HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE ADVANCED SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Masuda, H. Articles by Hiraoka, Y. Search for Related Content PubMed PubMed Citation Articles by Masuda, H. Articles by Hiraoka, Y.

¹ Cell Biology Group and CREST/JST, Kansai Advanced Research Center, National Institute of Information and Communications Technology, Kobe, 651-2492, Japan
² Laboratory of Cell Regulation, Cancer Research UK London Research Institute, Lincoln's Inn Fields Laboratories, London WC2A 3PX, UK

	Abstract

Top Abstract Introduction Results Discussion Experimental procedures References

 
The -tubulin complex acts as a nucleation unit for microtubule assembly. It remains unknown, however, how spatial and temporal regulation of the complex activity affects microtubule-mediated cellular processes. Alp4 is one of the essential components of the S. pombe-tubulin complex. We show here that overproduction of a carboxy-terminal form of Alp4 (Alp4C) and its derivatives tagged to a nuclear localization signal or to a nuclear export signal affect localization of -tubulin complexes and induces novel phenotypes that reflect distinct functions of nuclear and cytoplasmic -tubulin complexes. Nuclear Alp4C induces a Wee1-dependent G2 delay, reduces the levels of the -tubulin complex at the spindle pole body, and results in defects in mitotic progression including spindle assembly, cytoplasmic microtubule disassembly, and chromosome segregation. In contrast, cytoplasmic Alp4C induces oscillatory nuclear movement and affects levels of cell polarity markers, Bud6 and Tip1, at the cell ends. These results demonstrate that regulation of nuclear -tubulin complex activity is essential for cell cycle progression through the G2/M boundary and M phase, whereas regulation of cytoplasmic -tubulin complex activity is important for nuclear positioning and cell polarity control during interphase.

	Introduction

Top Abstract Introduction Results Discussion Experimental procedures References

 
Microtubules (MTs) are essential for many cellular processes including chromosome segregation, vesicle transport, nuclear positioning, and establishment of cell polarity. MTs are organized from MT-organizing centers (MTOCs) such as the centrosome of animal cells and the spindle pole body (SPB) of yeast. -Tubulin plays a central role in MT nucleation at MTOCs (reviewed in Job et al. 2003). -Tubulin exists in multiple protein complexes in the cytoplasm and is recruited to MTOCs to mediate MT nucleation (reviewed in Wiese & Zheng 1999).

The -tubulin ring complex (TuRC), an open ring structure of 25 nm (25–30S, > 2000 kDa), has been purified from Xenopus, Drosophila, and humans and shown to nucleate MTs in vitro (Zheng et al. 1995; Oegema et al. 1999; Murphy et al. 2001). The TuRCs consist of -tubulin and 5–7 other polypeptides termed Xgrips, Dgrips, or hGCPs. The -tubulin small complex (TuSC), which may be the functional and structural core of the TuRC, exists in Drosophila embryos and Xenopus eggs and consists of -tubulin and two other polypeptides that are homologous to hGCP2 and hGCP3 (Oegema et al. 1999; Zhang et al. 2000). In Saccharomyces cerevisiae, the -tubulin complex exists both as a small complex (300 kDa) that consists of Tub4p (-tubulin), Spc97p (hGCP2 homolog), and Spc98p (hGCP3 homolog) (Geissler et al. 1996; Knop et al. 1997; Vinh et al. 2002), and as a large complex that may be the equivalent of the higher eukaryotic TuRC (Vinh et al. 2002). In Schizosaccharomyces pombe, -tubulin (Gtb1), together with Alp4 (Spc97/hGCP2 homolog), Alp6 (Spc98/hGCP3 homolog), Alp16 (hGCP6 homolog), Gfh1 (hGCP4 homolog) and Mto1/Mbo1/Mod20 (Vardy & Toda 2000; Fujita et al. 2002; Sawin et al. 2004; Venkatram et al. 2004), forms a large complex (> 20S), comparable to that in higher eukaryotes. Except for Dgp71WD (Gunawardane et al. 2003) and Mto1 (Venkatram et al. 2004), all of the non-tubulin subunits that have been sequenced contain conserved structural motifs that are termed grip motifs 1 and 2 (Gunawardane et al. 2000).

In this paper we use S. pombe as a model system to study the functions of the -tubulin complex in MT-dependent cellular processes. S. pombe cells grow cylindrically and cytoplasmic MTs are required for polarized cell growth and nuclear positioning (reviewed in Hagan 1998). Cytoplasmic MTs are organized parallel to the long axis of the cell from iMTOCs (MTOCs for interphase MTs). iMTOCs are located at the SPB and a few other sites on the nuclear envelope (Drummond & Cross 2000; Tran et al. 2001; Sagolla et al. 2003; Sawin et al. 2004: Zimmerman et al. 2004). The nucleus maintains its position at the cell center by a balance of pushing forces exerted by MTs extending from iMTOCs on the nuclear envelope towards both cell ends (Tran et al. 2001). MTs are required for correct distribution of cortical actin patches and other cell polarity factors (reviewed in Chang & Peter 2003). Tea1 is a cell polarity factor that is transported to the cell end on the plus ends of MTs (Mata & Nurse 1997) together with a CLIP-170 homolog, Tip1 (Brunner & Nurse 2000), and a kinesin-like protein, Tea2 (Browning et al. 2000). Tea1 is thought to regulate cell polarity by associating with other factors such as SH-3 containing Tea4 (Martin et al. 2005); an actin binding protein, Bud6/Aip3 (Glynn et al. 2001; Jin & Amberg 2001); and an activator of actin filament nucleation, the formin For3 (Feierbach et al. 2004).

As revealed by immunofluorescence, in S. pombe the -tubulin complex is localized at the SPB throughout the cell cycle (Horio et al. 1991; Masuda et al. 1992; Vardy & Toda 2000; Fujita et al. 2002; Venkatram et al. 2004). Most of the complex, however, is observed at the nuclear face of the SPB (Ding et al. 1997). The MT-organizing activity of the nuclear complex is regulated during the cell cycle: no MTs are observed at the nuclear face of the SPB during interphase (Ding et al. 1997). In vitro experiments have shown that the nuclear complex is inactive for MT nucleation during interphase and is activated at the onset of mitosis (Masuda et al. 1992; Masuda & Shibata 1996; Takada et al. 2000). In addition to its essential role in spindle formation, the nuclear -tubulin region may be important for anchoring centromeres close to the SPB during interphase; S. pombe centromeres cluster close to the SPB (Funabiki et al. 1993; Kniola et al. 2001). In the cytoplasm, the -tubulin complex may be recruited to iMTOCs located on the nuclear envelope via Mto1 and Mto2 for cytoplasmic MT formation (Sawin et al. 2004; Venkatram et al. 2004, 2005; Janson et al. 2005; Samejima et al. 2005; Zimmerman & Chang 2005). The nature of iMTOCs, however, is not well understood. The -tubulin complex is also localized to the equatorial MTOC (eMTOC) (Horio et al. 1991; Vardy & Toda 2000; Fujita et al. 2002; Sawin et al. 2004; Venkatram et al. 2004). The eMTOC establishes a postanaphase array of MTs at septum formation (Heitz et al. 2001; Zimmerman et al. 2004) and plays a role in anchoring the contractile actin ring during cytokinesis (Pardo & Nurse 2003). These observations suggest that formation and activity of the -tubulin complex are regulated both spatially and temporally in the cell.

Temperature-sensitive mutations in genes encoding essential components of the -tubulin complex, -tubulin, Alp4, and Alp6 in S. pombe cells have pleiotropic effects on MT-dependent cellular processes both in the cytoplasm and in the nucleus, including MT dynamics and organization, mitotic spindle assembly, chromosome segregation, and cytokinesis (Paluh et al. 2000; Vardy & Toda 2000; Hendrickson et al. 2001). alp4 and alp6 mutant cells show a bent morphology that is associated with a ‘cut’ phenotype, displaced nuclei, formation of monopolar spindles, and abnormally long cytoplasmic MTs (Vardy & Toda 2000). Alp16, Gfh1 and Mto1 are nonessential components of the -tubulin complex (Fujita et al. 2002; Sawin et al. 2004; Venkatram et al. 2004). Cells deleted for genes encoding these proteins, however, show some defects in cell morphology and organization of cytoplasmic MT arrays, mitotic spindles, and astral MTs (Fujita et al. 2002; Sawin et al. 2004; Venkatram et al. 2004), suggesting that they are not required for MT nucleation, but rather modulate the functions of the complex. Overproduction of components of the -tubulin complex is also toxic and results in pleiotropic effects on MT organization, cell morphology, and cell cycle progression (Horio et al. 1999; Vardy & Toda 2000; Fujita et al. 2002; Venkatram et al. 2004). It remains largely unknown at the molecular level how compromised -tubulin complexes affect MT dynamics and organization and induce the defects found in the nucleus and in the cytoplasm.

