QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

Vol. 17, Issue 12, 5075-5093, December 2006

This Article Abstract Full Text (PDF) Supplemental Material All Versions of this Article: E05-11-1009v1 17/12/5075    most recent Alert me when this article is cited Alert me if a correction is posted 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 HighWire Citing Articles via Google Scholar Google Scholar Articles by Anders, A. Articles by Sawin, K. E. Search for Related Content PubMed PubMed Citation Articles by Anders, A. Articles by Sawin, K. E.

Noncore Components of the Fission Yeast -Tubulin Complex

Wellcome Trust Centre for Cell Biology, Edinburgh University, Edinburgh EH9 3JR, United Kingdom

Submitted November 3, 2005; Revised September 8, 2006; Accepted September 25, 2006
Monitoring Editor: Trisha Davis

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Relatively little is known about the in vivo function of individual components of the eukaryotic -tubulin complex (-TuC). We identified three genes, gfh1+, mod21+, and mod22+, in a screen for fission yeast mutants affecting microtubule organization. gfh1+ is a previously characterized -TuC protein weakly similar to human -TuC subunit GCP4, whereas mod21+ is novel and shows weak similarity to human -TuC subunit GCP5. We show that mod21p is a bona fide -TuC protein and that, like gfh1 mutants, mod21 mutants are viable. We find that gfh1 and mod21 mutants have qualitatively normal microtubule nucleation from all types of microtubule-organizing centers (MTOCs) in vivo but quantitatively reduced nucleation from interphase MTOCs, and this is exacerbated by mutations in mod22+. Simultaneous deletion of gfh1p, mod21p, and alp16p, a third nonessential -TuC protein, does not lead to additive defects, suggesting that all three proteins contribute to a single function. Coimmunoprecipitation experiments suggest that gfh1p and alp16p are codependent for association with a small "core" -TuC, whereas mod21p is more peripherally associated, and that gfh1p and mod21p may form a subcomplex independently of the small -TuC. Interestingly, sucrose gradient analysis suggests that the major form of the -TuC in fission yeast may be a small complex. We propose that gfh1p, mod21p, and alp16 act as facultative "noncore" components of the fission yeast -TuC and enhance its microtubule-nucleating ability.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
In eukaryotic cells, microtubule nucleation from both centrosomal and noncentrosomal sites is thought to be driven primarily by protein complexes containing -tubulin and associated proteins that are alternatively known as gamma-complex proteins (GCPs) or gamma-ring proteins (Grips) (Zheng et al., 1995; Murphy et al., 1998; Wilde and Zheng, 1999; Gunawardane et al., 2000; Murphy et al., 2001). For simplicity, in this work, we use the GCP nomenclature. In higher eukaryotes, two different -tubulin complexes (-TuCs) have been identified, and these may vary in their relative abundance in different types of cells. The smaller complex is known as the -tubulin small complex (-TuSC) and contains two copies of -tubulin and one copy each of GCP2 and GCP3 (Oegema et al., 1999; Gunawardane et al., 2000) The larger complex, the -tubulin ring complex (-TuRC), contains multiple copies of -tubulin, GCP2, and GCP3, plus the additional proteins GCP4, GCP5, and GCP6 (Fava et al., 1999; Oegema et al., 1999; Murphy et al., 2001). In vitro, the -TuRC is much more active for microtubule nucleation than the -TuSC (Oegema et al., 1999). Accordingly, the -TuRC has been observed to form a "lock-washer" structure that may be made up of several linked -TuSCs, allowing the -TuRC to act as a direct template for microtubule nucleation (Zheng et al., 1995; Gunawardane et al., 2000; Oakley, 2000; Schiebel, 2000; Job et al., 2003). However, to date there has been relatively little analysis of the relative contributions of the -TuRC and -TuSC to microtubule nucleation inside living cells; thus, it remains an open question to what extent both large and small -TuCs are directly involved in nucleation in vivo, and whether different complexes may be involved in different types of microtubule nucleation.

In the budding yeast Saccharomyces cerevisiae, -tubulin (Tub4p) and the GCP2 and GCP3 homologues Spc97p and Spc98p, respectively, are all essential proteins. They form a "Tub4 complex" that is recruited to the nucleoplasmic face of the spindle pole body (SPB; the yeast centrosome equivalent) by the protein Spc110p and to the cytoplasmic face of the SPB by Spc72p (Knop and Schiebel, 1998, and references therein). However, budding yeast homologues of GCP4, GCP5, and GCP6 have not been identified; thus, it is not clear whether budding yeast can serve as a useful model for understanding the function of the proteins that contribute to the formation of the larger -TuRC.

Unlike budding yeast, microtubule nucleation sites in the fission yeast Schizosaccharomyces pombe are not restricted to the SPB, but rather they are also found on the nuclear envelope and on microtubules themselves during interphase (so-called interphase microtubule-organizing centers, or iMTOCs), and at the cell division site during mitosis (the so-called equatorial microtubule-organizing center, or eMTOC; Hagan, 1998; Drummond and Cross, 2000; Tran et al., 2001; Sawin et al., 2004; Zimmerman et al., 2004; Janson et al., 2005; Sawin and Tran, 2006). The fission yeast homologues of -tubulin, GCP2, and GCP3 are known as gtb1p/tug1p, alp4p, and alp6p, respectively, and all three proteins are essential for viability (Horio et al., 1991; Stearns et al., 1991; Vardy and Toda, 2000). In addition, homologues of both GCP4 and GCP6 have recently been identified and characterized; these are known as gfh1p and alp16p, respectively (Fujita et al., 2002; Venkatram et al., 2004). Interestingly, although defects in microtubule distribution are apparent in both gfh1 and alp16 mutants, neither gfh1p nor alp16p is essential for viability. This suggests that in fission yeast, microtubule nucleation may not absolutely require an intact large -TuC (we reserve the term "ring" complex for those complexes where a ring complex has been observed directly in the electron microscope). More generally, the presence of such proteins in fission yeast suggests that this organism may provide a useful system for understanding the function of these components of the -TuC.

