Anatomy of the fungal microtubule organizing center, the spindle pole body

Abstract

The fungal kingdom is large and diverse, representing extremes of ecology, life cycles and morphology. At a cellular level, the diversity among fungi is particularly apparent at the spindle pole body (SPB). This nuclear envelope embedded structure, which is essential for microtubule nucleation, shows dramatically different morphologies between different fungi. However, despite phenotypic diversity, many SPB components are conserved, suggesting commonalities in structure, function and duplication. Here, I review the organization of the most well-studied SPBs and describe how advances in genomics, genetics and cell biology have accelerated knowledge of SPB architecture in other fungi, providing insights into microtubule nucleation and other processes conserved across eukaryotes.

Introduction

Microtubule organizing centers (MTOCs) are a diverse group of membrane-less organelles that serve as sites of microtubule nucleation in eukaryotes. In animal cells, centrosomes are the primary, nuclear-associated MTOC that form the poles of the mitotic spindle. Each centrosome is composed of a pair of centrioles, which are surrounded by a cloud of pericentriolar material from which microtubules emanate. The centriole is a cylindrical structure made of triplet microtubules organized in nine-fold symmetry by a central structure known as the cartwheel. Although centrioles, like those found in centrosomes, are found throughout all kingdoms of life, including fungi, most fungi evolved a centriole-less MTOC known as the spindle pole body (SPB) [1]. While SPBs and centrosomes are morphologically distinct, both organelles share components and regulators. Analysis of fungal SPBs have identified some of the most well conserved and important MTOC components, including γ-tubulin, first identified in the filamentous fungus Aspergillus nidulans [2] (Figure 1A).

Figure 1. Structure of the budding and fission yeast SPB.

A. Table of fungal SPB components. ^# Stu2 is not considered a structural component of the SPB. * Ppc89 contains the centrosome microtubule-binding domain motif found in Cep57 (PFAM 06657) and likely fulfills the function of ScSpc42 and ScSpc29. ** In S. cerevisiae, Mps3 is a dual component of the half-bridge and the SPIN [48]; Sad1 also forms ring-like structures at the S. pombe SPB [23], but it is unclear if this function is conserved at centrosomes. Sun1/2 and their KASH counterparts along with Ndc1 are thought to play a role in NPC insertion in metazoans [47]. B. Schematic of microtubule (green) organization in S. cerevisiae (top) and S. pombe in interphase (center) and mitotic (bottom) cells. An EM image of the SPB from each is shown together with a cartoon that depicts the organization of ScSPB and SpSPB core components. Not shown are components of the half-bridge or the pore, which are listed in A. Bars, 100 nm.

In the electron microscope (EM), SPBs appear as small, layered disc-shaped structures associated with the nuclear envelope (Figure 1B). Perhaps best characterized is the Saccharomyces cerevisiae SPB (ScSPB), which is embedded in the nuclear membrane throughout the lifecycle and contains five layers: the outer, central and inner plaques and two intermediate layers (IL1 and IL2) [3,4]. The Schizosaccharomyces pombe SPB (SpSPB) is more amorphous than its budding yeast counterpart and is only inserted in the nuclear envelope during mitosis; throughout interphase, the SpSPB is located on the cytoplasmic face of the nuclear envelope, tethered to an electron density at the inner nuclear membrane [5,6]. Adjacent to both the ScSPB and SpSPB is an electron-dense region of the nuclear envelope known as the half-bridge. The ScSPB and SpSPB have provided a foundation for analysis of SPB structure in other fungi, including yeasts and fungi of clinical and agricultural significance, many of which share a similar laminar structure. Equally important, analysis of yeast SPB structure has provided fundamental insights into the function of all MTOCs, their role in microtubule nucleation and contribution to nuclear envelope breakdown.

