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EMBO J. Sep 15, 2003; 22(18): 4779–4793.
PMCID: PMC212726

The XMAP215 homologue Stu2 at yeast spindle pole bodies regulates microtubule dynamics and anchorage

Abstract

The yeast protein Stu2 belongs to the XMAP215 family of conserved microtubule-binding proteins which regulate microtubule plus end dynamics. XMAP215-related proteins also bind to centrosomes and spindle pole bodies (SPBs) through proteins like the mammalian transforming acidic coiled coil protein TACC or the yeast Spc72. We show that yeast Spc72 has two distinct domains involved in microtubule organization. The essential 100 N-terminal amino acids of Spc72 interact directly with the γ-tubulin complex, and an adjacent non-essential domain of Spc72 mediates binding to Stu2. Through these domains, Spc72 brings Stu2 and the γ-tubulin complex together into a single complex. Manipulation of Spc72–Stu2 interaction at SPBs compromises the anchorage of astral microtubules at the SPB and surprisingly also influences the dynamics of microtubule plus ends. Permanently tethering Stu2 to SPBs by fusing it to a version of Spc72 that lacks the Stu2-binding site in part complements these defects in a manner which is dependent upon the microtubule-binding domain of Stu2. Thus, the SPB-associated Spc72–Stu2 complex plays a key role in regulating microtubule properties.

Keywords: centrosomes/microtubule dynamics/Spc72/Stu2/TACC

Introduction

Microtubules (MTs) are hollow cylinders that assemble from a heterodimer of α- and β-tubulin. MTs are inherently polar structures, so that the two ends of an MT are different. Only the MT minus ends are attached to MT organizing centres such as the centrosome of vertebrate cells and the spindle pole body (SPB) of yeast. MT plus ends are directed away from the centrosome towards the cell periphery. MTs are highly dynamic structures and the ends switch from states of growing to shrinking (called catastrophe) and from shrinking to growing (rescue). The plus ends of MTs are more dynamic than the minus ends (Howard and Hyman, 2003).

When measured in vivo, MTs are more dynamic than MTs assembled in vitro from pure tubulin, due to the presence of regulatory factors. Members of the conserved family of MT-binding proteins named Stu2 in budding yeast, XMAP215 in Xenopus, mini-spindles (Msps) in Drosophila, Dis1 and Alp14 in Schizosaccharomyces pombe, DdCP224 of Dictyostellium and the colonic-hepatic tumour-overexpressed gene (ch-TOG) in mammalian cells represent proteins that regulate MT dynamics (Nabeshima et al., 1995; Wang and Huffaker, 1997; Gräf et al., 2000).

XMAP215-like proteins regulate MT function by a number of mechanisms. One place of action is the MT plus end. Work in Xenopus extract has shown that XMAP215 stabilizes MT plus ends by opposing the destabilizing activity of the kinesin XKCM1 (Tournebize et al., 2000). In contrast, studies using stabilized MTs identified XMAP215 as an MT-destabilizing factor (Shirasu-Hiza et al., 2003). XMAP215-like proteins also bind to centrosomes and SPBs independently of MTs. In Drosophila, the centrosomal protein D-TACC, named after the human counterpart of the transforming acidic coiled coil (TACC) protein, mediates binding of Msps to centrosomes (Gergely et al., 2000; Lee et al., 2001). If D-TACC levels are reduced, Msps does not efficiently concentrate at centrosomes and the centrosomal MTs are destabilized (Lee et al., 2001). The interpretation of this result is complicated by the fact that Msps and D-TACC are also present at MT plus ends. The three human TACC proteins target ch-TOG to centrosomes and TACC-3 has a role in stabilizing centrosomal MTs (Gergely et al., 2003). A number of studies suggest that defects in ch-TOG and the three TACC proteins may lead to cancer; however, the function of these proteins in mammalian cells is poorly understood (Raff, 2002).

The budding yeast Stu2 is associated with SPBs, kinetochores, nuclear and astral MTs, whereas Stu2 probably fulfils diverse functions (Wang and Huffaker, 1997; Chen et al., 1998; He et al., 2001). It is not surprising, therefore, that total depletion of Stu2 leads to a mixture of phenotypes including fewer and less dynamic astral MTs, cell cycle arrest and a failure to elongate the mitotic spindle (Kosco et al., 2001; Severin et al., 2001). Although recent evidence suggests that Stu2 is a plus end-binding MT destabilizer (Kosco et al., 2001; van Breugel et al., 2003), the function of Stu2 at SPBs is not understood. It is only known that Stu2 interacts with the SPB component Spc72 (Chen et al., 1998).

The SPB is a large multi-layered structure that is embedded in the nuclear envelope throughout the cell cycle. The cytoplasmic side of the SPB organizes the astral MTs involved in nuclear positioning, whereas the SPB nuclear side assembles the nuclear MTs that have a role in chromosome segregation. The protein Spc72 is only associated with the cytoplasmic side of the SPB. The C-terminus of Spc72 targets the protein to this side of the SPB through binding to the SPB components Nud1 and Kar1 (Pereira et al., 1999; Gruneberg et al., 2000; Schaerer et al., 2001). Besides Nud1, Kar1 and Stu2, Spc72 also interact with the yeast γ-tubulin complex, named the Tub4 complex. Spc72 recruits the Tub4 complex to the cytoplasmic face of the SPB, thereby mediating nucleation of astral MTs (Knop and Schiebel, 1998).