Here, we describe the isolation of a novel gene, mod21+, which we show to be the fission yeast homologue of GCP5. Characterization of the mod21 phenotype, both singly and in combination with deletion mutants of gfh1+ and alp16+, suggests that gfh1p, mod21p, and alp16p act together as "noncore" subunits of the -TuC, promoting the ability of the complex to drive microtubule nucleation. Biochemical characterization of all three proteins suggests that gfh1p and alp16p may cooperate in the assembly of a large -TuC in fission yeast, and this is consistent with our finding that nonadditive phenotypes are observed when gfh1+, mod21+, and alp16+ are simultaneously deleted. Importantly, however, even in this triple-deletion strain, most aspects of -tubulin–dependent microtubule nucleation persist as normal. Because we find that -tubulin in wild-type fission yeast mostly seems to exist in the form of a small complex, we suggest that -TuCs containing noncore subunits and -TuCs lacking noncore subunits both contribute to microtubule nucleation in fission yeast.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Yeast Methods
General yeast methods were as described previously (Moreno et al., 1991). A complete strain list is presented in Table 1. Curved cell-shaped mutants were isolated by visual screening of yeast colonies after transformation with an insertional mutagenesis cassette containing the ura4+ gene, and sites of insertion were determined as described previously (Snaith and Sawin, 2003). Gene deletion and C-terminal epitope- or green fluorescent protein (GFP)-tagging was performed by polymerase chain reaction (PCR)-based gene targeting, by using the kanMX or natMX selectable marker for resistance to G418 and nourseothricin, respectively (Bahler et al., 1998; Goldstein and McCusker, 1999). gfh1::hphMX and mod21::hphMX hygromycin-resistant strains were generated by transforming gfh1::kanMX and mod21kanMX strains with a PCR-amplified hphMX cassette containing flanked regions of homology (to promoter and terminator regions) also found within the kanMX cassette (Hentges et al., 2005). Gene deletions and tagging were confirmed by yeast colony PCR and Western blotting as appropriate. The functionality of tagged versions of gfh1p, mod21p, alp16p, alp4p, and alp6p was determined by immunofluorescence analysis of interphase microtubule organization in both the mod22+ and the more sensitive mod22-1 background (Supplemental Tables 2 and 3), and this was complemented by a cell shape assay (see below). From these, gfh1-Myc, mod21-Myc, and mod21-GFP were judged to be fully functional, gfh1-GFP to be partially functional, and gfh1-HA likely to be defective or have altered function. Other strains bearing one or more tagged proteins were judged to have intermediate levels of function, to varying degrees, based on comparisons to untagged (positive control) and deletion (negative control) alleles (Supplemental Tables 2 and 3). It should be emphasized that the assay of function on the basis of microtubule organization is very sensitive and independent of assays for physical interaction by coimmunoprecipitation; in several cases, we observed what may be physiologically relevant protein–protein interactions with tagged proteins that might otherwise be considered "defective."

Immunofluorescence and Microscopy
For anti-tubulin immunofluorescence, cells were fixed in methanol at –70°C, processed as described above, and imaged by laser scanning confocal microscopy (Sawin and Nurse, 1998; Sawin et al., 2004). Figures show maximum projections of Z-stacks, including the entire cell volume. Live cell imaging was performed on a Nikon TE300 wide-field inverted microscope system as described previously (Snaith and Sawin, 2003; Sawin et al., 2004). For live cell three-channel imaging of GFP fused to -TuC components (either alp4p, gfh1p, mod21p, or alp16p) together with sad1-dsRed and 4,6-diamidino-2-phenylindole (DAPI), eight Z-sections at 0.6-µm intervals were collected at a single time point (800-ms exposure for GFP, 400-ms exposure for sad1 and DAPI, with appropriate neutral density filters to minimize photobleaching), and maximum projections were generated. For quantitation of microtubule bundle number in live cells expressing GFP-atb2, 10 Z-sections were collected at 0.6-µm intervals at a single time point (800-ms exposure, with neutral density filters). These images were deconvolved using softWoRx (Applied Precision, Issaquah, WA), and maximum projections of Z-sections were then generated. For time-lapse two-channel imaging of GFP-atb2p and sad1-dsRed, eight Z-sections at 0.6-µm intervals were collected every 20 s (400-ms exposure for GFP and 200 ms for sad1-dsRed, with neutral density filters) and deconvolved, and maximum projections of Z-sections were generated. For display of total SPB movement in these sequences, maximum projections from all time points of a given sequence were combined into a single average "time projection." For time-lapse single-channel imaging of GFP-atb2p (assays of astral microtubule release), eight Z-sections at 0.6-µm intervals were collected every 30 s (800-ms exposure per section, with neutral density filters). After deconvolution, maximum projections were generated. For live-cell imaging of alp4-GFP in various mutant strains, eight Z-sections at 0.6-µm intervals were collected at a single time point (400-ms exposure, with neutral density filters), and maximum projections of Z-sections were generated without deconvolution.

Physiological Experiments
For morphology experiments, cell shape defects were determined by growing cells on YE5S plates for 2 d, replica plating to fresh plates, and examining cell shape after 3 h at 32°C (Snaith and Sawin, 2003). For imaging of cell shape by differential interference contrast (DIC) microscopy, cells were washed off the plates with deionized water and immediately fixed in 3% formaldehyde.

For microtubule regrowth experiments, exponentially growing cells were chilled in an ice water bath for 30 min and transferred to 32°C for the specified time before collection by rapid filtration, fixation in methanol at –70°C, and processing for immunofluorescence and confocal imaging (Sawin et al., 2004). For quantitation, nucleation sites were counted in at least 150 cells per strain, at the 30-s time point.

Biochemical Methods
For immunoprecipitations, native cell extracts were prepared by freezing pelleted cells in liquid nitrogen and grinding to a powder while frozen. Frozen powder was resuspended in buffer H50 (50 mM Na HEPES, pH 7.5, 75 mM KCl, 1 mM EDTA, and 0.1% Triton X-100, plus a protease inhibitor cocktail). Extracts were clarified by microfuge centrifugation (13,000 rpm) for 15 min at 4°C, and the protein concentration was adjusted to 8 mg/ml. One milliliter of each extract was incubated with 10 µl of protein G Dynabeads suspension, preloaded with 2 µg of either hemagglutinin (HA)-antibody 12CA5, or an anti-GFP antibody. After incubation with rotation for 1 h at 4°C, beads were washed six times in 1 ml of buffer H50 and resuspended in Laemmli sample buffer for SDS-PAGE and Western blotting. In all immunoprecipitation experiments, immunoprecipitation lanes were 50x overloaded relative to total extract lanes.

For sucrose gradients, clarified extracts were prepared as for immunoprecipitation by using buffer H50 also containing 1 mM -mercaptoethanol and 0.1 mM GTP. Sucrose gradients (5–40%) were generated in the same buffer. Gradients were loaded with 100 µl of clarified extract and centrifuged at 50,000 rpm for 4 h at 4°C in a TLS-55 rotor. Fractions (100 µl) were collected from the top of the gradient with a cut pipette tips and analyzed by Western blotting. For higher resolution gradients (Supplemental Figure 6), 200 µl of clarified extracts was loaded onto 13.2 ml 5–40% sucrose gradients and centrifuged at 28,000 rpm for 20 h at 4°C in a SW40 rotor. Fractions of 550 µl were taken from the top of the gradient by using a gradient-uploader and a fraction collector. Sedimentation coefficients were determined by parallel centrifugation of sucrose gradients containing protein standards.