Molecular Composition of the SPB Core

The ScSPB contains eighteen components present in multiple copies (reviewed in [7]) (Figure 1A). The locations of proteins within the SPB have been mapped using immunoEM and more recently structured-illumination microscopy (SIM) [8,9] (Figure 1B). Molecular modeling and in vitro reconstitution have further expanded our understanding of ScSPB architecture [10]. The ScSPB is built around a hexagonal lattice of Spc42 [11,12] (Figure 2). Drennan and colleagues reconstituted the Spc42 lattice in vitro using lipid monolayers, finding that the trimeric and antiparallel coiled-coil region was sufficient for array formation [13]. In yeast, this C-terminal domain is also needed for Spc42 oligomerization. Interaction between the C-termini of Spc42 and Cnm67 may help stabilize the Spc42 lattice [8,14]. Contrary to previous models, Spc42’s N-terminal dimeric coiled-coil was generally not needed for lattice formation and could be replaced by a similar coiled-coil from myosin [13]. Most likely the N-terminal dimeric coiled-coil is involved in connecting IL2 and the central plaque (Figure 2).

Figure 2. Spc42 hexagonal lattice.

Spc42 adopts three distinct coiled-coiled conformations, which are predicted to form the hexagonal lattice at the ScSPB core. The dimeric (DCC), trimeric (TCC) and antiparallel (ACC) coiled-coil domains are separated by unstructured regions, shown in gray. Two Spc42 molecules dimerize through the dimeric and antiparallel coiled-coils, while the trimeric coiled-coil interacts with other pairs to form the hexagonal array. Adapted from [13].

In the central plaque, the N-terminus of Spc42 interacts with Spc29 and with the C-terminus of Spc110, a protein related to pericentrin found in the pericentriolar material of centrosomes [8,14,15] (Figure 1B). The C-terminus of Spc110 also binds to Spc29 and calmodulin (Cmd1) using partially overlapping sites in its conserved PACT (pericentrin-AKAP450 centrosomal targeting) domain [16]. Analysis of sites in Spc110 required for SPB targeting and function suggest that the PACT domain extends into the last heptad repeat of the Spc110 coiled-coil domain [10,17], possibly allowing trimeric bundles of Spc110 to align with Spc42 coiled-coils to transmit the hexagonal symmetry of IL2 into the central plaque.

The C-terminus of Cnm67 folds into a novel globular domain that is sufficient for ScSPB localization in yeast, supporting the idea that it binds directly to Spc42 [15,18]. The coiled-coils of Cnm67 are involved in the spacing of IL2 and IL1 [19], and the N-terminus of Cnm67 binds to the outer plaque protein Nud1 [8,15,20] (Figure 1B). Nud1 plays a role in signaling via the mitotic exit network (MEN), and it is involved in the regulated recruitment of Spc72, which binds to a central region of Nud1 in vitro [21].

The SpSPB is built using similar connections (reviewed in [7]) (Figure 1B). For example, Sid4-Cdc11-Mto1 interact in much the same manner as their orthologs Cnm67-Nud1-Spc72, and Cdc11 is involved in signaling via the septation initiation network (SIN) [22,23]. However, as Spc42 and Spc29 are only conserved in closely related Saccharomycetaceae, it is assumed that a single protein, Ppc89 serves as a structural scaffold in fission yeast and likely other fungi (Figure 1A). Like Spc42, Ppc89 oligomerizes in vivo [22]. Ppc89 contains a ~300-amino acid N-terminal extension (Spc29-like) followed by multiple coiled-coils and a Cep57 microtubule binding domain, which interacts with Sid4 [22]. An interaction between Ppc89 and Pcp1 (the Spc110 ortholog) has not been reported, however, the N-terminus of Ppc89 is located near Pcp1 and calmodulin (Cam1) by SIM [23]. This orientation of Ppc89 is identical to that of Spc42. Homologs of Ppc89 can be found across Ascromycota, Pezizomycotina and Taphrinomycotina, suggesting that a single ancestral protein split into two proteins (Spc29 and Spc42) during evolution.

