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Vol. 16, Issue 6, 3040-3051, June 2005

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Fission Yeast mto2p Regulates Microtubule Nucleation by the Centrosomin-related Protein mto1p

Wellcome Trust Centre for Cell Biology, School of Biological Sciences, University of Edinburgh, Edinburgh EH9 3JR, United Kingdom

Submitted November 16, 2004; Revised January 3, 2005; Accepted January 5, 2005
Monitoring Editor: Trisha Davis

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION ACKNOWLEDGMENTS REFERENCES

 
From an insertional mutagenesis screen, we isolated a novel gene, mto2+, involved in microtubule organization in fission yeast. mto2 strains are viable but exhibit defects in interphase microtubule nucleation and in formation of the postanaphase microtubule array at the end of mitosis. The mto2 defects represent a subset of the defects displayed by cells deleted for mto1+ (also known as mod20+ and mbo1+), a centrosomin-related protein required to recruit the -tubulin complex to cytoplasmic microtubule-organizing centers (MTOCs). We show that mto2p colocalizes with mto1p at MTOCs throughout the cell cycle and that mto1p and mto2p coimmunoprecipitate from cytoplasmic extracts. In vitro studies suggest that mto2p binds directly to mto1p. In mto2 mutants, although some aspects of mto1p localization are perturbed, mto1p can still localize to spindle pole bodies and the cell division site and to "satellite" particles on interphase microtubules. In mto1 mutants, localization of mto2p to all of these MTOCs is strongly reduced or absent. We also find that in mto2 mutants, cytoplasmic forms of the -tubulin complex are mislocalized, and the -tubulin complex no longer coimmunoprecipitates with mto1p from cell extracts. These experiments establish mto2p as a major regulator of mto1p-mediated microtubule nucleation by the -tubulin complex.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION ACKNOWLEDGMENTS REFERENCES

 
The -tubulin complex is a large, conserved multisubunit protein complex involved in microtubule nucleation in eukaryotic cells (Stearns and Kirschner, 1994; Zheng et al., 1995; for reviews, see Gunawardane et al., 2000; Oakley, 2000; Schiebel, 2000; Job et al., 2003). In a variety of cell types, the -tubulin complex is found both localized to microtubule-organizing centers (MTOCs) and also in significant levels in cytoplasmic pools. Although much of the characterization of -tubulin function has involved work on soluble complexes, most MTOCs in vivo are associated with largely insoluble compartments or subcellular organelles, and as a result we currently understand very little of the mechanisms controlling the intracellular localization and activity of the -tubulin complex and how these mechanisms might be regulated. Recently, a small number of proteins have been identified that may interact with the -tubulin complex and recruit it to structures such as the centrosome, the Golgi apparatus, or other MTOCs (Takahashi et al., 2002; Terada et al., 2003; Kawaguchi and Zheng, 2004; Rios et al., 2004; Sawin et al., 2004; Thompson et al., 2004; Venkatram et al., 2004; Zimmerman et al., 2004a,b). However, in this area much still remains to be learned.

The fission yeast Schizosaccharomyces pombe has recently become a useful model system for understanding microtubule nucleation and dynamics. Three distinct patterns of microtubule nucleation take place during the mitotic cell cycle in fission yeast (Hagan, 1998). During interphase, dynamic microtubules tend to run along the long axis of the cylindrically shaped cells and terminate at cell tips. These microtubules are nucleated from cytoplasmic interphase MTOCs (iMTOCs) in the middle of the cell that may be associated with the nuclear envelope or with microtubule bundles themselves (Drummond and Cross, 2000; Tran et al., 2001; Sawin et al., 2004; Zimmerman et al., 2004a). Nucleation sites on the surface of the nucleus are easily recognized in microtubule regrowth experiments after microtubule depolymerization, but whether they exist in the same form during steady-state growth is unclear. By contrast, although de novo nucleation of microtubules from microtubule bundles has not yet been directly observed in growing cells, components of the -tubulin complex are seen not only at the spindle pole body (SPB, the yeast centrosome equivalent) but also in the form of small satellite particles moving along microtubules (Sawin et al., 2004; Zimmerman et al., 2004a). Against this background of incomplete understanding, in this work we apply the term "iMTOC" somewhat freely, both to nuclear surface nucleation sites and to the aforesaid microtubule-associated particles. During mitosis, duplicated SPBs nucleate an intranuclear mitotic spindle, as well as cytoplasmic astral microtubules, which may help to position the spindle and/or coordinate mitotic progression with cytokinesis (Gachet et al., 2001; Oliferenko and Balasubramanian, 2002; Gachet et al., 2004). At the end of mitosis, a specialized microtubule structure, termed the postanaphase array (PAA), is nucleated from an equatorial MTOC (eMTOC) at the cell division site (Heitz et al., 2001; Pardo and Nurse, 2003).

There is good evidence that -tubulin plays a role in microtubule nucleation at all fission yeast MTOCs (Horio et al., 1991; Paluh et al., 2000; Heitz et al., 2001; Sawin et al., 2004; Venkatram et al., 2004; Zimmerman et al., 2004a). Thus far, nearly all of the subunits of the higher eukaryotic -tubulin complex have been identified in fission yeast (Horio et al., 1991; Stearns et al., 1991; Vardy and Toda, 2000; Fujita et al., 2002; Venkatram et al., 2004), and recent efforts have begun to identify fission yeast proteins regulating -tubulin function. The nonessential protein mod20p/mbo1p seems to play a particularly important role in -tubulin complex-mediated microtubule nucleation (Sawin et al., 2004; Venkatram et al., 2004). To establish a common nomenclature, from hereon in this article we refer to mod20p/mbo1p as microtubule organizer 1 (mto1p). In mto1 mutants, new microtubule nucleation by iMTOCs is profoundly crippled, such that only the SPB is (poorly) active for microtubule nucleation. In addition, during mitosis, mto1 mutants fail to nucleate cytoplasmic astral microtubules, although the assembly of the intranuclear mitotic spindle seems to be normal. Moreover, at the end of mitosis, when wild-type cells form a PAA, mto1 mutants fail to nucleate any microtubules from the cell division site. The role of mto1p in these various manifestations of spatially restricted microtubule nucleation seems to be to recruit the -tubulin complex to MTOCs, as components of the -tubulin complex are coimmunoprecipitated with mto1p, and in mto1 cells, the -tubulin complex is specifically absent from the sites at which microtubule nucleation should but does not occur.