For sucrose-gradient analysis of higher-eukaryotic -tubulin complexes, Xenopus egg extract was prepared as described previously (Sawin and Mitchison, 1991) and diluted fourfold in buffer H50 containing 1 mM -mercaptoethanol and 0.1 mM GTP before loading onto gradients. Drosophila embryo extract was prepared by homogenizing 0–16 h Drosophila embryos in buffer H50 containing 1 mM -mercaptoethanol and 0.1 mM GTP. Crude extract was clarified by centrifugation in a tabletop centrifuge (13,000 rpm for 15 min at 4°C) before loading onto sucrose gradients.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Mod21p Is a Novel Component of the Fission Yeast -TuC
Previously we identified two genes, mto1+ and mto2+, in an insertional mutagenesis screen for cell-morphology mutants; mto1+ was initially known as mod20+/mbo1+ (Sawin et al., 2004; Venkatram et al., 2004, 2005; Janson et al., 2005; Samejima et al., 2005). Both mto1p and mto2p are involved in microtubule nucleation and organization via interactions with the -TuC, and mutants produce a curved cell phenotype after return-to-growth from stationary phase (Sawin et al., 2004; Samejima et al., 2005). In the same screen, we identified two additional mutants, mod12 and mod21, that also had a curved cell phenotype but showed different characteristics relative to mto1 and mto2 mutants after back-crossing (our unpublished data; Figure 1A). Although mto1 and mto2 mutations segregated as single Mendelian traits in relation to the curved cell phenotype, both mod12 and mod21 mutants were found to produce curved cells only when combined with a mutation in an additional gene, which we have termed mod22 (our unpublished data). mod22-1 single mutants, which can be identified only by tetrad analysis, have no morphological mutant phenotype on their own (Figure 1A); we surmise that the mod22-1 mutation, which is not in our standard "wild-type" laboratory strain, spontaneously arose in the strain used for mutagenesis.

View larger version (109K): [in this window] [in a new window]   Figure 1. Cell shape and microtubule distribution in gfh1, mod21, and mod22-1 single and double mutants. (A) Cell shape in strains of the indicated genotypes after growth to stationary phase on solid media, replica-plating to fresh media, and subsequent growth for 3 h. (B) Anti-tubulin immunofluorescence of asynchronous, exponentially growing cells of the indicated genotypes.

gfh1 mod22-1

gfh1 mod21

gfh1 mod21 mod22-1

To confirm that mod21p is a bona fide component of the fission yeast -TuC, we examined its potential associations with known proteins of the -TuC as well as its intracellular localization. In coimmunoprecipitation experiments from fission yeast extracts, we found that Myc-tagged mod21p was physically associated with HA-tagged -TuC proteins alp4p (GCP2 homologue) and alp6p (GCP3 homologue), in the same manner as Myc-tagged gfh1p (Figures 2, A and B, and 10; see Materials and Methods and Supplemental Tables 1–3 for an assessment of the functionality of these and other tagged strains).

View larger version (33K): [in this window] [in a new window]   Figure 2. mod21p is a component of the fission yeast -TuC. (A and B) Anti-HA coimmunoprecipitation experiments from cell extracts expressing (A) Myc-tagged mod21p and (B) Myc-tagged gfh1p as well as HA-tagged alp4p or HA-tagged alp6p, as indicated. (C) Localization of GFP-tagged alp4p, gfh1p, mod21p, and alp16p (green in merge) in live cells coexpressing the SPB marker sad1-dsRed (red in merge), with DAPI counterstaining (blue in merge). In each set, interphase cells are shown above and mitotic cells below. Note interphase satellites of alp4-GFP (yellow arrow) and relatively weak eMTOC localization of gfh1p, mod21p, and alp16p relative to alp4p (white arrows). All nonenhanced images were collected and processed under identical conditions, allowing direct comparison of intensities. Enhanced images were individually altered to highlight faint eMTOC localization.

In general, the fluorescence intensity of alp4-GFP at SPBs and eMTOCs was higher than that of gfh1-GFP, mod21-GFP, and alp16-GFP. This could indicate that alp4-GFP is more abundant than gfh1-GFP, mod21-GFP, and alp16-GFP at these sites. Alternatively, it is possible that GFP-tagging might negatively affect the localization of gfh1-GFP, mod21-GFP, and alp16-GFP to SPBs and eMTOCs. Indeed, additional experiments suggest that gfh1-GFP and possibly alp16-GFP (but not mod21-GFP) may have somewhat impaired function relative to untagged proteins (see Materials and Methods and Supplemental Tables 1–3).

Previously it has been shown that GCP4 is likely present in more than one copy in the human -TuC, whereas GCP5 is single-copy (Murphy et al., 2001). We therefore performed coimmunoprecipitations from diploid strains in which the different alleles of gfh1p and mod21p were fused to both GFP- and Myc-tags. In these experiments, gfh1-GFP was able to coimmunoprecipitate both mod21-Myc and gfh1-Myc, whereas mod21-GFP was able to coimmunoprecipitate gfh1-Myc but not mod21-Myc (Supplemental Figure 3). Subject to the caveat that gfh1-GFP may have impaired function, this suggests that gfh1p may be multicopy in the fission yeast -TuC, whereas mod21p is only single copy.

gfh1p, mod21p, and alp16p All Contribute to a Single Function in Interphase Microtubule Organization and Nucleation
Neither gfh1p nor alp16p, the fission yeast homologue of the mammalian -TuC protein GCP6, is essential for viability, but deletion of either gene leads to defects in microtubule organization (Fujita et al., 2002; Venkatram et al., 2004). Because there has generally been relatively little in vivo characterization of the specific roles of GCP4, GCP5, or GCP6 in any eukaryote, we wanted to determine whether all or a subset of these proteins might be functionally redundant. Interestingly, we were able to construct a viable quadruple mutant gfh1 mod21 alp16 mod22-1 strain, as well as all possible double and triple mutants, without difficulty. We also tested in more detail whether multiple mutations in any of these genes might produce a stronger microtubule-defective phenotype than single mutations. Immunofluorescence experiments in exponentially-growing cells showed that all possible combinations of deletions of gfh1+, mod21+, and alp16+ were roughly similar to each other with regard to microtubule distribution; in addition, we found that when, and only when, any of these deletions or combination of deletions was combined with the mod22-1 mutation, the microtubule phenotype became significantly worse (Figure 1B, Supplemental Figure 4, and Supplemental Table 1; also see Figure 3).

