Description

Faithful chromosome segregation during mitosis requires the formation of the mitotic spindle. A key component of the spindle apparatus is microtubules, α- and β-tubulin dimers arranged into thirteen protofilaments. Nucleation of microtubules is catalyzed by microtubule organizing centers (MTOCs) (Moritz et al. 1995; Zheng et al. 1995). MTOCs are structurally diverse, but all are defined by enrichment of the γ-tubulin ring complex (γ-TuRC) composed of repeating tetrameric units of γ-tubulin and γ-tubulin complex proteins (GCPs) arranged in a helical fashion which mimics the microtubule geometry and serves as a template for microtubule nucleation (Figure 1A). Most organisms have five related GCPs (GCP2-6), but budding yeast lacks homologs of GCP4-6 and instead nucleates microtubules with a minimal version of the complex known as the γ-tubulin small complex (γ-TuSC) (reviewed in (Kollman et al. 2011; Lin et al. 2015; Liu et al. 2021; Teixido-Travesa et al. 2012)). Although the fission yeast Schizosaccharomyces pombe contains genes encoding all five GCPs (GCP2/alp4+, GCP3/alp6+, GCP4/gfh1+, GCP5/mod21+ and GCP6/alp16+) in addition to γ-tubulin (GCP1/gtb1+), only the genes encoding subunits corresponding to the γ-TuSC are essential for mitotic growth (Anders et al. 2006; Fujita et al. 2002; Horio et al. 1991; Vardy and Toda 2000; Venkatram et al. 2004). If the γ-TuSC proteins are sufficient for microtubule nucleation, what is the function of the γ-TuRC? Structural analysis of γ-TuRC suggests that it is a better match for microtubule geometry; biochemical studies confirmed the γ-TuRC has an enhanced nucleation activity compared to the γ-TuSC (Erlemann et al. 2012; Oegema et al. 1999; Stearns et al. 1991; Vardy and Toda 2000). In addition, there is also evidence for differential localization of γ-TuSC and γ-TuRC complexes (Gao et al. 2019). The combined effect could translate into molecularly distinct MTOCs with differential microtubule nucleation potential, leading to unique microtubule arrays in different regions of a cell.

In yeast, the nuclear envelope (NE) remains intact during mitosis and the MTOC (known as the spindle pole body, SPB) is embedded in the membrane during all or part of the cell cycle so that it can nucleate both nuclear microtubules involved in chromosome segregation and cytoplasmic microtubules needed for spindle positioning. Most yeast, including filamentous fungi, budding and fission yeast have an increased number of nuclear microtubules compared to cytoplasmic microtubules (reviewed in (Jaspersen 2021)). Analysis of γ-TuSC and γ-TuRC distribution at the SPB of the filamentous fungus Aspergillus nidulans showed that γ-TuRC is recruited to the SPB nuclear surface while γ-TuSC binds to the cytoplasmic side (Gao et al. 2019). This differential localization is mediated by the γ-TuRC activating subunit, MztA (the ortholog of MOZART1/mzt1+) (Gao et al. 2019). The presence of distinct forms of microtubule nucleators on the nuclear and cytoplasmic sides of the SPB provides an example illustrating how microtubule nucleation may be controlled by γ-TuSC and γ-TuRC to result in a greater number of nuclear microtubules compared to cytoplasmic microtubules. Analysis of Mzt1 in fission yeast suggests that it plays an essential role in mitotic spindle formation likely through its role in stabilization of Gcp3 within the γ-TuSC (Leong et al. 2019; Masuda and Toda 2016). However, it is unknown if the distribution of γ-TuSC and γ-TuRC is regulated at the SPB in fission yeast as it is in A. nidulans.

To test if differential localization of γ-TuSC and γ-TuRC is a ubiquitous method used by yeast to regulate the number of nuclear and cytoplasmic microtubules, we used structured illumination microscopy (SIM) to examine SPBs in asynchronously growing S. pombe strains containing GFP-tagged versions of Gcp2 (γ-TuSC), Gcp4, Gcp5 or Gcp6 (γ-TuRC); Ppc89-mCherry marked the SPB core. SIM improves resolution roughly two-fold over widefield and confocal imaging methods, and it has previously allowed us to visualize the distribution of the γ-TuSC and Mzt1 in asynchronously growing cells on the nuclear and cytoplasmic facing sides of the SPB (Bestul et al. 2017). Under all conditions tested, Gcp2 and Gcp3 behaved identically (Bestul et al. 2017). Similar results were also obtained in work examining Gcp2/Gcp3 localization and binding (Anders et al. 2006; Leong et al. 2019). Therefore, we used Gcp2-GFP as a proxy for the γ-TuSC in this study. The intensity of Ppc89-mCherry was used to determine the old SPB from the pre-existing cell cycle and the newly formed SPB, while cell morphology was used to determine cell cycle position and to determine the nuclear and cytoplasmic orientation of the SPBs (Figure 1B). In G2 phase cells (9.5-11 µm with SPBs ~200 nm apart), both Mzt1 and the γ-TuSC proteins Gcp2-GFP and Gcp3-GFP accumulate strongly at the nuclear facing side of the old and new SPB with some additional material on the bridge that connects the two SPBs (Figure 1C) (Bestul et al. 2017). The γ-TuRC components in wild-type cells mirrored that of the γ-TuSC (Figure 1C), suggesting that the γ-TuRC is present at both the old and new SPBs.

