Published In Centrosomes and Spindle Pole Bodies (R.E. Palazzo and T. Davis, Editors); Method in
Cell Biology Volume 67 (2000)
Revised on June 11,2002

In Vitro Analysis of Microtubule Nucleation at the Fission Yeast Spindle
Pole Body.
Hirohisa Masuda1, Saeko Takada2, Takehiko Shibata2, W. Zacheus Cande3 and Yasushi Hiraoka1.
1)Structural Biology Section, CREST Research Project, Kansai Advanced Research Center, Communcations
Research laboratory, Kobe 651-2492, Japan. 2) Cellular and Molecular Biology Laboratory, RIKEN (TheInstitute of Physical and Chemical Research), 2-1 Hirosawa, Wako, Saitama 351-0198. 3) Department of
Molecular and Cell Biology, University of California, Berkeley, CA94720, USA.
Contents:
I.
Introduction
II.
Protocol for permeabilization of S. pombe cells
A. Solutions
B. Cell culture
C. Digestion of the cell wall
D. Permeabilization
III.
Protocol for microtubule nucleation at the spindle pole body
A. Solutions
B. Tubulin preparation
C. Preparation of Xenopus egg mitotic extracts
D. Microtubule nucleation
E. Immunofluorescence
IV.
Results
A. Microtubule nucleation at the mitotic spindle pole body
B. Activation of the interphase spindle pole body-tubulin in activation of the spindle pole body
D. Identification of an activator of the spindle pole bodyI.

Introduction

The yeast spindle pole body (SPB), the structure responsible for promoting microtubule nucleationand spindle formation, is the functional equivalent of the animal centrosome. In the fission yeast
Schizosaccharomyces pombe, the SPB is an oblate ellipsoidal structure that resides on the cytoplasmic
surface of the nuclear envelope during interphase (see Chapter I-3). As the cell enters mitosis, the nuclear
envelope invaginates beneath the SPB and forms an opening into which the SPB settles (Ding et al., 1997).
Microtubules are then nucleated at the nuclear surface of the SPB for spindle assembly. During anaphase, theSPBs move back into the cytoplasm.
Microtubule-nucleating activity of the S. pombe SPB is regulated during the cell cycle (Hagan andHyams, 1988; Masuda et al., 1992), although -tubulin, a protein responsible for microtubule nucleation, is-tubulin is found on the osmiophilic material that lies
near the inner surface of the nuclear envelope immediately adjacent to the SPB (Ding et al., 1997). ). During
interphase, a few microtubules attach to or terminate at the region close to the cytoplasmic face of the SPB.
-tubulin associated with the osmiophilic material is not active for microtubule nucleation during-tubulin on the osmiophilic material begins to nucleate spindle
microtubules. At metaphase through telophase, the cytoplasmic face of the SPB nucleates cytoplasmic
microtubules that are required for nuclear positioning (Hagan and Yanagida, 1997).
The activity of the centrosome in animal cells has been studied using permeabilized cells (Snyder and
McIntosh, 1975; Joshi et al., 1992) and isolated centrosomes (Kuriyama and Borisy, 1981; Mitchison and
Kirschner, 1984; Bornens et al., 1987). For preparation of permeabilized cells, mammalian cultured cells aretreated with microtubule inhibitors to depolymerize microtubules, followed by permeabilization with non-tubulin heterodimers.

