Centrioles are microtubule-based cylindrical structures that assemble the centrosome and template the formation of cilia. The proximal part of centrioles is associated with the pericentriolar material, a protein scaffold from which microtubules are nucleated. This activity is mediated by the γ-tubulin ring complex (γTuRC) whose central role in centrosomal microtubule organization has been recognized for decades. However, accumulating evidence suggests that γTuRC activity at this organelle is neither restricted to the pericentriolar material nor limited to microtubule nucleation. Instead, γTuRC is found along the entire centriole cylinder, at subdistal appendages, and inside the centriole lumen, where its canonical function as a microtubule nucleator might be supplemented or replaced by a function in microtubule anchoring and centriole stabilization, respectively. In this Opinion, we discuss recent insights into the expanded repertoire of γTuRC activities at centrioles and how distinct subpopulations of γTuRC might act in concert to ensure centrosome and cilia biogenesis and function, ultimately supporting cell proliferation, differentiation and homeostasis. We propose that the classical view of centrosomal γTuRC as a pericentriolar material-associated microtubule nucleator needs to be revised.

The centrosome is a major microtubule-organizing center (MTOC) in many animal cells and thus plays a central role in a variety of microtubule-dependent processes, such as intracellular transport, cell migration and chromosome segregation during cell division (Lüders and Stearns, 2007; Paz and Lüders, 2018; Sanchez and Feldman, 2017). The core of the centrosome is formed by microtubule-based cylindrical structures termed centrioles. Centrioles are associated with pericentriolar material (PCM), a layered scaffold assembled and organized by a large number of proteins with diverse functions (Arquint et al., 2014; Fry et al., 2017) (Fig. 1A). The most-prominent and best-characterized function of the PCM is the nucleation of microtubules (Gould and Borisy, 1977; Lüders and Stearns, 2007; Moritz et al., 1995a,b; Paz and Lüders, 2018). Microtubules are composed of 13 protofilaments that are formed by a head-to-tail arrangement of α-tubulin–β-tubulin (hereafter α-β-tubulin) heterodimers and that build the microtubule wall through lateral association (Nogales, 2000). The head-to-tail configuration of tubulin subunits in the lattice provides microtubules with an intrinsic polarity. α-Tubulin is exposed at the so-called minus-end and β-tubulin at the plus-end. At the plus-end, α-β-tubulin dimers are added or removed during microtubule growth and shrinkage, respectively (Desai and Mitchison, 1997), whereas the minus-end is less dynamic and frequently anchored at MTOCs. Apart from microtubules that populate the cytoplasm, a set of highly stable microtubules forms the wall of the centriole cylinder (Kochanski and Borisy, 1990; Wang and Stearns, 2017) (Fig. 1A). In mammalian cells, these centriolar microtubules are arranged with a nine-fold radial symmetry as triplets in the proximal and central regions and as doublets in the distal part (Greenan et al., 2018, 2020; Wang and Stearns, 2017) (Fig. 1A). Each microtubule triplet consists of one complete microtubule, the A-tubule, and two 10-protofilament-containing microtubules, the B- and C-tubule, which share their wall with the A- and B-tubule, respectively. The microtubule doublets are composed of only A- and B-tubules (Fig. 1A). The inner and outer surfaces of the centriole wall are associated with a plethora of proteins. Although in many cases their exact roles remain elusive, some have been shown to contribute to the assembly and stabilization of the centriole cylinder and, as centrioles mature, to the recruitment of PCM and the formation of specific appendage structures (Kumar and Reiter, 2021; LeGuennec et al., 2021; Nigg and Holland, 2018). Indeed, as a consequence of their semi-conservative duplication mode and the resulting difference in age and maturity, the two centrioles that each cell is born with are structurally and functionally distinct (Sullenberger et al., 2020; Vorobjev and YuS, 1982) (Fig. 1A). While in G1, both centrioles are able to recruit PCM, only one, the older mother centriole, carries distal and subdistal appendages, which are required for ciliogenesis and anchoring of microtubule minus-ends, respectively. At their proximal end, the two centrioles are connected by a proteinaceous tether, also called centrosomal linker, that allows for some variability in the distance and relative orientation between the centrioles (Remo et al., 2020). Typically, however, both centrioles are in close proximity to form a single MTOC (Fig. 1A). As cells progress into S-phase, duplication of the two centrioles (now both called mother centrioles) is initiated (Fig. 1B). This process is controlled by the kinase PLK4 (Bettencourt-Dias et al., 2005; Habedanck et al., 2005; Kleylein-Sohn et al., 2007). PLK4 promotes the ordered recruitment of various centriole-duplication factors, such as CPAP and the cartwheel components SAS-6 (also known as SASS6 in mammals) and CEP135, to assemble a daughter centriole on the wall of the mother centriole (Kleylein-Sohn et al., 2007). Each mother centriole templates the formation of exactly one daughter, which grows in perpendicular orientation, remaining engaged with its mother (Nigg and Holland, 2018) (Fig. 1B). In G2 or early mitosis, the centrosomal linker is disassembled, allowing the separation of the two mother–daughter centriole pairs (Remo et al., 2020). During mitosis, each centrosome associates with one of the two spindle poles, ensuring faithful segregation into the daughter cells during cell division. After passing through mitosis, the daughter centrioles, now fully elongated and disengaged from their mother, acquire the capacity to recruit their own PCM (Sullenberger et al., 2020). This process is known as ‘centriole-to-centrosome conversion’ (Fu et al., 2016; Izquierdo et al., 2014; Wang et al., 2011). However, whereas the mother centriole is fully mature and carries distal and subdistal appendages, the daughter centriole acquires these structures only one cell cycle later (Sullenberger et al., 2020; Vorobjev and YuS, 1982). Thus, it takes 1.5 cell cycles from birth to full maturation of centrioles.