Common ancestral features between SPBs and centrosomes were highlighted in recent work examining ectopic expression of Drosophila centriole components in S. pombe [24]. Despite the distinct morphology of SpSPBs and centrosomes, multiple centriole components independently localize to the fission yeast spindle pole. A conserved interaction motif was mapped to the PACT domain in the C-terminus of Pcp1, which recruited SAS-6 to the SpSPB in a cell cycle dependent manner in much the same way that SAS-6 is regulated in animal cells. This observation in fission yeast revealed a previously unknown role for the Pcp1/Spc110 ortholog pericentrin in SAS-6 targeting to centrioles, highlighting both the utility of the yeast system and the evolutionary conservation between MTOCs.

Microtubule Nucleation Complexes

Microtubule nucleation in fungi, as in other eukaryotes, is mediated by the multisubunit γ-tubulin complex (γ-TuC). The γ-TuC is connected to the SPB core by two linker proteins, Spc72/Mto1 and Spc110/Pcp1, orthologs of human CDK5RAP2 and pericentrin, respectively [25–27] (Figure 1A). Conserved C-terminal motifs target Spc72/Mto1 (MASC, for Mto1 and Spc72 C-terminus) and Spc110/Pcp1 (PACT) to the cytoplasmic and nuclear faces of the SPB, respectively, while their N-termini contain a conserved centrosomin motif 1 (CM1), which likely serves as a γ-TuC docking site, interacting directly with components of the γ-TuC [16,28–31]. Spc110/Pcp1 have a second shared motif (SPM) that is thought to further enhance microtubule nucleation [16] (Figure 3A).

Figure 3. γ-tubulin complex receptors and microtubule nucleation.

A. Substructure of outer and inner plaque receptors from S. pombe, A. nidulans and S. cerevisiae. B. Two copies of γ-tubulin and one copy of GCP2 and GCP3 is the minimal module required for formation of a ring-structure that nucleates microtubules under low salt conditions. In fission yeast and Aspergillus, other paralogs are thought to form complexes that also bind to γ-tubulin and incorporate into the γ-TuRC. C. The fission yeast Mto1 N-termini fragment (Mto1[bonsai]), Mto1, γ-tubulin complex components and Mzt1 co-assemble into a ring-like MGM holocomplex that is a potent nucleator of microtubules in vitro. Mzt1 stabilizes GCP3/Alp6 in this complex. D. Model of N-terminal binding region of receptors and the γ-TuC at the SPB inner and outer plaque in S. cerevisiae and A. nidulans. Based on data from [16,30,34,35,59].

S. cerevisiae have a minimal version of the γ-TuC called the γ-tubulin small complex (γ-TuSC), composed of two molecules of γ-tubulin/Tub4 and one molecule each of GCP2/Spc97 and GCP3/Spc98 [32]. Reconstituted γ-TuSCs appear as Y-shaped molecules in cryoEM reconstructions, with N-termini of Spc97 and Spc98 at the base of the Y and split C-terminal arms that each interact with a molecule of Tub4 [33]. Under low salt conditions, the γ-TuSC can oligomerize into ring-like structures with a similar (but not identical) pitch and diameter to the microtubule protofilament (Figure 3B). Cryo-electron tomography on purified ScSPBs revealed a ‘closed’ γ-TuSC structure at the inner plaque; a conformational bend in the flexible region of Spc98 shifts the position of Tub4 to more closely match that of the microtubule in this structure [34]. A key unresolved question is how the γ-TuSC is converted to this closed state observed at the ScSPB.