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-tubulin dimers
purified from bovine or porcine brains. Centrosomes also can be isolated from cultured cells, after
incubation of the cells in drugs that disrupt the cytoskeleton. After this treatment, the centrosome is no longer
anchored to other components of the cytoplasm, and subsequent lysis in hypotonic solution releases thecentrosomes into the medium.
These same kinds of approaches can be used for studying the activity of the yeast SPB in vitro. In
Saccharomyces cerevisiae, the SPB is a disk-shaped, multilayered structure that is embedded into the nuclear
envelope throughout the cell cycle. The isolation procedure requires fractionation and lysis of the nuclei torelease the SPB from the nuclear envelope (Rout and Kilmartin, 1990; see Chapter I-1). These isolated
preparations were used for defining the composition (Rout and Kilmartin, 1990; Wigge et al., 1998) and thestructure of the SPB (Bullitt et al., 1997). Microtubule-nucleating activity of the budding yeast SPB has also
been studied using the partially purified SPB (Byers et al., 1978; Hyams and Borisy, 1978). An isolation
procedure for purifying fssion yeast SPBs has not yet been developed.
We have developed a procedure to permeabilize S. pombe cells for studying the mechanisms ofspindle elongation (Masuda et al., 1990) and of microtubule nucleation at the SPB (Masuda et al., 1992,
1996; Takada et al., 2000). Using permeabilized cells we have shown that the mitotic SPB, but not theinterphase SPB is capable of nucleating microtubules in vitro. This result indicates th-tubulin localized at
the SPB is in an inative form during interphase. The interphase SPB can be activated for microtubule
nucleation by incubation in mitotic extracts prepared from unfertilized Xenopus eggs. This activation seems
to occur through inte-tubulin located at the SPB. Finally,
the activator has been isolated from the extracts based on the in vitro activity, and identified as theribonucleotide reductase, large subunit R1.
II. Protocol for Permeabilization
A. Solutions
YPD: 1% yeast extract, 2% polypeptone, 2% glucose
YPDS: YPD + 1M sorbitol
0.1 M Tris-H2SO4 (pH9.4): 0.1 M Tris, adjusted to pH9.4 with H2SO4
SSD: 0.67% yeast nitrogen base without amino acid (DIFCO), 2% glucose, 1 M sorbitol, adjusted to pH7.2
with NaOH
MDS: 0.1 M MES (pH6.5), 5 mM EDTA, 1 mM spermidine, 0.5 mM spermine, 20% DMSO, 1 M sorbitol,
0.1 mM trolox (Fluka Chemical Co.), 5 mM DTT, 0.2 mM PMSF, 1 ug/ml aprotinin, and 1/200 of PI
MD: MDS without sorbitol
PI (protease inhibitor cocktail): 10 mg/ml BAME, 10 mg/ml TPCK, 10 mg/ml TAME, 1 mg/ml leupeptin, 1
mg/ml pepstatin A in DMSO
B. Cell Culture
Wild type cells (h-972) are grown at 26 ~33°C in rich media such as YPD and YEA to a density at 2 ~4 x
10 /ml. 40 ml of cells (0.8~1.6 x 108 cells) are used for one preparation. Since the composition of the cellwall is known to vary with growth conditions, cells grown at early log phase are recommended. Synthetic
media such as EMM2 may be used instead of rich media, although we have not tested it.
Wild type cells treated with drugs, or cell division cycle mutants arrested at the restrictive temperatures,are used to obtain cells that are in a similar stage of the cell cycle. Wild type cells are arrested at S phase by
incubation at 29°C for 3-4 hr in the presence of 10 mM hydroxyurea. Mutant strains arrested in M phase at
the restrictive temperatures are used for obtaining mitotic cells. Nuc2 cells (nuc2-663) are used for obtaining
mitotic cells arrested at metaphase (Hirano et al., 1988). They have a defect in APC/C or cyclosome function
at the restrictive temperature (36°C) (Yamada et al., 1997). Nuc2 cells are grown at 26°C in YPD to a densityof 2-4 x106 cells/ml and then incubated at 36°C for 4 hr. Nda3 cells (nda3-KM311) are used for obtaining
mitotic cells arrested in mitosis without spindle assembly. They have a mutation on the -tubulin gene andshow a defect in microtubule nucleation at early stages of mitosis at the restrictive temperature (20°C)(Hiraoka et al., 1984). They are grown at 30°C in YPD to the density of 2-3 x106 cells/ml and then incubatedat 20°C for 6 hrs and a
microtubule depolymerizing drug. MBC is dissolved in 100% DMSO at 1.5 mg/ml, and added 1/100 vol. tothe culture medium. The addition of MBC is required for obtaining permeabilized mitotic cells without
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microtubule assembly. We found that a short spindle is formed even at the restrictive temperature during theperiod when the cell wall is being digested for spheroplasting.
C. Digestion of the Cell WallDigestion of the cell wall with lytic enzymes is required prior to permeabilization of the cells with a nonionic detergent. The cell wall consists mainly of polysaccharides of glucose, mannose and Nacetylglucosamine (Cabib et al., 1982). In addition, chitin constitutes the primary septum. We use
Zymolyase-100T (Seikagaku Corp.) for digestion. An essential enzyme responsible for lysis in the product is-1,3-1, 3-linkages.
1. Wash cells briefly in 40 ml of 0.1 M Tris-H2SO4 (pH9.4) prewarmed to 29°C (36°C for nuc2, and 20°C fornda3). Incubate cells with 0.1 M Tris-H2SO4 (pH9.4) containing 10 mM DTT for 4-5 min at 29°C (36°C for
nuc2, and 20°C
lutions.
Mannan is depolymerized by mild alkaline treatment. In addition, digestion of the cell wall by lytic enzymes
is facilitated by preincubation with sulfhydryl compounds (Cabib et al., 1982).
2. Wash cells twice with 10 ml of YPDS. Resuspend cells in 2 ml of YPDS and add 60-100
Zymolyase 100T (final concentration 0.6-1.0 mg/ml). Incubate at 29°C (36°C for nuc2) with shaking. Checkthe extent of digestion every ten minutes under a microscope by diluting a small amount of the cellsuspension into water. Incubate a further 30 min, after more than half of the cells diluted burst in water. We
usually incubate for 40-60 min total. For nda3 cells, wash cells twice with 10 ml of SSD at 20°C. Incubate
cells at 20°C with 1.5 mg/ml Zymolyase 100T in SSD (pH7.2) containing MBC, for 40-60 min.
The digestion requires the presence of 1 M sorbitol for preventing cells from bursting due to the osmoticpressure. The solubility of Zymolyase is limited. Add the enzyme using the 20 mg/ml suspension in H 2O.
The quality of the enzyme depends on the product batches. The optimal pH of the enzyme activity at 35°C is
pH7.5, whereas the pH of YPD is around 6. Digestion of the cell wall at 20°C requires increasing the pH ofculture media to near the opitimal pH for enzymatic activity.
D. Permeabilization with Triton X-100
1. Wash cells three times with 7 ml of ice-cold MDS. Resuspend cells in 4 ml of MDS and add 4 ml of MDS
containing 1% Triton X-100. Incubate cells for 7-10 min on ice.
Addition of DMSO, Trolox, and protease inhibitors (PI) to the medium is critical for preserving the structureand function of the SPB. Trolox is a water-soluble derivative of Vitamin E, which acts as a radical scavenger.
For studying the mechanism of spindle elongation, spindle microtubules are preserved by washing twice with
MDS at room temperature, followed by a wash with cold MDS. The quality of permeabilization needs to bechecked by an in vitro activation assay of microtubule nucleation (see section III).
2. Wash cells three times with 10 ml of MD. Resuspend cells in MD at 2.5 x 107/ml. Freeze cells as aliquots
in liquid N2, and store at –80°C.
Permeabilized cells used for SPB activation are stored at –80°C up to 1 year. For spindle elongation, use of
freshly prepared permeabilized cells is required. Freezing cells may inactivate the motor proteins required forspindle elongation.
III. Protocol for Microtubule Nucleation
A. Solutions
Make 2x solutions and freeze as aliquots. Before use, dilute with H 2O and mix with additives.
Wash: 75 mM HEPES, 2.5 mM MgAcetate, 5 mM EGTA, 10% DMSO, adjusted to pH7.6 with KOH; add 1
mg/ml ovalbumin, 3 mM GTP, 5 mM DTT, and 1/200 vol. of PI before use.