Fig. 1.

The structure of centrioles and their decoration with γTuRC throughout interphase. (A) Overview of the centrosome in G1 phase of the cell cycle. Both centrioles are composed of a microtubule-based cylinder, assembled from A-, B-, and C-tubules, that is associated with PCM in its proximal part. The daughter centriole, which has been templated by the mother centriole in the previous cell cycle, lacks appendage structures. Both centrioles are connected by a proteinaceous linker, which allows them to act as one MTOC. (B) The centriole duplication cycle is depicted and γTuRC localization to specific sub-centrosomal sites is shown according to the cell cycle stage. As cells exit the cell cycle and differentiate, the centrosomal MTOC may become inactive through loss of γTuRC at the outside of centrioles.

Fig. 1.

The structure of centrioles and their decoration with γTuRC throughout interphase. (A) Overview of the centrosome in G1 phase of the cell cycle. Both centrioles are composed of a microtubule-based cylinder, assembled from A-, B-, and C-tubules, that is associated with PCM in its proximal part. The daughter centriole, which has been templated by the mother centriole in the previous cell cycle, lacks appendage structures. Both centrioles are connected by a proteinaceous linker, which allows them to act as one MTOC. (B) The centriole duplication cycle is depicted and γTuRC localization to specific sub-centrosomal sites is shown according to the cell cycle stage. As cells exit the cell cycle and differentiate, the centrosomal MTOC may become inactive through loss of γTuRC at the outside of centrioles.

An essential component of the centrosome is the microtubule nucleator γ-tubulin ring complex (γTuRC; Farache et al., 2018; Liu et al., 2020b; Tovey and Conduit, 2018), a multi-protein complex assembled from γ-tubulin, γ-complex proteins (GCPs) 2 to 6 (also known as TUBGCP2–TUBGCP6), MZT1, MZT2 and an actin-like protein. γTuRC has been implicated in both centriole biogenesis (Bahtz et al., 2012; Chi et al., 2021; Cota et al., 2017; Dammermann et al., 2004; Haren et al., 2006; Kleylein-Sohn et al., 2007; Ruiz et al., 1999; Shang et al., 2002) and acquisition of MTOC activity (Paz and Lüders, 2018). However, while γTuRC was discovered more than two decades ago (Moritz et al., 1995b; Zheng et al., 1995), its mode of action has remained obscure. New insight comes from recent structural analyses of native and recombinant γTuRC by cryo-electron microscopy (cryo-EM) (Consolati et al., 2020; Liu et al., 2020a; Wieczorek et al., 2020; Würtz et al., 2021; Zimmermann et al., 2020) (Box 1) and from assaying γTuRC-mediated microtubule nucleation in vitro by single-molecule total internal reflection fluorescence (TIRF) microscopy (Consolati et al., 2020; Liu et al., 2020a; Thawani et al., 2018, 2020; Wieczorek et al., 2021). One important conclusion from these studies is that γTuRC appears to require specific activation and/or cooperation with other factors at the centrosome and other MTOCs to become an efficient microtubule nucleator (Box 1). Such factors may control not only the nucleation activity of γTuRC, but also its targeting to subcellular sites, the anchoring of the minus-end of nucleated microtubules and potentially additional γTuRC functions (Paz and Lüders, 2018). Although the widely accepted view is that centrosomal γTuRC nucleates microtubules as part of the PCM, γTuRC has also been detected in the centriole lumen (Bahtz et al., 2012; Fang et al., 2019; Fuller et al., 1995; Moudjou et al., 1996; Schweizer et al., 2020 preprint; Sullenberger et al., 2020; Vasquez-Limeta et al., 2020 preprint) and at the subdistal appendages of mature centrioles (Chong et al., 2020; Clare et al., 2014; Hagiwara et al., 2000; Nguyen et al., 2020). Whereas the earlier of the aforementioned studies relied on EM of immunogold-labeled samples, the later work confirmed the findings through immunofluorescence imaging using super-resolution and expansion microscopy (ExM). Although accumulating evidence suggests that centrosomal γTuRC may form subpopulations with potentially distinct functions (Fig. 1B; Table 1), this is not commonly appreciated.