The little knowledge we have of the function played by XMAP215-like proteins at centrosomes and SPBs is due in large part to the lack of mutants that specifically affect the centrosome/SPB-associated function while leaving the other functions unaffected. Here, we show that the Tub4 complex and Stu2 bind to adjacent but distinct domains in Spc72. Stu2 and the Tub4 complex are tethered together through their independent associations with Spc72, and they cooperate in organizing astral MTs. Analysis of a SPC72 mutant that fails to bind Stu2 demonstrates that SPB-associated Stu2 is required for astral MTs anchorage and regulation of MT dynamics.

Results

The 35 most C-terminal amino acids of Stu2 confer binding to Spc72 and astral MT plus ends

In order to understand how Stu2 binds to SPBs, we investigated which domain of Stu2 confers binding to the SPB component Spc72. In the yeast two-hybrid system, the 35 most C-terminal amino acids of Stu2 (Stu2853–888) were sufficient to mediate interaction with Spc72 (Figure 1A, lane 3). Consistently, deletion of C-terminal amino acids of Stu2 (Stu21–855) completely abolished interaction with Spc72 (Figure 1A, lane 2). The Stu21–855 two-hybrid construct was functional, since it interacted strongly with STU2648–888 (Figure 1A). The two-hybrid data were confirmed by an in vitro approach. The C-terminal truncated Stu21–855 from insect cells had only a weak interaction with recombinant Escherichia coli-expressed N-Spc72 (Figure 1B, lane 6), whereas Stu2 showed strong interaction (lane 4). Thus, amino acids 853–888 of Stu2 are essential for Spc72 binding.

figure cdg459f1
Fig. 1. The 35 C-terminal amino acids of Stu2 mediate binding to Spc72. (A) The 35 C-terminal amino acids of Stu2 interact with Spc72 in the yeast two-hybrid system. The domain structure of Stu2 is shown. The MT-binding domain is from amino acids ...

We then asked whether the Spc72-interaction domain of Stu2 is essential for viability and whether STU21–855 cells are specifically defective in Stu2 binding to SPBs. Using a PCR approach, the chromosomal STU2 was terminated after codon 855. Although STU21–855 cells were viable when grown between 14–37°C (data not shown), cells showed nuclear and astral MT defects (see Supplementary figure 1 available at The EMBO Journal Online). Moreover, localization studies showed that the Stu21–855 protein not only failed to bind to SPBs but also associated with strongly reduced efficiency (79.6% in wild-type and 16.7% in STU21–855 cells) with MT plus ends (see Supplementary figure 2C). Thus, the astral MT defect of STU21–855 cells is likely to arise from the failure of Stu2 to function at both ends of astral MTs.

Distinct domains of Spc72 facilitate interaction with the fully assembled Tub4 complex and with Stu2

The requirement for the C-terminal 35 amino acids of Stu2 for its function at both the plus and minus ends of astral MTs probably explains why we failed to obtain STU2 mutants with defects in only one of the two activities. Based on two-hybrid and co-immunoprecipitation experiments, it was suggested that Spc72 interacts with the Tub4 complex and Stu2 (Chen et al., 1998; Knop and Schiebel, 1998; Souès and Adams, 1998). If Stu2 and the Tub4 complex bind to distinct regions of Spc72, it should be possible to construct SPC72 mutants in which the association with Stu2 is defective yet the interaction with the Tub4 complex is unaffected. We therefore mapped the binding sites of Spc72 for the Tub4 complex subunit Spc98 (Geissler et al., 1996) and Stu2.

As reported previously (Chen et al., 1998; Knop and Schiebel, 1998), full-length Spc72 interacted with Spc98 and Stu2 in the two-hybrid system (Figure 2A, lane 1). Interaction of Stu2 with Spc72 was relatively weak, which is explained by the recruitment of the Stu2 construct to MTs. Analysis of Spc72 subfragments then showed that the C-terminal Spc72231–622 fragment (Spc72SPB), which interacts with the SPB components Nud1 and Kar1 by two-hybrid (Pereira et al., 1999; Gruneberg et al., 2000), failed to bind to Spc98 and Stu2 (lane 7). This suggests that the N-terminus of Spc72 mediates interaction with Spc98 and Stu2. Indeed, the 267 N-terminal amino acids of Spc72 (N-Spc72) showed two-hybrid interactions with Spc98 and Stu2 (lane 4). N-terminal deletion constructs of Spc72 were then used to address whether Spc98 and Stu2 bind to distinct or overlapping regions of Spc72. Spc72 constructs lacking amino acids 116–230 (Spc72ΔStu2*, line 2) or 176–230 (Spc72ΔStu2, line 3) interacted with Spc98 but not Stu2. Similarly, Spc721–116 (N-Spc72ΔStu2, line 6) bound to Spc98 but not Stu2. In contrast, Spc7299–267 (N-Spc72ΔTub4, line 5) only interacted with Stu2 but not Spc98. These data suggest that the N-terminus of Spc72 carries distinct binding sites for the Tub4 complex and Stu2.

figure cdg459f2afigure cdg459f2b
Fig. 2. The N-terminus of Spc72 has distinct binding sites for Stu2 and the Tub4 complex. (A) Two-hybrid interactions of STU2 and SPC98 with indicated fragments of SPC72. Abbreviations are as in Figure 1A. (B) Anti-HA immunoprecipitations ...