View larger version (163K): [in this window] [in a new window]   Figure 3. Microtubule renucleation in wild-type and mod21, mod22-1, and multiple mutant cells. Fixed time-point images of representative strains after cold-induced microtubule depolymerization and regrowth. Note that the number of apparent nucleation sites is reduced in mod21 (C) relative to wild-type (A) and mod22-1 (B) and further reduced in mod21 mod22-1 (D) and gfh1 mod21 alp16 mod22-1 (E) strains.

mto1

mto2

mto2

mto1

mto1

mto2

In all combinations of gfh1, mod21, alp16, and mod22-1 mutants, we observed microtubule nucleation from multiple sites on the nuclear surface upon recovery from cold treatment, indicating that these genes are not essential for formation of the multiple, distributed nucleation sites seen in wild-type cells (Figure 3; our unpublished data). However, mutants that showed more aberrant microtubule distributions during exponential growth (e.g., mod21 mod22-1 double mutants, or gfh1 mod21 alp16 mod22-1 quadruple mutants) were also more impaired in the earliest stages of microtubule regrowth (Figure 3, D and E). In these experiments, we also quantitated the number of apparent microtubule nucleation sites at the earliest time points (Figure 4). Relative to wild-type cells, we observed reductions in the number of nucleation sites in gfh1, mod21 and alp16 single mutants, and similar reductions in multiple mutants. Due to inherent difficulties in scoring nucleation sites at the light-microscopic level, there was some variation in the number of apparent sites in different mutants, but we did not observe any strong synthetic effects after multiple deletion of gfh1+, mod21+, and alp16+. Interestingly, when single- or multiple-deletion mutants were combined with the mod22-1 mutation, the number of nucleation sites was typically further reduced (Figure 4), whereas mod22-1 single mutants themselves were not significantly different from wild-type cells. Together, these results suggest that gfh1p, mod21p, and alp16p are likely to contribute to a single common function in microtubule behavior and/or nucleation, and that mod22+ is also important for nucleation.

View larger version (32K): [in this window] [in a new window]   Figure 4. Quantitation of microtubule renucleation in wild-type and mutant cells. Number of microtubule nucleation sites after 30 s of microtubule regrowth in the strains shown in Figure 3 as well as in eleven additional mutant strains, as indicated. At least 150 cells were scored for each genotype.

View larger version (50K): [in this window] [in a new window]   Figure 5. Reduced numbers of interphase microtubule bundles in gfh1, mod21, alp16, and multiple-mutant cells. (A and B) Examples of live wild-type (A) and mod21 mod22-1 mutants (B), expressing GFP-atb2p together with endogenous untagged atb2p. (C) Number of interphase microtubule bundles per cell in the strains indicated, expressing either GFP-tagged atb2p in conjunction with endogenous untagged atb2p, or GFP-atb2p alone. One hundred cells were scored for each strain.

Because the most likely candidate for such a hypothetical "unique nucleation site" was the SPB, we coimaged GFP-atb2 and the SPB marker sad1-dsRed in wild-type cells; single-mutant gfh1, mod21, and alp16 cells; and triple-mutant gfh1 mod21 alp16 cells, both in mod22+ and mod22-1 backgrounds, by time-lapse microscopy. Interestingly, we found that in all mutants, new cytoplasmic microtubules could be nucleated at sites away from the SPB, as also occurs in wild-type cells (Figure 6, C, D, E, F, I, J, K, and L, white arrows; and Supplemental Movies 1–10; additional data not shown). This directly discounts the simple model that the SPB might be the only cytoplasmic microtubule nucleation site in the mutants.

View larger version (82K): [in this window] [in a new window]   Figure 6. Microtubule nucleation and SPB behavior in wild-type and mutant cells. Stills from movies imaging GFP-atb2p (expressed together with endogenous untagged atb2p) and sad1-dsRed, at 20-s intervals, in cells of the indicated genotypes (see Materials and Methods). Yellow arrows indicate representative (but not all) examples of nucleation from the SPB, and white arrows indicate nucleation from non-SPB

View larger version (62K): [in this window] [in a new window]   Figure 7. Microtubule bundle dynamics and SPB oscillations in wild-type and mutant cells. (A) Microtubule bundle appearance and lifetimes. Each graph shows the times of appearance and the lifetimes of microtubule bundles in a representative single cell of the indicated genotype, from a 30-min time-lapse sequence imaging both GFP-atb2 and sad1-dsRed (see Materials and Methods). Two cells are shown for each genotype, with mod22+ strains on the left-hand side and mod22-1 strains on the right-hand side. Microtubule bundles associated with the SPB are shown in red. (B) Schematic of how time-projection images were created to display SPB oscillations. (C) Average time-projection images of wild-type and mutant cells, from 30-min time-lapse sequences imaging both GFP-atb2 and sad1-dsRed. In wild-type and mod22-1 cells, only small movements are observed.

An additional indicator of non-SPB–mediated microtubule nucleation in fission yeast is the presence of iMTOC satellites on microtubules themselves (Sawin et al., 2004; Zimmerman et al., 2004; Janson et al., 2005; Samejima et al., 2005). For example, microtubule-associated satellites of GFP-tagged alp4p are visible in wild-type cells, but not in mto2 mutants, which are defective in non-SPB–mediated microtubule nucleation (Janson et al., 2005; Samejima et al., 2005), and it has recently been observed that new microtubules can be nucleated from such satellites, although this may be difficult to detect routinely (Janson et al., 2005). We observed alp4-GFP satellites in gfh1 and mod21 mutants, both in mod22+ and mod22-1 backgrounds (Figure 8), consistent with our observations of microtubule behavior in vivo.

View larger version (117K): [in this window] [in a new window]   Figure 8. Alp4-GFP interphase satellites are present in gfh1 and mod21 mutants. Alp4-GFP localization in live wild-type (A) and mutant (B–F) cells of the indicated genotypes. Bright spots are SPBs; arrows indicate representative satellites. All images were collected and processed under identical conditions.

gfh1, mod21, and Multiple Mutants Do Not Release Astral Microtubules

gfh1 mod22-1

gfh1 mod21 alp16 mod22-1

View larger version (58K): [in this window] [in a new window]   Figure 9. Microtubule behavior at the end of mitosis. Stills from movies of wild-type (A) and mutant (B–D) cells toward the end of mitosis. Time indicates minutes and seconds relative to the first time point shown; unless indicated, the time between successive frames is 30 s. Note that astral microtubules tend to be short-lived. Arrow in B indicates rare release of an astral microtubule from the spindle pole body. Note also that microtubules are sporadically "fired" from the equatorial MTOC in the center of the cell before formation of a well-formed postanaphase array. Colored dots indicate the presumed minus ends of these microtubules, with a single color for each such microtubule. In some cases, these microtubules seem to translocate away from the nucleation site, both in wild-type and mutant cells. All cells shown express both GFP-atb2p and endogenous atb2p.

