited polarized F-actin compared with
80% of wild-type cells. With extended exposure (3 h), this number dropped to
10 and
15% in cbk1
and mob2
cultures, respectively, whereas
85% of wild-type cells remained polarized. Expression of a pheromone-induced fus1-lacZ reporter (Trueheart et al., 1987), measured as ß-galactosidase activity, was similar in pheromone-treated mob2
cells, cbk1
cells, and wild-type cells (unpublished data). Furthermore, neither budding nor nuclear division occurred with elevated frequency in pheromone-treated mob2
and cbk1
strains relative to wild-type cells (unpublished data). Together, these findings indicate that Mob2p and Cbk1p have a shared function in sustaining polarized growth of the mating projection and that the proteins are not required for cell cycle arrest or transcriptional response upon pheromone treatment.
Mob2p associates with Cbk1p
Two-hybrid experiments have suggested that Mob2p and Cbk1p can interact physically (Racki et al., 2000). To test whether functional Mob2p and Cbk1p interact under more physiologically relevant conditions, we performed coimmunoprecipitation experiments using strains expressing epitope-tagged Cbk1p and Mob2p. CBK1-HA, MOB2-myc, and CBK1-myc, MOB2-HA strains, which express these epitope-tagged proteins instead of native Cbk1p and Mob2p, were phenotypically wild type (unpublished data). We immunoprecipitated tagged Mob2p and Cbk1p from extracts of exponentially growing cells and found that Cbk1p coimmunoprecipitated with Mob2p and vice versa, regardless of affinity tag (Fig. 3 A). Thus, Mob2p and Cbk1p associate as a complex.
|
Cbk1p kinase activity is Mob2p dependent and varies over the cell cycle
In vitro analysis suggests that Dbf2p protein kinase activity requires association with Mob1p (Lee et al., 2001; Mah et al., 2001). To determine if the same is true for Cbk1p and Mob2p, we characterized the in vitro kinase activity of HA- and myc-tagged Cbk1p isolated through both direct immunoprecipitation and in association with HA and myc-tagged Mob2p. Immunoprecipitated Cbk1p phosphorylated the exogenous substrate histone H1 in vitro (Fig. 3 C, filled arrowhead). In addition, a protein of 90 kD was phosphorylated by immunoprecipitated Cbk1p-HA, and a
120-kD species was phosphorylated by immunoprecipitated Cbk1p-myc (Fig. 3 C, open arrowheads). The difference in apparent molecular weight of these species corresponds to the difference between the sizes of the HA and myc epitope tags, suggesting that these bands represent autophosphorylated Cbk1p. We isolated similar kinase activity in association with Mob2p as predicted by coimmunoprecipitation of Mob2p and Cbk1p. Interestingly, Cbk1p immunoprecipitated from mob2
cells had very low in vitro activity (Fig. 3 C). Corresponding immunoblots indicate that similar amounts of Cbk1p were immunoprecipitated from each strain (Fig. 3 A). These findings establish that Mob2p is necessary for Cbk1p activity.
We asked if Mob2pCbk1p interaction or Cbk1p kinase activity varied across the cell cycle. We immunoprecipitated Cbk1p-myc from extracts of CBK1-myc, MOB2-HA cells synchronized in G1 by exposure to mating pheromone and examined Cbk1-mycMob2-HA coimmunoprecipitation and Cbk1p protein kinase activity. Immunoblots revealed no dramatic variation in protein levels of Cbk1p or associated Mob2p (Fig. 3 D). However, the protein kinase activity of immunoprecipitated Cbk1p varied significantly over the cell cycle (Fig. 3 E). In G1-arrested cells (time zero), Cbk1p kinase activity was relatively high. Given that the kinase is required for mating projection formation, this is not unexpected. Cbk1p activity dropped after release from G1 and rose as cells proceeded into the budding cycle. Late in the cell division cycle, when the majority of cells in culture were large budded, Cbk1p kinase activity was maximal. This peak of activity is consistent with Cbk1p control of cell separation, which is among final events of the cell cycle.