Immunoprecipitation experiments were performed to confirm the two-hybrid data. Co-immunoprecipitation of Tub4 complex subunits with Spc72 (Figure 2B, lane 6), Spc72ΔStu2* (lane 8) and Spc72ΔStu2 (data not shown) but not with Spc7299–622 (Spc72ΔTub4, lane 12) was consistent with the notion that the Tub4 complex binding site of Spc72 resided in the 100 N-terminal amino acids and that deletion of the Stu2-binding domain did not affect Tub4 complex binding to Spc72. Moreover, confirming that Spc72ΔStu2 fails to associate with Stu2 in vivo, Stu2-3HA co-immunoprecipitated Spc72-9Myc (Figure 2C, lane 6) but not Spc72ΔStu2-9Myc (lane 8).

Although the two-hybrid and immunoprecipitation experiments clearly demonstrated that the Tub4 complex and Stu2 interacted with Spc72, it remained unclear whether these interactions were direct or indirect. In vitro binding experiments were performed to show direct binding of the Tub4 complex and Stu2 to Spc72. Reconstituted Tub4 complex from insect cells (Vinh et al., 2002) interacted strongly with recombinant N-Spc72 (Figure 2D, lane 6). In contrast, Spc72 association with individual subunits (Spc97 and Spc98) and heterodimers (Spc97–Tub4 and Spc98–Tub4) was much weaker (lanes 7-12). Thus, only the fully assembled Tub4 complex binds with high efficiency to Spc72. When N-Spc72 subfragments were tested, Stu2 but not the Tub4 complex interacted with N-Spc72ΔTub4 (Figure 2E, lane 5). In contrast, only the Tub4 complex but not Stu2 bound to N-Spc72ΔStu2 (lane 4). These binding data suggest that the N-terminal 100 amino acids of Spc72 interact directly with the Tub4 complex and amino acids 99–267 of Spc72 with Stu2. The Stu2-binding site is not required for Tub4 complex binding and vice versa.

The Tub4 complex but not the Stu2-binding region of Spc72 is essential

In our S288c strain background SPC72 is an essential gene (Knop and Schiebel, 1998). This allowed us to address whether the Stu2 or Tub4 complex binding domains of Spc72 were required for viability. Cells from which the chromosomal SPC72 was deleted and which were kept alive by SPC72 on a URA3-based plasmid were transformed with LEU2-based plasmids containing SPC72 derivatives. Cells were then tested for growth on 5-FOA. As the 5-FOA selects against the URA3-based SPC72 plasmid, the LEU2-based SPC72 construct was the only source of SPC72 activity in the 5-FOA resistant colonies. Despite its expression, SPC72ΔTub4 failed to support growth on 5-FOA plates (Figure 3A, sector 3). Only after long incubation did a few, poor-growing colonies expressing SPC72ΔTub4 but not SPC72 appear (Figure 3B, lane 2). Similarly SPC72176–622 cells lacking the Tub4 complex and part of the Stu2-binding regions of Spc72 were unable to grow on 5-FOA (sector 4). Thus, the Tub4 complex binding region of Spc72 is essential for viability. However, the Tub4 complex binding domain by itself was not sufficient to support viability (sectors 7 and 8). Therefore, this domain has to be attached to the C-terminal SPB targeting region of Spc72 (Knop and Schiebel, 1998) in order to fulfil its essential function. Finally, both SPC72ΔStu2* and SPC72ΔStu2 constructs allowed growth on 5-FOA (Figure 3A, sectors 5 and 6, 5-FOA), indicating that the Stu2-binding domain of Spc72 is non-essential. Together, the Tub4 but not the Stu2-binding domain of Spc72 is required for cell viability.

figure cdg459f3
Fig. 3. Only the Tub4 complex binding domain but not the Stu2-binding site of Spc72 is essential for viability. (AΔspc72 pRS316-SPC72 cells were transformed with the indicated LEU2-based plasmids. Transformants were grown on YPD and ...

Spc72ΔStu2 is specifically defective in Stu2 interaction at SPBs

Spc72ΔStu2 was characterized to ensure that it only affected the interaction between Spc72 and Stu2 and not the association of Stu2 with the plus ends of astral MTs. The functional Stu2–4GFP localized in SPC72 wild-type (Figure 4A, arrow head) and SPC72ΔStu2 cells (Figure 4B, arrow) to astral MT plus ends. Analysis of the Stu2–4GFP signal intensity then revealed that Stu2 binding to astral MT plus ends was not altered in SPC72ΔStu2 cells compared with SPC72 wild-type cells (Figure 4C). Thus, the binding of Stu2 to astral MT plus ends is not affected by the SPC72ΔStu2 mutation.

figure cdg459f4
Fig. 4. Stu2 binds to the plus ends of astral MTs of SPC72ΔStu2 cells. Wild-type (A) and SPC72ΔStu2 cells (B) with STU2-4GFP CFP-TUB1 were analysed for Stu2 localization by fluorescence microscopy. The arrowhead in (A) ...