To quantitate the relative levels of γ-TuSC and γ-TuRC at the two SPBs, we performed single particle averaging of SIM images (SPA-SIM). Using Ppc89-mCherry as a fiducial marker, this method allows us to compare distribution over multiple images, increasing resolution and eliminating artifacts that are frequently observed in SIM images (see Bestul et al. 2017; Burns et al. 2015). This analysis showed that both the γ-TuSC and γ-TuRC localize over a wide area spanning both the old and new SPBs in wild-type cells, particularly on the nuclear side of the SPB along the SPB-SPB axis (Figure 1D-E). In cells lacking gcp4+, the distribution of γ-TuSC along the SPB-SPB axis was reduced, based on analysis of individual and merged images and measurement of the full-width half maximum (FWHM) values of protein distribution (Figure 1D-F). Both Gcp2-GFP and Gcp6-GFP were enriched in the bridge area between the two SPBs in most gcp4∆ mutant cells compared to the distribution to both SPBs in wild-type cells. Loss of gcp5+ did not have a statistically significant effect on the spread of γ-TuSC and γ-TuRC along the SPB-SPB axis based on FWHM values (Figure 1F). In gcp6∆ mutants, the spread of Gcp2-GFP along the SPB-SPB axis wa similar to wild-type cells (Figure 1F), however, inspection of individual and averaged images revealed an important difference in the placement of Gcp2. Wild-type cells had Gcp2-GFP spread between both SPBs whereas Gcp2-GFP was concentrated at the old SPB in gcp6∆ mutants (Figure 1C-E). This observation could explain why gcp6∆ cells, but not gcp4∆ or gcp5∆ mutants, give rise to monopolar spindles in prometaphase (Masuda and Toda 2016). At a mechanistic level, it is unknown why γ-TuSC is maintained at the old SPB in cells lacking gcp6+ function but this might reflect a role for Gcp6 or Mzt1 in recruitment or stabilization of the γ-TuSC; previous work showed that Gcp6 plays a synergistic role with Mzt1 in early spindle assembly (Masuda and Toda 2016).

A comparison of γ-TuSC and γ-TuRC levels at the nuclear and cytoplasmic facing sides of the SPB, known as the inner (IP) and outer (OP) plaques respectively, showed an asymmetric distribution similar to that of microtubules themselves. Although we observed some variation in levels of individual components, most were present in 2.5-4 fold greater levels on the nuclear face compared to the cytoplasmic side in wild-type cells (Figure 1F). Based on EM analysis of microtubule distribution in fission yeast, approximately 12-15 microtubules form at the inner plaque compared to the 1-3 astral microtubules observed at the outer plaque (Ding et al. 1993). The observed ratio of 2.5-4 γ-TuRC on the nuclear side of the SPB compared to the cytoplasmic face suggests γ-TuRC distribution is a major factor controlling microtubule distribution. However, given that levels of γ-TuRC on the cytoplasmic face were higher than expected based on microtubule number, it seems likely that other factors beyond γ-TuRC abundance control the amount of microtubules. Leading candidates include SPB receptors, microtubule polymerases and perhaps modifications of the γ-TuRC that affect its function. Curiously, Gcp6-GFP had a lower IP:OP ratio than other components of the γ-TuSC or γ-TuRC in wild-type cells (2.5±0.9 versus 3.5-4) due to reduced levels of protein at the IP (Figure 1F). It is unknown if this is an artifact of tagging or fixation, or if it reflects compositional heterogeneity in γ-TuRC not previously appreciated in biochemical studies. A reduced IP:OP ratio was also observed for γ-TuSC in mutants in the γ-TuRC components due to loss of protein at the OP (Figure 1F).

The ability to visualize theγ-TuSC andγ-TuRC in vivo and to genetically manipulate the composition of complexes opens up new avenues to investigate how structural heterogeneity at an MTOC translates into differences in microtubule nucleation, stability and attachment. In A.nidulans, asymmetric distribution of γ-TuSC and γ-TuRC may underlie differences in microtubule number (Gao et al. 2019), while studies in budding yeast highlight the important role of theγ-TuSC receptors (Geymonat et al. 2020; Knop and Schiebel 1998). In vitro analysis of microtubule nucleation highlighted several key mechanistic differences for Mzt1 and Gcp3 in fission yeast (Leong et al. 2019) that will be interesting to follow-up with in vivo analyses of γ-TuSC andγ-TuRC assembly and distribution. Together, these results highlight the diverse mechanisms used by cells to ensure that microtubules are formed at the right time, in the correct place and in the proper number to form a bipolar spindle capable of chromosome segregation.