Cbk1p kinase activity is required for cell separation and sustained polarized growth
To evaluate the importance of Cbk1p's protein kinase activity in cell separation and polarized growth, we introduced point mutations predicted to affect catalytic activity. A cbk1D475A allele, in which an aspartic acid shown to be essential for catalytic activity was changed to alanine (Hanks and Hunter, 1995), failed to complement the cbk1 phenotype for both cell separation and mating projection formation (Fig. 4 A). To allow for a conditional block of Cbk1p kinase activity, we replaced methionine 429 with alanine, a substitution corresponding to -as2 alleles that render other protein kinases inhibitable by cell permeable compounds that do not effectively inhibit wild-type kinases (Bishop et al., 2000; Weiss et al., 2000). We found that the cbk1-as2 allele complemented cbk1
for cell separation and mating projection formation, although a small number of aggregated cells were present. We then allowed cbk1-as2 and wild-type cells to grow for 3 h in the presence of varying concentrations of the cbk1-as2 inhibitor 1NA-PP1. At 1NA-PP1 concentrations
1 µM in cbk1-as2 cells, mother/daughter separation was blocked. Cell separation was unaffected in similarly treated wild-type cells (Fig. 4 B). As with cbk1D475A cells, inhibitor-treated cbk1-as2 cells were rounder than wild-type cells (Fig. 4 B compared with Fig. 1 A) and formed mating projections poorly (unpublished data). Thus, Cbk1p activity is required for cell separation and polarized growth.
|
Mob2p and Cbk1p colocalize at sites of cell growth, the bud neck, and the daughter cell nucleus
Cbk1p was shown previously to localize to the cell cortex during bud growth and to the bud neck at the end of mitosis (Racki et al., 2000; Bidlingmaier et al., 2001). To investigate the subcellular distribution of Mob2p and its relationship to Cbk1p localization, we expressed COOH-terminally tagged Mob2GFP and Cbk1GFP in live cells. Both Mob2GFP and Cbk1GFP were expressed under the control of their endogenous promoters as the only forms of Mob2p or Cbk1p in these cells. Mob2GFP and Cbk1GFP strains exhibited no obvious growth defects (unpublished data). In pheromone-treated cells, both Mob2GFP and Cbk1GFP were clearly visible at the tips of mating projections, consistent with the proteins' role in morphogenesis (Fig. 5 A, right). Early in the budding cycle, both Mob2GFP and Cbk1GFP localized to incipient bud sites and the cortex of small and medium sized buds (Fig. 5 A, arrowheads). In large budded cells, Mob2GFP and Cbk1GFP strongly localized to a bright band at the bud neck with no apparent bias toward the mother or daughter side.
|
As our report was in preparation, it was reported that Mob2p and Cbk1p localize to bud cortex, bud neck, and daughter cell nucleus in budded cells (Colman-Lerner et al., 2001). However, several of the published images from Colman-Lerner et al. (2001) are ambiguous, particularly with respect to nuclear localization of Mob2p and Cbk1p. It is not clear if this ambiguity is due to poor preservation after fixation, limitations in signal detection, or poor image reproduction. For these reasons, we therefore present our independent analysis of Mob2p and Cbk1p localization in live cells.
Mob2p and Cbk1p are interdependent for localization
To assess the possibility that Cbk1p and Mob2p mediate one another's localization, we examined Mob2GFP localization in cbk1 cells and Cbk1GFP localization in mob2
cells. We found that Mob2GFP fluorescence was uniformly cytoplasmic in cbk1
cells and exhibited no accumulation in nuclei, the cell cortex, or the bud neck (Fig. 6 A), indicating that Mob2p localization is Cbk1p dependent. Similarly, Cbk1p localization was Mob2p dependent. Cbk1GFP was absent from the cell cortex and bud necks in the vast majority of mob2
cells (Fig. 6 B). However, we could detect extremely faint Cbk1GFP at the bud neck in <1% of cells (not depicted). Thus, our data indicate that Mob2p and Cbk1p are interdependent for cortical localization and that nuclear localization of Mob2p requires Cbk1p. In contrast, the nuclear localization of Cbk1p is not strictly Mob2p dependent. Mob2p was required for temporal and spatial restriction of Cbk1p to the daughter nucleus in late M/early G1. Colman-Lerner et al. (2001) also examined interdependency of Mob2pCbk1p localization. However, they concluded that Mob2p localizes to nuclei in cbk1
cells. In our studies of live cells, we never observed this despite examining >500 cells. Moreover, our studies indicate that Cbk1GFP does not localize normally to bud neck and cortex in mob2
cells, in contrast to conclusions drawn by Colman-Lerner et al. (2001).