Box 1. γTuRC as template for microtubule nucleation

The γTuRC core structure is assembled from γ-tubulin, GCPs 2 to 6, MZT1, MZT2 and an actin-like protein (Consolati et al., 2020; Liu et al., 2020a; Wieczorek et al., 2020; Zimmermann et al., 2020). Together these proteins form a cone-shaped complex with a width of ∼30 nm and a height of ∼25 nm, in which 14 γ-tubulin molecules, exposed at the open face of the cone, are arranged in a left-handed helix, with a partial overlap of positions 1 and 14 (see figure; PDB: 7AS4). The ‘template model’ of microtubule nucleation proposes that the arrangement of γ-tubulin molecules in γTuRC, which resembles a microtubule in cross-section, mimics a microtubule seed to which α-β-tubulin heterodimers can be added. This would facilitate the formation of a tubular polymer, a process that is otherwise slow and energetically unfavorable (Kollman et al., 2011). Curiously, the configuration of γ-tubulins at the microtubule-nucleation interface of γTuRC resembles, but does not perfectly match, the 13-fold symmetry of a microtubule. In contrast to α-β-tubulin in the microtubule, γ-tubulins in γTuRC have variable lateral spacing and do not follow a perfectly circular trajectory. At one end, the γ-tubulin helix is splayed open and the helical pitch is lower (Zupa et al., 2021). This may explain why cytosolic γTuRC has a relatively low nucleation activity (Consolati et al., 2020; Liu et al., 2020a). Turning γTuRC into an active nucleator may require a conformational change in its structure (Kollman et al., 2015; Liu et al., 2020a), possibly triggered by γTuRC-activating factors that concentrate at MTOCs (Paz and Lüders, 2018) or cooperation with accessory factors, such as members of the XMAP215 family, which promote addition of α-β-tubulin to the γTuRC template (King et al., 2020; Thawani et al., 2018).

Table 1.

Studies that have observed γTuRC at distinct sub-centrosomal sites

Studies that have observed γTuRC at distinct sub-centrosomal sites
Studies that have observed γTuRC at distinct sub-centrosomal sites

Here, we dissect distinct γTuRC subpopulations at centrioles according to cell cycle stage and centriole age, and outline how spatially and temporally separable γTuRC activities are linked to the biogenesis and function of the centrosome and cilium. Apart from the canonical role of γTuRC as nucleator of centrosomal microtubules that extend into the cytoplasm, we also discuss less well-established functions of γTuRC in generating microtubules that form the centriole wall and in promoting centriole integrity, as well as its potential involvement in the formation of ciliary microtubules. We propose a revised view of centrosomal γTuRC that acknowledges its intricate organization and function. This has important implications for the interpretation of phenotypes caused by γTuRC deficiency, both in experimental models and in human disease.

An initial step in centriole biogenesis at the beginning of S-phase is the formation of the cartwheel (Fig. 1B). The cartwheel, composed of a central hub with nine radially extending spokes that each terminate in a pinhead structure, dictates the arrangement and nine-fold radial symmetry of the microtubule triplets that constitute the proximal-central part of the centriolar wall (Anderson and Brenner, 1971; Cavalier-Smith, 1974; Gibbons and Grimstone, 1960; Vakonakis, 2021) (Fig. 2). Although it is poorly understood how the structure of the centriole cylinder is established, several observations suggest that this process relies on γTuRC activity. Deficiency in the γTuRC-targeting factor NEDD1, γ-tubulin, MZT1, or any of the GCPs 2–6, blocks centriole assembly in different systems (Bahtz et al., 2012; Chi et al., 2021; Cota et al., 2017; Dammermann et al., 2004; Haren et al., 2006; Kleylein-Sohn et al., 2007; Ruiz et al., 1999; Shang et al., 2002). One possibility is that γTuRC promotes centriole assembly by nucleating centriolar wall microtubules (Fig. 2). This is supported by the observation of cone-shaped, γTuRC-resembling structures at the minus-end of the A-tubules by cryo-electron tomography (Guichard et al., 2010). Alternatively or additionally, γTuRC might stabilize the minus-end of A-tubules through its capping function (Zheng et al., 1995). This activity might only be required during early stages, as the cone-shaped structures at the A-tubule minus-end were not observed in fully assembled centrioles (Guichard et al., 2010). Curiously, cap structures were also absent at the minus-end of B- and C-tubules (Guichard et al., 2010), suggesting that these form independently of γTuRC-mediated nucleation and/or stabilization. The γTuRC-targeting factor NEDD1 and the GCP6 subunit of γTuRC have been identified as substrates of the kinase PLK4, the master regulator of centriole duplication (Bahtz et al., 2012; Chi et al., 2021). How phosphorylation of GCP6 promotes centriole duplication is unclear, but in the case of NEDD1, phosphorylation was shown to promote interaction with and recruitment of the central cartwheel component SAS-6 (Chi et al., 2021). SAS-6 also requires γ-tubulin for its recruitment to the centriole assembly site (Kleylein-Sohn et al., 2007) and interacts with other γTuRC core subunits (Gupta et al., 2020). While the pinhead protein CEP135 was not found to interact with γTuRC, SAS-6 binds γTuRC through its C-terminal part, which is located in the region of the pinheads (Gupta et al., 2020). Thus, SAS-6 may position γTuRC near the A-tubule assembly site (Fig. 2). Apart from PLK4, γTuRC activity during centriole formation may also be under the control of the kinase SADB (also known as BRSK1), but the underlying mechanism remains obscure (Alvarado-Kristensson et al., 2009). Together, the data demonstrate a requirement for γTuRC during the early stage of centriole biogenesis, through co-assembly with SAS-6 and possibly through the nucleation and/or stabilization of centriolar microtubules.