D-TACC, the functional homologue of Spc72, and Msps interact not only at centrosomes but also at MT plus ends (Lee et al., 2001). A similar interaction of Spc72 and Stu2 at astral MT plus ends would complicate the interpretation of the SPC72ΔStu2 phenotype. Previous localization studies using a SPC72-GFP gene fusion may have missed a weak Spc72 signal at astral MT plus ends (Chen et al., 1998; Knop and Schiebel, 1998). We therefore re-investigated the localization of the fully functional Spc72–4GFP in CFP-TUB1 cells. Under conditions that allowed detection of Stu2–4GFP protein at astral MT plus ends (Figure 4A), it was only detected at SPBs but not along astral MTs or MT plus ends (Figure 4D). Spc72ΔStu2 behaved as Spc72 (data not shown). Thus, Stu2 and Spc72 only co-localize at SPBs. It is likely, therefore, that the SPC72ΔStu2 mutation only affects Stu2 at SPBs but not astral MT plus ends.

Astral MTs detach from the SPB of SPC72ΔStu2 cells

The phenotype of SPC72ΔStu2 cells was determined to address the function of Stu2 at SPBs. Because astral MT behaviour is cell cycle dependent, we analysed cells in different phases of the cell cycle (Carminati and Stearns, 1997; Vogel et al., 2001). Wild-type SPC72 and SPC72ΔStu2 cells were synchronized in the G1 phase of the cell cycle by α-factor block and release. In G1, SPBs of SPC72ΔStu2 and SPC72 cells had similar numbers of MTs (Figure 5A). A more detailed analysis by time-lapse video microscopy, however, showed that astral MTs detached from the SPB of SPC72ΔStu2 (Figure 5E, arrows) but not SPC72 cells (data not shown). Thus, binding of Stu2 to Spc72 is important for the anchoring of astral MTs to SPBs.

figure cdg459f5afigure cdg459f5b
Fig. 5. The Stu2-binding site of Spc72 fulfils distinct functions during the cell cycle. (AD) Astral MT number and morphology of synchronized SPC72 wild-type and SPC72ΔStu2 cells with GFP-TUB1 were analysed throughout the cell ...

Astral MT defects were also observed in pre-anaphase cells. Twenty-three per cent of pre-anaphase SPC72ΔStu2 cells showed astral MTs that were disconnected from the SPB (Figure 5B, arrow). Time-lapse analysis revealed that these MTs were initially organized from the SPB but subsequently detached (see Supplementary figure 3A). Moreover, 60% of SPC72ΔStu2 cells either lacked (Figure 5B, arrow head) or had fewer astral MTs than SPC72 cells (Figure 5B). These astral MT defects resulted in spindles that were no longer oriented along the mother–bud axis in around 80% of SPC72ΔStu2 cells (Figure 5B, arrow head).

SPC72ΔStu2 cells in anaphase did not show detached astral MTs. This either means that the detachment of astral MT is restricted to pre-anaphase cells or that detached astral MTs of anaphase cells are highly unstable. Despite the lack of MT detachment, astral MTs of anaphase SPC72ΔStu2 cells were far from normal. Instead, the SPC72ΔStu2 mutation affected the two astral MT bundles organized of the two SPBs in a different and distinct manner. There was only a slight reduction in the number of astral MTs associated with the SPB, which was located within the bud (Figure 5C). However, in 40–50% of these cells, the MTs were about 10 times longer and curved around the bud tip back into the mother cell (Figure 5C, arrows). Time-lapse video microscopy analysis of these SPC72ΔStu2 cells showed that the astral MTs extended normally into the bud at the beginning of anaphase (Figure 5F, 0–285 s, arrows). Instead of the normal depolymerization that is seen in wild-type cells (Carminati and Stearns, 1997), these bud proximal astral MTs of SPC72ΔStu2 cells did not depolymerize, but slid along the bud cortex as anaphase progressed. In contrast, the mother ward directed SPBs of SPC72ΔStu2 cells were not associated with elongated astral MTs. In fact, these SPBs were more often devoid of detectable astral MTs than the bud-ward directed SPB (Figure 5D; 20% versus 45%). In summary, the Stu2-binding domain of Spc72 fulfils multiple functions during the cell cycle. It is required for the anchorage of astral MTs to SPBs in pre-anaphase cells, for formation of sufficient astral MTs throughout the cell cycle, and for depolymerization of astral MTs in late anaphase.