Potential homologues of mto1p are found in fungi and in higher eukaryotes. These are all large proteins, with extensive regions of predicted -helical coiled-coil, and they share in their amino-terminus a roughly 60-amino acid region of limited sequence conservation. Some of these proteins, such as Aspergillus nidulans apsB and Drosophila centrosomin (cnn), have been characterized functionally (Suelmann et al., 1998; Megraw et al., 1999; Terada et al., 2003), and this analysis is consistent with the proposed function of mto1p. Other proteins, such as human myomegalin/PDE4DIP or CDK5RAP2, are to date only little characterized (Ching et al., 2000; Verde et al., 2001; Andersen et al., 2003), but they are likely, even on the basis of limited analysis, to be equally interesting. Amino acid sequence analysis also suggests that in some organisms, mto1p-like proteins may exist as pairs of paralogs. For example, in fission yeast, the putative mto1p paralog pcp1p, which may be orthologous to budding yeast Spc110p (Knop and Schiebel, 1998; Flory et al., 2002), has been hypothesized to be responsible for intranuclear mitotic spindle microtubule nucleation, which is independent of mto1p (Sawin et al., 2004; Venkatram et al., 2004).

From this perspective, a deeper understanding of how mto1p localization and activity are regulated should provide further insights into the mechanisms controlling microtubule nucleation by the -tubulin complex in fission yeast. Here, we describe a novel nonessential gene, mto2+, which is involved in mto1p-mediated microtubule nucleation. Mto2p is physically associated with mto1p and colocalizes with mto1p throughout the cell cycle. Interestingly, mto2 mutants display a subset of the microtubule-nucleation–defective phenotypes seen in mto1 mutants. Collectively, our results suggest that mto2p promotes the ability of mto1p to recruit the -tubulin complex to a subset of MTOCs away from the SPB.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION ACKNOWLEDGMENTS REFERENCES

 
Yeast Strain Construction and Growth Conditions
Standard yeast genetic techniques were used throughout (Moreno et al., 1991; Alfa et al., 1993). Insertional mutagenesis was performed as described previously (Snaith and Sawin, 2003). Deletion and tagging of genes at both N and C termini was achieved using polymerase chain reaction (PCR)-based targeting methods (Bahler et al., 1998), and deletions and tagging were confirmed by PCR and Western blotting as appropriate. See Table 1 for a list of strains used. The cyan fluorescent protein (CFP)-atb2 plasmid pRL72 (Glynn et al., 2001) was used to transform strains for the experiments shown in Figure 7. Growth conditions were as described previously (Moreno et al., 1991), except that sodium glutamate was used as nitrogen source for minimal medium. Although this is unrelated to mto2+ function per se, we note for interested investigators that the mto2+ locus is closely linked (distance 10 kb) to alp6+, an essential component of the -tubulin complex (Vardy and Toda, 2000).

View larger version (108K): [in this window] [in a new window]   Figure 7. Mto1p localization in mto2 mutants. (A–D) Mto1p does not redistribute to the nuclear surface upon cold-shock in mto2 mutants. Immunofluorescent staining with affinity-purified anti-mto1p antibody. Wild-type and mto2 cells were fixed and stained (A and B) before and (C and D) after 30 min cold-shock at 0°C. Note interphase (i), mitotic SPB (m) and eMTOC (e) localization of mto1p. Nuclear position also is marked (N). (E and F) Localization of mto1-YFP in living wild-type and mto2 cells. Projections of Z-sections through the entire cell volume. Smaller panels show single Z-section examples of mto1-YFP in cells coexpressing CFP-atb2 tubulin. Note that not all microtubules in mto2 cells have associated mto1-YFP. Same labeling convention as above. (G and H) Localization of mildly overexpressed nmt81:GFP-mto1p in wild-type and mto2 cells. Projections of Z-sections through the entire cell volume. Small panels show examples of single Z-sections in interphase and mitotic cells, allowing visualization of the GFP-mto1p localized to the nuclear envelope that is obscured in projections. In all panels, images comparing wild-type and mto2 cells were always acquired and processed under identical conditions, allowing direct comparison of intensities. Bar, 5 µm (large panels); 7 µm (small panels).

Antibodies
Antibodies to mto2p were raised in sheep against recombinant glutathione S-transferase (GST)-mto2p purified from Escherichia coli. Anti-mto2p antibody was affinity purified from serum by using standard methods (Sawin et al., 1992). First, anti-GST antibodies were removed by passing serum through a column of GST coupled to Affigel (Bio-Rad, Hercules, CA) until all anti-GST reactivity was removed. Anti-mto2p antibodies were then affinity purified on a column of GST-mto2p coupled to Affigel and eluted at pH 3.0. The anti-mto1p polyclonal antibody and TAT1 anti-tubulin (Woods et al., 1989), 9E10 anti-myc (Evan et al., 1985), and GTU-88 anti--tubulin (Sigma-Aldrich, St. Louis, MO) monoclonal antibodies were used as described previously (Sawin et al., 2004). Secondary antibodies were Alexa labeled (Molecular Probes, Eugene, OR) for immunofluorescence and horseradish peroxidase labeled (Amersham Biosciences, Piscataway, NJ; and Sigma-Aldrich) for Western blotting.

Immunofluorescence and Physiology Experiments
For anti-tubulin and anti-mto1p immunofluorescence, cells were fixed in methanol at –70°C and processed exactly as described previously (Sawin and Nurse, 1998; Sawin et al., 2004).