|
Mob2p and Cbk1p restrict Ace2p to daughter cell nuclei
Cbk1p likely controls Ace2p function (Racki et al., 2000; Bidlingmaier et al., 2001). Using overexpression constructs, Ace2p has been found to shuttle between nucleus and cytoplasm in mother and daughter cells (O'Conallain et al., 1999; Jensen et al., 2000). Using Ace2GFP integrated at the endogenous locus, we found that Ace2p localized specifically to daughter cell nuclei of large budded cells (Fig. 7 A). To assess the cell cycle kinetics of Ace2GFP localization, we synchronized Ace2GFPexpressing cells in S phase using hydroxyurea. Ace2GFP was not readily detectable during and immediately after release from arrest. As cells proceeded into nuclear division, Ace2GFP initially became visible in both mother and daughter nuclei. As the population progressed into late stages of division, Ace2GFP accumulated strongly in daughter nuclei and was not apparent in mother cell nuclei (Fig. 7 B). These findings are in agreement with those of Colman-Lerner et al. (2001), who used strains expressing GFP-tagged Ace2p from low copy plasmids.
|
Mob2p asymmetry is generated by a novel mechanism
The transcription factor Ash1p accumulates in daughter cell nuclei late in cell division and is absent from mother cell nuclei, a localization pattern similar to that of Mob2p, Ace2p, and Cbk1p (Bobola et al., 1996; Sil and Herskowitz, 1996). To determine if Ash1p localization requires MOB2 function, we examined localization of Ash1pGFP in asynchronous mob2 cells. Ash1GFP localized specifically to daughter cell nuclei in mob2
cells (Fig. 8 A). Therefore, Ash1p asymmetry is Mob2p independent. Asymmetric localization of Ash1p was shown to require Myo4p and Bni1pdependent localization of ASH1 mRNA to the daughter cell cortex. We found Mob2GFP localization unaffected in myo4
or bni1
cells (Fig. 8 B). Ace2GFP was also unaffected in myo4
cells (C. Herbert, personal communication). Thus, asymmetric nuclear localization of Mob2p and Ace2p represents a novel mechanism for asymmetric segregation of proteins that ultimately control gene expression.
|
Nuclear localization of Mob2p and Ace2p require Mob1p-mediated MEN signaling
Our findings indicate that Mob2p, Cbk1p, and Ace2p control processes that are executed as cells pass from M to G1, coincident with other events controlled by the MEN (McCollum and Gould, 2001). Mob1p, a Mob2p-related protein, and Cdc14p phosphatase are essential components of the MEN. Cells carrying conditional mob177 or cdc141 alleles arrest in telophase at 34°C due to loss of MEN function (Visintin et al., 1998; Luca et al., 2001). To determine if Mob2p and Ace2p localization require the MEN, we examined their localizations in mob177 and cdc141 cells. Mob2GFP localization was clearly abnormal in mob177 cells arrested by a 23-h shift to 34°C: 65% (n = 90) arrested with Mob2GFP localized diffusely in the cytoplasm, whereas 10% displayed weak Mob2GFP cortical localization and 25% exhibited weak Mob2GFP fluorescence at bud necks (Fig. 9 A). Significantly, neither Mob2GFP nor Ace2GFP localized to nuclei in telophase-arrested mob177 cells. The same is true in cdc141 mutants at restrictive temperature (unpublished data).
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
Our results address an interesting additional layer of Mob2pCbk1pdependent regulation of Ace2p. In wild-type cells, Ace2p accumulates specifically in daughter cell nuclei during late mitosis. This localization pattern was also observed recently by others and found to drive expression of Ace2p-dependent genes specifically in the daughter cell (Colman-Lerner et al., 2001). Also in agreement with recently reported results, we found that deletion of either MOB2 or CBK1 permits distribution of Ace2p to both mother and daughter cell nuclei (Colman-Lerner et al., 2001). Interestingly, however, we found that markedly lower amounts of Ace2p accumulate in nuclei when Cbk1p kinase activity is inhibited. This is not due to a simple two-fold dilution of available Ace2p: the transcription factor accumulates to relatively high levels in both nuclei when its nuclear export is blocked. Therefore, the low levels of Ace2p present in nuclei of cells lacking Cbk1p function likely reflect a steady-state level that is reached in the absence of a signal that causes Ace2p nuclear retention.