View this table: [in this window] [in a new window]   Table 3. Astral microtubules are not released from spindle poles in gfh1, mod21, and additional mutants

In summary, our results from both live and fixed cell experiments indicate that deletion of gfh1+, mod21+ and/or alp16+, either individually or in combination, does not affect major qualitative aspects of microtubule nucleation and dynamics, including the appearance of interphase microtubule nucleation sites, mitotic spindle assembly, astral microtubule behavior and postanaphase array formation. Rather, deletion of these genes, especially in combination with the mod22-1 mutation, leads to a more quantitative reduction in the number of apparent interphase microtubule nucleation sites or iMTOCs—specifically, non-SPB sites. We interpret this to indicate that gfh1p, mod21p, and alp16p may function to promote the efficiency of microtubule nucleation by the -TuC, thereby increasing the number of active microtubule nucleation sites in vivo.

Organizational State of the Fission Yeast -TuC In Vivo
One potential explanation for the nonadditive defective microtubule phenotype seen in the gfh1 mod21 alp16 multiple mutant relative to single or double mutants, in both mod22+ and mod22-1 backgrounds, is that each of the nonessential -TuC proteins might be required for the others to be stable in vivo or for them to associate with a small -TuC containing -tubulin, alp4p, and alp6p. For example, in Xenopus -TuRC reconstitution experiments, immunodepletion of GCP6 from salt-dissociated -TuRC has been shown to prevent the reassembly of large complexes (Zhang et al., 2000). We therefore examined association dependencies of gfh1p, mod21p, and alp16p with the -TuC, by performing coimmunoprecipitation experiments in a variety of mutant backgrounds (Figure 10).

View larger version (87K): [in this window] [in a new window]   Figure 10. Association of mod21p with -TuC requires both gfh1p and alp16p, whereas association of gfh1p with -TuC requires only alp16p. Anti-HA coimmunoprecipitations of Myc-tagged mod21p (A) or Myc-tagged gfh1p with HA-tagged -TuC components alp4p or alp6p (B), in strains with the indicated genotypes. alp4-HA and alp6-HA strains were used in separate experiments, with the results from alp4-HA strains shown in the top panels of A and B, and the results from alp6-HA strains shown in the bottom panels of A and B. For alp4-HA and alp6-HA, "–" indicates negative control strains with untagged protein; for mod22, "–" indicates the mod22-1 allele. For other genes, wild-type or deletion alleles are as indicated.

In further experiments, we found that the coimmunoprecipitation of alp16-Myc with alp4-HA was dependent on gfh1p but not on mod21p (Figure 11A), and also that levels of alp16p were not altered in gfh1 or mod21 strains. Subject to the caveats associated with using tagged strains (see Materials and Methods and Supplemental Tables 1–3), we conclude from these results that association dependencies can account for at least some of the similar phenotypes seen among single and multiple deletions of gfh1+, mod21+ and alp16+, although, notably, both gfh1p and alp16p still associate with the small -TuC proteins in mod21 mutants.

View larger version (62K): [in this window] [in a new window]   Figure 11. gfh1p and mod21p can associate independent of alp16p or the -TuC. (A) Anti-HA coimmunoprecipitation of Myc-tagged alp16p with HA-tagged alp4p in strains with the indicated genotypes. alp16-Myc is only weakly associated with alp4-HA in gfh1 strains. Asterisk marks a nonspecific band that is occasionally but not always enriched in immunoprecipitates, depending on time of exposure of Western blots (compare with Figures 2 and 10). (B) Anti-HA coimmunoprecipitation of Myc-tagged mod21p with HA-tagged gfh1p in alp16+ and alp16 mutants. Mod21p and gfh1p coimmunoprecipitate in alp16, i.e., even when they are not associated with the -TuC (see Figure 10). The two identical central lanes represent two different strain isolates of gfh1-HA mod21-Myc alp16. (C) Anti-HA coimmunoprecipitation of Myc-tagged gfh1p or Myc-tagged mod21p with HA-tagged alp16p in strains of the indicated genotypes. Mod21p does not associate with alp16p in gfh1 strains (i.e., when mod21p and alp16p are not associated with -TuC), whereas gfh1p does associate with alp16p in mod21 strains (i.e., when both gfh1p and alp16p are associated with -TuC).

As described above, our analysis of mutant phenotypes indicates that many -TuC–dependent microtubule behaviors are essentially intact in fission yeast when multiple subunits and/or regulators of the -TuC are absent (gfh1, mod21, and alp16) or mutated (mod22-1). This led us to wonder whether these subunits might not always be tightly associated with the -TuC in vivo; that is, whether a significant fraction of functional -TuCs in wild-type cells might in fact be small complexes. Previous analysis of the fission yeast -TuC by gel filtration chromatography has produced ambiguous and/or conflicting results concerning the size of wild-type and mutant complexes (Vardy and Toda, 2000; Fujita et al., 2002; Venkatram et al., 2004; see Discussion). We therefore followed a complementary approach to analyze the -TuC, by using sucrose gradient sedimentation.

Using the buffer conditions of our coimmunoprecipitation experiments, we found that nearly all -tubulin in wild-type fission yeast extracts was present in a relatively low S-value complex, 8–9S, and this was not significantly altered in a gfh1 mod21 alp16 mod22-1 strain (Figure 12A). As controls, we prepared Drosophila embryo extracts and Xenopus egg extracts, using the same buffers as for fission yeast (see Materials and Methods). As reported previously, Drosophila -tubulin sedimented in both small and large complexes, whereas Xenopus -tubulin sedimented mostly as a large complex (Stearns and Kirschner, 1994; Oegema et al., 1999). Both gfh1p and mod21p also sedimented with low S-values, albeit reproducibly differently from each other (Figure 12B and Supplemental Figure 6). From these data, it could not be confirmed that either gfh1p or mod21p was comigrating with -tubulin. Overall, our results suggest that, in contrast to -tubulin from higher eukaryotes, most of the -tubulin in fission yeast cell extracts is not detected in large protein complexes, even when the homologues of GCP4 (gfh1p), GCP5 (mod21p), and GCP6 (alp16p) are present. This could reflect a lower abundance of large complexes in vivo or a reduced stability of large complexes in vitro.

