Noncore components of the fission yeast gamma-tubulin complex

Abstract

Relatively little is known about the in vivo function of individual components of the eukaryotic gamma-tubulin complex (gamma-TuC). We identified three genes, gfh1+, mod21+, and mod22+, in a screen for fission yeast mutants affecting microtubule organization. gfh1+ is a previously characterized gamma-TuC protein weakly similar to human gamma-TuC subunit GCP4, whereas mod21+ is novel and shows weak similarity to human gamma-TuC subunit GCP5. We show that mod21p is a bona fide gamma-TuC protein and that, like gfh1Delta mutants, mod21Delta mutants are viable. We find that gfh1Delta and mod21Delta 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 gamma-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" gamma-TuC, whereas mod21p is more peripherally associated, and that gfh1p and mod21p may form a subcomplex independently of the small gamma-TuC. Interestingly, sucrose gradient analysis suggests that the major form of the gamma-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 gamma-TuC and enhance its microtubule-nucleating ability.

Figure 1.

Cell shape and microtubule distribution in gfh1Δ, mod21Δ, 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.

Figure 2.

mod21p is a component of the fission yeast γ-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.

Figure 3.

Microtubule renucleation in wild-type and mod21Δ, 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 mod21Δ (C) relative to wild-type (A) and mod22-1 (B) and further reduced in mod21Δ mod22-1 (D) and gfh1Δ mod21Δ alp16Δ mod22-1 (E) strains.

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.

Figure 5.

Reduced numbers of interphase microtubule bundles in gfh1Δ, mod21Δ, alp16Δ, and multiple-mutant cells. (A and B) Examples of live wild-type (A) and mod21Δ 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.

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

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.

Figure 8.

Alp4-GFP interphase satellites are present in gfh1Δ and mod21Δ 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.

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.

Figure 10.

Association of mod21p with γ-TuC requires both gfh1p and alp16p, whereas association of gfh1p with γ-TuC requires only alp16p. Anti-HA coimmunoprecipitations of Myc-tagged mod21p (A) or Myc-tagged gfh1p with HA-tagged γ-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.

Figure 11.

gfh1p and mod21p can associate independent of alp16p or the γ-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 gfh1Δ 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 alp16Δ mutants. Mod21p and gfh1p coimmunoprecipitate in alp16Δ, i.e., even when they are not associated with the γ-TuC (see Figure 10). The two identical central lanes represent two different strain isolates of gfh1-HA mod21-Myc alp16Δ. (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 gfh1Δ strains (i.e., when mod21p and alp16p are not associated with γ-TuC), whereas gfh1p does associate with alp16p in mod21Δ strains (i.e., when both gfh1p and alp16p are associated with γ-TuC).

Figure 12.

Fission yeast γ-tubulin is mostly present in a small complex on sucrose gradients. Western blots of cell extracts after sucrose gradient sedimentation. (A) Untagged γ-tubulin in extracts from wild-type and gfh1Δ mod21Δ alp16Δ 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.

Figure 13.

γ-TuC organization and function in fission yeast. Schematic view that attempts to reconcile the phenotypic consequences of deletion of noncore γ-TuC components gfh1p, mod21p, and/or alp16p (i.e., reduced interphase microtubule nucleation) with our inability to detect a significant pool of large γ-TuCs on sucrose gradients. In this view, the fission yeast γ-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.