Cell morphology assays after growth to stationary phase on YE5S agar plates were performed as described previously (Snaith and Sawin, 2003). Cells were washed off plates and fixed in 3% formaldehyde before imaging. Assays of microtubule regrowth after cold-shock were as described previously (Sawin et al., 2004). Exponentially growing cells were chilled in ice water bath for 30 min and transferred to a prewarmed flask and incubated at 32°C for the specified time before collection by filtration and fixation. Mitotic block-and- release experiments by using reversible cold-sensitive nda3 mutants also were exactly as described previously (Sawin et al., 2004).

Biochemical Methods
For coimmunoprecipitation experiments of mto2p with mto1p, yeast cells were disrupted by bead-beating by using 0.5-mm zirconium beads in a buffer containing 50 mM Tris, pH 8.0, 150 mM NaCl, 1 mM EDTA, and 1 mM phenylmethylsulfonyl fluoride (PMSF). Cleared cell extract was incubated with anti-myc antibody (9E10) or affinity-purified anti-mto2p antibody for 30 min. Protein A- or protein G-Sepharose was added to the cell extract/antibody mixture and incubated for a further 30 min at 4°C. The Sepharose beads were washed four times with the buffer before being boiled in 2x SDS PAGE buffer and used for SDS-PAGE and Western blotting.

For coimmunoprecipitation experiments of mto1p with components of the -tubulin complex, frozen yeast cells were disrupted by grinding with a mortar and pestle. These and further manipulations were carried out exactly as described previously (Sawin et al., 2004).

A TnT T7 coupled in vitro transcription/translation kit (Promega, Madison, WI) was used to synthesize mto1p polypeptides for use in GST pull-down assays. GST-mto2p was expressed in E. coli BL21(DE3)pLysS and purified on glutathione (GSH)-Sepharose (Amersham Biosciences) following manufacturer's instructions. Truncated mto1 genes were amplified by PCR from a plasmid template, and the resulting PCR products were used as the template for in vitro transcription/translation. All 5' primers start with a common 40-base sequence (5'-CCGCGGGGCCCTAATACGACTCACTATAGGGAGAACCATG-3', which contains T7 primer, Kozak sequence, and an initiation methionine codon) followed by the appropriate specific mto1 sequence. Fifteen microliters of a 50-µl reaction was incubated with GST-mto2p bound to GSH-Sepharose (Sigma-Aldrich) in 500 µl of GST binding buffer (50 mM Tris, pH 7.5, 100 mM NaCl, 1% Triton X-100, 10% glycerol, and 1 mM PMSF) at 4°C for 1 h. Beads were washed four times with the GST binding buffer before boiling in 2x SDS-PAGE buffer.

In Vivo Fluorescence Imaging
Single time-point and time-lapse imaging of green fluorescent protein (GFP)-, CFP-, and yellow fluorescent protein (YFP)-fusion proteins in fission yeast was essentially as described previously (Snaith and Sawin, 2003; Sawin et al., 2004). Cells were mounted on medium-agarose pads before imaging and sealed with VALAP. Images were collected on a Nikon TE300 inverted microscope with automated filter and z-axis control, running MetaMorph software (Universal Imaging, Downingtown, PA). Appropriate neutral density filters were used to reduce photobleaching and photodamage. Some sequences were further deconvolved using Softworx (Applied Precision, Issaquah, WA).

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION ACKNOWLEDGMENTS REFERENCES

 
Aberrant Cell Shape and Microtubule Organization in mto2 Mutants
From an insertional mutagenesis screen designed to identify genes regulating cell polarity in fission yeast (Snaith and Sawin, 2003), we isolated a mutant that produces a curved cell shape upon return to growth from stationary phase (Figure 1A). Sequencing of the disrupted gene showed it to be SPBC902.06, a previously uncharacterized open reading frame, which we have termed mto2+. Database searches did not reveal any obvious eukaryotic homologues of mto2p at the primary sequence level. Targeted deletion of the mto2+ open reading frame yielded viable cells (mto2) with a phenotype identical to the original disruption mutant, and the mto2 strain was used for all subsequent experiments.

View larger version (70K): [in this window] [in a new window]   Figure 1. Phenotype of mto2 cells. (A) Morphology of wild-type and mutant cells 3 h after return to fresh medium from stationary phase. Only wild-type cells are straight. Bar, 10 µm. (B) Microtubule organization in wild-type and mutant cells, as shown by anti-tubulin immunofluorescent staining of asynchronous cultures. Note that mutants tend to have fewer microtubule bundles, and bundles often curve around cell tips. Bar, 2 µm.

We next examined the behavior of mto2 mutants in microtubule regrowth assays after a cold shock at 0°C. In these experiments, wild-type interphase cells rapidly renucleate microtubules from numerous independent sites in the cell middle, probably in contact with the nuclear surface, whereas mto1 mutants renucleate microtubules extremely slowly and then only from the SPB (Figure 2; Sawin et al., 2004). Comparing wild-type, mto1, and mto2 cells in this assay, we found that mto2 mutants showed a phenotype distinct from both wild-type cells and mto1mutants. Although, as in mto1 mutants, microtubule nucleation in mto2 mutants was restricted to a single bundle growing from the SPB, the rate of regrowth was considerably faster in mto2 mutants, which reached a steady-state distribution 10 min after warming, in contrast to 30–60 min for mto1 cells and 4 min for wild-type cells (Figure 2). In these experiments, mto1 mto2 double mutants showed the strong defects characteristic of mto1 single mutants, further supporting the notion that mto1p and mto2p act in a common genetic pathway.

View larger version (94K): [in this window] [in a new window]   Figure 2. Microtubule regrowth after cold shock in wild-type and mutant cells. Antitubulin staining of fixed cells. Microtubules were depolymerized by a 30-min cold shock and then fixed at the indicated times after being returned to 32°C. Note that all mutants initially nucleate a single microtubule bundle, but mto2 mutants nucleate much faster and reach steady state more quickly than mto1 and mto2 mto1 mutants. The position of the nucleus in cells can be identified by faint negative staining. Bar, 2 µm.