Fig. 2.

Centrosomal subpopulations of γTuRC. γTuRC is recruited to the outside of the centriole wall, the PCM, the centriole lumen and subdistal appendages. Known recruitment factors are indicated. During centriole biogenesis, γTuRC might also be present at the minus-end of A-tubules, which are arranged around a central cartwheel structure. The cartwheel dictates the nine-fold symmetry of centrioles and is formed by the protein SAS-6. The C-terminus of SAS-6 may recruit and position γTuRC for nucleating the A-tubules. The pinheads at the outer end of the cartwheel spokes contain CEP135 and link the cartwheel to the A-tubules. Distinct γTuRC subpopulations are involved in microtubule nucleation, minus-end stabilization and anchoring, and, in the case of luminal γTuRC, centriole stabilization.

Fig. 2.

Centrosomal subpopulations of γTuRC. γTuRC is recruited to the outside of the centriole wall, the PCM, the centriole lumen and subdistal appendages. Known recruitment factors are indicated. During centriole biogenesis, γTuRC might also be present at the minus-end of A-tubules, which are arranged around a central cartwheel structure. The cartwheel dictates the nine-fold symmetry of centrioles and is formed by the protein SAS-6. The C-terminus of SAS-6 may recruit and position γTuRC for nucleating the A-tubules. The pinheads at the outer end of the cartwheel spokes contain CEP135 and link the cartwheel to the A-tubules. Distinct γTuRC subpopulations are involved in microtubule nucleation, minus-end stabilization and anchoring, and, in the case of luminal γTuRC, centriole stabilization.

Using super-resolution microscopy and ExM, γ-tubulin and NEDD1 were found to associate with nascent daughter centrioles, accumulating at the outer side of their wall during S-phase (Schweizer et al., 2020 preprint; Sonnen et al., 2012; Vasquez-Limeta et al., 2020 preprint) (Fig. 1B). γTuRC recruitment to the outside of centrioles not only depends on NEDD1, but also on CEP192, a protein with a similar localization pattern (Schweizer et al., 2020 preprint; Sonnen et al., 2013; Vasquez-Limeta et al., 2020 preprint; Yoshiba et al., 2019). Consistent with this, CEP192 depletion by RNAi was demonstrated to reduce centrosomal γTuRC levels and impair centrosomal microtubule nucleation (Chi et al., 2021; Gavilan et al., 2018; O'Rourke et al., 2014; Zhu et al., 2008). However, newly formed centrioles acquire PCM and robust microtubule-nucleating activity only after passing through mitosis (Wang et al., 2011). Thus, γTuRC on the wall of nascent daughter centrioles likely represents a population that is distinct from PCM-associated γTuRC. Whether it also engages in microtubule nucleation or has another function, remains to be determined.