S. pombe, like metazoans, has paralogs of GCP2/alp4+, GCP3/alp6+, including GCP4/gfh1+, GCP5/mod21+ and GCP6/alp16+, as well as the small regulator Mozart/mzt1+. Although fission yeast contains subunits to assemble the γ-tubulin ring complex (γ-TuRC) seen in higher eukaryotes, genetic analysis of mutants has shown that only γ-TuSC components (gtb1+, alp4+, alp6+) and mzt1+ are essential for mitotic growth. Furthermore, reconstitution of γ-TuC activity revealed that potent (nanomolar/subnanomolar) microtubule nucleation activity resulted from six proteins: Mzt1, γ-TuSC, a fragment of Mto1 (Mto1[bonsai]) and the Mto1 interacting partner Mto2 [31] (Figure 3C). This ensemble, known as the MGM holocomplex, formed 34–40S ring-like structures and appeared as crescent shaped lock-washers by negative stain, similar to the γ-TuRC.

Mozart is a highly conserved protein present in most eukaryotes, except S. cerevisiae. This somewhat mysterious protein has been proposed to facilitate γ-TuC recruitment and oligomerization. A microtubule nucleation assay using reconstituted proteins derived from the dimorphic yeast Candida albicans suggested that Mzt1 promotes γ-TuC assembly by serving as a bridge between GCP3/Spc98 and Spc110/Spc72 [29]. A similar assay using fission yeast derived proteins indicated that Mzt1 interacts directly with GCP3/Alp6 and prevents GCP3/Alp6 aggregation [31]. This suggests that Mzt1’s role in microtubule nucleation in fission in yeast is to stabilize GCP3/Alp6 so that Mto1-induced conformational changes can convert the MGM complex into an effective nucleator (Figure 3C). In this system Mzt1 did not interact directly with Mto1, and Mzt1’s role is in nucleation with the full constellation of γ-TuRC subunits was not examined. In A. nidulans, MztA interacts with the inner plaque receptor PcpA but not with the outer plaque receptor ApsB [35] (Figure 3D). Interestingly, only subunits of γ-TuSC localize to the outer plaque while GCPB-F (orthologs of GCP2–6) can be detected at the inner plaque in this fungus, suggesting that Mozart may recognize specific receptors and/or versions of the γ-TuC, an idea also suggested in metazoans [36]. The differential localization of MztA and the γ-TuC is thought to underlie asymmetric microtubule nucleation at the AnSPB, with more nuclear compared to cytoplasmic microtubules (Figure 3D).

The idea that the nuclear and cytoplasmic sides of the SPB differ in microtubule nucleation dates back decades and has been attributed to multiple factors. In A. nidulans, α/β-tubulin dimers are specifically imported into the nucleus during mitosis to promote the formation of spindle microtubules from the AnSPB inner plaque [37]. Mitotic phosphorylation of SpMto2 disrupts assembly of the Mto1-Mto2-γTuC as well as nuclear transport of spindle assembly factors. This is thought inactivate cytoplasmic microtubule formation and ensure that the SpSPB creates nuclear microtubules following its insertion into the nuclear envelope [38]. EM and fluorescence microscopy also show that the ScSPB inner plaque nucleates 5–10-fold more microtubules than the outer plaque [3,39,40]. Analysis of the γTuC distribution by SIM showed a ~2-fold difference, a qualitatively similar result to that obtained by other super-resolution imaging methods [21,41]. Together, these data suggest that the activity of the γTuC is differentially regulated at the inner and outer plaques. The obvious candidates to regulate microtubule nucleation are the receptors themselves: Spc110 has a higher affinity for γTuC in vitro than does Spc72, is more effective at microtubule formation in vitro and in vivo and is twice as abundant as Spc72 [25,29,30,40]. However, the role of the microtubule polymerase Stu2/Alp14/chTOG/XMAP215 as well as phosphorylation of the receptors and the γTuC need to be considered as well. In the multi-nucleate filamentous fungus Ashbya gossypii, deletion of AgSPC72 or AgSTU2 both resulted in defects associated with loss of astral microtubules, but the phenotypes were distinct: Agspc72∆ lacked cytoplasmic microtubules whereas Agstu2∆ contained many short microtubule fragments [42]. These data suggest that the role of Stu2 is to lengthen microtubules formed at the outer plaque formed by Spc72-dependent nucleation, a finding that is consistent with data from fission yeast showing that Alp14 stabilizes nascent microtubules [43].