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-glycerophosphate, 35 mM sucrose, 0.1
mM trolox, adjusted to pH7.6 with KOH; add 1 mg/ml ovalbumin, 3 mM GTP, 4.8 mM glutathione, 5 mM
DTT, and 1/200 vol. of PI before use.
-glycerophosphate, 0.2 mM trolox, adjusted
T, and 1/200 vol. of PI, and centrifuge before
use.
PME: 80 mM PIPES, 1 mM MgSO4, 1 mM EGTA, adjusted to pH6.8 with KOH-glycerophosphate, 0.1 mM trolox,
adjusted to pH6.8 with KOH; add 3 mM GTP, 10%
before use.
-glycerophosphate, 0.1 mM trolox,
adjusted to pH7.4 with KOH; add 6 mM ATP, 5 mM DTT, and 1/200 vol. of PI before use.
Fixative: 75 mM PIP-glycerophosphate, 0.1 mM trolox(adjusted to pH6.8 with KOH); add 10% DMSO, 4% paraformaldehyde, 0.2% glutaraldehyde.
PEMBL: 0.1 M PIPES (pH7.2), 1 mM EGTA, 1 mM MgSO4, 1.83% L-lysine, 1% BSA, and 0.1% sodium
azide.
B. Tubulin Preparation
The protocol for tubulin isolation from S. pombe cells has not been developed yet. Instead, neuro-tubulin
is purified from bovine or porcine brains using DEAE-Sepharose or phosphocellulose. Purified tubulin
dissolved in PME and 1 mM GTP is frozen in liquid N2 as aliquots and stored at –80°C.
C. Preparation of Xenopus Egg Mitotic Extracts
Mitotic crude extracts are prepared from Xenopus laevis unfertilized eggs according to the method of
Murray (1991) with slight modifications (Masuda et al., 1992; Masuda and Shibata, 1996). The unfertilized-speed extracts(HSE) are prepared by diluting the crude extract 1.5-fold with XEB containing protease inhibitors and energy
mixture (7.5 mM creatine phosphate, 1 mM ATP, 0.1 mM EGTA, 1 mM MgCl 2, pH7.7), followed bycentrifugation at 80,000 rpm for 60 min with Beckman TLA100 rotor. The clear central zone of thesupernatant is collected as HSE, frozen in liquid N2, and stored at -80°C.
D. Microtubule Nucleation
1. Thaw frozen permeabilized cells on ice, and aliquot 10 6 cells per reaction in 500-cold Wash solution. For nuc2 cells, depolymerize microtubules by incubating for 10
min with HMEG containing 6 mM ATP at room temperature. For SPB activation in interphase cells, incubate
HSE prepared from Xenopus unfertilized eggs or purified fractions from HSE for 5 min atroom temperature.
incubate for 10 min at room temperature. Centrifu