The defects in interphase microtubule nucleation observed in mto2 mutants in microtubule regrowth experiments after cold shock also were observed during live-cell imaging of mto2 cells expressing the GFP-tubulin fusion protein GFP-atb2 from the low-strength nmt81 promoter. In contrast to wild-type cells, which nucleate microtubules from multiple independent sites (see Movie 01; Brunner and Nurse, 2000; Drummond and Cross, 2000; Tran et al., 2001; Sawin et al., 2004), mto2 mutants nucleated microtubules from only a single site on the nuclear surface, which we interpret to be the SPB (see Movies 02–05). mto2 mutants also contained fewer microtubule bundles, in particular when expressing GFP-atb2 from the medium-strength nmt41 promoter (see Movie 05). In addition, microtubules in mto2 mutants displayed unusual dynamics not seen in wild-type cells, including bending around the cell tip and then sometimes breaking, generating a new microtubule bundle as a result (see Movies 02–05). Similar bend-breakage behavior has been observed previously in mto1 mutants (Sawin et al., 2004; see Discussion).

View larger version (78K): [in this window] [in a new window]   Figure 3. PAA formation of microtubules in mto2 mutants. Anti-tubulin staining of fixed cells. (A) Mitotic spindle, PAA, and interphase microtubule organization in wild-type and mto2 cells after release from nda3 mitotic arrest. Note that spindles and PAAs occur at similar times in both strains, but mto2 mutants reach an interphase organization more quickly. (B) Quantitation of early (top) and late (bottom) microtubule structures at different times after release from the nda3 arrest in the experiment in A. Filled circles, wild-type cells; open circles, mto2. Early structures (classes 1–3) appear and disappear with comparable kinetics in the two strains, whereas well defined PAAs (class 4) are not sustained in mto2 mutants and are more quickly replaced by more disorganized microtubules (classes 5 and 6). (C) PAAs of microtubules in asynchronous wild-type and mto2 cells. Note aberrant PAA and cytoplasmic astral microtubules (arrowheads) in mto2 mutants. Bar, 2 µm.

To further understand the basis for this complexity, we followed the appearance of PAAs in live mto2 cells expressing nmt81:GFP-atb2 (see Movies 01–05). Although wild-type cells showed robust PAAs with continuous microtubule nucleation from eMTOCs (see Movie 01), mto2 mutants typically showed obvious but less frequent nucleation from the eMTOC, such that PAAs were either weak or highly transient. In addition, astral microtubules were clearly observed in mto2 mutants, and after nucleation, both PAA and astral microtubules were typically more stable dynamically, with astral microtubules often making long excursions into their sister cell before septation (see Movies 02–04). As with interphase microtubule dynamics, these effects of mto2+ deletion seemed somewhat stronger in cells expressing nmt41: GFP-atb2 (see Movie 05). Collectively, these results suggest that, although mto2p is not absolutely required for PAA formation, it may nevertheless contribute to the stability of the eMTOC and PAA formation. The apparent stability of the PAA in mto2 nda3 mutants may be a consequence of an extended mitotic arrest (see Discussion).

Together, the above-mentioned analyses of interphase and mitotic microtubule nucleation suggest that mto2p is involved in the regulation of microtubule nucleation in the same genetic network as mto1p and that mto2p is required for a specific subset of mto1p-dependent microtubule nucleation functions.

Localization of mto2p to Microtubule-organizing Centers
To determine the intracellular localization of mto2p, we tagged the carboxy-terminus of the endogenous mto2+ open reading frame with GFP, such that the resulting fusion protein is expressed from its endogenous promoter and is the sole source of mto2p in the cell (see, for example, Figure 6). In living cells, mto2-GFP was localized to the SPBs during both interphase and mitosis, to the eMTOC, and to satellite particles of varying intensity on cytoplasmic interphase microtubules (Figure 4A; see Movie 06). A similar localization has been previously observed for mto1-GFP, with mto1-GFP satellite particles showing rapid back-and-forth movements on microtubules (Sawin et al., 2004; Venkatram et al., 2004). Mto2-GFP satellites showed similar movements by time-lapse videomicroscopy, on both interphase and PAA microtubules (see Movie 07). Attempts to localize untagged mto2p in fixed cells with affinity-purified anti-mto2p antibodies (Figure 5) were unsuccessful, due to high cytoplasmic background (our unpublished data).

View larger version (46K): [in this window] [in a new window]   Figure 6. Mto1p and mto2p protein levels in wild-type and mutant strains. Extracts from wild-type cells (lane 1), as well as mto1-GFP (lane 2), mto2 (lane 3), mto1 (lane 4), mto1-GFP mto2 (lane 5), mto2-GFP mto1 (lane 6), and mto2-GFP (lane 7) were blotted and probed with antibodies to mto1p (top) and mto2p (middle). -Tubulin also was probed, as a loading control (bottom). Note that deletion of mto2p does not affect mto1p levels, or vice-versa. Also note that GFP-tagging of mto1p slightly destabilizes the resulting fusion protein, whereas GFP-tagging of mto2p may slightly stabilize the resulting fusion.

View larger version (101K): [in this window] [in a new window]   Figure 4. Mto2p localization in wild-type cells. (A) Mto2-GFP in live cells. Projection of Z-sections through the entire cell volume. Note localization to mitotic SPBs (arrow m) and eMTOC (arrow e) in mitotic and postmitotic cells, and satellite particles in interphase cells (arrow i). (B) Coexpression of mto2-CFP and mto1-YFP in live cells. Single Z-sections. The two signals colocalize during interphase (top three rows) as well as during mitosis (fourth row) and cell division (bottom row). Labeling convention as in A. Bars, 5 µm.