In S. cerevisiae, asymmetric cell division results in a larger mother cell and a smaller daughter cell or bud. Astral microtubules, together with a network of extrinsic factors, ensure asymmetric inheritance of the poles such that the ‘old’ SPB is invariantly inherited by the bud while the ‘new’ SPB remains in the mother (reviewed in [44,45]). Analysis of ScSPB assembly by SIM suggests that intrinsic SPB asymmetry plays a critical role in this inheritance pattern. While Spc42, Spc29, Cnm67 and Nud1 are added during SPB duplication in G1 phase [9], incorporation of Spc72 and the γTuC onto the outer plaque of the new ScSPB is delayed [21] (Figure 4A).

Figure 4. Membrane remodeling at the SPB.

A. Schematic depicting SPB duplication intermediates in budding yeast (top) and fission yeast (bottom) during the cell cycle. Delayed outer plaque maturation at the new SPB is denoted by a star. B. Localization of Nbp1-mTurquoise2 (magenta) and Ndc1-YFP (green) in S. cerevisiae by SIM shows rings of both proteins around the side-by-side ScSPB, as depicted in the cartoon below. SIM, top-down view. Cartoon, side view. Scale, 100 nm. C. Proposed model of membrane fusion by the budding yeast SPIN components. A non-canonical interaction between the N-termini of Mps2 and Mps3 may drive or stabilize the highly curved pore membrane. D. Localization of Sad1-mCherry (magenta) and Cut11-GFP (green) in S. pombe by SIM shows rings of both proteins around mitotic SpSPBs following insertion, as depicted in the cartoon below. SIM, top-down view. Cartoon, side view. Scale, 100 nm. E. In S. pombe, the centromere binding protein Csi1 (blue) mediates centromere attachment to the N-terminus of Sad1, which leads to SPB insertion likely through the reorganization of Sad1 into a ring-like structure similar to that seen in budding yeast. Mutations (X) in sad1+ or csi1+ lead to defects in centromere attachment, SPB ring formation and SPB insertion, which ultimately results in SPB dissociation from the nuclear envelope. For simplicity, one of the two SpSPBs is depicted.

Thus, the ‘old’ and ‘new’ SPBs have differential microtubule nucleation capabilities that work together with extrinsic factors to promote inheritance. Although other work points to a central role for these extrinsic factors [41], the idea that ScSPB maturation is linked to cell cycle regulators is consistent with other genetic and cell biological data, and it provides an interesting paradigm as to how centrosome maturation may be temporally and spatially regulated during asymmetric cell divisions common in development and differentiation in many organisms.

Association of the SPB with the nuclear membrane

Most fungi undergo a closed or semi-closed mitosis where the nuclear envelope remains intact. Integration of the SPB into the nuclear envelope is considered necessary for nucleation of spindle microtubules. In S. cerevisiae, the ScSPB is inserted into the nuclear membrane during SPB duplication, and the pole remains membrane-associated for the duration of the yeast life cycle [46] (Figure 4A). In other fungi such as S. pombe, the pole is associated with the cytoplasmic face of the nuclear envelope throughout interphase and incorporates into the membrane at mitotic onset through a process known as polar fenestration [5,6] (Figure 4A). Membrane attachment is also thought to occur in basal fungi such as chytrids, whose centriole-based spindle poles reside in nuclear envelope fenestrae during mitosis [1].