We searched for a probe that modulates, but does not totally inhibit, -tubulin complex function as it can be a useful tool to spatially and temporally dissect the roles of the complex in MT-dependent cellular processes. We show here that a carboxy-terminal form of Alp4 and its derivatives, tagged to a nuclear localization signal or to a nuclear export signal, induce novel phenotypes in S. pombe cells by affecting localization and function of the -tubulin complex. Distinct functions of the nuclear and cytoplasmic -tubulin complex are revealed.

	Results

Top Abstract Introduction Results Discussion Experimental procedures References

 
Effects of overproduction of Alp4 and Alp6 fragments

In an attempt to dissect the roles of the -tubulin complex in MT-dependent cellular processes we studied the effects of overproduction of Alp4 and Alp6 fragments. In a two-hybrid assay, S. cerevisiae Tub4p (-tubulin) and Spc97p (the Alp4 homolog) bound to the carboxy-terminal and central domains of Spc98p (the Alp6 homolog), respectively (Pereira et al. 1998). Based on a sequence alignment of grip motifs (Gunawardane et al. 2000; Fujita et al. 2002), we created Alp4 and Alp6 fragments by cutting the proteins at positions corresponding to that used for producing the C-terminal portion of Spc98p. The fragments were overproduced using the pREP1 vector, a multicopy plasmid with the thiamine-repressible nmt1 promoter (Maundrell 1990).

We examined the effects of Alp4, Alp6, and their fragments on cell growth, cell polarity control, cell cycle progression, nuclear positioning, and chromosome segregation in a histone H3-GFP (hht2-GFP) strain (Wang et al. 2002) (Fig. 1). We assumed that bent cell morphology is a typical phenotype for defects in cell polarity control and that elongated cell morphology is a common phenotype for delays or arrests in cell cycle progression. We also assumed that elongated cell morphology with septa is a typical phenotype for defects in mitotic progression, since some types of mitotic mutants such as nuc2.663 (Hirano et al. 1988) show similar phenotypes. As shown in Fig. 1, full length Alp4 and Alp6 and an amino-terminal fragment of Alp6 (1–581 amino acids) had pleiotropic effects: they induced growth defects, bent and elongated morphology with and without septa, and chromosome missegregation. In addition to these phenotypes which are similar to those observed in temperature-sensitive alp4 and alp6 mutants (Vardy & Toda 2000), they also induced oscillatory nuclear movements in some of the cells. In contrast, a carboxy-terminal fragment of Alp4 (566–784 amino acids; termed Alp4C) more specifically affected cell cycle progression and nuclear positioning in interphase cells. It induced cell elongation without septa and oscillatory nuclear movement (Fig. 1B-c) but was much less effective for induction of bent morphology or chromosome missegregation. An amino-terminal fragment of Alp4 (1–565 amino acids) induced cell elongation with and without septa and nuclear movement in a small portion of cells. A carboxy-terminal fragment of Alp6 (582–832 amino acids) had almost no effect except that it induced nuclear movements in some of the cells. Because the Alp4C fragment gave rise to the most specific phenotypes we analyzed the effects of Alp4C overproduction in more detail.

View larger version (91K): [in this window] [in a new window]   Figure 1  Alp4C induces cell elongation and nuclear movement. (A) Effects of Alp4, Alp6 and their fragments on cell morphology. Alp4, Alp4N, Alp4C, Alp6, Alp6N and Alp6C were overproduced from pREP1, a multicopy plasmid carrying the thiamine-repressible nmt1 promoter, in hht2-GFP cells by incubating the transformed cells for 18–20 h at 30 °C in EMM2 media without thiamine. Bright-field images are shown. OP, overproduced. (B) Behavior of chromosomes. Chromosomes were observed in hht2-GFP cells overproducing Alp4 (a), Alp6N (b), or Alp4C (c). In (c) live cell images with time-lapse sequences of about 18 min at 1 min 17 s intervals were obtained. Projection images are shown at every 2 time points; projection images were reconstituted from images of 11 different focal planes at 0.3 µm spacing. (C) Summary of effects of overproduction of Alp4, Alp6, and their fragments. Effects of overproduction on cell morphology, nuclear movement, and chromosome missegregation were examined by incubating hht2-GFP cells for 18–24 h at 30 °C in EMM2 media without thiamine. Effects on cell growth were examined by incubating the cells for 3 days at 30 °C on agar plates containing EMM2 without thiamine. Bars, 10 µm.

The level of -tubulin complexes at the SPB is reduced in Alp4C-overproducing cells

We first studied localization of Alp4C using the pREP1 vector harboring GFP-tagged Alp4C. GFP-Alp4C fluorescence was observed in the nucleus and in the cytoplasm in the early stages of overproduction, with signals in the nucleus a little brighter than those observed in the cytoplasm. In the late stages of overproduction, signals were observed in the nucleus, at the SPB, and in the cytoplasm as diffuse protein or as aggregates (Fig. 2A).

View larger version (48K): [in this window] [in a new window]   Figure 2  Alp4C reduces levels of the -tubulin complex at the SPB. (A) Localization of GFP-Alp4C. GFP-Alp4C was overproduced in sid4-mRFP cells. Bar, 10 µm. (B) -Tubulin levels are reduced at the SPB. Alp4C-OP cells (Alp4C OP) and control cells (Control) were processed for immunofluorescence with anti--tubulin (red) and DAPI (blue). Bar, 5 µm. (C, D) Levels of Alp4 and Alp6 are reduced at the SPB in Alp4C-OP cells. Localization of endogenous Alp4 and Alp6 was visualized in alp4-GFP and alp6-GFP-HA cells overproducing Alp4C (Alp4C OP) or harboring pREP1 (Control). Bar, 10 µm. (E) Quantification of the reduction in levels of the -tubulin complex. Maximum fluorescence intensities of anti--tubulin, Alp4-GFP, and Alp6-GFP signals were obtained from projection images and are represented as percentages of the intensities of control cells. Error bars represent standard deviation. Levels of components of the -tubulin complex were significantly reduced compared to control cells (the Student's t-test; P < 0.0001).

Wee1-dependent G2 delays

Most of the Alp4C-OP cells elongated without septa, suggesting that these cells experienced a delay in cell cycle progression. Measurement of DNA content by flow cytometry showed that most of the Alp4C-OP cells were in G2 phase and not in G1 or S (data not shown). To determine which checkpoint pathway is required for inducing the delay, we examined the effects of Alp4C overproduction in mad2 (He et al. 1997; Kim et al. 1998), rad3 (Bentley et al. 1996), and wee1-50 (Lundgren et al. 1991) cells. Alp4C overproduction delayed cell cycle progression of mad2 and rad3 cells to a similar extent as wild-type cells (Fig. 3), suggesting that spindle assembly and DNA structure checkpoints are not involved in the delay. In contrast, Alp4C did not delay cell cycle progression of wee1-50 cells at 30 °C, the semipermissive temperature, or at 26 °C, the permissive temperature (Fig. 3). This suggests that Alp4C induces a G2 delay through the action of Wee1, an inhibitory tyrosine kinase of Cdc2. Consistent with this result, Alp4C induced extensive cell elongation at 26 °C and 30 °C, permissive temperatures in cdc25.22 cells (Fig. 3), which have a temperature-sensitive mutation in Cdc25, a tyrosine phosphatase that is required for Cdc2 activation to counteract Wee1 (Russell & Nurse 1986).

View larger version (138K): [in this window] [in a new window]   Figure 3  Wee1-dependent cell cycle delays are induced by Alp4C overproduction. Wild-type cells, wee1-50, cdc25.22, rad3d, and mad2d cells overproducing Alp4C (Alp4C OP) or harboring pREP1 (Control) were incubated for 1 day at 30 °C on EMM2-leucine-thiamine plates. Wild-type, wee1-50, and cdc25.22 cells were also incubated for 2 days at 26 °C. A cdc13-GFP sid4-mRFP cell overproducing Alp4C is shown. Cdc13-GFP is green and Sid4-mRFP is red. Bars, 10 µm.

Alp4C affects cytoplasmic MT organization, and localization of cell-end markers Tip1 and Bud6 at the cell ends

We observed MTs in GFP-2-tubulin (GFP-atb2) cells overproducing Alp4C. Three to five MT bundles were found in Alp4C-OP cells, similar to control cells, but some of the MTs were curved or curled around the cell ends (Fig. 4A). Live cell observation of MT end behaviors showed that cytoplasmic MTs are less dynamic in Alp4C-OP cells than in control cells (see Masuda et al. in this issue). Spindle formation and elongation and formation of postanaphase arrays of MTs seemed normal in Alp4C-OP cells (data not shown), although the percentage of mitotic cells decreased relative to control cells due to delays in cell cycle progression.

View larger version (63K): [in this window] [in a new window]   Figure 4  Organization of cytoplasmic MTs and localization of cell end markers at the cell ends of Alp4C-OP cells. (A) Organization of cytoplasmic MTs in Alp4C-OP cells. MTs were visualized using GFP-atb2 cells. (B) Bud6 levels are decreased and Tip1 levels are increased at the cell ends of Alp4C-OP cells. Localization of cell end markers, Tea1, Bud6, and Tip1 was examined in tea1-GFP, bud6-GFP, and tip1-YFP cells, respectively, overproducing Alp4C (Alp4C OP) and harboring pREP1 (Control). (C) Quantification of Bud6-GFP, Tip1-YFP, and Tea1-GFP levels at the cell end. See Experimental procedures for detail. Bars, 10 µm.