Altered astral MTs dynamics of SPC72ΔStu2 cells

The dynamic properties of astral MTs of pre-anaphase SPC72 and SPC72ΔStu2 cells were determined from time-lapse data (see Supplementary figure 3B). Although the astral MTs of pre-anaphase SPC72ΔStu2 cells depolymerized at the same rate as seen in wild-type SPC72 cells (Table I), their growth rate was moderately increased by a factor of 1.3. The most significant change was the 1.5- and 2.3-fold reduction in catastrophe and rescue frequencies, respectively (Table I). These changes resulted in more continuous growth and shrinkage of astral MTs of SPC72ΔStu2 cells (see Supplementary figure 3B). Thus, Stu2 binding to Spc72 at the SPB has a significant and important impact upon the dynamics of astral MTs.

Table I.
Astral microtubule dynamics

Stu2, the Tub4 complex and Spc72 assemble into a complex and cooperate

Both the Tub4 complex and Stu2 are recruited to the cytoplasmic side of the SPB by binding to Spc72 (Chen et al., 1998; Knop and Schiebel, 1998), where they have important roles in astral MT organization (Figures (Figures33 and 5). The close spatial link of the two binding sites in Spc72 (Figure 2) suggests that the Tub4 complex and Stu2 may, in a large complex, cooperate in astral MT organization. To address this possibility, we first asked whether the Tub4 complex and Stu2 are present in common complexes. In such a case, the Tub4 complex subunit Spc97 would be expected to co-immunoprecipitate with Stu2. An anti-HA immunoprecipitate from cells expressing HA-tagged Spc97 contained not only Spc97-3HA but also the Tub4 complex components Spc98 and Tub4, Spc72 and Stu2-3Myc independently of whether logarithmically growing or G1 arrested (α-factor) cells were used (Figure 6A). Thus, the Tub4 complex, Spc72 and Stu2 are part of a larger complex.

figure cdg459f6
Fig. 6. The Tub4 complex, Stu2 and Spc72 assemble into one complex and act in a cooperative manner. (A) Tub4 complex, Stu2 and Spc72 are part of common complexes. Lysates of logarithmically growing or α-factor arrested SPC97 and SPC97-3HA ...

In vitro binding studies were performed to address how the Tub4 complex, Spc72 and Stu2 interact. Recombinant Tub4 complex and Stu2-3HA were incubated with GST or GST–N-Spc72, followed by the precipitation of Stu2-3HA with anti-HA antibodies. In the presence of GST, the Tub4 complex did not co-precipitate with Stu2-3HA (Figure 6B, lane 8). Addition of N-Spc72 enabled co-immunoprecipitation of the Tub4 complex and Stu2 (Figure 6B, lane 9). Similar results were obtained with N-Spc72-6His (data not shown). This result has two important implications. First, it suggests that the Tub4 complex and Stu2 do not interact directly. Secondly, the Tub4 complex and Stu2 assemble into a larger complex via their association with Spc72. However, since in the yeast two-hybrid assay N-Spc72 still interacted with itself (data not shown), our data do not enable us to determine whether Stu2 and the Tub4 complex bind to the same Spc72 molecule or to different Spc72 molecules, which subsequently form dimers (Chen et al., 1998).

The above experiments show Spc72-mediated complex formation between the Tub4 complex and Stu2. This interaction and the common function in astral MT organization suggest that the Tub4 complex and Stu2 cooperate at SPBs. Functional cooperation of Stu2 and the Tub4 complex should result in genetic interaction between SPC72ΔStu2 and alleles of components of the Tub4 complex. spc98-2 SPC72ΔStu2 cells were less viable at 23°C and 30°C compared with spc98-2 or SPC72ΔStu2 cells (Figure 6C, top). The tub4-1 allele also weakly interacted with SPC72ΔStu2 (Figure 6C, 35°C). A genetic interaction was not observed when the conditional lethal spc97-1 and spc97-3 alleles (Knop et al., 1997) were combined with SPC72ΔStu2 (data not shown). This allele specific interaction suggests functional cooperation between the Tub4 complex and Stu2 in astral MT organization.

Intramolecular complementation of SPC72 mutants

Our data indicate that the Tub4 complex and Stu2 cooperate in astral MT organization. For this cooperation, the Tub4 complex and Stu2 have to bind to either the same Spc72 molecule or to distinct Spc72 molecules, which are then brought together by Spc72 dimerization (Chen et al., 1998). In the latter case, two Spc72 derivatives, one able to bind only the Tub4 complex and the other only Stu2, should show trans-complementation when coexpressed in cells lacking any other form of Spc72.

The position of the nucleus is dependent on astral MTs (Palmer et al., 1992). To test for trans-complementation, we investigated whether SPC72ΔTub4 suppressed the nuclear position defect of pre-anaphase SPC72ΔStu2 cells. In 45% of SPC72ΔStu2 cells, the nucleus was not seen close to the bud neck (Figure 7A, lane 1). This defect was reduced to the same extent by either SPC72 (lane 2) or SPC72ΔTub4 (lane 4), but was not significantly reduced by SPC72176–622 (lane 3), SPC72ΔStu2* (lane 5) or SPC72ΔStu2 (lane 6), all lacking at least part of the Stu2-binding domain. Thus, SPC72ΔTub4 suppresses the nuclear position defect of SPC72ΔStu2 cells.

figure cdg459f7
Fig. 7. Intramolecular complementation of Spc72 molecules. (A) The nuclear position defect of SPC72ΔStu2 cells was rescued by SPC72ΔTub4. Logarithmically growing SPC72ΔStu2 cells with chromosomally integrated pRS306 control ...