E. Immunofluorescence
Incubate permeabilized cells for 90anti- -tubulin (Sigma, DM1A or B-5-1-2; diluted to 1/250) and affinity-purified rabbit anti-S. pombe 90-120 min with FITC (or4Alexa488)-labeled anti-mouse IgG and Cy3-labeled anti-rabbit IgG (Jackson ImmunoResearch). Wash 3 x
2O. Put a small amount of cellsuspension on multiwell coverslips, dry them out completely and seal in 90% glycerol, 10% PBS, and 100
mg/ml 1, 4-diazabicyclo(2.2.2)octane.
IV.

Results

A. Microtubule nucleation at the mitotic SPB
Mitotic and interphase S. pombe cells were processed for permeabilization, and microtubulenucleating activity of the SPB in the cells was tested by addition of brain tubulin. After fixation, microtubules
and the SPB were detected by immunofluorescence with anti- -tubulin and anti- -tubulin antibody,respectively.
Microtubule nucleation at the SPB was induced in permeabilized mitotic cells (Masuda et al., 1992).
The permeabilized nuc2 cells that retained a metaphase spindle were further incubated with 6 mM ATP at
pH7.6 to depolymerize spindle microtubules. After this treatment, most of the SPBs still were able tonucleate microtubules in the presence of exogeneous tubulin (Fig. 1a, b). We also examined the ability of
mitotic SPBs in nda3 cells that arrested in mitosis with no microtubules nucleated at the SPBs. The SPBs
also promoted microtubule assembly (Fig. 1c, d).
In contrast, the SPBs in most of cells grown asynchronously, and the SPBs in most of cells arrested
at S phase (Fig. 1e, f) did not nucleate microtubules in the presence of exogenous tubulin, showing thatmicrotubule-nucleating activity of the SPB at G2 and S phase is low (Masuda et al., 1992).