Oscillatory nuclear movements are led by the SPB and are dependent on MTs

Overproduction of Alp4C induced nuclear movements in more than 70% of the cells (Fig. 1C). Oscillatory movement of the deformed and enlarged nucleus (Fig. 1B-c) was observed in most of the elongated Alp4C-OP cells, and oscillatory movement of the normal-sized nucleus with smaller amplitudes was observed in more than half of normal-sized cells. Using Sid4-mRFP as an SPB marker (Chang & Gould 2000) and Hht2-GFP as a chromosome marker, we found that the SPB was located at the leading edge of the moving nucleus (see Masuda et al.in this issue). The nuclear and SPB movements were suppressed by addition of 100 µg/mL thiazendazole, a MT inhibitor, but not by addition of 200 µM Latrunculin A, an actin inhibitor (Ayscough 1998) (data not shown). These observations indicate that the nucleus is agitated by SPB movement, and this movement is mediated by MTs.

Effects of NLS- and NES-tagged Alp4C

Since the -tubulin complex is present in both the nucleus and the cytoplasm, we sought to determine whether the phenotypes observed with Alp4C overproduction, Bud6 reduction and Tip1 accumulation at the cell ends, nuclear oscillation, and Wee1-dependent G2 delays, were caused by nuclear or cytoplasmic Alp4C. We examined this by tagging the N-terminal of Alp4C with a nuclear localization signal (NLS) or a nuclear export signal (NES). The C-terminus of Xenopus nucleoplasmin (146–197 amino acids) (Hayashi et al. 1998) and a peptide of 29 amino acids including the NES of S. pombe Dsk1 (308–320 amino acids) (Fukuda et al. 1997) were used as the NLS and the NES, respectively. Localization of NLS-Alp4C to the nucleus and NES-Alp4C to the cytoplasm was confirmed by immunofluorescence using HA-tagged NLS-Alp4C and NES-Alp4C visualized with an anti-HA antibody (Fig. 5A).

View larger version (80K): [in this window] [in a new window]   Figure 5  Effects of NLS- and NES-tagged Alp4C. (A) Localization of NLS-Alp4C and NES-Alp4C. Cells overproducing NLS-Alp4C-HA or NES-Alp4C-HA were processed for immunofluorescence with anti-HA and DAPI. (B) Bud6 levels are reduced and Tip1 levels are increased by NES-Alp4C but not by NLS-Alp4C. NLS-Alp4C or NES-Alp4C was overproduced in bud6-GFP or tip1-YFP cells. (C) NES-Alp4C induces nuclear movement. NES-Alp4C was overproduced in hht2-GFP cells. Time-lapse images at time 0 and at 1 min 17 s are shown. (D) NLS-Alp4C induces cell elongation and septation in wee1-50 cells. Bright-field images are shown of wee1–50 cells overproducing NLS-Alp4C, NES-Alp4C, or harboring pREP1 (Control). Bar, 10 µm.

To test the possibility that NLS-Alp4C induces defects in mitotic progression, we examined the dynamic behavior of chromosomes, MTs, and the SPB, and localization of the -tubulin complex in mitotic NLS-Alp4C-OP cells. We observed defects in four events required for mitotic progression: spindle assembly, cytoplasmic MT disassembly, spindle elongation, and chromosome segregation. We also found reduction in levels of -tubulin complexes at the SPB of cells showing these mitotic defects.

NLS-Alp4C arrests cells at a stage prior to spindle assembly and cytoplasmic MT disassembly

Observing NLS-Alp4C-OP cells expressing GFP-Atb2 and Sid4-GFP, we found that some of the elongated cells were arrested with duplicated SPBs at a stage prior to spindle assembly and cytoplasmic MT disassembly (Fig. 6). Several lines of evidence show that spindle formation occurs following SPB maturation (Ding et al. 1997; Uzawa et al. 2004) and is accompanied by disassembly of cytoplasmic MTs (Hagan & Hyams 1988; Sagolla et al. 2003). In control cells, at the onset of mitosis, a dot of Sid4-GFP signal that was observed during interphase elongated and separated into two and cytoplasmic MTs started to disassemble (Fig. 6A-c). Within the next two minutes a spindle formed between the two dots. Disassembling cytoplasmic MTs were still seen with a short spindle and gradually disappeared over a few minutes. Thus, elongation and separation of the Sid4-GFP signal in control cells occurred at the G2/M boundary and was followed by formation of a mitotic spindle and initiation of cytoplasmic MT disassembly. In contrast, some (18%, n = 49) of the NLS-Alp4C-OP cells were arrested at stages following elongation or duplication of the Sid4-GFP signal. Cytoplasmic MTs did not disassemble and a spindle did not form between the SPBs (Fig. 6A-a,b). Observations of Alp4-GFP and Sid4-mRFP showed that the intensity of Alp4-GFP signals in the cells arrested with one elongated signal or two adjacent signals of Sid4-mRFP at the SPBs was reduced to 24% (n = 5) of that of control cells (Fig. 6B). To determine the arrest point of these cells, we examined the localization of Polo-like kinase Plo1 (Ohkura et al. 1995) using plo1-GFP-HA sid4-mRFP cells. Plo1 is localized at the SPB following Cdc2/Cyclin B activation through early anaphase and therefore can be used as a marker for entry into mitosis (Tanaka et al. 2001) (as a control, see Fig. 6C-b). In NLS-Alp4C-OP cells, Plo1 was localized at the SPBs of elongated cells arrested with one elongated Sid4 signal, or two adjacent Sid4 signals (Fig. 6C-a). In contrast, in the cells overproducing untagged Alp4C, Plo1 was not localized at the SPB of elongated cells in late G2 (data not shown). These results suggest that elongated cells with one elongated Sid4 signal or two adjacent Sid4 signals are arrested before disassembly of cytoplasmic MTs, but after Cdc2/Cyclin B activation.

View larger version (45K): [in this window] [in a new window]   Figure 6  NLS-Alp4C arrests spindle assembly and cytoplasmic MT disassembly at a stage when Plo1 is localized on the duplicated SPB. (A) Spindle assembly and cytoplasmic MT disassembly are defective in NLS-Alp4C-OP cells. The behavior of the MTs and SPBs in a GFP-atb2 sid4-GFP cell overproducing NLS-Alp4C (a, b) or harboring pREP1 (c) are shown as time-lapse sequences. The SPB visualized with Sid4-GFP was observed as a particularly bright spot on GFP-labeled MTs. The SPB regions of (a) are enlarged (b). Both spindle assembly and cytoplasmic MT disassembly were suppressed in the NLS-Alp4C-OP cell with a duplicated Sid4-GFP signal. (B) The levels of -tubulin complex are reduced at the duplicated SPBs of the cells arrested prior to spindle assembly. The -tubulin complex and the SPBs were visualized in alp4-GFP sid4-mRFP cells overproducing NLS-Alp4C (a–f) or harboring pREP1 (g–i). The SPB regions are enlarged. In NLS-Alp4C-OP cells, the intensity of Alp4-GFP signals (b, e) was reduced at the duplicated SPBs with a pair of Sid4-mRFP signals (c) and with an elongated Sid4-mRFP signal (f) compared to that in control cells (h). (C) Plo1 is localized at the SPBs of the cells arrested before spindle assembly. Plo1 was visualized in plo1-GFP-HA sid4-mRFP cells overproducing NLS-Alp4C (a) or harboring pREP1 (b). In NLS-Alp4C-OP cells (a), Plo1-GFP was localized at the duplicated SPB arrested before spindle assembly. The SPB regions of (a) at time 0 are enlarged. In control cells (b), Plo1-GFP was localized at the SPB at early prophase, prior to duplication of Sid4-mRFP dots through early anaphase. A weak Plo1-GFP signal localized at the SPB is visible at 4 : 29 (arrowhead). Short bars, 1 µm. Long bars, 10 µm.

Although some NLS-Alp4C-OP cells arrested at a stage prior to spindle assembly, as mentioned above, other mitotic cells did form a spindle. Most of the spindles formed, however, seemed to have reduced GFP-Atb2 fluorescence intensities compared to those in control cells, and were defective in spindle elongation and/or chromosome segregation. Live observation of mitotic spindles in elongated cells showed that half of the mitotic cells (n = 10) were arrested with a partially elongated spindle (Fig. 7A). To observe the behavior of centromeres during arrest, we used Mis6-GFP (Saitoh et al. 1997) as a centromere marker. Multiple Mis6-GFP dots were observed between the two SPBs of the partially elongated spindles (Fig. 7B). The Mis6-GFP dots dispersed along the spindle did not move to the SPBs and lost intensity during arrest. Levels of the -tubulin complex visualized with Alp4-GFP were reduced at the SPBs of the cells arrested with a spindle (35% of that of control cells, n = 20) (Fig. 7C,D), and this reduction was accompanied by a defect in spindle elongation. These results suggest that the cells arrested with a partially elongated spindle were defective in both anaphase A (chromosome-to-pole movement) and anaphase B (spindle elongation).