The number of astral MTs per cell was determined to confirm that the SPC72ΔTub4 construct rescued the astral MT defect of SPC72ΔStu2 cells. SPC72ΔTub4 increased the number of astral MTs per SPC72ΔStu2 cell by a factor of 1.5. Wild-type SPC72 was with a two-fold increase, slightly more efficient (Figure 7B). However, full complementation of the astral MT defect of SPC72ΔStu2 cells by SPC72ΔTub4 was hardly expected, considering that the functional entity of Spc72 is a homodimer (Chen et al., 1998). The active Spc72ΔStu2–Spc72ΔTub4 dimer would therefore only be expected to occur in 50% of Spc72–Spc72 dimer pairings. Together, the correction of the nuclear position defect and the increase in astral MT number of SPC72ΔStu2 cells by SPC72ΔTub4 show that the Tub4 complex and Stu2 do not have to bind to the same Spc72 molecule in order to effectively organize astral MTs.

SPB-associated Stu2 requires the MT-binding domain

Given that the Stu2 and Tub4 complex binding domains do not have to be encoded by the same Spc72 molecule, a fusion between STU2 and the SPB targeting domain of SPC72 might be expected to complement astral MT defects of SPC72ΔStu2 cells if the main defect in these cells lies in the recruitment of Stu2 to SPBs. If is the case, deletion analysis will reveal the domains of Stu2 that are essential for its function at the SPB.

We fused the entire STU2 gene (codons 1–888) to the SPC72277–622 subfragment, which lacked the Stu2 and Tub4 complex binding sites but encoded the SPB-binding domain (Knop and Schiebel, 1998). The fusion protein was named Stu21–888–Spc72SPB*. To ensure that Stu21–888–Spc72SPB* functions at SPBs, we first studied the subcellular localization of the GFP-tagged protein. In 96% of cells Stu2–Spc72SPB*–GFP only associated with SPBs (Spc42-RFP) but not with astral MTs or the central spindle (Figure 8A). A weak astral MT-like staining was only apparent in ~4% of cells. The STU2-SPC72SPB* gene fusion did not complement a deletion of STU2 (Figure 8B), indicating that it fulfilled only a subset of Stu2 functions. Thus, Stu2–Spc72SPB* is predominantly associated with SPBs.

figure cdg459f8
Fig. 8. The MT-binding domain of Stu2 is important for its function at SPBs. (A) A Stu21–888–Spc72SPB*–GFP fusion protein is predominantly associated with SPBs. Cells with the SPB marker SPC42-RFP and STU2 ...

The nuclear position defect of SPC72ΔStu2 cells (Figure 8C, lane 1) was reduced by STU21–888-SPC72SPB* (lane 8) as strongly as by SPC72 (lane 2). This suppression was a direct consequence of altered astral MTs. First, in the presence of STU21–888-SPC72SPB*, more astral MTs were associated with SPBs of SPC72ΔStu2 cells (Figure 8D) but not wild-type cells (data not shown). Secondly, the strong elongation of bud-ward directed astral MTs in anaphase SPC72ΔStu2 cells was suppressed by STU21–888-SPC72SPB* (Figure 8E). Thirdly, STU21–888-SPC72SPB* restored, in part, the dynamic behaviour of astral MTs of SPC72ΔStu2 cells (Table I).

Complementation of SPC72ΔStu2 defects by STU21–888-SPC72SPB* allowed us to investigate the region of Stu2 that was important for Stu2 function at SPBs. The C-terminal 33 amino acids of Stu2 were required for MT plus end binding (see Supplementary figure 2C). However, deletion of these same 33 amino acids still enabled a 70–80% complementation of the fusion protein (Figure 8C, lane 7). Deletion of the N-terminal HEAT repeats reduced complementation by 30–40% (lane 4). Hardly any complementation was observed when the MT-binding domain of Stu2 was removed (lanes 5, 6 and 9). All Stu2–Spc72SPB* derivatives were expressed similarly (data not shown). The MT-binding domain is therefore the part that is most important for the function of Stu2 at SPBs.

Discussion

In this report, we investigated the binding and function of Stu2 at SPBs and its interaction with the Tub4 complex. A model of how Stu2 and the Tub4 complex bind to SPBs via Spc72 is given in Figure 9. Consistent with previous results (Chen et al., 1998; Knop and Schiebel, 1998), Stu2 and the Tub4 complex bind directly to the SPB component Spc72. The first 100 amino acids of Spc72 are essential and interact with the Tub4 complex. Only the Tub4 complex but not individual subunits or subcomplexes bind with high efficiency to Spc72. This ensures that only the fully assembled Tub4 complex that is competent to promote MT nucleation is targeted to SPBs. The second, Stu2-binding domain of Spc72, encoded by amino acids 100–230, is non-essential. Interaction of Stu2 and the Tub4 complex via Spc72 (Figure 6) suggests that Spc72 joins Stu2 and the Tub4 complex together in a single, larger complex. This can be achieved in two ways. The Tub4 complex and Stu2 could interact through the spatially distinct binding sites with the same Spc72 molecule. Alternatively, considering that Spc72 forms homodimers (Chen et al., 1998), the Tub4 complex could bind to one Spc72 molecule while Stu2 binds to the other. In either case, complementation of the SPC72ΔStu2 phenotype by SPC72ΔTub4 (Figure 7) indicates that Stu2 and the Tub4 complex do not have to interact with the same Spc72 molecule in order to be functional (as illustrated in Figure 9).

figure cdg459f9
Fig. 9. Model for the cooperation of Stu2 and the Tub4 complex at the cytoplasmic side of the SPB. See text for details.