View larger version (52K): [in this window] [in a new window]   Figure 12. Fission yeast -tubulin is mostly present in a small complex on sucrose gradients. Western blots of cell extracts after sucrose gradient sedimentation. (A) Untagged -tubulin in extracts from wild-type and gfh1 mod21 alp16 mod22-1 fission yeast as well as from Drosophila embryos and Xenopus eggs. (B) Myc-tagged gfh1p, and, independently, Myc-tagged mod21p, in extracts from wild-type fission yeast. The top of the gradient is at the left, and the positions of S-value standards are indicated above the top panel. The gap in staining near the bottom of the Drosophila extract gradient is due to a loading error.

gfh1 mod21 alp16 mod22-1

gfh1 mod21 alp16 mod22-1

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Understanding how the various components of the multisubunit -TuC contribute to its function is a major outstanding question in microtubule nucleation. Our characterization of mod21p, together with gfh1p and alp16p (Fujita et al., 2002; Venkatram et al., 2004), supports the notion that fission yeast can form a large -TuC similar to that found in higher eukaryotes and that this complex is important for maintaining normal levels of microtubule nucleation in vivo. At the same time, however, two findings in particular lead us to propose that the nonessential proteins gfh1p, mod21p, and alp16p should be considered as noncore components of the -TuC, in contrast to the essential core components -tubulin, alp4p and alp6p. First, we have found that most qualitative aspects of microtubule nucleation persist in vivo when all noncore components of the -TuC are deleted, the major observed difference being a quantitative reduction in interphase microtubule nucleation activity. This stands in marked contrast to the very strong nucleation-defective phenotypes observed upon loss of nonessential proteins that recruit the -TuC to prospective cytoplasmic nucleation sites, such as mto1p or mto2p (Sawin et al., 2004; Venkatram et al., 2004, 2005; Janson et al., 2005; Samejima et al., 2005). Second, we have not found evidence for an abundant high S-value -TuC containing the noncore components, even in wild-type cells; rather, we observe nearly all -tubulin in low S-value forms (see below for further discussion).

Roles of Novel Components mod21+ and mod22+
Because both gfh1p and alp16p are required for association of mod21p with the -TuC, it is difficult to judge whether the three noncore subunits have distinct functions. However, we note that the association of both gfh1p and alp16p with the -TuC is independent of mod21p, whereas the association of mod21p with the -TuC depends on both gfh1p and alp16p, and the loss of mod21p phenocopies simultaneous loss of all three noncore subunits. In this light, mod21p may merit special attention as the most "peripheral" and the least "architectural" of the noncore subunits. Further mutational analysis of noncore components will help to address these issues.

In this work, we also identified genetically an additional regulator of -TuC function, mod22+. The identification of mod22-1 was not only fortuitous but also essential to our identification of mod21+ in a morphological mutant screen. Because the mod22-1 phenotype is strongest when noncore components are deleted, it seems plausible that mod22+ may function to promote the nucleation efficiency of a small core -TuC. Although mod22+ has not yet been cloned, our results to date indicate that mod22 is not allelic to any known components of the -TuC or to any of the three tubulin genes in fission yeast (our unpublished data).

Function of gfh1p, mod21p, and alp16p in Microtubule Nucleation and Organization
Our analysis of the noncore subunits suggests that they can be treated together as a single functional class in relation to regulating microtubule behavior. In the area of microtubule nucleation, we have shown that loss of one, two, or all three noncore subunits does not disrupt microtubule nucleation from any of the three different types of MTOCs normally present in vivo. This in turn indicates that an intact large -TuC is broadly dispensable for microtubule nucleation in fission yeast (Fujita et al., 2002; Venkatram et al., 2004). It is possible that the reduced iMTOC nucleation observed in deletion mutants is due to the loss of a specific but as yet unrecognized subclass of iMTOCs; further higher-resolution work will be required to address this.

Altered microtubule dynamics have been observed in many fission yeast mutants affecting microtubule nucleation, particularly -TuC mutants (Paluh et al., 2000; Vardy and Toda, 2000; Fujita et al., 2002; Sawin et al., 2004; Venkatram et al., 2004, 2005; Janson et al., 2005; Zimmerman and Chang, 2005). In all of our mutants, microtubules within bundles were seen to turn over, but the bundles themselves had longer lifetimes than in wild-type cells, accompanied by oscillations of SPBs. The mechanistic reasons for these differences in bundle behavior and/or structure are not yet clear. Such differences could be an indirect consequence of reduced microtubule nucleation (Sawin et al., 2004; Samejima et al., 2005), driven, for example, by a higher ratio of bundling proteins relative to bundles. Alternatively, if microtubule minus-ends were normally capped by -TuCs in wild-type fission yeast, but not in our mutants, minus-ends might be more free to elongate and become bundled. Although we observed the -TuC marker alp4-GFP associated with microtubules in our mutants, we do not know whether its localization may be subtly altered.

In our experiments, we did not quantitate parameters of dynamic instability of individual microtubules, partly because important dynamic instability transitions may occur deep within bundles rather than at the ends of bundles (making them very difficult to observe) and also because the bundles in mutant cells may themselves have a different polarity and/or organization relative to wild-type cells. It is still unclear whether the alterations in dynamics that have been observed in microtubule-nucleation mutants arise primarily as an indirect consequence of nucleation defects or whether nucleation complexes themselves play a more direct role in modulating microtubule dynamics (Paluh et al., 2000; Sawin et al., 2004; Zimmerman and Chang, 2005). Further work is needed to resolve this important issue.

In contrast to the previous results of Venkatram et al. (2004), we did not observe significant release of astral microtubules from SPBs during mitosis in any of our mutants. The reason for this discrepancy is not clear. Very early PAA microtubules that translocate from their nucleation sites could be mistaken for released astral microtubules, but we observed such microtubules equally frequently in wild-type and mutant cells. The results of Venkatram et al. (2004) could be due to aberrantly high levels of GFP-tubulin expression. In their work, GFP-atb2p was expressed from a multi-copy plasmid, using the very high-strength nmt1 promoter under fully derepressed conditions (Venkatram, personal communication), whereas we used two integrated versions of GFP-atb2p, expressed at near to or slightly lower than physiological levels. In previous work, we demonstrated that some mutant strains defective in microtubule nucleation can be supersensitive to GFP-tubulin levels (Sawin et al., 2004).