View larger version (64K): [in this window] [in a new window]   Figure 7  Overproduction of NLS-Alp4C induces defects in spindle elongation and chromosome segregation. (A) Spindle elongation is defective in NLS-Alp4C-OP cells. MTs and SPBs were observed in GFP-atb2 sid4-GFP cells overproducing NLS-Alp4C. A mitotic spindle formed between the duplicated Sid4-GFP signals, and cytoplasmic MTs were disassembled, but spindle elongation was suppressed. (B) NLS-Alp4C-OP cells are defective in chromosome-to-pole movements. Centromeres (green) and the SPBs (red) were visualized in mis6-GFP sid4-mRFP cells. Time-lapse sequences of approximately 40 min are shown. (C) Levels of the -tubulin complex are reduced at the SPBs of bipolar spindles defective in spindle elongation. -tubulin complexes and SPBs were visualized in alp4-GFP sid4-mRFP cells harboring pREP1 (a–c) or overproducing NLS-Alp4C (d–h). (a, d) Bright-field cell images; (b e, g) Alp4-GFP; (c, f, h) Sid4-mRFP. Time-lapse images of an NLS-Alp4C-OP cell are shown at time 0 (e, f) and at 23 min (g, h). (D) Quantification of -tubulin complex levels at the SPBs of spindles defective in spindle elongation. The reductions in the levels of Alp4-GFP were quantified and are represented as a percentage of levels in control cells. Error bars represent standard deviation. (E) Cdc13 accumulates in NLS-Alp4C-OP cells arrested with a partially elongated spindle. Time-lapse sequences of 12 min and 22 min are shown in cdc13-GFP sid4-mRFP cells harboring pREP1 (a–g) and overproducing NLS-Alp4C (h–n), respectively. (b, d, f, i, k, m) Cdc13-GFP; (c, e, g, j, l, n) Sid4-mRFP. (F) Plo1-GFP is localized at the SPBs of NLS-Alp4C-OP cells arrested during spindle elongation. Time-lapse sequences of about 23 min are shown in a plo1-GFP sid4-mRFP cell overproducing NLS-Alp4C. (G) Chromosomes are missegregated in NLS-Alp4C-OP cells. (a) Time-lapse image sequences are shown in mitotic hht2-GFP cells. Duration of anaphase spindle elongation was approximately 14 min for the upper cell and approximately 11 min for the lower cell, times similar to those of wild-type cells (Nabeshima et al. 1998). Note that anaphase spindle elongation was not delayed, but that chromosomes were missegregated in these cells. (b) Septated cells with missegregated chromosomes. Bars, 10 µm.

Live observation of chromosomes in hht2-GFP cells overproducing NLS-Alp4C showed that some of the spindles elongated at rates similar to those in control cells, but the chromosomes were frequently missegregated (Fig. 7G-a). Mis-segregated chromosomes were also observed in many of the elongated cells with septa (Fig. 7G-b), indicating that the cells were arrested at a stage between septum formation and cytokinesis after chromosome missegregation.

Comparison of phenotypes induced by Alp4C overproduction with those of temperature-sensitive alp4-1891 mutant cells

We examined whether any of the phenotypes induced by Alp4C overproduction were displayed by alp4-1891 temperature-sensitive mutant cells (Vardy & Toda 2000) that were incubated for 2–6 h at the restrictive temperature (36 °C). We found all of the phenotypes in the mutant cells (Table 1). Using alp4-1891 GFP-atb2 sid4-GFP cells, we observed oscillatory SPB movement at both permissive and restrictive temperatures (41%, n = 69 at 26 °C; 26%, n = 81 at 3–4 h after temperature shift-up to 36 °C) (Fig. 8A), defects in spindle assembly and cytoplasmic MT disassembly (9%, n = 81 at 3–4 h after shift-up) (Fig. 8D), and defects in spindle assembly and elongation (23%, n = 48 at 5–6 h after shift-up) (Fig. 8E,F) at the restrictive temperature. In addition to these phenotypes, similar to those observed by Alp4C overproduction, in 9% (n = 69) of the mutant cells at 26 °C and in 22% (n = 81) at 3–4 h after shift-up to 36 °C (Fig. 8B) we observed detachment of the SPB from cytoplasmic MTs and little movement of the SPB along the long cell axis. In these cells the SPB occasionally reattached to the MTs and started to move again (Fig. 8C). The observation that interaction of the SPB with cytoplasmic MTs mediates nuclear oscillation suggests that the -tubulin complex is required for this interaction. Tip1-YFP accumulation and Bud6-GFP reduction at the cell ends in the alp4-1891 genetic background were also observed at the restrictive temperature, although Bud6 reduction was only detectable at later stages of incubation at 36 °C. We also examined the effect of temperature shift-up on alp4-1891 wee1-50 cells, and found that the cells incubated for 2–6 h at 36 °C, the restrictive temperature, did not elongate, but showed wee phenotypes similar to those observed in wee1-50 cells at 36 °C. This suggests that the cell elongation observed in interphase alp4-1891 cells is dependent on Wee1. Taken together, we suggest that the phenotypes observed by Alp4C overproduction are induced by disturbing functions of the -tubulin complex.

View larger version (66K): [in this window] [in a new window]   Figure 8  Phenotypes of alp4-1891 mutant cells incubated at the restrictive temperature. alp4-1891 GFP-atb2 sid4-GFP cells were incubated at 36 °C for 3 h. (A) Oscillation of an SPB attached to MTs. (B) An SPB showing little movement. The SPB remained detached from the cytoplasmic MTs. (C) An SPB showing little movement (at time 0 through 1 min 58 s) attached back to MTs and started to move on the MTs (at 2 min 29 s). (D) Suppression of both spindle assembly and cytoplasmic MT disassembly. A duplicated Sid4-GFP signal and cytoplasmic MTs were observed. The SPB region was enlarged at 1 min 54 s. White bar, 2 µm. (E) Suppression of spindle assembly. A short spindle was assembling, and cytoplasmic MTs were disassembling at 2 min 29 s, but a stable bipolar spindle failed to assemble. (F) A bipolar spindle defective in elongation. Bar, 10 µm.

	Discussion

Top Abstract Introduction Results Discussion Experimental procedures References

Modulation of Alp4 function by Alp4C affects the localization and function of the -tubulin complex. Levels of the -tubulin complex localized at the SPB in cells overproducing untagged Alp4C or nuclear Alp4C (NLS-Alp4C) were reduced relative to control cells. This reduction could be attributed to a reduction in the levels of the nuclear complex, since most of the -tubulin complex is localized at the nuclear face of the SPB (Ding et al. 1997) and cytoplasmic Alp4C (NES-Alp4C) does not reduce levels of the complex at the SPB. In contrast, the effects of Alp4C on localization of the -tubulin complex in the cytoplasm cannot be easily determined by fluorescence microscopy. Nevertheless, the effects of Alp4C on the dynamics and organization of cytoplasmic MT arrays suggest that Alp4C affects functions of the cyoplamic -tubulin complex. In Alp4C-overproducing (Alp4C-OP) cells, some of cytoplasmic MTs are curved or curl around the cell ends. These phenotypes are consistent with those observed with conditional -tubulin mutants (Paluh et al. 2000; Tange et al. 2004) and temperature-sensitive mutants of alp4 and alp6 (Vardy & Toda 2000). Phenotypes of enhanced cytoplasmic MT assembly have been observed in some Aspergillus-tubulin mutants (Jung et al. 2001), in S. cerevisiae-tubulin-deletion strains, and in conditional -tubulin and Spc97 (Alp4 homolog) mutants (Sobel & Snyder 1995; Marschall et al. 1996; Spang et al. 1996; Knop et al. 1997). These observations are consistent with the hypothesis that the -tubulin complex, in addition to nucleating MTs, has a role in regulating MT dynamics and organization (Jung et al. 2001).