View larger version (40K): [in this window] [in a new window]   Figure 5. Physical interactions between mto2p and mto1p. (A) Immunoprecipitation of mto1p with mto2p from cell extracts. Affinity-purified anti-mto2p antibody was used for immunoprecipitation. Cell extracts and immunoprecipitates of wild-type (lanes 1 and 3) and mto2 (lanes 2 and 4) were blotted and probed with antibodies to mto1p (top) and mto2p (bottom). (B) Immunoprecipitation of mto2p with mto1-myc. Anti-myc antibody was used for immunoprecipitation. Cell extracts and immunoprecipitates of mto1+ (i.e., no tag on mto1p; lanes 1 and 4), mto1-myc (lanes 2 and 5), and mto1-myc mto2 (lanes 3 and 6) were blotted and probed with antibodies to mto2p (top) and mto1p (bottom). (C) Some truncated mto1 proteins cannot immunoprecipitate mto2p. Anti-myc antibody was used to immunoprecipitate carboxyl-terminal truncations of mto1-myc. Cell extracts and immunoprecipitates of full-length mto1-myc (1-1115; lanes 1 and 6), mto1(1-285)-myc (lanes 2 and 7), mto1(1-500)-myc (lanes 3 and 8), mto1(1-800)-myc (lanes 4 and 9), and mto1(1-1051)-myc (lanes 5 and 10) were blotted and probed with antibodies to mto2p (top) and mto1p (bottom). Immunoprecipitate lanes represent 20x extract-equivalent loading relative to total cell extract lanes. (D) In vitro binding of mto1p truncations to GST-mto2p by pull-down assays. Mto1 proteins expressed in reticulocyte lysate (left) were tested for binding to GST-mto2 (right). Reticulocyte lysates and pull-downs of mto1(1-500) (lanes 1 and 5), mto1(1-600) (lanes 2 and 6), mto1(1-700) (lanes 3 and 7), and mto1(1-800) (lanes 4 and 8) were blotted and probed with antibodies to mto1p. (E) Summary of results of additional GST-mto2p pull-down assays, showing central region of mto1p required for binding to mto2p.

Mto2p Interacts Directly with mto1p
Given the colocalization of mto2p with mto1p at the light microscope level, we investigated whether mto2p and mto1p interact physically (Figure 5). We found that affinity-purified anti-mto2p antibodies were able to coimmunoprecipitate mto1p with mto2p in native fission yeast extracts (Figure 5A) and that anti-myc antibodies were able to coimmunoprecipitate mto2p with myc-tagged mto1p (Figure 5B). These results indicate that mto1p and mto2p are likely to exist in a common complex in vivo. To further define the regions of mto1 required for interaction with mto2p, we repeated coimmunoprecipitation experiments by using strains containing C-terminal truncations of myc-tagged mto1p. Both mto1(1-800)-myc and mto1(1-1051)-myc were able to coimmunoprecipitate mto2p, whereas mto1(1-285)-myc and mto1(1-500)-myc were not (Figure 5C). This suggests that a central portion of mto1p is likely to be important for its association with mto2p (full-length mto1p is 1115 amino acids; Figure 5E).

To determine whether the observed interaction between mto2p and mto1p is direct and to further map the regions of mto1p required for interaction with mto2p, we performed GST pull-down experiments by using bacterially expressed GST-mto2p and in vitro-translated mto1p. Initial experiments showed that GST-mto2p was able to pull down mto1(1-600), mto1(1-700), and mto1(1-800) but not mto1(1-500), suggesting that mto1p and mto2p bind directly and also that the GST pull-down experiments reproduced the nature of the interaction observed in our coimmunoprecipitation experiments (Figure 5D). A series of amino- and carboxyl-terminal deletions of in vitro-translated mto1p further showed that an 90-amino acid sequence in the central portion of mto1p, between amino acids 461 and 549, is required for interaction with mto2p (Figure 5E; additional data not shown); this is distinct from the region of mto1p conserved among centrosomin-related proteins (mto1p amino acids 249–308).

Role of mto2p in mto1p Localization
Together with the physical association between mto2p and mto1p, the observation that mto2 mutants display only a subset of the microtubule nucleation defects seen in mto1 suggested that the association of mto2p with mto1p may be required to regulate specific aspects of mto1p function. We therefore investigated possible mechanisms by which mto2p might exert this effect.

We first explored the possibility that mto1p protein levels might be altered in mto2 mutants, because interphase and PAA microtubule nucleation might be more sensitive than intranuclear spindle microtubule nucleation to mto1p levels. However, mto1p levels were not altered in mto2 mutants nor were mto2p levels altered in mto1 mutants (Figure 6).

Our previous work showed that during regrowth of interphase microtubules after cold shock, the nucleation from multiple sites around the cell nucleus that occurs in wild-type cells (Figure 2) is driven by a redistribution of mto1p to the nuclear surface, as a specific result of the cold treatment (Sawin et al., 2004). Because microtubule regrowth in mto2 mutants is restricted to the SPB in this assay, we examined mto1p localization upon cold-shock in mto2 mutants. Before cold-shock, anti-mto1p staining revealed mto1p at SPBs and eMTOCs in both wild-type and mto2 cells (Figure 7, A and B). Mto1p was also visible along microtubules in wild-type cells (Figure 7A; Sawin et al., 2004), although this was less apparent in mto2 cells (Figure 7B). On cold shock, in contrast to wild-type cells, no accumulation of mto1p was observed at the nuclear surface in mto2 mutants (Figure 7D), consistent with the mto2 microtubule nucleation phenotype. This suggested that deletion of mto2+ might affect the behavior of mto1p in vivo. We therefore further analyzed whether normal mto1p localization depended on mto2p.