In agreement with Colman-Lerner et al. (2001), we found that Mob2p and Cbk1p also accumulate in daughter cell nuclei late in cell division and that this localization is ACE2 dependent. Ace2p carries putative nuclear localization sequences (Jensen et al., 2000), whereas Mob2p and Cbk1p do not. Therefore, it is possible that the three proteins form a complex that is cotransported into nuclei. In cells that lack Mob2p, Cbk1p localizes to both mother and daughter nuclei. Colman-Lerner et al. (2001) concluded that Mob2p localizes to mother and daughter nuclei in cbk1 cells. Contrary to this conclusion, we found that Mob2p clearly does not accumulate in nuclei of cbk1
cells. These findings suggest that Cbk1p and Ace2p are no longer restricted to the daughter bud in mob2
cells and can associate and accumulate in both nuclei. On the other hand, Mob2p likely associates with Ace2p through mutual interaction with Cbk1p.
The role of nuclear transport in Ace2p asymmetry
How might Mob2pCbk1p promote accumulation of Mob2p, Ace2p, and Cbk1p in daughter cell nuclei, with concomitant disappearance of the transcription factor from mother cell nuclei? Our results indicate that localization of these proteins is separate from Myo4pBni1pmediated sequestration of ASH1 mRNA to the daughter cell cortex, the only previously described pathway for asymmetric partitioning of cell fate determinants in budding yeast (Bobola et al., 1996; Sil and Herskowitz, 1996). Specific localization of Ace2p to daughter cell nuclei could be achieved through regulation of nuclear entry or exit or through degradation of the protein in the mother cell nucleus. Dephosphorylation of three conserved CDK consensus sites on Ace2p permits the protein's nuclear import at the end of mitosis (O'Conallain et al., 1999). Ace2p nuclear export requires Xpo1p, a Crm1-related exportin family protein (Jensen et al., 2000). We found that Ace2p accumulates abundantly in both mother and daughter nuclei when its nuclear export is blocked. Therefore, asymmetric Ace2p accumulation is most likely attributable to suppression of its nuclear export in the daughter cell, rather than differential nuclear import or stability. We propose that Mob2p and Cbk1p mediate suppression of Ace2p nuclear export. Furthermore, we suggest that sequestration of active Mob2pCbk1p to the growing daughter cell is ultimately responsible for biased accumulation of Ace2p in daughter nuclei.
Mob2pCbk1p may suppress Ace2p export from daughter cell nuclei by various mechanisms. It is unlikely that Mob2pCbk1p kinase inhibits Ace2p nuclear export by direct inhibition of Xpo1p, since Xpo1p is an essential protein required for a wide range of nuclear export (Stade et al., 1997). Instead, we favor the idea that Mob2pCbk1pmediated phosphorylation of Ace2p inhibits Ace2p nuclear export, perhaps by making Ace2p unable to interact with nuclear export machinery. Alternately, Mob2pCbk1pmediated phosphorylation might promote anchoring of Ace2p to fixed objects within the nucleus. We found that Ace2p accumulation in nuclei is much lower when Cbk1p kinase activity is inhibited, suggesting that mere association of Mob2Cbk1p with Ace2p does not block Ace2p nuclear export. Ace2p localization is analogous to that of the transcription factor prospero, which determines ganglion mother cell fate during Drosophila nervous system development (Spana and Doe, 1995). Interestingly, prospero contains a domain that antagonizes the protein's nuclear export in cis, possibly through nuclear export sequence masking (Demidenko et al., 2001). Evaluation of Cbk1p phosphorylation of Ace2p will be an important step toward determining if Ace2p nuclear traffic is controlled in an analogous manner.