The Fission Yeast -TuC in Relation to Higher Eukaryotes
In spite of extensive biochemical studies of the -TuC in several different organisms, there are still questions as to which form(s) of the complex may be active for nucleation in vivo, and in what contexts. In budding yeast, where homologues of GCP4, GCP5 and GCP6 have not been identified, the active form of -TuC is thought to be the 11S Tub4 complex, anchored to the SPB (Knop et al., 1997; Vinh et al., 2002). In metazoan cells, both small and large -TuCs can be identified in cell extracts to varying extents, the 30–35S -TuRC being a much more potent nucleator in vitro than the smaller 10S -TuSC subcomplex, perhaps by 2 orders of magnitude (Oegema et al., 1999). Because the -TuRC has been seen to form a cap on nucleated microtubules (Moritz et al., 2000) and is by far the predominant form of -tubulin in vertebrate cells, a general view has emerged that the -TuRC is the primary microtubule nucleator in higher eukaryotes, for both centrosomal and noncentrosomal nucleation. However, while our paper was under review, Verrolet et al. (2006) published a study on the role of the Drosophila homologues of GCP4, GCP5, and GCP6 in microtubule nucleation in vivo (Verollet et al., 2006). In this work, -tubulin targeting and microtubule nucleation at the centrosome were still preserved after combined RNA interference knockdowns of these three -TuRC–specific proteins, suggesting that the -TuSC is sufficient for at least some aspects of microtubule nucleation in higher eukaryotes. However, it is unclear whether -TuSC–mediated nucleation would normally function alongside -TuRC–mediated nucleation in untreated cells (Verollet et al., 2006).

What is the situation in fission yeast? Our sucrose gradient data suggest that a large proportion of the -TuC in wild-type fission yeast may be in the form of small complexes, with perhaps only a very small fraction in larger, canonical complexes containing noncore subunits. How can we reconcile this with our phenotype analysis showing that cells without noncore subunits nucleate microtubules reasonably well from all types of MTOCs but are nevertheless partially defective in interphase nucleation? One interesting, albeit speculative, possibility is that there may be a "division of labor" between large and small -TuCs in fission yeast (Figure 13). According to this view, wild-type cells may contain a relatively small number of large -TuCs, which contain noncore subunits and are highly active for microtubule nucleation, and a much greater number of small -TuCs, which lack noncore subunits and are less active for nucleation. In this manner, the two pools of -TuCs could each contribute a significant amount of the total interphase microtubule nucleation activity. In relation to other systems, this view would put fission yeast somewhere "in between" higher eukaryotes, which rely primarily or exclusively on the -TuRC (possibly because of the need to nucleate hundreds of microtubules), and budding yeast, which have only the equivalent of the small -TuSC. Because budding yeast nucleate cytoplasmic microtubules only from the SPB, it is also noteworthy that loss of noncore components in fission yeast does not restrict cytoplasmic nucleation to the SPB, because this indicates that the differences between budding and fission yeast with regard to nucleation are not simply explained by the presence/absence of noncore components. Further work will be necessary to test these ideas in detail.

View larger version (14K): [in this window] [in a new window]   Figure 13. -TuC organization and function in fission yeast. Schematic view that attempts to reconcile the phenotypic consequences of deletion of noncore -TuC components gfh1p, mod21p, and/or alp16p (i.e., reduced interphase microtubule nucleation) with our inability to detect a significant pool of large -TuCs on sucrose gradients. In this view, the fission yeast -TuC may exist as two populations: a small, abundant, but weakly active complex lacking noncore components (A); and a larger, much less abundant, but much more active complex containing noncore components, including a subcomplex of gfh1p and mod21p (B). In wild-type cells, both types of complexes could contribute to total microtubule nucleation.

	ACKNOWLEDGMENTS

 
We thank T. Toda and O. Niwa for providing yeast strains, S. MacNeill and A. Carr for tagging cassettes, and K. Gull and I. Stancheva for antibodies. We also thank H. Ohkura, I. Samejima, H. Snaith, and S. Venkatram for useful discussions and/or comments on the manuscript. K.E.S. is a Wellcome Trust Senior Research Fellow in Basic Biomedical Sciences. This work was supported by the Wellcome Trust.

	Footnotes

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

This article was published online ahead of print in MBC in Press (http://www.molbiolcell.org/cgi/doi/10.1091/mbc.E05-11-1009) on October 4, 2006.

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

	REFERENCES

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Bahler, J., Wu, J. Q., Longtine, M. S., Shah, N. G., McKenzie, A. 3rd, Steever, A. B., Wach, A., Philippsen, P., Pringle, J. R. (1998). Heterologous modules for efficient and versatile PCR-based gene targeting in Schizosaccharomyces pombe. Yeast 14, 943–951.[CrossRef][Medline]

Basi, G., Schmid, E., Maundrell, K. (1993). TATA box mutations in the Schizosaccharomyces pombenmt1 promoter affect transcription efficiency but not the transcription start point or thiamine repressibility. Gene 123, 131–136.[CrossRef][Medline]

Chen, C. R., Li, Y. C., Chen, J., Hou, M. C., Papadaki, P., Chang, E. C. (1999). Moe1, a conserved protein in Schizosaccharomyces pombe, interacts with aRas effector, Scd1, to affect proper spindle formation. Proc. Natl. Acad. Sci. USA 96, 517–522.[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]

Cormack, B. P., Valdivia, R. H., Falkow, S. (1996). FACS-optimized mutants of the green fluorescent protein (GFP). Gene 173, 33–38.[CrossRef][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]

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

Fava, F., Raynaud-Messina, B., Leung-Tack, J., Mazzolini, L., Li, M., Guillemot, J. C., Cachot, D., Tollon, Y., Ferrara, P., Wright, M. (1999). Human 76p: a new member of the gamma-tubulin-associated protein family. J. Cell Biol 147, 857–868.[Abstract/Free Full Text]

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

Goldstein, A. L. and McCusker, J. H. (1999). Three new dominant drug resistance cassettes for gene disruption in Saccharomyces cerevisiae. Yeast 15, 1541–1553.[CrossRef][Medline]

Gunawardane, R. N., Lizarraga, S. B., Wiese, C., Wilde, A., Zheng, Y. (2000). gamma-tubulin complexes and their role in microtubule nucleation. Curr. Top. Dev. Biol 49, 55–73.[Medline]

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

Hentges, P., Van Driessche, B., Tafforeau, L., Vandenhaute, J., Carr, A. M. (2005). Three novel antibiotic marker cassettes for gene disruption and marker switching in Schizosaccharomyces pombe. Yeast 22, 1013–1019.[CrossRef][Medline]

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

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

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

Keating, T. J. and Borisy, G. G. (1999). Centrosomal and non-centrosomal microtubules. Biol Cell 91, 321–329.[CrossRef][Medline]

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

Knop, M. and Schiebel, E. (1998). Receptors determine the cellular localization of a gamma-tubulin complex and thereby the site of microtubule formation. EMBO J 17, 3952–3967.[CrossRef][Medline]

Masuda, H., Miyamoto, R., Haraguchi, T., Hiraoka, Y. (2006). The carboxy-terminus of Alp4 alters microtubule dynamics to induce oscillatory nuclear movement led by the spindle pole body in Schizosaccharomyces pombe. Genes Cells 11, 337–352.[Abstract/Free Full Text]

Mata, J. and 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]