Effects of Alp4C on the function of the nuclear -tubulin complex during mitosis

NLS-Alp4C induced defects in mitotic progression including assembly of a mitotic spindle, disassembly of cytoplasmic MTs, spindle elongation, and chromosome segregation. These defects seemed to be correlated with reduction in levels of the -tubulin complex at the SPB. One of the phenotypes, mitotic arrest with a duplicated SPB signal at a stage prior to spindle assembly and cytoplasmic MT disassembly is novel and suggest that initiation of cytoplasmic MT disassembly is linked to the initiation of spindle assembly but not to SPB duplication, Cdc2 kinase activation, or SPB localization of Plo1 kinases. At the onset of mitosis in wild-type cells formation of new cytoplasmic MTs is suppressed and existing cytoplasmic MTs gradually disappear, suggesting that cytoplasmic MT disassembly is induced by suppressing the activities of proteins that promote assembly of cytoplasmic MTs (Sagolla et al. 2003). The signal for suppression of cytoplasmic MT assembly seems to be produced in the nucleus, since cytoplasmic Alp4C does not interfere with assembly of a mitotic spindle or disassembly of cytoplasmic MTs. Spindle assembly may produce the signal required for promoting cytoplasmic MT disassembly in a positive feedback pathway. Alternatively, initiation of cytoplasmic MT disassembly may not be directly linked to spindle assembly, but to SPB maturation, which is required for initiation of spindle assembly. SPB maturation occurs in several steps at late G2 phase and throughout prophase (Ding et al. 1997; Uzawa et al. 2004). The steps for SPB maturation following entry into mitosis includes insertion of the duplicated SPB into the nuclear envelope (Ding et al. 1997), physical separation of the two daughter SPBs, and activation of the -tubulin complex (Masuda et al. 1992; Masuda & Shibata 1996). EM studies have shown that cytoplasmic MTs are disassembled after SPB insertion into the nuclear envelope (Ding et al. 1997). Nuclear Alp4C may arrest SPB maturation at a stage prior to SPB insertion into the nuclear envelope, which could result in suppression of both cytoplasmic MT disassembly and spindle assembly.

Some of the NLS-Alp4C-OP cells arrested with a partially elongated spindle and dispersed centromeres prior to the metaphase/anaphase transition. Observation of this phenotype suggests that there was a failure in the establishment of bipolar kinetochore MT attachment. It is likely that the decrease in the MT-nucleating activity of the SPB that results from reduced -tubulin complex levels affects attachment of MTs to kinetochores by decreasing the probability of MT capture by kinetochores. Regulation of MT dynamics also seems to be important for bipolar kinetochore MT attachment. For instance, noscapine, a drug that alters the steady-state dynamics of MT assembly, but that does not significantly affect MT polymerization, causes chromosome alignment failure and loss of tension across kinetochore pairs in mammalian cells (Zou et al. 2002). Although we have not examined the effect of nuclear Alp4C on spindle MT dynamics, MT dynamics in the nucleus may be altered in the same way as was observed in the cytoplasm and contribute to loss of bipolar kinetochore MT attachment. Even when mitosis proceeds with normal kinetics in NLS-Alp4C-OP cells, chromosomes are frequently missegregated. The phenotypes with unequally divided nuclei are similar to those of cells carrying mutations in the centromere proteins, Mis6 and Mis12 (Saitoh et al. 1997; Goshima et al. 1999), and may be indicative of an inability to establish correct bi-orientation of sister kinetochores.

In addition to arrests in mitosis, cells with multiple septa are observed as a result of NLS-Alp4C overproduction. Some conditional -tubulin mutants have been shown to form abnormal spindles, followed by the formation of multiple septa (Hendrickson et al. 2001). A temperature-sensitive alp4 mutant was shown to form a monopolar spindle and activate the Mad2-dependent spindle checkpoint with high levels of Cdc13. However, septation proceeds through activation of the septation initiation network (SIN) (Vardy et al. 2002), suggesting that the -tubulin complex provides a link between spindle function and cytokinesis. Our results also support a role of the -tubulin complex in the coordination of chromosome segregation and cytokinesis.

Effects of Alp4C on the function of the nuclear -tubulin complex during interphase

Although it is clear that the nuclear -tubulin complex localized at the mitotic SPB has a role in spindle MT nucleation, it is not known whether the nuclear -tubulin complex has any function during interphase. Nuclear -tubulin complexes localized at the interphase SPB do not function in MT nucleation as the MT-nucleating activity of the interphase SPB is very low compared to that of the mitotic SPB (Masuda et al. 1992) and no MTs are observed in the nucleus during interphase (Ding et al. 1997). This is in contrast to the nuclear -tubulin complex in S. cerevisiae, which nucleates MTs throughout the cell cycle. We have shown that nuclear Alp4C, but not cytoplasmic Alp4C, induces both Wee1-dependent G2 delays and a reduction in the levels of the -tubulin complex localized at the SPB. These results suggest that the G2 delays may be linked to the reduction in -tubulin complex levels. A Wee1-dependent checkpoint may directly detect reduction in levels of the complex or defects in any function involving the complex.

The nuclear -tubulin complex during interphase may be essential for SPB duplication and maturation, although the relationship between them has not been studied. The SPB is duplicated and maturated in several steps during late G2 phase and throughout prophase (Ding et al. 1997; Uzawa et al. 2004). The -tubulin complex needs to be recruited to the duplicated or maturating SPB. Reduction in levels of the -tubulin complex suggests that recruitment of the -tubulin complex to the SPB may be affected by nuclear Alp4C. Interestingly, S. cerevisiae temperature-sensitive mutants of Spc97 (Alp4 homolog) but not Spc98 (Alp6 homolog) or Tub4 (-tubulin homolog) are defective in SPB duplication (Knop et al. 1997). Mutant S. cerevisiae cells defective in SPB duplication generally exhibit transient G2 arrest. Nuclear Alp4C may affect SPB duplication or maturation, which activates a checkpoint monitoring SPB duplication or maturation.

Alternatively, the -tubulin complex localized at the SPB may be a component of the machinery for regulating cell cycle progression and directly affect the function of Wee1. Association of cell cycle regulators with centrosomes and the SPBs is an essential step in cell cycle control (reviewed in Doxsey et al. 2005). In Drosophila, Wee1 physically interacts with components of the -tubulin complex (Stumpff et al. 2005). In S. pombe, Wee1 is a nuclear protein (Wu et al. 1996), and Wee1 truncated at the C-terminus and fused with GFP is localized in the nucleus and concentrated at one or two sites, which may be the positions of the SPBs (Ding et al. 2000). It is tempting to speculate that localization of Wee1 at the SPB through interaction with the -tubulin complex is important for cell cycle progression through G2/M, and nuclear Alp4C may dislocate Wee1 from the SPB, leading to a G2 delay.

Effects of Alp4C on the function of the cytoplasmic -tubulin complex

We have shown that cytoplasmic Alp4C affects the dynamic behavior of MT ends, and induces Bud6 reduction and Tip1 accumulation at the cell ends, and SPB/nuclear oscillatory movement. These phenotypes may be related to each other, and caused through the same pathway by disturbing functions of the cytoplasmic -tubulin complex. Tip1 is a CLIP170 family protein, which accumulates at the growing MT plus ends and is required for cell-end targeting of cytoplasmic MTs (Brunner & Nurse 2000). In the absence of Tip1, MT catastrophe is not restricted to the cell end, but occurs at any region where MTs reach. Accumulation of Tip1 at the cell end may influence MT dynamics by stabilizing MT ends, which could affect nuclear positioning mechanisms based on the dynamic behavior of MTs (Tran et al. 2001; see Masuda et al. in this issue).

Alp4C induces cell elongation with bent morphology only in a small proportion of the cells, suggesting that it does not significantly affect cell polarity control. However, the decrease in the levels of Bud6 and increase in the levels of Tip1 at the cell ends suggests that Alp4C may affect some aspects of polarity control. We have also shown that in contrast to Bud6 and Tip1, levels of another cell end marker, Tea1, are not reduced at the cell ends. Bud6 is a cell polarity factor required for bipolar growth. Localization of Bud6 at the cell ends is dependent on Tea1, and Bud6 and Tea1 associate in multiple complexes (Glynn et al. 2001). Tea1 anchored at the cell end is thought to regulate cell polarity at the cell end through its association with other cell polarity factors including Bud6. Our results suggest that localization of Tea1 at the cell end is not sufficient for localization of Bud6 at the cell end. We suggest that changes in MT dynamics and organization caused by Alp4C overproduction may affect the distribution or function of other factors required for Bud6 localization. Alternatively, levels of Bud6 protein may be decreased, resulting in a reduction in the amount of Bud6 observed at the cell end. Altered MT dynamics and organization may affect protein synthesis of Bud6, since association of RNA with the cytoskeleton, MTs, and microfilaments is required for mRNA transport and localization and for efficient protein synthesis (reviewed in Jansen 1999).

In conclusion, we have shown here that the nuclear and cytoplasmic -tubulin complexes play distinct roles. Coherent progression of these nuclear and cytoplasmic functions may be important for cell cycle regulation.

	Experimental procedures

Top Abstract Introduction Results Discussion Experimental procedures References

 
Cell culture and strains

S. pombe strains used in this study are shown in Table 2. The cells harboring pREP vectors were maintained in EMM2 media containing appropriate amino acids, supplements, and 10 µM thiamine. rad3D, wee1–50, and mad2d strains were generously provided by Anthony M. Carr, Paul Nurse, and Tomohiro Matsumoto, respectively. The GFP-atb2 strain was created by integrating the GFP-atb2⁺ gene under the control of the nda3 promoter at the lys1⁺ locus using the pYC36 vector (Chikashige et al. 2004). Construction of mis6-GFP and sad1-GFP strains was previously described (Chikashige et al. 2004). sid4-GFP, sid4-mRFP, bud6-GFP, tea1-GFP, cdc13-GFP and hht2-GFP strains were generously provided by Kathleen L. Gould, Yuji Chikashige, Fred Chang, Paul Nurse, Mitsuhiro Yanagida, and Mohan K. Balasubramanian, respectively. Other GFP- and GFP-HA-tagged strains were created by fusion of the tags to the 3' ends of their chromosomal loci using PCR-mediated gene-tagging methods (Bahler et al. 1998).