Mitotic exit network coordination of Mob2p and Ace2p localization
Septum destruction occurs in earnest only after completion of cytokinesis, reflecting an important temporal coordination of events that occur as cells pass from mitosis into G1. The final events of cell division are controlled by the MEN, a conserved signaling pathway that links initiation of cytokinesis with the completion of nuclear division (McCollum and Gould, 2001). We have shown that Mob2p and Ace2p do not localize to the nucleus when MEN signaling is blocked by mob177 or cdc141 conditional loss-of-function alleles. More significantly, a partial bypass of mitotic exit block that allows progression into G1 without cytokinesis does not restore nuclear localization of these proteins. Therefore, MEN signaling may directly control Mob2pCbk1p's ability to promote nuclear localization and activity of Ace2p. Alternatively, since MEN signaling is required for initiation of cytokinesis, Mob2pCbk1p activation may be linked to the actual progress of actomyosin ring contraction and septum deposition. Importantly, cortical and bud neck localization of Mob2p are not strictly dependent on MEN signaling but rather are promoted by progression into subsequent cell cycle stages. These findings further support distinct and separable roles for Mob2pCbk1p complex in cortical remodeling and transcriptional control.
The role of Mob2pCbk1p in cell surface remodeling
Mob2pCbk1p has an important Ace2p-independent role in cell cortex remodeling and polarized growth. Cells lacking Mob2pCbk1p function do not sustain polarized growth during budding or mating projection formation, although polarity establishment is not abrogated. The presence of Mob2p and Cbk1p at sites of active cell growth, such as the tips of buds and mating projections, suggest that the proteins perform some function critical for ongoing cortical expansion at these sites. Bud neck localization of Mob2pCbk1p late in cell division is consistent with a function in cortical remodeling; this is a site of active cell wall reorganization as cells undergo cytokinesis and cell separation. In contrast to conclusions drawn elsewhere (Colman-Lerner et al., 2001), we found that Mob2p and Cbk1p are clearly interdependent for proper localization to cortical sites. Interestingly, although Cbk1p's kinase activity is required both for maintenance of polarized growth and septum destruction, suppression of Cbk1p kinase activity does not disrupt Mob2p localization to the bud cortex and bud neck. Therefore, recruitment of Mob2pCbk1p to sites of cell wall remodeling and polarized growth is likely independent of Cbk1p kinase activity.
The relevant targets of Mob2pCbk1p at the cell cortex are not known presently. However, Mob2pCbk1p localizes similarly to other components of cell growth machinery, such as the exocyst complex, which directs the targeting of secretory vesicles to the cell cortex (TerBush et al., 1996; Finger and Novick, 1998), and the ß-1,3-glucan synthase complex, which is important in cell wall construction (Qadota et al., 1996). These complexes are important for polarized cell morphogenesis and are therefore candidates for regulation by Mob2pCbk1p.
Our results indicate that Cbk1p itself may be regulated by phosphorylation and that this control requires Mob2p. Interestingly, the Mob2p-related protein Mob1p is required for regulatory phosphorylation of the Cbk1p-related kinase Dbf2p, most likely by the kinase Cdc15p (Mah et al., 2001). It is not presently clear how Mob2p-dependent phosphorylation of Cbk1p influences the kinase's function. It is plausible that Mob2pCbk1p acts within a regulatory pathway organized similarly to the MEN that coordinates events at the cell cortex with cell cycle progression and transcriptional control. Given the conservation of Mob1p-like proteins and Dbf2/Cbk1p kinases, our findings may anticipate a role for related proteins in linking the regulation of cell morphology and cell cycle transitions with cell fate determination and development.
![]() |
Materials and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
Protein and immunological techniques
Yeast lysis buffers, wash buffers, and kinase assay conditions were as described previously (Mah et al., 2001) with the addition of 10% glycerol to lysis and wash buffers. To prepare lysates for immunoprecipitations, 20 OD600 equivalents of log phase cells were resuspended in 2.5 ml lysis buffer plus 5 ml glass beads in 15 ml conical tubes and vortexed on ice eight times for 30 s. Lysates were clarified by centrifugation, assayed for protein concentration using the Bradford method (Bradford, 1976), adjusted to 8 mg/ml total protein, and preincubated 30 min with 100 µl protein A beads. 25 µg of either anti-HA (14b11; Covance) or anti-myc (9E10) monoclonal antibody was added to 1 ml lysate and incubated on ice, then added to 100 µl of protein A beads (Amersham Biosciences) and rotated 1 h at 4°C. Washing of beads, preparation for kinase assays, SDS-PAGE, and immunoblotting were performed as described previously (Mah et al., 2001). Immunoblots were probed with appropriate primary antibody and detected by sheep antimouse peroxidase-conjugated secondary antibody and ECL (Amersham Biosciences). Nonreducing sample buffer was used for Western blot analysis of Mob2p-myc. Kinase assays were quantified using a PhosphorImager (Molecular Dynamics).