Because mto1-GFP is localized to SPBs, eMTOCs, and dynamic microtubule-associated interphase satellites in living cells (Sawin et al., 2004), we sought to image mto1-GFP in mto2 mutants. Initial experiments suggested that mto1-GFP was at SPBs and eMTOCs in mto2 mutants, but cellular autofluorescence interfered with our ability to detect potentially faint mto1-GFP signals from interphase satellites (our unpublished data). We therefore focused our analysis on mto1-YFP localization, which is better separated spectrally from the autofluorescence (our unpublished data). In mto2 mutants, mto1-YFP was observed at SPBs and eMTOCs (Figure 7, E and F; see Movies 08 and 09), consistent with observed nucleation of cytoplasmic astral microtubules from the SPB and PAA microtubules from the eMTOC in mto2 mutants. However, mto1-YFP signal at eMTOCs was reduced in mto2 mutants relative to wild-type cells; this may contribute to the reduced PAA nucleation characteristic of mto2 mutants.

We also observed mto1-YFP interphase satellite particles in mto2 mutants, although relative to wild-type cells, the mto1-YFP signal was faint and prone to photobleaching, and its localization to microtubules was more easily recognized in single sections than in projections encompassing the entire cell volume (compare Figure 7 with Movie 09). We confirmed this microtubule localization by coexpressing CFP-atb2 in double-label experiments (Figure 7, E and F); in some mto2 cells, microtubules without associated mto1-YFP were seen (compare also Movies 08 and 09). Under optimal imaging conditions, we observed dynamic movement of mto1-YFP interphase satellites on microtubules in mto2 mutants, although this was difficult to detect routinely and was not easily seen in all cells (Movies 10 and 11).

We also examined the localization of moderately overexpressed GFP-mto1p in wild-type and mto2 strains (Figure 7, G and H). Expressed at three to four times wild-type levels from the nmt81 promoter (our unpublished data), GFP-mto1p showed localization to the SPB and eMTOC, as well as noticeable localization to the nuclear envelope (Figure 7G) and apparently uniform decoration of cytoplasmic microtubules, in contrast to the satellite-particle localization observed when mto1-GFP is expressed from its endogenous promoter. Interestingly, none of these localizations required the presence of mto2p (Figure 7H).

Together, the above-mentioned results indicate that mto2p is not absolutely required for localization of mto1p to the SPB or to eMTOCs or for localization to interphase cytoplasmic microtubules; however, the degree to which mto1p localizes to eMTOCs and interphase microtubules does seem to depend partially on mto2p. Moreover, in the cold shock assay, relocalization of mto1p to the nuclear surface depends on mto2p. These results suggest that although mto2p may affect some aspects of mto1p localization under specific experimental conditions, this is unlikely to be the sole cause of all of the observed microtubule nucleation defects in mto2 mutants (see Discussion for further details).

Mto2p Is Required for Coimmunoprecipitation of the -Tubulin Complex with mto1p and for Proper In Vivo Localization of the -Tubulin Complex
Because the major molecular role for mto1p in microtubule nucleation seems to be to bind and recruit the -tubulin complex to different MTOCs (Sawin et al., 2004; Venkatram et al., 2004), we tested whether mto1p could still bind to the -tubulin complex in the absence of mto2p. Strikingly, whereas cytoplasmic extracts prepared from cells expressing mto1-myc could coimmunoprecipitate mto2p, -tubulin, and hemagglutinin (HA)-tagged alp4p (an essential component of the -tubulin complex; Vardy and Toda, 2000) with mto1-myc, neither -tubulin nor alp4-HA was coimmunoprecipitated with mto1-myc to any extent in mto2 cells, even after very long exposures of Western blots (Figure 8; additional data not shown). This indicates that soluble forms of mto1p are not associated with the -tubulin complex in mto2 mutants; we address this further below in relation to mto1 and mto2 phenotypes (see Discussion).

View larger version (56K): [in this window] [in a new window]   Figure 8. The -tubulin complex is no longer coimmunoprecipitated with mto1p in mto2 cells. Anti-myc antibody was used for immunoprecipitation. Cell extracts and immunoprecipitates of mto1-myc alp4-HA (lanes 1 and 4), negative control mto1+ alp4-HA (i.e., no tag on mto1p; lanes 2 and 5), and mto1-myc alp4-HA mto2 (lanes 3 and 6) were blotted and probed with antibodies to myc (top), mto2p (second), -tubulin (third), and HA (bottom). Asterisk marks the position of anti-myc IgG heavy chain in immunoprecipitates; -tubulin is the band underneath. Immunoprecipitate lanes represent 40x extract equivalent loading relative to total cell extract lanes.

View larger version (93K): [in this window] [in a new window]   Figure 9. The -tubulin complex is not properly localized to eMTOCs or interphase satellite particles in mto2 mutants. Alp4-GFP localization in live wild-type (A) and mto2 mutant (B) cells. Large panels show interphase cells (with one just-divided cell pair in B); small panels show dividing cells. Bar, 5 µm.

Mto1p Is Required for mto2p Localization
We also examined mto2-GFP localization in mto1 mutants. Strikingly, mto2-GFP interphase satellites were no longer observed in mto1 cells, and mto2-GFP localization to SPBs and eMTOCs was almost abolished (Figure 10B). Approximately one-third of cells showed a faint GFP signal suggestive of the SPB, and dividing cells showed only a very faint signal at the septum, or no signal at all. The faint signals were always small and punctate rather than band-like (compare Figure 10, A and B), suggesting that this small amount of mto2-GFP might be recruited to the division site only at the very end of septation; alternatively, it is possible that low amounts of mto2-GFP are present throughout septation in mto1 mutants but are visible only when concentrated into a small volume. Because mto1p and mto2p interact, we tried to test more directly whether mto2p localization to microtubules occurs specifically via its interaction with mto1p. We found that in mto1+ cells, overexpressed GFP-mto2p (nmt41: GFP-mto2p) was easily visualized at SPBs and at eMTOCs but was not seen on microtubules above levels of cytoplasmic GFP-mto2p background fluorescence; some protein aggregation also was observed (Figure 10C). However, when nmt81:mto1p was simultaneously overexpressed in the GFP-mto2p–overexpressing cells, GFP-mto2p now localized to interphase microtubules, as well as to the nuclear envelope, mirroring the localization of nmt81:GFP-mto1p (Figure 10D; compare with Figure 7G). These results indicate not only that mto1p is required for normal mto2p localization to most subcellular structures but also that mto1p protein levels are limiting for mto2p localization, suggesting that mto2p localization to microtubules occurs in the form of a mto1p/mto2p protein complex in which mto1p provides the microtubule targeting.