The plasmid for expression of Alp4C, pREP1HM.alp4C was constructed as follows: a DNA fragment coding for Alp4 (566–784 amino acids) (Alp4C) was PCR-amplified from pURalp4 (Vardy & Toda 2000) and subcloned into the pREP41HM vector with the thiamine-repressive nmt41 promoter and a combined tag of six histidine residues and two copies of the myc epitope (Craven et al. 1998). The DNA fragment containing the tag and the Alp4C DNA was PCR-amplified and subcloned into pREP1, a multicopy expression vector with the thiamine-repressive nmt1 promoter (Maundrell 1990). Other plasmids, pREP2HM.alp4C (pREP2 vector for expressing Alp4C), pREP1HM.alp4 (pREP1 for expressing full length Alp4), pREP1HM.alp4N (pREP1 for expressing Alp4 (1–565 amino acids)), pREP1HM.alp6 (pREP1 for expressing full length Alp6), pREP1HM.alp6N (pREP1 for expressing Alp6 (1–581 amino acids)), and pREP1HM.alp6C (pREP1 for expressing Alp6 (582–832 amino acids)) were constructed by similar methods. The pREP1.GFP.alp4C vector was constructed by subcloning the Alp4C fragment into pTN178 with an nmt81 promoter and an N-terminal GFP (Nakamura et al. 2001), followed by subcloning the DNA fragment encoding GFP-Alp4C into the pREP1 vector. The pREP1NLS.alp4C was constructed by subcloning an Alp4C DNA fragment into the vector pREP1NLS, which contains the Xenopus nucleoplasmin C-terminus (146–197 amino acids). The pREP1NES.alp4C vector was constructed by subcloning Alp4C and an N-terminal NES fragment, encoding a peptide of 29 amino acids (MHGMDELYKSTLGSLEGAVSEISLRDKST) including a NES of S. pombe Dsk1 (308–320 amino acids) (Fukuda et al. 1997), into the pREP1 vector.

Live cell imaging

Cells grown to late-exponential phase in EMM2 in the presence of thiamine were washed three times with thiamine-free EMM2. The cells were then added to thiamine-free EMM2 at a density of 2 x 10⁵ cells/mL and incubated at 30 °C for 18–24 h. One hundred microlitre of cells were transferred to a glass-bottom dish (MatTek Corp., Ashland, MA, USA) that had been coated with 0.2% (w/v) concanavalin A and incubated for 30 min or more at 25–26 °C for cell adhesion. Live cell imaging was carried out at 25–26 °C using a DeltaVision microscope system (Applied Precision Inc., Seattle, WA, USA), a computer-operated microscope system with a cooled CCD camera, as previously described (Chikashige et al. 1994; Ding et al. 1998; Haraguchi et al. 1999). Typically, images of 10–13 different focal planes at 0.3–0.35 µm were collected at each time point, and projection images were reconstituted from these. For quantification of -tubulin, Alp4-GFP, and Alp6-GFP levels at the SPB, projection images of maximum intensity were obtained and maximum fluorescence intensities at the SPB were used for statistic data analysis. For quantification of levels of cell end markers, Bud6-GFP, Tip1-YFP, and Tea1-GFP, projection images of summed intensity were reconstituted. Fluorescence intensity over the background intensity (intensity in the cytoplasm) was integrated per each cell end and used for statistic data analysis.

Indirect immunofluorescence microscopy

Cells were fixed in –80 °C methanol and treated with 0.1 mg/mL Zymolyase 100T for 10 min at 36 °C. For anti--tubulin staining the cells were incubated for 2 h with rabbit anti-S. pombe-tubulin (Masuda & Shibata 1996) and then incubated with Cy3-conjugated goat anti-rabbit IgG antibody. For anti-HA staining the cells were incubated with monoclonal anti-HA (BABCO: 16B12) and then incubated with Cy3-conjugated donkey anti-mouse IgG.

	Acknowledgements

 
We thank Mohan Balasubramanian, Anthony Carr, Fred Chang, Yuji Chikashige, Tomohiro Matsumoto, Kathleen Gould, Osami Niwa, Paul Nurse, and Mitsuhiro Yanagida for providing fission yeast strains, Iain Hagan and Taro Nakamura for providing vectors, and David Alexander for critical reading of the manuscript. This work was supported by grants from the Japan Science and Technology Corporation to T.H and Y.H and Human Frontier Science Program to H.M. T.T. is supported by Cancer Research UK.

	Footnotes

 
Communicated by: Masayuki Yamamoto

^* Correspondence: E-mail: hmasuda{at}nict.go.jp

	References

Top Abstract Introduction Results Discussion Experimental procedures References

 
Ayscough, K. (1998) Use of latrunculin-A, an actin monomer-binding drug. Methods Enzymol. 298, 18–25.[Medline]

Bahler, J., Wu, J.Q., Longtine, M.S., et al. (1998) Heterologous modules for efficient and versatile PCR-based gene targeting in Schizosaccharomyces pombe. Yeast 14, 943–951.[CrossRef][Medline]

Bentley, N.J., Holtzman, D.A., Flaggs, G., et al. (1996) The Schizosaccharomyces pombe rad3 checkpoint gene. EMBO J. 15, 6641–6651.[Medline]

Browning, H., Hayles, J., Mata, J., Aveline, L., Nurse, P. & McIntosh, J.R. (2000) Tea2p is a kinesin-like protein required to generate polarized growth in fission yeast. J. Cell Biol. 151, 15–28.[Abstract/Free Full Text]

Brunner, D. & Nurse, P. (2000) CLIP170-like tip1p spatially organizes microtubular dynamics in fission yeast. Cell 102, 695–704.[CrossRef][Medline]

Chang, L. & Gould, K.L. (2000) Sid4p is required to localize components of the septation initiation pathway to the spindle pole body in fission yeast. Proc. Natl. Acad. Sci. USA 97, 5249–5254.[Abstract/Free Full Text]

Chang, F. & Peter, M. (2003) Yeasts make their mark. Nature Cell Biol. 5, 294–299.[CrossRef][Medline]

Chikashige, Y., Ding, D.Q., Funabiki, H., et al. (1994) Telomere-led premeiotic chromosome movement in fission yeast Schizosaccharomyces pombe. Science 264, 270–273.[Abstract/Free Full Text]

Chikashige, Y., Kurokawa, R., Haraguchi, T. & Hiraoka, Y. (2004) Meiosis induced by inactivation of Pat1 kinase proceeds with aberrant nuclear positioning of centromeres in the fission yeast Schizosaccharomyces pombe. Genes Cells 9, 671–684.[Abstract/Free Full Text]

Craven, R.A., Griffiths, D.J., Sheldrick, K.S., Randall, R.E., Hagan, I.M. & Carr, A.M. (1998) Vectors for the expression of tagged proteins in Schizosaccharomyces pombe. Gene 221, 59–68.[CrossRef][Medline]

Decottignies, A., Zarzov, P. & Nurse, P. (2001) In vivo localisation of fission yeast cyclin-dependent kinase cdc2p and cyclin B cdc13p during mitosis and meiosis. J. Cell Sci. 114, 2627–2640.[Medline]

Ding, D.-Q., Chikashige, Y., Haraguchi, T. & Hiraoka, Y. (1998) Oscillatory nuclear movement in fission yeast meiotic prophase is driven by astral microtubules as revealed by continuous observation of chromosomes and microtubules in living cells. J. Cell Sci. 111, 701–712.[Abstract]

Ding, D.-Q., Tomita, Y., Yamamoto, A., Chikashige, Y., Haraguchi, T. & Hiraoka, Y. (2000) Large-scale screening of intracellular protein localization in living fission yeast cells by the use of a GFP-fusion genomic DNA library. Genes Cells 5, 169–190.[Abstract]

Ding, R., West, R.R., Morphew, D.M., Oakley, B.R. & McIntosh, J.R. (1997) The spindle pole body of Schizosaccharomyce pombe enters and leaves the nuclear envelope as the cell cycle proceeds. Mol. Biol. Cell 8, 1461–1479.[Abstract]

Doxsey, S., Zimmerman, W. & Mikule, K. (2005) Centrosome control of the cell cycle. Trends Cell Biol. 15, 303–311.[CrossRef][Medline]

Drummond, D.R. & Cross, R.A. (2000) Dynamics of interphase microtubules in Schizosaccharomyces pombe. Curr. Biol. 10, 766–775.[CrossRef][Medline]