Cytology, microscopy, and image analysis
Staining of F-actin with rhodamine-phalloidin, staining of chitin with calcofluor, and FITC-ConA pulse labeling were performed as described (Tkacz and Lampen, 1972; Pringle, 1991). Fluorescence/DIC microscopy and image acquisition were either performed as described previously (Luca et al., 2001) or using a Nikon Eclipse TE300 fluorescence microscope equipped with a Hamamatsu ORCA CCD camera. Cell wall digestion was performed on formaldehyde-fixed cells by treatment with 0.1 mg/ml zymolyase (ICN Biomedicals) in PBS. In scoring cell separation defects upon inhibitor treatment, cells sonicated for 2 s at low power were allowed to grow in indicated conditions, fixed in 5% formaldehyde, and calcofluor stained. Each cluster of more than two attached cell bodies was counted as cell separation failure.
![]() |
Footnotes |
---|
* Abbreviations used in this paper: CDK, cyclin-dependent kinase; MEN, mitotic exit network.
![]() |
Acknowledgments |
---|
This work was supported by National Institutes of Health grant RO1 GM60575 to F.C. Luca., National Institutes of Health grant RO1 GM50399 to D.G. Drubin, and National Institutes of Health grant RO1 AI44009 to K. Shokat. E.L. Weiss is supported as a Research Special Fellow of the Leukemia and Lymphoma Society.
Submitted: 20 March 2002
Revised: 25 June 2002
Accepted: 9 July 2002
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Bi, E., P. Maddox, D.J. Lew, E.D. Salmon, J.N. McMillan, E. Yeh, and J.R. Pringle. 1998. Involvement of an actomyosin contractile ring in Saccharomyces cerevisiae cytokinesis. J. Cell Biol. 142:13011312.
Bidlingmaier, S., E.L. Weiss, C. Seidel, D.G. Drubin, and M. Snyder. 2001. The Cbk1p pathway is important for polarized cell growth and cell separation in Saccharomyces cerevisiae. Mol. Cell. Biol. 21:24492462.
Bobola, N., R.P. Jansen, T.H. Shin, and K. Nasmyth. 1996. Asymmetric accumulation of Ash1p in postanaphase nuclei depends on a myosin and restricts yeast mating-type switching to mother cells. Cell. 84:699709.[Medline]
Colman-Lerner, A., T.E. Chin, and R. Brent. 2001. Yeast Cbk1 and Mob2 activate daughter-specific genetic programs to induce asymmetric cell fates. Cell. 107:739750.[Medline]
Demidenko, Z., P. Badenhorst, T. Jones, X. Bi, and M.A. Mortin. 2001. Regulated nuclear export of the homeodomain transcription factor Prospero. Development. 128:13591367.
Dohrmann, P.R., G. Butler, K. Tamai, S. Dorland, J.R. Greene, D.J. Thiele, and D.J. Stillman. 1992. Parallel pathways of gene regulation: homologous regulators SWI5 and ACE2 differentially control transcription of HO and chitinase. Genes Dev. 6:93104.[Abstract]
Finger, F.P., and P. Novick. 1998. Spatial regulation of exocytosis: lessons from yeast. J. Cell Biol. 142:609612.
Guthrie, C., and G.R. Fink. 1991. Guide to yeast genetics and molecular biology. In Methods Enzymol. Vol. 194. 1933.
Hanks, S.K., and T. Hunter. 1995. Protein kinases 6. The eukaryotic protein kinase superfamily: kinase (catalytic) domain structure and classification. FASEB J. 9:576596.
Jensen, T.H., M. Neville, J.C. Rain, T. McCarthy, P. Legrain, and M. Rosbash. 2000. Identification of novel Saccharomyces cerevisiae proteins with nuclear export activity: cell cycle-regulated transcription factor ace2p shows cell cycle-independent nucleocytoplasmic shuttling. Mol. Cell. Biol. 20:80478058.