View larger version (135K): [in this window] [in a new window]   Figure 10. Mto1p is required for mto2p localization. (A and B) Localization of mto2-GFP in live wild-type (A) and mto1 (B) cells. Projections of Z-sections through the entire cell volume. Images in A and B were acquired and processed under identical conditions, allowing direct comparison of intensities. (C and D) Localization of overexpressed nmt41:GFP-mto2p in wild-type cells (C) and cells mildly overexpressing nmt81:mto1+ (i.e., untagged mto1p) (D). Projections of Z-sections through the entire cell volume. Smaller panels show single Z-section examples of nmt41:GFP-mto2p localizing to the nuclear envelope only when mto1p is overexpressed (arrow). Images in C and D were acquired and processed under identical conditions, allowing direct comparison of intensities. Bar, 5 µm.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION ACKNOWLEDGMENTS REFERENCES

 
Model for mto2p Function
In this work, we have identified and characterized a novel protein, mto2p, which plays an important role in the regulation of fission yeast microtubule nucleation. Most microtubule nucleation in eukaryotic cells is thought to be mediated by the -tubulin complex (Gunawardane et al., 2000; Oakley, 2000; Schiebel, 2000; Job et al., 2003). In fission yeast, normal cytoplasmic microtubule nucleation from the SPB, eMTOC, and iMTOCs requires the centrosomin-related protein mto1p to recruit the -tubulin complex to these MTOCs (Sawin et al., 2004; Venkatram et al., 2004). Given the spatio-temporal diversity of fission yeast MTOCs (Hagan, 1998; Drummond and Cross, 2000; Tran et al., 2001; Sawin et al., 2004; Venkatram et al., 2004; Zimmerman et al., 2004a), it might be expected that regulators of mto1p exist within cells. Here, we have shown that mto2p is required for cytoplasmic microtubule nucleation from iMTOCs but not from the SPB. Mto2p also contributes significantly to eMTOC nucleation. In conjunction with our biochemical and localization studies, these data suggest a provisional model in which mto2p regulates microtubule nucleation mediated by mto1p and the -tubulin complex. We propose the following four main points, which are discussed in more detail below: 1) Mto2p is a stable component of an mto1p-containing protein complex that recruits the -tubulin complex to MTOCs. 2) Mto1p interphase satellite particles, which may represent the soluble population of mto1p complexes that can be probed by immunoprecipitation, do not bind the -tubulin complex in the absence of mto2p. 3) Mto1p complexes at SPBs and eMTOCs, which may not be identifiable in immunoprecipitation experiments because they are largely insoluble, can associate with the -tubulin complex even in the absence of mto2p. 4) Mto2p is not required for the general ability of the mto1p complex to be targeted to SPB, eMTOC, and interphase satellite iMTOCs but may contribute to the efficient recruitment of mto1p to the eMTOC and the behavior and/or dynamics of mto1p satellites.

With regard to the first point, we have shown that mto1p and mto2p are very closely colocalized at MTOCs throughout the cell cycle and that mto2p is likely to bind directly to a central portion of mto1p. In addition, mto2p is coimmunoprecipitated with mto1-myc much more efficiently than the -tubulin complex (Figure 8). This suggests to us that it may be most appropriate to think of -tubulin complex recruitment to MTOCs as involving an interaction between the -tubulin complex and an mto1p/mto2p complex.

The second two points of the model raise several related issues concerning protein–protein interactions between mto1p and the -tubulin complex. The -tubulin complex is no longer found in mto1-myc immunoprecipitates from cytoplasmic extracts in mto2 strains. This could be interpreted to mean that in this instance, absolutely no mto1p in the cell is capable of interacting with the -tubulin complex. However, our current understanding of the role of -tubulin in fission yeast microtubule nucleation (Horio et al., 1991; Stearns et al., 1991; Paluh et al., 2000; Vardy and Toda, 2000; Heitz et al., 2001; Fujita et al., 2002; Zimmerman et al., 2004a) and previous and current analyses of mto1 and mto2 mutant phenotypes (Sawin et al., 2004; Venkatram et al., 2004) lead us to suggest that small amounts of solid-phase mto1p interacting with the -tubulin complex at SPBs and eMTOCs may be sufficient for the nucleation activities observed in cells. This view is supported by our observation of extremely low levels of alp4-GFP at eMTOCs in mto2 mutants, which correlates with their reduced microtubule nucleation activity. In general, essentially all biochemical work on -tubulin in eukaryotes has been done on soluble complexes, and very little is known about solid-phase -tubulin complexes in eukaryotes (see, for example, Salas, 1999; Terada et al., 2003; Zimmerman et al., 2004b).

A second issue concerns the role of mto2p in promoting association of mto1p with the -tubulin complex. One possibility is that mto2p forms a "bridge" between an mto1p/mto2p complex and the -tubulin complex. If this were the case, we might expect a functional analog of mto2p to be similarly associated with mto1p at SPBs and eMTOCs, in the place of mto2p. Although this has not yet been investigated, we note that mto2p seems to remain associated with mto1p at all MTOCs, throughout the cell cycle (even when, in our view, it may not be very important for mto1p function). Thus, we speculate that the role of mto2p in promoting -tubulin complex binding to mto1p may be more indirect, possibly allosteric, and that supplementary mechanisms may promote mto2p-independent -tubulin complex binding to mto1p at SPBs and eMTOCs. Future experiments to dissect the molecular interactions governing localization of mto1p and -tubulin complex association will help to resolve this issue.