Feierbach, B., Verde, F. & Chang, F. (2004) Regulation of a forming complex by the microtubule plus end protein tea1p. J. Cell Biol. 165, 697–707.[Abstract/Free Full Text]

Fujita, A., Vardy, L., Garcia, M.A. & Toda, T. (2002) A fourth component of the fission yeast -tubulin complex, Alp16, is required for cytoplasmic microtubule integrity and becomes indispensable when -tubulin function is compromised. Mol. Biol. Cell 13, 2360–2373.[Abstract/Free Full Text]

Fukuda, M., Asano, S., Nakamura, T., et al. (1997) CRM1 is responsible for intracellular transport mediated by the nuclear export signal. Nature 390, 308–311.[CrossRef][Medline]

Funabiki, H., Hagan, I., Uzawa, S. & Yanagida, M. (1993) Cell cycle-dependent specific positioning and clustering of centromeres and telomeres in fission yeast. J. Cell Biol. 121, 961–976.[Abstract/Free Full Text]

Geissler, S., Pereira, G., Spang, A., et al. (1996) The spindle pole body component Spc98p interacts with the -tubulin-like Tub4p of Saccharomyces cerevisiae at the sites of microtubule attachment. EMBO J. 15, 3899–3911. (Erratum in: EMBO J. 15, 5124.)[Medline]

Glynn, J.M., Lustig, R.J., Berlin, A. & Chang, F. (2001) Role of bud6p and tea1p in the interaction between actin and microtubules for the establishment of cell polarity in fission yeast. Curr. Biol. 11, 836–845.[CrossRef][Medline]

Goshima, G., Saitoh, S. & Yanagida, M. (1999) Proper metaphase spindle length is determined by centromere proteins Mis12 and Mis6 required for faithful chromosome segregation. Genes Dev. 13, 1664–1677.[Abstract/Free Full Text]

Gunawardane, R.N., Martin, O.C., Cao, K., et al. (2000) Characterization and reconstitution of Drosophila -tubulin ring complex subunits. J. Cell Biol. 151, 1513–1523.[Abstract/Free Full Text]

Gunawardane, R.N., Martin, O.C. & Zheng, Y. (2003) Characterization of a new TuRC subunit with WD repeats. Mol. Biol. Cell 14, 1017–1026.[Abstract/Free Full Text]

Hagan, I.M. (1998) The fission yeast microtubule cytoskeleton. J. Cell Sci. 111, 1603–1612.[Abstract]

Hagan, I.M. & Hyams, J.S. (1988) The use of cell division cycle mutants to investigate the control of microtubule distribution in the fission yeast Schizosaccharomyces pombe. J. Cell Sci. 89, 343–357.[Abstract/Free Full Text]

Haraguchi, T., Ding, D.Q., Yamamoto, A., Kaneda, T., Koujin, T. & Hiraoka, Y. (1999) Multiple-color fluorescence imaging of chromosomes and microtubules in living cells. Cell Struct. Funct. 24, 291–298.[CrossRef][Medline]

Hayashi, A., Ogawa, H., Kohno, K., Gasser, S. & Hiraoka, Y. (1998) Meiotic behaviors of chromosomes and microtubules in budding yeast: relocalization of centromeres and telomeres during meiotic prophase. Genes Cells 3, 587–601.[Abstract]

He, X., Patterson, T.E. & Sazer, S. (1997) The Schizosaccharomyces pombe spindle checkpoint protein mad2p blocks anaphase and genetically interacts with the anaphase-promoting complex. Proc. Natl. Acad. Sci. USA 94, 7965–7970.[Abstract/Free Full Text]

Heitz, M.J., Petersen, J., Valovin, S. & Hagan, I.M. (2001) MTOC formation during mitotic exit in fission yeast. J. Cell Sci. 114, 4521–4532.[Medline]

Hendrickson, T.W., Yao, J., Bhadury, S., Corbett, A.H. & Joshi, H.C. (2001) Conditional mutations in -tubulin reveal its involvement in chromosome segregation and cytokinesis. Mol. Biol. Cell 12, 2469–2481.[Abstract/Free Full Text]

Hirano, T., Hiraoka, Y. & Yanagida, M. (1988) A temperature-sensitive mutation of the Schizosaccharomyces pombe gene nuc2+ that encodes a nuclear scaffold-like protein blocks spindle elongation in mitotic anaphase. J. Cell Biol. 106, 1171–1183.[Abstract/Free Full Text]

Horio, T., Basaki, A., Takeoka, A. & Yamato, M. (1999) Lethal level overexpression of -tubulin in fission yeast causes mitotic arrest. Cell Motil. Cytoskeleton 44, 284–295.[CrossRef][Medline]

Horio, T., Uzawa, S., Jung, M.K., Oakley, B.R., Tanaka, K. & Yanagida, M. (1991) The fission yeast -tubulin is essential for mitosis and is localized at microtubule organizing centers. J. Cell Sci. 99, 693–700.[Abstract]

Jansen, R.-P. (1999) RNA–cytoskeletal association. FASEB J. 13, 455–466.[Abstract/Free Full Text]

Janson, M.E., Setty, T.G., Paoletti, A. & Tran, P.T. (2005) Efficient formation of bipolar microtubule bundles requires microtubule-bound -tubulin complexes. J. Cell Biol. 169, 297–308.[Abstract/Free Full Text]

Jin, H. & Amberg, D.C. (2001) Fission yeast Aip3p (spAip3p) is required for an alternative actin-directed polarity program. Mol. Biol. Cell 12, 1275–1291.[Abstract/Free Full Text]

Job, D., Valiron, O. & Oakley, B. (2003) Microtubule nucleation. Curr. Opin. Cell Biol. 15, 111–117.[CrossRef][Medline]

Jung, M.K., Prigozhina, N., Oakley, C.E., Nogales, E. & Oakley, B.R. (2001) Alanine-scanning mutagenesis of Aspergillus -tubulin yields diverse and novel phenotypes. Mol. Biol. Cell 12, 2119–2136.[Abstract/Free Full Text]

Kim, S.H., Lin, D.P., Matsumoto, S., Kitazono, A. & Matsumoto, T. (1998) Fission yeast Slp1: an effector of the Mad2-dependent spindle checkpoint. Science 279, 1045–1047.[Abstract/Free Full Text]

Kniola, B., O'Toole, E., McIntosh, J.R., et al. (2001) The domain structure of centromeres is conserved from fission yeast to humans. Mol. Biol. Cell 12, 2767–2775.[Abstract/Free Full Text]

Knop, M., Pereira, G., Geissler, S., Grein, K. & Schiebel, E. (1997) The spindle pole body component Spc97p interacts with the -tubulin of Saccharomyces cerevisiae and functions in microtubule organization and spindle pole body duplication. EMBO J. 16, 1550–1564.[CrossRef][Medline]

Lundgren, K., Walworth, N., Booher, R., Dembski, M., Kirschner, M. & Beach, D. (1991) mik1 and wee1 cooperate in the inhibitory tyrosine phosphorylation of cdc2. Cell 64, 1111–1122.[CrossRef][Medline]

Marschall, L.G., Jeng, R.L., Mulholland, J. & Stearns, T. (1996) Analysis of Tub4p, a yeast gamma-tubulin-like protein: implications for microtubule-organizing center function. J. Cell Biol. 134, 443–454.[Abstract/Free Full Text]

Martin, S.G., McDonald, W.H., Yates, J.R. 3rd & Chang, F. (2005) Tea4p links microtubule plus ends with the formin for3p in the establishment of cell polarity. Dev. Cell 8, 479–491.[CrossRef][Medline]

Masuda, H., Miyamoto, R., Haraguchi, T. & Hiraoka, Y. (2006) The caboxy-terminus of Alp4 alters microtubule dynamics to induce oscillatory nuclear movement led by the spindle pole body in Schizosaccharomyces pombe. Genes Cells, 11, 000–000.