Moreno, S., Klar, A., Nurse, P. (1991). Molecular analysis of the fission yeast Schizosaccharomyces pombe. Methods Enzymol 194, 795–823.[Medline]

Moritz, M., Braunfeld, M. B., Guenebaut, V., Heuser, J., Agard, D. A. (2000). Structure of the gamma-tubulin ring complex: a template for microtubule nucleation. Nat. Cell Biol 2, 365–370.[CrossRef][Medline]

Murphy, S. M., Preble, A. M., Patel, U. K., O'Connell, K. L., Dias, D. P., Moritz, M., Agard, D., Stults, J. T., Stearns, T. (2001). GCP5 and GCP6, two new members of the human gamma-tubulin complex. Mol. Biol. Cell 12, 3340–3352.[Abstract/Free Full Text]

Murphy, S. M., Urbani, L., Stearns, T. (1998). The mammalian gamma-tubulin complex contains homologues of the yeast spindle pole body components spc97p and spc98p. J. Cell Biol 141, 663–674.[Abstract/Free Full Text]

Oakley, B. R. (2000). gamma-Tubulin. Curr. Top. Dev. Biol 49, 27–54.[Medline]

Oegema, K., Wiese, C., Martin, O. C., Milligan, R. A., Iwamatsu, A., Mitchison, T. J., Zheng, Y. (1999). Characterization of two related Drosophila gamma-tubulin complexes that differ in their ability to nucleate microtubules. J. Cell Biol 144, 721–733.[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. and Nurse, P. (2005). The nuclear rim protein Amo1 is required for proper microtubule cytoskeleton organisation in fission yeast. J. Cell Sci 118, 1705–1714.[Abstract/Free Full Text]

Samejima, I., Lourenco, P. 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]

Sawin, K. E. and Mitchison, T. J. (1991). Mitotic spindle assembly by two different pathways in vitro. J. Cell Biol 112, 925–940.[Abstract/Free Full Text]

Sawin, K. E. and Nurse, P. (1996). Identification of fission yeast nuclear markers using random polypeptide fusions with green fluorescent protein. Proc. Natl. Acad. Sci. USA 93, 15146–15151.[Abstract/Free Full Text]

Sawin, K. E. and Nurse, P. (1998). Regulation of cell polarity by microtubules in fission yeast. J. Cell Biol 142, 457–471.[Abstract/Free Full Text]

Sawin, K. E. and Snaith, H. A. (2004). Role of microtubules and tea1p in establishment and maintenance of fission yeast cell polarity. J. Cell Sci 117, 689–700.[Abstract/Free Full Text]

Sawin, K. E. and Tran, P. T. (2006). Cytoplasmic microtubule organization in fission yeast. Yeast 23, 1001–1014.[CrossRef][Medline]

Schiebel, E. (2000). gamma-Tubulin complexes: binding to the centrosome, regulation and microtubule nucleation. Curr. Opin. Cell Biol 12, 113–118.[CrossRef][Medline]

Snaith, H. A. and Sawin, K. E. (2003). Fission yeast mod5p regulates polarized growth through anchoring of tea1p at cell tips. Nature 423, 647–651.[CrossRef][Medline]

Stearns, T., Evans, L., Kirschner, M. (1991). Gamma-Tubulin is a highly conserved component of the centrosome. Cell 65, 825–836.[CrossRef][Medline]

Stearns, T. and Kirschner, M. (1994). In vitro reconstitution of centrosome assembly and function: the central role of gamma-tubulin. Cell 76, 623–637.[CrossRef][Medline]

Tanaka, K., Kohda, T., Yamashita, A., Nonaka, N., Yamamoto, M. (2005). Hrs1p/Mcp6p on the meiotic SPB organizes astral microtubule arrays for oscillatory nuclear movement. Curr. Biol 15, 1479–1486.[CrossRef][Medline]

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.[Abstract/Free Full Text]

Vardy, L. and Toda, T. (2000). The fission yeast gamma-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 gamma-tubulin complex function. Mol. Biol. Cell 15, 2287–2301.[Abstract/Free Full Text]

Verollet, C., Colombie, N., Daubon, T., Bourbon, H. M., Wright, M., Raynaud-Messina, B. (2006). Drosophila melanogaster gamma-TuRC is dispensable for targeting gamma-tubulin to the centrosome and microtubule nucleation. J. Cell Biol 172, 517–528.[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 gamma-tubulin complex. Mol. Biol. Cell 13, 1144–1157.[Abstract/Free Full Text]

Wilde, A. and Zheng, Y. (1999). Stimulation of microtubule aster formation and spindle assembly by the small GTPase Ran. Science 284, 1359–1362.[Abstract/Free Full Text]

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., Mitchison, T. (1995). Nucleation of microtubule assembly by a gamma-tubulin-containing ring complex. Nature 378, 578–583.[CrossRef][Medline]

Zimmerman, S. and Chang, F. (2005). Effects of [gamma]-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]

This article has been cited by other articles:

				Y. Xiong and B. R. Oakley In vivo analysis of the functions of {gamma}-tubulin-complex proteins J. Cell Sci., November 15, 2009; 122(22): 4218 - 4227. [Abstract] [Full Text] [PDF]

				A. Bouissou, C. Verollet, A. Sousa, P. Sampaio, M. Wright, C. E. Sunkel, A. Merdes, and B. Raynaud-Messina {gamma}-Tubulin ring complexes regulate microtubule plus end dynamics J. Cell Biol., November 2, 2009; 187(3): 327 - 334. [Abstract] [Full Text] [PDF]

				C.J. T. Zeng, Y.-R. J. Lee, and B. Liu The WD40 Repeat Protein NEDD1 Functions in Microtubule Organization during Cell Division in Arabidopsis thaliana PLANT CELL, April 1, 2009; 21(4): 1129 - 1140. [Abstract] [Full Text] [PDF]

				I. Samejima, V. J. Miller, L. M. Groocock, and K. E. Sawin Two distinct regions of Mto1 are required for normal microtubule nucleation and efficient association with the {gamma}-tubulin complex in vivo J. Cell Sci., December 1, 2008; 121(23): 3971 - 3980. [Abstract] [Full Text] [PDF]

				C. Wiese Distinct Dgrip84 Isoforms Correlate with Distinct {gamma}-Tubulins in Drosophila Mol. Biol. Cell, January 1, 2008; 19(1): 368 - 377. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) Supplemental Material All Versions of this Article: E05-11-1009v1 17/12/5075    most recent Alert me when this article is cited Alert me if a correction is posted 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 HighWire Citing Articles via Google Scholar Google Scholar Articles by Anders, A. Articles by Sawin, K. E. Search for Related Content PubMed PubMed Citation Articles by Anders, A. Articles by Sawin, K. E.