The functional analysis of Stu2 at SPBs was complicated by the fact that Stu2 is associated not only with SPBs but also with astral MT plus ends, kinetochores, the spindle midzone and along MTs (Wang and Huffaker, 1997; He et al., 2001; Kosco et al., 2001). Truncation of Stu2 at the C-terminus abolished not only Stu2 binding to SPBs but also localization to astral MT plus ends (see Supplementary figure 2) where Stu2 probably destabilizes MTs (van Breugel et al., 2003). An alternative approach was to study SPC72 mutants that specifically fail to interact with Stu2. For the interpretation of the phenotype of such mutants, it was important to know whether the Spc72 protein is only important at SPBs or, as is the case of D-TACC (Lee et al., 2001), also associates with MTs and MT plus ends. The fully functional Spc72 protein tagged with four copies of GFP was only detected at SPBs but not along MTs or at MT plus ends (Figure 4D). The conditions used were such that they allowed the detection of Stu2–4GFP at astral MT plus ends (Figure 4A, B and C). In addition, the Tub4 complex and Stu2-binding domains of Spc72 fulfil their function in astral MT organization when fused to the integral SPB components Kar1 and Cnm67 (Pereira et al., 1999; Gruneberg et al., 2000). When taken together, these results support the view that Spc72 only functions at SPBs.

To impair the binding of Stu2 to Spc72 at SPBs, we deleted the non-essential Stu2-binding domain of Spc72. Spc72ΔStu2 is specifically defective in Stu2 interaction at SPBs, but is normal with regards to the interaction with the Tub4 complex (Figure 2), Stu2 localization to MT plus ends (Figure 4A, B and C) and other functions of Spc72 in astral MT organization. This notion is further supported by the finding that Stu2 permanently tethered to SPBs strongly reduced the astral MT defect of SPC72ΔStu2 cells (Figure 8).

The dynamic properties of astral MTs were altered in SPC72ΔStu2 cells. There was a marked reduction in catastrophe and rescue frequencies. Similarly, defects in the budding and fission yeast γ-tubulin complexes (which are like the Spc72–Stu2 complex, associated with MT minus ends) also affected astral MT dynamics (Spang et al., 1996; Paluh et al., 2000; Vogel et al., 2001; Fujita et al., 2002). Since astral MTs in budding yeast are only dynamic at their plus ends (Maddox et al., 2000), these observations suggest that proteins associated with the MT minus ends influence MT plus end dynamics. Two models are consistent with this phenotype. Stu2 and Tub4 complex binding to the MT minus ends could impose an altered conformation onto interacting tubulin subunits, which then would be transmitted from the minus ends onward toward the dynamic plus ends of MTs through tubulin–tubulin interactions. Alternatively, MT plus end regulators could be loaded onto SPBs before travelling along MTs to their plus ends. Recent work has provided evidence that a complex of Kar9, the MT-binding protein Bim1 and yeast Cdk1, Cdc28, are transported from SPBs to MT plus ends by the kinesin motor Kip2 (Liakopoulos et al., 2003; Maekawa et al., 2003). Since Kar9 and Stu2 are present in common complexes (Miller et al., 2000), Kar9 loading onto the SPB could require complex formation between Spc72 and Stu2. To this end, known MT regulators such as Bik1 (Berlin et al., 1990), Kip2 (Huyett et al., 1998), Kip3 (Miller et al., 1998), Cdk1/Cdc28 and Kar9 still associate with MT plus ends in SPC72ΔStu2 cells (see Supplementary figure 4; data not shown). However, our data do not exclude the possibility that interaction of an MT regulator with Stu2 at SPBs is required for them to obtain activity.

We also observed that the two SPBs of anaphase SPC72ΔStu2 cells were affected differently. It is possible that the selective loading of the Bim1–Kar9 complex onto the bud-ward directed pre-existing old SPB, accompanied by the transport of the Bim1–Kar9 complex along astral MTs to the MT plus ends (Pereira et al., 2001; Liakopoulos et al., 2003; Maekawa et al., 2003), stabilizes the astral MTs organized by the old SPB. In contrast, the astral MTs generated in the mother cell body by the new SPB do not carry the Bim1–Kar9 complex and remain unstable.