In G2 phase, as newly assembled daughter centrioles further elongate, γTuRC is recruited to yet another site, the centriole lumen (Fig. 1B). Similar to γTuRC localization at the outer surface of the centriole, lumen accumulation depends on the targeting factor NEDD1 (Schweizer et al., 2020 preprint). Although lumen localization of γTuRC subunits has been described before (Bahtz et al., 2012; Fang et al., 2019; Fuller et al., 1995; Moudjou et al., 1996; Sonnen et al., 2012; Sullenberger et al., 2020; Vasquez-Limeta et al., 2020 preprint), the functional implications of this observation were unclear. Surprisingly, γTuRC is recruited to the centriole lumen by augmin (Schweizer et al., 2020 preprint) (Fig. 2). The eight-subunit augmin complex is known to mediate the nucleation of microtubule branches laterally from other microtubules, by binding to the microtubule lattice and by recruiting γTuRC (Alfaro-Aco et al., 2020; Goshima et al., 2008; Ho et al., 2011; Petry et al., 2013; Tariq et al., 2020). The localization of augmin–γTuRC in the centriole lumen is confined to the central region and requires POC5 (Schweizer et al., 2020 preprint) (Fig. 2), a component of the recently discovered centriole inner scaffold that safeguards the structural integrity of the centriole wall (Le Guennec et al., 2020, 2021; Steib et al., 2020). Removal of augmin–γTuRC from the centriole lumen by depletion of the augmin subunit HAUS6 impairs centriole stability during experimentally induced prolonged mitotic arrest, a phenotype that is also observed after depletion of POC5 (Schweizer et al., 2020 preprint). This suggests that augmin and potentially also γTuRC participate in the stabilizing function of the centriole inner scaffold. This model is supported by previous studies in Tetrahymena, which demonstrated a role of γ-tubulin in centriole maintenance (Shang et al., 2002). Depletion of γ-tubulin not only inhibited centriole formation, but also resulted in the gradual loss of assembled centrioles. Consistent with a role in centriole structural maintenance, overexpression of Tetrahymena γ-tubulin carrying a serine 129 to aspartic acid mutation, mimicking the constitutive phosphorylation of this conserved residue, resulted in centrioles that lacked B- and C-tubules, or even entire triplets, in their wall (Joachimiak et al., 2018). However, it is not known whether γ-tubulin localizes to the centriole lumen in this organism and it remains possible that the phosphorylation of serine 129 affects non-luminal roles of γ-tubulin. How augmin–γTuRC promotes centriole stability is still unknown, but both the luminal localization and the loading to this site at late stages of centriole assembly suggest that this function is unrelated to the nucleation of centriole cylinder microtubules. While recruitment of augmin–γTuRC to the centriole lumen requires POC5, augmin may also interact with the inner centriole wall through its ability to directly bind to the microtubule lattice (Hsia et al., 2014; Song et al., 2018). Curiously, the shape and dimension of native (Song et al., 2018) and reconstituted augmin (Hsia et al., 2014) have a striking similarity to a Y-shaped structure of the inner centriole scaffold (Li et al., 2012), which might promote centriole cylinder cohesion by linking the A- and B-tubules within each microtubule triplet (LeGuennec et al., 2021). Multiple augmin-bound γTuRCs protruding into the centriole lumen might engage in lateral and/or longitudinal interactions with each other to provide additional stabilization. A similar luminal role has been previously proposed for γ-tubulin in Tetrahymena (Shang et al., 2002). Further support for such a model comes from the observation that γ-tubulin and γTuRC subcomplexes can form filamentous assemblies both in vitro (Aldaz et al., 2005; King et al., 2020; Kollman et al., 2010, 2015; Thawani et al., 2020) and in cells (Lindström and Alvarado-Kristensson, 2018), although this has not been shown for γTuRC. Curiously, the microtubule-doublet-containing centrioles in somatic Drosophila cells not only lack the Y-shaped linker (Greenan et al., 2018), but likely also luminal γ-tubulin (Fu and Glover, 2012; Mennella et al., 2012). Here, centriole stabilization from within the lumen might entirely depend on the cartwheel, which, in contrast to human cells, is present throughout the cell cycle (Callaini et al., 1997). Cartwheel-dependent centriole stabilization has been described previously in Tetrahymena (Bayless et al., 2012) and human cells (Izquierdo et al., 2014; Yoshiba et al., 2019), and might at least partially compensate for the loss of luminal γTuRC after augmin RNAi (Schweizer et al., 2020 preprint).

Recently, it has been shown that knockdown of POC5 or of the inner scaffold protein WDR90, which recruits POC5, impairs roundness and wall integrity of the centriole cylinder (Steib et al., 2020). Centriole stabilization by the inner scaffold may not be crucial in cycling cells, since depletion of POC5 or of augmin subunits does not seem to impair centriole duplication and maintenance (Azimzadeh et al., 2009; Lawo et al., 2009; Schweizer et al., 2020 preprint). Instead, the stabilizing function could be important in differentiated cells for maintaining the capacity of the centriole to template the formation of a cilium, which we will discuss further below. Notably, in some cases of cell differentiation, for example in muscle cells or neurons, the centrosome loses or downregulates its MTOC activity, and this correlates with a reduction in centrosomal γ-tubulin (Becker et al., 2020; Leask et al., 1997; Muroyama and Lechler, 2017). Interestingly, removal of γ-tubulin from the centrosome in neurons appears to mostly affect the γ-tubulin populations on the outer surface of centrioles, whereas the luminal population remains in place (Sánchez-Huertas et al., 2016; Schweizer et al., 2020 preprint). Together, these observations suggest a novel function of γTuRC that does not involve interaction with microtubule minus ends but participation in a centriole-stabilizing luminal scaffold, and this activity may be important for centrioles beyond their role as MTOC.