5Figure 1. Microtubule nucleation at the SPB in permeabilized S. pombe cells. (a, b) nuc2 cells arrested at
metaphase, (c, d) nda3 cells arrested in mitosis, (e, f) interphase cells arrested at S phase, and (g, h)interphase cells treated with Xenopus egg mitotic extracts. Cells were double-stained with anti-S. pombe tubulin antibody (a, c, e, g), and anti- B. Activation of the interphase SPB
The interphase SPB was converted into a state competent for microtubule nucleation by incubation
with mitotic extracts prepared from Xenopus unfertilized eggs (Fig.1g, h) (Masuda et al., 1992). The
conversion was not induced by interphase extracts, but by an interphase extract driven into mitosis byaddition of exogenous cyclin B (GST-cyclin B) in the presence of cycloheximide. The conversion was ATPdependent, and suppressed by protein kinase inhibitors or alkaline phosphatase. Purified, active cdc2/cyclin
B complex, however, was not effective for SPB activation. These results suggest that SPB activation occurs
through a downstream pathway that is regulated by cdc2 kinase.
-tubulin in SPB activation-tubulin in SPB activation, we examined the effect of anti- -tubulin
antibodies on microtubule nucleation (Masuda and Shibata, 1996). Preincubation of permeabilized interphasecells with anti-S. pombe -tubulin prior to incubation in the Xenopus mitotic extract suppressed microtubule-tubulin also is involved in microtubule
nucleation at the activated SPB. Using antibodies that were affinity-purified with an N-terminal, middle, andC-tubulin, we have found that anti-N-terminal fragment antibody was effective ininhibiting microtubule nucleation at the interphase SPB incubated in Xenopus mitotic extracts, but not at the
mitotic SPB of nda3 cells. Anti-C-terminal fragment antibody suppressed microtubule assembly at the SPB
incubated in Xenopus mitotic extracts, and at the mitotic SPB of nda3 cells. Anti-middle fragment antibody
was not effective for inhibiting microtubule nucleation. These results suggest that the N-terminal region of-tubulin is important for SPB activation, and the C-terminal region is involved in microtubule assembly.
We also examined the effect of recombinant S. pombe -tubulin on SPB activation (Masuda andShibata, 1996). Addition of full length or an N-terminal-tubulin to Xenopus mitotic extracts
suppressed SPB activation in a concentration-dependent manner.
To partially purify the activator, we first fractionated the mitotic extracts with Q-Sepharose column
chromatography (Masuda and Shibata, 1996). The flow-through fraction retained the SPB converting activity.
-tubulin from the extract is not
required for SPB activation. The active fraction was further fractionated by affinity column chromatography
with an N-tubulin. The bound fraction had the SPB converting activity. Taken
tubulin located on the SPB for SPB activation.
D. Identification of an SPB activator
Based on the in vitro activity monitored by the SPB activation assay, we have purified an 85kDa
SPB activator from Xenopus egg mitotic extracts (Takada et al., 2000). The 85 kDa protein was identified as
the ribonucleotide reductase large subunit R1 by amino acid sequence analysis. The ribonucleotide reductase(RNR) consists of a complex formed by a dimer of the large subunit R1 and a dimer of the small subunit R2.
It catalyses the synthesis of deoxyribonucleotides, and provides substrates required for DNA synthesis andrepair. Recombinant mouse R1 was also effective for SPB activation. In contrast, R2 was not required for
SPB activation. SPB activation by R1 was inhibited by antibodies against R1, and by a partial oligopeptide of
R1. GST-mouse R1 bound to R2, -tubulin dimers in Xenopus extracts.
R1 also seems to be involved in regulation of the centrosome activity. The partial oligopeptide of R1
inhibited aster formation at the centrosome reconstituted from Xenopus sperm centrioles in Xenopus egg
mitotic extracts (see chapter V-25, 26 for detail). R1 was detected in the animal centrosome byimmnofluorescence and immunoblotting with anti-R1 antibodies.
Our results suggest that R1 is a bifunctional protein that acts as the subunit of ribonucleotide
reductase and as an activator of the SPB. This bifunctionality may be important for regulating the onset ofmitosis. For instance, DNA damage increases the activity of ribonucleotide reductase, which may suppress
the SPB activator function of R1 and subsequent spindle formation. Further in vitro and in vivo studies on

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regulation of R1 dual functions will clarify the molecular mechanisms of microtubule nucleation at the SPB,
and its regulation during the cell cycle.
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