The final point of the model concerns targeting of an mto1p/mto2p complex to MTOCs and its consequences for nucleation. Overall, our data from GFP and YFP fusion proteins suggest that mto1p targeting is independent of mto2p and that mto2p targeting depends on mto1p. However, in the two cases where mto2 microtubule nucleation is not completely normal (i.e., iMTOC and eMTOC nucleation), levels of mto1-YFP are somewhat reduced and/or more difficult to detect at the corresponding MTOCs. This could be due to unrecognized defects in the mto1p complex itself or alternatively to the defects in microtubule organization known to exist in mto2 mutants. Aberrantly organized microtubules might fail to provide an optimal distribution of tracks or loading sites for mto1p complexes (for example, in the form of microtubule minus ends). In either case, it is worth noting that achieving a full complement of mto1p at eMTOCs could depend on microtubule-based movement of mto1p on PAA microtubules, which are themselves abnormal in mto2 mutants. Further work toward understanding the nature of satellite particle movements, particularly using in vitro systems, will help to clarify these issues.

Complicating our understanding of the role of mto2p in the subcellular targeting of mto1p is the fact that mto1p does not redistribute properly to the nuclear surface in cold shock experiments in mto2 mutants. This result stands in apparent opposition to our observations with GFP- and YFP-fusions to mto1p in live mto2 cells, especially with regard to nmt81:GFP-mto1p localization, for reasons which remain unclear. The fact that a significant fraction of mto1p may not remain in situ after fixation (Sawin et al., 2004) may be a contributory factor. In any case, the microtubule nucleation defects seen in mto2 mutants both in the cold shock experiment and during live cell analysis are most easily understood by the failure of cytoplasmic mto1p complexes to associate with the -tubulin complex, as shown in our immunoprecipitation and alp4-GFP localization studies.

Microtubule Nucleation and Dynamics in mto2 Mutants
Under steady-state growth conditions, mto1 mutants do not nucleate new interphase microtubules, and during recovery from cold shock they nucleate microtubules very poorly, and only from the SPB (Sawin et al., 2004; Venkatram et al., 2004). By contrast, mto2 cells efficiently nucleate interphase microtubules both during steady-state growth and after cold shock, but in both cases apparently only from the SPB. How do we account for these different microtubule nucleation phenotypes relative to each other and to wild-type cells? In fission yeast, the discoid SPB is associated with the nuclear envelope (Ding et al., 1997). It has been proposed previously that mto1p might be localized to the cytoplasmic face of the SPB in wild-type cells and that the poor SPB-mediated microtubule nucleation seen in mto1 mutants after cold shock may be driven by an mto1p paralog, pcp1p, from the nucleoplasmic face of the SPB (Sawin et al., 2004; Venkatram et al., 2004). We suggest that the efficient nucleation of cytoplasmic microtubules from the SPB in mto2 mutants is driven not by pcp1p but by active mto1p localized to the cytoplasmic face of the SPB. This mto2p-independent localization of mto1p also would account for the nucleation of cytoplasmic astral microtubules seen during mitosis in mto2 mutants and not mto1 mutants. In wild-type (i.e., mto2+) interphase cells, the additional ability to recruit the -tubulin complex to mto1p satellites would drive non-SPB–mediated microtubule nucleation.

At the end of mitosis in asynchronous cultures, mto1 mutants completely fail to form a PAA (Sawin et al., 2004; Venkatram et al., 2004), whereas mto2 mutants nucleate PAA microtubules but at a reduced frequency relative to wild-type cells. This can be explained by the presence of active mto1p at eMTOCs in mto2 mutants, but at a reduced level (see comments above). In contrast, after release from an nda3 mitotic arrest, mto2 cells show a more robust PAA, similar to wild-type cells. To explain this, we suggest that during an extended mitotic arrest in nda3 mto2 mutants, additional mto1p may be able to slowly accumulate at the cell division site. The mechanism of mto1p targeting to the division site is not known but may involve an interaction with the cytokinetic actin ring (Heitz et al., 2001; Pardo and Nurse, 2003; Sawin et al., 2004), which is formed and maintained during the nda3 arrest (Chang et al., 1996).

Finally, we note that the dynamics of interphase, PAA, and astral microtubules in mto2 mutants are different from wild-type cells. In particular, mto2 interphase microtubule dynamics look like those in mto1 cells, with abnormally bundled microtubules curling around cell tips and bend-breakage events generating new microtubule fragments (Sawin et al., 2004). We have previously suggested for mto1 mutants that these phenomena may represent an indirect consequence of altering the number of microtubule nucleation sites in a system with a fixed volume and tubulin concentration (see Sawin et al., 2004 for a further discussion). We believe an identical case can be made for mto2 mutants. Although we will not repeat the detailed arguments here, we note that theoretical calculations based on a simplified model of dynamic instability in a closed system arrive at conclusions consistent with this view (Mitchison and Kirschner, 1987).

	ACKNOWLEDGMENTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION ACKNOWLEDGMENTS REFERENCES

 
We thank A. Douglas and P. Sasajala for contributions to this work during summer projects; F. Chang, K. Gull, I. Stancheva, and T. Toda for antibodies, strains, and plasmids; H. Ohkura for comments and discussion; and K. Gould, S. Venkatram, and P. Tran for alerting us to similar work on mto2p in the respective laboratories. K.E.S. is a Wellcome Trust Senior Research Fellow in Basic Biomedical Sciences. H.A.S. is a Caledonian Research Fellow. This work was supported by the Wellcome Trust.

	Footnotes

 
This article was published online ahead of print in MBC in Press (http://www.molbiolcell.org/cgi/doi/10.1091/mbc.E04-11-1003) on January 19, 2005.

Abbreviations: eMTOC, equatorial microtubule-organizing center; iMTOC, interphase microtubule-organizing center; PAA, postanaphase array; SPB, spindle pole body.

The online version of this article contains supplemental material at MBC Online (http://www.molbiolcell.org).

^* Present address: National CJD Surveillance Unit, Western General Hospital, Crewe Rd., Edinburgh EH4 2XU, United Kingdom.

Address correspondence to: Kenneth E. Sawin (ken.sawin{at}ed.ac.uk).

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