Following cell division, each cell inherits one mother–daughter centriole pair. Daughters disengage from their mothers and recruit their own PCM including additional γTuRCs, providing them with a robust microtubule-nucleation activity and the ability to function as mothers during centriole biogenesis in the following S-phase (Fig. 1B). Although some γTuRC is already recruited to the outer surface of daughter centrioles as they assemble (see above), centriole-to-centrosome conversion significantly increases the amount of wall-associated γTuRC (Vasquez-Limeta et al., 2020 preprint; Wang et al., 2011). Thus, it is likely that recruitment of this additional γTuRC involves PCM proteins that assemble in more proximal regions of the centriole cylinder (Schweizer et al., 2020 preprint) (Fig. 2). Indeed, whereas earlier super-resolution studies have imaged mostly end-on views of centrioles, to reveal the layered organization of PCM proteins rather than their distribution along the centriole cylinder (Fang et al., 2019; Lawo et al., 2012; Sonnen et al., 2012), more recent studies confirmed an enrichment of γTuRC in proximal centriole regions (Chong et al., 2020; Schweizer et al., 2020 preprint; Vasquez-Limeta et al., 2020 preprint). Importantly, CEP192 was shown to be evenly distributed along the centriole wall during interphase (Sonnen et al., 2013; Yoshiba et al., 2019), suggesting that there are at least two modes of recruiting γTuRC to the outer centriole surface – first, through interaction with CEP192, starting during centriole biogenesis, and second, through interaction with PCM proteins, after centriole-to-centrosome conversion (Figs 1B and 2). Although centrosomal microtubule nucleation is generally believed to occur within the PCM, mainly based on initial EM studies that traced the origin of microtubules to this site (Gould and Borisy, 1977; Moritz et al., 1995a), ExM has revealed that microtubule nucleation also occurs from distal centriole regions in microtubule-regrowth experiments (Schweizer et al., 2020 preprint). Moreover, single or combined knockout of the PCM proteins pericentrin (PCNT) and CDK5RAP2, which have been implicated in γTuRC recruitment and stimulation of its microtubule nucleation activity (Choi et al., 2010; Lin et al., 2014), did not or only marginally reduced centrosomal γ-tubulin levels and microtubule nucleation in interphase (Gavilan et al., 2018). In contrast, depletion of CEP192, whose localization is not restricted to the PCM, efficiently removed γTuRC from the outer surface of the centriole (Schweizer et al., 2020 preprint) and severely compromised centrosome-associated microtubule nucleation activity (Gavilan et al., 2018; O'Rourke et al., 2014; Zhu et al., 2008). Together, these findings challenge the current view that γTuRC-dependent microtubule nucleation at the interphase centrosome is confined to the PCM. It is noteworthy, that the Drosophila homologs of pericentrin and CDK5RAP2, Plp and Cnn, respectively, and the Caenorhabditis elegans functional counterpart of human CDK5RAP2, SPD-5, appear to be crucial for γ-tubulin centrosome accumulation in these organisms (Hamill et al., 2002; Martinez-Campos et al., 2004; Megraw et al., 1999; Ohta et al., 2021). However, whereas human and C. elegans centrioles recruit γ-tubulin and nucleation activity during interphase (Bobinnec et al., 2000; Hannak et al., 2002; Strome et al., 2001), in Drosophila, this occurs only during G2 and mitosis, despite SPD-2 (the fly homologue of CEP192) and Plp being present at centrioles throughout interphase (Dobbelaere et al., 2008; Fu and Glover, 2012; Giansanti et al., 2008; Rogers et al., 2008). These observations suggest species-specific differences and a specific regulation of γTuRC recruitment and nucleation activity at interphase centrosomes.

While it is not essential for the nucleation of cytoplasmic microtubules during interphase, the PCM is required for centriole duplication by recruiting centriole assembly factors (Dammermann et al., 2004; Nigg and Holland, 2018). SAS-6 targeting to the proximal wall of mother centrioles, for instance, is partially mediated by Plp in Drosophila, likely through a direct, highly conserved interaction between these two proteins (Ito et al., 2019). In human cells, the accumulation of SAS-6 at the centriole assembly site has been demonstrated to depend on NEDD1 (Chi et al., 2021) and γ-tubulin (Kleylein-Sohn et al., 2007). Thus, it may be specifically the PCM-associated γTuRC fraction that is used to mediate centriole formation. The PCM could then have a dual role in this process in that it might not only concentrate, but also activate, γTuRC at the centriole assembly site. In line with the absence of γ-tubulin from S-phase centrosomes in Drosophila cells, its depletion did not inhibit centriole duplication in this system (Dobbelaere et al., 2008; Raynaud-Messina et al., 2004). Curiously, centriole cylinders in γ-tubulin-depleted cells were structurally aberrant, showing hyper-elongated centriolar microtubules (Raynaud-Messina et al., 2004), a phenotype that was also observed after γ-tubulin depletion in C. elegans embryos (O'Toole et al., 2012). Whether this centriolar defect is directly linked to a function of γ-tubulin at centrioles is unclear. It may also be an indirect effect caused by mitotic delay (Kong et al., 2020), since γ-tubulin is required for mitotic spindle assembly (Barbosa et al., 2000; Hannak et al., 2002).