EM analysis shows that the inner and outer nuclear membrane (INM and ONM) are fused at SPB insertion sites, similar to the pore-like fusions observed surrounding nuclear pore complexes (NPCs), the conserved multi-subunit molecular structures that facilitate bidirectional transport of macromolecules across the nuclear envelope in all eukaryotes [4,5]. How the INM and ONM fuse to create the SPB and NPC pores is poorly understood compared to our molecular understanding of both complexes and the process of microtubule nucleation. Genetic analysis of budding and fission yeast identified a series of mutants that underwent SPB duplication but failed to insert the nascent pole into the nuclear envelope (reviewed in [47]). These genes, which in budding yeast are referred to as the SPB insertion network (SPIN), include Nbp1 and Ndc1 and are ideal candidates to mediate membrane remodeling associated with SPB insertion, forming a partially redundant, ring-like structure surrounding the ScSPB core that, like the NPC, expands and contracts via radial dilation [48–50] (Figure 4B). The SPIN components Mps2 and Mps3 are the budding yeast KASH (Klarshicht-ANC-1-Syne-1) and SUN (Sad1-UNC-84) domain containing proteins that form a canonical linker of nucleoskeleton and cytoskeleton (LINC) complex connected through their C-terminal luminal domains [51] (Figure 4C). However, Mps2 and Mps3 N-termini also interact during SPB duplication, suggesting that a non-canonical extraluminal interaction of the LINC complex facilitates INM-ONM fusion and/or stabilization [48].

A similar ring-like structure of Cut11 and Sad1 (the Ndc1 and Mps3 orthologs) is also observed in fission yeast during SpSPB insertion (Figure 4D), which involves a partial breakdown of the nuclear envelope in the region underneath the pole [23]. This localized nuclear breakdown is controlled, at least in part, by the connection of centromeres to the N-terminus of Sad1 through the centromere-binding protein Csi1 (Figure 4E). Centromere attachment is hypothesized to recruit mitotic regulators [52]. Candidates include other genes implicated in SpSPB insertion, such as the mitotic regulator Cut12, the KASH proteins Kms1/2, Cut11, as well as mitotic kinases. The fungal-specific transmembrane protein, Brr6, is also involved the SpSPB insertion [53]. This enigmatic protein contains four conserved cysteine resides thought to form disulfide bonds in the luminal region, possibly leading to membrane curvature at SPB and NPCs [54]. During meiosis, blocking SPB insertion through a mutation in both Sad1 and telomere contacts (which substitute for the centromere) resulted in the formation of an acentrosomal spindle similar to those found in metazoan oocytes that have undergone centrosome elimination during oogenesis [55]. The ability of fission yeast to form acentrosomal spindles hints at the conserved nature of components needed for this pathway. It will be interesting to determine if nuclear envelope-associated factors involved in interphase cytoplasmic microtubule nucleation in fission yeast also play a role in acentrosomal spindle formation.

In both budding and fission yeast, deletion of the membrane nucleoporins suppresses the growth defect associated with mutations in the dual SPB and NPC component, NDC1/cut11+ [56–58]. One interpretation of this finding is that the NPC and SPB share a subset of insertion factors needed to remodel the INM and ONM. In budding yeast, NPCs are often observed by EM in the region near ScSPB insertion [3,8,46], leading to the idea that the NPC itself may be involved in membrane remodeling. Localization of NPCs near duplicating ScSPBs was recently validated by fluorescence microscopy [50]; however, it is unclear what, if any, role the NPC plays during SPB insertion particularly given that NPCs are not typically visible near the SPB in most fungi as they are in S. cerevisiae. One possibility is that the NPC is indirectly involved in SPB insertion through changes in membrane rigidity.

Conclusion

The diverse architecture and unique ecology of yeasts provides a framework to understand the evolution of MTOCs, including their role in mitosis, nuclear envelope remodeling and asymmetric cell division. Key insights into the mechanisms of microtubule nucleation have originated in these tractable model systems, along with their cognate of microtubule regulatory factors. The closed mitosis of fungi necessitates a coordination between nuclear and cytoplasmic microtubule formation that we are just beginning to dissect. With readily accessible genomes and a simple molecular toolkit, fungal cytoskeletons are ripe systems for cell and structural biologists interested in understanding the breadth of microtubule and MTOC biology.