Masuda, H., Sevik, M. & Cande, W.Z. (1992) In vitro microtubule-nucleating activity of spindle pole bodies in fission yeast Schizosaccharomyces pombe: cell cycle-dependent activation in Xenopus cell-free extracts. J. Cell Biol. 117, 1055–1066.[Abstract/Free Full Text]

Masuda, H. & Shibata, T. (1996) Role of -tubulin in mitotic specific microtubule nucleation from the Schizosaccharomyces pombe spindle pole body. J. Cell Sci. 109, 165–177.[Abstract]

Mata, J. & Nurse, P. (1997) tea1 and the microtubular cytoskeleton are important for generating global spatial order within the fission yeast cell. Cell 89, 939–949.[CrossRef][Medline]

Maundrell, K. (1990) Nmt1 of fission yeast: a highly transcribed gene completely repressed by thiamine. J. Biol. Chem. 265, 10857–10864.[Abstract/Free Full Text]

Murphy, S.M., Preble, A.M., Patel, U.K., et al. (2001) GCP5 and GCP6: two new members of the human gamma-tubulin complex. Mol. Biol. Cell 12, 3340–3352.[Abstract/Free Full Text]

Nabeshima, K., Nakagawa, T., Straight, A.F., et al. (1998) Dynamics of centromeres during metaphase-anaphase transition in fission yeast: Dis1 is implicated in force balance in metaphase bipolar spindle. Mol. Biol. Cell 9, 3211–3225.[Abstract/Free Full Text]

Nakamura, T., Nakamura-Kubo, M., Hirata, A. & Shimoda, C. (2001) The Schizosaccharomyces pombe spo3+ gene is required for assembly of the forespore membrane and genetically interacts with psy1(+)-encoding syntaxin-like protein. Mol. Biol. Cell 12, 3955–3972.[Abstract/Free Full Text]

Oegema, K., Wiese, C., Martin, O.C., et al. (1999) Characterization of two related Drosophila -tubulin complexes that differ in their ability to nucleate microtubules. J. Cell Biol. 144, 721–733.[Abstract/Free Full Text]

Ohkura, H., Hagan, I.M. & Glover, D.M. (1995) The conserved Schizosaccharomyces pombe kinase plo1, required to form a bipolar spindle, the actin ring, and septum, can drive septum formation in G1 and G2 cells. Genes Dev. 9, 1059–1073.[Abstract/Free Full Text]

Paluh, J.L., Nogales, E., Oakley, B.R., McDonald, K., Pidoux, A.L. & Cande, W.Z. (2000) A mutation in gamma-tubulin alters microtubule dynamics and organization and is synthetically lethal with the kinesin-like protein pkl1p. Mol. Biol. Cell 11, 1225–1239.[Abstract/Free Full Text]

Pardo, M. & Nurse, P. (2003) Equatorial retention of the contractile actin ring by microtubules during cytokinesis. Science 300, 1569–1574.[Abstract/Free Full Text]

Pereira, G., Knop, M. & Schiebel, E. (1998) Spc98p directs the yeast -tubulin complex into the nucleus and is subject to cell cycle-dependent phosphorylation on the nuclear side of the spindle pole body. Mol. Biol. Cell 9, 775–793.[Abstract/Free Full Text]

Russell, P. & Nurse, P. (1986) cdc25+ functions as an inducer in the mitotic control of fission yeast. Cell 45, 145–153.[CrossRef][Medline]

Sagolla, M.J., Uzawa, S. & Cande, W.Z. (2003) Individual microtubule dynamics contribute to the function of mitotic and cytoplasmic arrays in fission yeast. J. Cell Sci. 116, 4891–4903.[Abstract/Free Full Text]

Saitoh, S., Takahashi, K. & Yanagida, M. (1997) Mis6, a fission yeast inner centromere protein, acts during G1/S and forms specialized chromatin required for equal segregation. Cell 90, 131–143.[CrossRef][Medline]

Samejima, I., Louren, P.C.C., Snaith, H.A. & Sawin, K.E. (2005) Fission yeast mto2p regulates microtubule nucleation by the centrosomin-related protein mto1p. Mol. Biol. Cell 16, 3040–3051.[Abstract/Free Full Text]

Sawin, K.E., Lourenco, P.C. & Snaith, H.A. (2004) Microtubule nucleation at non-spindle pole body microtubule-organizing centers requires Fission yeast centrosomin-related protein mod20p. Curr. Biol. 14, 763–775.[CrossRef][Medline]

Sobel, S.G. & Snyder, M. (1995) A high divergent -tubulin gene is essential for cell growth and proper microtubule organization in Saccharomyces cerevisiae. J. Cell Biol. 131, 1775–1788.[Abstract/Free Full Text]

Spang, A., Geissler, S., Grein, K. & Schiebel, E. (1996) -Tubulin-like Tub4p of Saccharomyces cerevisiae is associated with the spindle pole body substructures that organize microtubules and is required for mitotic spindle formation. J. Cell Biol. 134, 429–441.[Abstract/Free Full Text]

Stumpff, J., Kellogg, D.R., Krohne, K.A. & Su, T.T. (2005) Drosophila Wee1 interacts with members of the gammaTURC and is required for proper mitotic-spindle morphogenesis and positioning. Curr. Biol. 15, 1525–1534.[CrossRef][Medline]

Takada, S., Shibata, T., Hiraoka, Y. & Masuda, H. (2000) Identification of ribonucleotide reductase protein R1 as an activator of microtubule nucleation in Xenopus egg mitotic extracts. Mol. Biol. Cell 11, 4173–4187.[Abstract/Free Full Text]

Tanaka, K., Petersen, J., MacIver, F., Mulvihill, D.P., Glover, D.M. & Hagan, I.M. (2001) The role of Plo1 kinase in mitotic commitment and septation in Schizosaccharomyces pombe. EMBO J. 20, 1259–1270.[CrossRef][Medline]

Tange, Y., Fujita, A., Toda, T. & Niwa, O. (2004) Functional dissection of the gamma-tubulin complex by suppressor analysis of gtb1 and alp4 mutations in Schizosaccharomyces pombe. Genetics 167, 1095–1107.[Abstract/Free Full Text]

Tran, P.T., Marsh, L., Doye, V., Inoue, S. & Chang, F. (2001) A mechanism for nuclear positioning in fission yeast based on microtubule pushing. J. Cell Biol. 153, 397–411. (Erratum in: J. Cell Biol. 153, 889.)[Abstract/Free Full Text]

Uzawa, S., Li, F., Jin, Y., et al. (2004) Spindle pole body duplication in fission yeast occurs at the G1/S boundary but maturation is blocked until exit from S by an event downstream of cdc10+. Mol. Biol. Cell 15, 5219–5230.[Abstract/Free Full Text]

Vardy, L., Fujita, A. & Toda, T. (2002) The gamma-tubulin complex protein Alp4 provides a link between the metaphase checkpoint and cytokinesis in fission yeast. Genes Cells 7, 365–373.[Abstract]

Vardy, L. & Toda, T. (2000) The fission yeast -tubulin complex is required in G(1) phase and is a component of the spindle assembly checkpoint. EMBO J. 19, 6098–6111.[CrossRef][Medline]

Venkatram, S., Jennings, J.L., Link, A. & Gould, K.L. (2005) Mto2p, a novel fission yeast protein required for cytoplasmic microtubule organization and anchoring of the cytokinetic actin ring. Mol. Biol. Cell 16, 3052–3063.[Abstract/Free Full Text]

Venkatram, S., Tasto, J.J., Feoktistova, A., Jennings, J.L., Link, A.J. & Gould, K.L. (2004) Identification and characterization of two novel proteins affecting fission yeast -tubulin complex function. Mol. Biol. Cell 15, 2287–2301.[Abstract/Free Full Text]

Vinh, D.B., Kern, J.W., Hancock, W.O., Howard, J. & Davis, T.N. (2002) Reconstitution and characterization of budding yeast -tubulin complex. Mol. Biol. Cell 13, 1144–1157.[Abstract/Free Full Text]

Wang, H., Tang, X., Liu, J., et al. (2002) The multiprotein exocyst complex is essential for cell separation in Schizosaccharomyces pombe. Mol. Biol. Cell 13, 515–529.[Abstract/Free Full Text]

Wiese, C. & Zheng, Y. (1999) -Tubulin complexes and their interaction with microtubule-organizing centers. Curr. Opin. Struct. Biol. 9, 250–259.[CrossRef][Medline]

Wu, L., Shiozaki, K., Aligue, R. & Russell, P. (1996) Spatial organization of the Nim1-Wee1-Cdc2 mitotic control network in Schizosaccharomyces pombe. Mol. Biol. Cell 7, 1749–1758.[Abstract]

Zhang, L., Keating, T.J., Wilde, A., Borisy, G.G. & Zheng, Y. (2000) The role of Xgrip210 in gamma-tubulin ring complex assembly and centrosome recruitment. J. Cell Biol. 151, 1525–1536.[Abstract/Free Full Text]

Zheng, Y., Wong, M.L., Alberts, B. & Michison, T. (1995) Nucleation of microtubule assembly by -tubulin-containing ring complex. Nature 378, 578–583.[CrossRef][Medline]

Zimmerman, S. & Chang, F. (2005) Effects of -tubulin complex proteins on microtubule nucleation and catastrophe in fission yeast. Mol. Biol. Cell 16, 2719–2733.[Abstract/Free Full Text]

Zimmerman, S., Tran, P.T., Daga, R.R., Niwa, O. & Chang, F. (2004) Rsp1p, a J domain protein required for disassembly and assembly of microtubule organizing centers during the fission yeast cell cycle. Dev. Cell 6, 497–509.[CrossRef][Medline]

Zou, J., Panda, D., Landen, J.W., Wilson, L. & Joshi, H.C. (2002) Minor alteration of microtubule dynamics causes loss of tension across kinetochore pairs and activates the spindle checkpoint. J. Biol. Chem. 277, 17200–17208.[Abstract/Free Full Text]

Received: 14 September 2005
Accepted: 22 December 2005

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Masuda, H. Articles by Hiraoka, Y. Search for Related Content PubMed PubMed Citation Articles by Masuda, H. Articles by Hiraoka, Y.