At mitotic entry, the PCM expands and this correlates with an increase in the amounts of centrosomal CEP192, pericentrin, CDK5RAP2 and γTuRC, and with enhanced microtubule nucleation activity (Bobinnec et al., 2000; Fu and Glover, 2012; Giansanti et al., 2008; Kemp et al., 2004; Khodjakov and Rieder, 1999; Lee and Rhee, 2011; Palazzo et al., 1999; Pelletier et al., 2004; Zhu et al., 2008). This ‘maturation’ of the PCM promotes efficient formation of the mitotic spindle. Interestingly, in the absence of pericentrin and CDK5RAP2, PCM expansion does not occur, but centriolar wall-associated CEP192 is sufficient to recruit γTuRC and organize mitotic spindle poles (Chinen et al., 2021). Together, the data reveal CEP192, localized along the centriole wall, rather than proteins specifically associated with PCM, as a crucial factor for the recruitment of γTuRC-dependent microtubule nucleation activity.

After passing through the second mitosis, centrioles acquire two types of appendage structures that extend radially from their surface – distal appendages, which are present as a set of nine near the distal tip, and subdistal appendages, which are found proximal to the distal appendages, but can display variability in number and precise position depending on the organism and cell type (Anderson, 1972; Paintrand et al., 1992; Uzbekov and Alieva, 2018). Interestingly, analysis of immunogold-labeled samples by EM and, more recently, by super-resolution immunofluorescence microscopy, has revealed the presence of NEDD1 and γ-tubulin at the periphery of subdistal appendages (Chong et al., 2020; Clare et al., 2014; Hagiwara et al., 2000; Nguyen et al., 2020). Subdistal appendages mediate the anchoring of microtubules, but the origin of these microtubules and the anchoring mechanism are unknown (Bornens, 2002). One possibility is that following nucleation at more proximal centriole regions or at non-centrosomal MTOCs, such as the Golgi, microtubules with γTuRC-capped minus-ends might be transferred to the subdistal appendages for anchoring. Alternatively or additionally, microtubules might be nucleated directly at the subdistal appendages. Ninein, which localizes to subdistal appendages and interacts with γTuRC, could be a candidate for mediating this activity (Delgehyr et al., 2005; Lin et al., 2006; Nguyen et al., 2020), but there is no evidence that it promotes microtubule nucleation. In contrast, both of these activities have been observed for the ninein-like protein NINL (Casenghi et al., 2003). However, while NINL is a mother centriole-specific protein (Rapley et al., 2005), subdistal appendage localization has not been unequivocally demonstrated. Together, the current data shows that γTuRC is present at subdistal appendages, but whether it nucleates microtubules at this site or merely participates in their minus-end anchoring has not been tested. It should also be noted that several subdistal appendage proteins additionally localize to the proximal end of centrioles (Mazo et al., 2016). Whether this region represents yet another microtubule nucleation or anchoring site remains to be determined.

After the acquisition of appendages, the centriole is fully mature and competent to template the formation of a cilium, a hair-like structure that is assembled during G1/G0 phase and protrudes from the surface of most animal cells (Kumar and Reiter, 2021; Mirvis et al., 2018). Only distal appendages are essential for ciliogenesis, by connecting the distal centriole end to the plasma membrane (Anderson, 1972), whereas subdistal appendages participate in correct cilium positioning (Mazo et al., 2016). Cilia are composed of an axoneme, a microtubule-based scaffold that is continuous with the distal end of the older of the two centrioles (here termed basal body), and a surrounding ciliary membrane (Fig. 3). The transition zone, a compartment between the basal body and the axoneme, controls the traffic of molecules into and out of the cilium (Garcia-Gonzalo and Reiter, 2017; Gonçalves and Pelletier, 2017). While primary cilia are sensory organelles that can respond to external chemical and mechanical stimuli by relaying this signal to the nucleus to regulate gene expression, motile cilia power cell motility and fluid flow on the cell surface through beating (Legendre et al., 2021; Wheway et al., 2018). Assembly and function of cilia may be scenarios where coordinated activities of distinct centrosomal subpopulations of γTuRC are particularly important (Fig. 3). Defects in these activities might also be linked to human disease. For example, fibroblasts with mutations in the γTuRC subunit GCP4 obtained from a patient with microcephaly and chorioretinopathy (Scheidecker et al., 2015) are severely impaired in their capacity to mount a cilium, despite having normal centriole numbers (Schweizer et al., 2020 preprint).