Komarnitsky, S.I., Y.C. Chiang, F.C. Luca, J. Chen, J.H. Toyn, M. Winey, L.H. Johnston, and C.L. Denis. 1998. DBF2 protein kinase binds to and acts through the cell cycle-regulated MOB1 protein. Mol. Cell. Biol. 18:21002107.
Lippincott, J., K.B. Shannon, W. Shou, R.J. Deshaies, and R. Li. 2001. The Tem1 small GTPase controls actomyosin and septin dynamics during cytokinesis. J. Cell Sci. 114:13791386.
Luca, F.C., and M. Winey. 1998. MOB1, an essential yeast gene required for completion of mitosis and maintenance of ploidy. Mol. Biol. Cell. 9:2946.
Luca, F.C., M. Mody, C. Kurischko, D.M. Roof, T.H. Giddings, and M. Winey. 2001. Saccharomyces cerevisiae Mob1p is required for cytokinesis and mitotic exit. Mol. Cell. Biol. 21:69726983.
Mah, A.S., J. Jang, and R.J. Deshaies. 2001. Protein kinase Cdc15 activates the Dbf2-Mob1 kinase complex. Proc. Natl. Acad. Sci. USA. 98:73257330.
Moll, T., G. Tebb, U. Surana, H. Robitsch, and K. Nasmyth. 1991. The role of phosphorylation and the CDC28 protein kinase in cell cycle-regulated nuclear import of the S. cerevisiae transcription factor SWI5. Cell. 66:743758.[Medline]
Pringle, J.R. 1991. Staining of bud scars and other cell wall chitin with calcofluor. Methods Enzymol. 194:732735.[Medline]
Pruyne, D., and A. Bretscher. 2000. Polarization of cell growth in yeast. I. Establishment and maintenance of polarity states. J. Cell Sci. 113(Pt 3):365375.
Qadota, H., C.P. Python, S.B. Inoue, M. Arisawa, Y. Anraku, Y. Zheng, T. Watanabe, D.E. Levin, and Y. Ohya. 1996. Identification of yeast Rho1p GTPase as a regulatory subunit of 1,3-beta-glucan synthase. Science. 272:279281.[Abstract]
Racki, W.J., A.M. Becam, F. Nasr, and C.J. Herbert. 2000. Cbk1p, a protein similar to the human myotonic dystrophy kinase, is essential for normal morphogenesis in Saccharomyces cerevisiae. EMBO J. 19:45244532.
Rappaport, R. 1996. Cytokinesis in Animal Cells. Cambridge University Press, Cambridge, U.K. 386 pp.
Sil, A., and I. Herskowitz. 1996. Identification of asymmetrically localized determinant, Ash1p, required for lineage-specific transcription of the yeast HO gene. Cell. 84:711722.[Medline]
Spana, E.P., and C.Q. Doe. 1995. The prospero transcription factor is asymmetrically localized to the cell cortex during neuroblast mitosis in Drosophila. Development. 121:31873195.
Spellman, P.T., G. Sherlock, M.Q. Zhang, V.R. Iyer, K. Anders, M.B. Eisen, P.O. Brown, D. Botstein, and B. Futcher. 1998. Comprehensive identification of cell cycle-regulated genes of the yeast Saccharomyces cerevisiae by microarray hybridization. Mol. Biol. Cell. 9:32733297.
Stegmeier, F., R. Visintin, and A. Amon. 2002. Separase, polo kinase, the kinetochore protein Slk19, and Spo12 function in a network that controls Cdc14 localization during early anaphase. Cell. 108:207220.[Medline]
TerBush, D.R., T. Maurice, D. Roth, and P. Novick. 1996. The Exocyst is a multiprotein complex required for exocytosis in Saccharomyces cerevisiae. EMBO J. 15:64836494.[Abstract]
Trueheart, J., J.D. Boeke, and G.R. Fink. 1987. Two genes required for cell fusion during yeast conjugation: evidence for a pheromone-induced surface protein. Mol. Cell. Biol. 7:23162328.[Medline]
Weiss, E.L., A.C. Bishop, K.M. Shokat, and D.G. Drubin. 2000. Chemical genetic analysis of the budding-yeast p21-activated kinase Cla4p. Nat. Cell Biol. 2:677685.[CrossRef][Medline]