Our study indicates that the full function of SPB-associated Stu2 is dependent upon its MT-binding site (Figure 8) (Wang and Huffaker, 1997). Moreover, Stu2 at SPBs promotes MT formation and prevents the detachment of astral MTs from SPBs (Figure 5). To explain these phenotypes, we propose that Spc72 binds the Tub4 complex that initiates MT formation from tubulin subunits. Stu2 associated via its C-terminus (Figure 1) either to the same Spc72 or to a second Spc72 molecule within the Spc72–Spc72 homodimer (Figure 9) (Chen et al., 1998). The MT-binding domain of Stu2 first stabilizes short MT seeds nucleated by the Tub4 complex. When the MT cylinder is extended, the MT-binding site of Stu2 cooperates with the Tub4 complex to anchor astral MTs to SPBs. The data presented here also indicate the astral MT promoting function of Stu2 at SPBs is not essential for the viability of yeast cells. The essential function of Stu2 is therefore either at kinetochores, the formation of anaphase spindles or both (He et al., 2001; Severin et al., 2001).

In human cells, ch-TOG and the three TACC proteins have been implicated in cancer, but the transforming activity of these proteins is unclear (Raff, 2002). Our finding now suggests that many astral MT defects observed in Stu2 depleted cells (Kosco et al., 2001) are actually caused by the failure of Stu2 to function at SPBs. In particular, we present the first evidence that the Spc72–Stu2 complex at SPBs contributes to the regulation of MT plus end dynamics. This finding makes XMAP215/Stu2-like proteins and their centrosomal anchors, TACC proteins and Spc72, important regulators of astral MT properties. OP18/stathmin, another protein with a role in cancer development, also regulates MT dynamics (Cassimeris, 2002). It will therefore be interesting to investigate whether a change in the MT regulatory activity of the TACC/ch-TOG complex at centrosomes is the basis of the transforming activity.

Materials and methods

Plasmids and yeast strains

Strains and plasmids are listed in Table II. Yeast strains were derivatives of YPH499 (Sikorski and Hieter, 1989). Yeast strains were constructed by PCR-based methods (Knop et al., 1999; Maekawa et al., 2003). Two-hybrid interactions were determined as described previously (Maekawa et al., 2003).

Table II.
Yeast strains and plasmids

Cell cycle analysis and growth conditions

Yeast strains were grown in yeast extract, peptone, dextrose medium containing 100 µg/l adenine (YPAD medium) (Sherman, 1991). Cells were blocked in G1 by the addition of 10 µg ml–1 mating pheromone, α-factor. After 2.5 h at 23°C or 2 h at 30°C, cells were released from the block by washing and re-suspension in YPAD medium.

Expression of recombinant proteins

Recombinant TUB4, SPC98, SPC97, STU2, STU21–855 and STU2-3HA were expressed in Sf9 cells by baculovirus expression system. The Tub4 complex was purified as described previously (Vinh et al., 2002). Fragments of Spc72 were expressed in E.coli BL21 (DE3) as GST fusion proteins following induction by the addition of 0.5 mM IPTG for 4 h at 30°C.

In vitro binding experiments

For assessment of the in vitro binding between Spc72 and the Tub4 complex or Stu2, GST-fused Spc72 proteins bound to glutathione–Sepharose (GSH–Sepharose, AmershamPharmacia Biotech) were mixed with Sf9 cell extracts that expressed Tub4 complex components or Stu2. After 60 min incubation, GSH–Sepharose was washed with extraction buffer and proteins were eluted with SDS sample buffer. The samples were analysed by immunoblotting with affinity-purified rabbit anti-Tub4, anti-Spc98, anti-Spc97, anti-Stu2, and monoclonal mouse anti-HA (12C5) antibodies. For in vitro binding of Tub4 complex, Stu2 and Spc72, purified GST or GST–N-Spc72 was incubated for 60 min in Sf9 cell extract in which the Tub4 complex, Stu2-3HA or both were expressed. Stu2-3HA was immunoprecipitated with anti-HA antibody (12CA5).

Antibodies and immunoprecipitation

Antibodies specific for Stu2 were produced and affinity purified using recombinant 6His-Stu2504–888. Spc72 antibodies were raised against TrpE-Spc72p1–428. Rabbit anti-C-Spc72 antibodies were affinity purified using 6His-Spc72231–622 purified from E.coli. Rabbit anti-Tub4, anti-Spc72, anti-Spc98 and anti-Spc97 antibodies have been described previously (Knop and Schiebel, 1998). Immunoprecipitation of Spc72-3HA was performed using a standard protocol (Knop and Schiebel, 1998).

Image analysis

GFP or RFP-labelled cells were analysed by fluorescence microscopy after fixation with 3.7% formaldehyde. DNA was stained with 4′,6-diamino-2-phenylindole (DAPI) (Pereira et al., 2000). Time-lapse analysis of living cells was as described previously (Pereira et al., 2001). Time-lapse series were acquired in Z-series 11 planes 0.4 µm apart. At each time point, the length of MT was measured. Growth, shrinkage and pause were defined as described by Kosco et al. (2001).

Supplementary data

Supplementary data are available at The EMBO Journal Online.

Acknowledgements

We thank Dr A.Hyman for communicating results before publication and Dr I.Hagan for comments on the manuscript. Dr Steve Bagley is acknowledged for helping us with the microscopy. This study was supported by the human frontier of science program and a program grant from CR UK. T.U. is financed by a grant of the Bioarchitect program of the RIKEN institute.

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