Fig. 3.

Possible contributions of γTuRC populations to cilium assembly and function. γTuRC might localize at or close to the transition zone to nucleate the central-pair microtubules, which are required for cilium motility. γTuRC at the outside of the centriole wall, at the PCM and at subdistal appendages might promote cilium formation and function through microtubule nucleation and potentially also by contributing to microtubule anchoring, facilitating trafficking and cilium positioning. γTuRC inside the centriole lumen promotes axoneme formation and/or maintenance by ensuring centriole integrity.

Fig. 3.

Possible contributions of γTuRC populations to cilium assembly and function. γTuRC might localize at or close to the transition zone to nucleate the central-pair microtubules, which are required for cilium motility. γTuRC at the outside of the centriole wall, at the PCM and at subdistal appendages might promote cilium formation and function through microtubule nucleation and potentially also by contributing to microtubule anchoring, facilitating trafficking and cilium positioning. γTuRC inside the centriole lumen promotes axoneme formation and/or maintenance by ensuring centriole integrity.

γTuRC-dependent microtubule nucleation from the PCM or outer centriole surface, subdistal appendages or both could contribute to cilium formation, maintenance and function in various ways. During ciliogenesis, centrosome-associated microtubules provide tracks for molecular motors that shuttle ciliogenesis-promoting factors and ciliary building blocks towards the mature centriole or remove negative regulators from this site (Mirvis et al., 2018). Such trafficking is also involved in maintaining ciliary homeostasis and might occur in the form of vesicles or centriolar satellites, membrane-less granules that contain many centrosome and cilia proteins (Gheiratmand et al., 2019; Gupta et al., 2015; Odabasi et al., 2019; Quarantotti et al., 2019). Moreover, centrosome-associated microtubules could directly or indirectly regulate cilia-associated signaling pathways (Gundersen and Cook, 1999; Moujaber and Stochaj, 2020). During ciliogenesis, the centrosome migrates towards the cell surface, where the cilium ultimately protrudes. Although the initial steps of this migratory behavior appear to depend on microtubules, the precise mechanism, which also involves the actin cytoskeleton, has not been elucidated (Pitaval et al., 2017). γTuRC-dependent microtubule nucleation and/or anchoring at subdistal appendages may also be important for positioning of basal bodies and consequently cilia (Mazo et al., 2016), as well as for the coordinated beating of motile cilia in multi-ciliated cells (Kunimoto et al., 2012).

In addition to γTuRC populations on the outer surface of the centriole, luminal γTuRC may also aid in ciliogenesis. As part of the inner scaffold, luminal γTuRC may help centrioles to maintain their structural integrity and capacity to assemble the axoneme (Hamel et al., 2017; Le Guennec et al., 2020; Schweizer et al., 2020 preprint; Steib et al., 2020). Consistent with this, depletion of POC5 or WDR90, the inner scaffold components required for luminal recruitment of augmin-γTuRC, impairs ciliogenesis (Hamel et al., 2017; Schweizer et al., 2020 preprint). Moreover, while mutations in POC5 have been associated with adolescent idiopathic scoliosis (Hassan et al., 2019; Xu et al., 2018) and retinitis pigmentosa (Weisz Hubshman et al., 2018), a mutation in the augmin subunit HAUS7 has been linked to male infertility (Li et al., 2018), diseases that can all be caused by impaired cilia assembly and/or function. Ciliogenesis is also impaired in γTuRC-depleted human fibroblasts (Scheidecker et al., 2015; Schweizer et al., 2020 preprint), but the current data cannot attribute this defect to any specific centrosomal subpopulation of this complex.

While the axoneme of primary cilia is assembled from nine microtubule doublets, in motile cilia it contains an additional central pair of microtubules, which is vital for motility (Ishikawa, 2017). However, it is currently unknown how these microtubules are generated. EM analyses in different organisms have demonstrated that the minus-ends of the central pair microtubules are positioned at the distal border of the transition zone (Szymanska and Johnson, 2012). Immunogold labeling in Chlamydomonas, which possesses two motile cilia, revealed an association of γ-tubulin with the transition zone (Silflow et al., 1999), which may suggest a role of γTuRC in central microtubule pair assembly (Fig. 3). In line with this hypothesis, depletion of γ-tubulin, GCP2 or GCP3 in Trypanosoma causes loss of the central pair microtubules (McKean et al., 2003; Zhou and Li, 2015). However, in this organism, γ-tubulin was only detectable at the basal body, well below the transition zone (Dean et al., 2019; Zhou and Li, 2015). Nevertheless, a minimal number of only two γTuRCs in the transition zone might have evaded detection, but would theoretically be sufficient for nucleating and/or stabilizing the central pair microtubules. How would γTuRCs end up in the transition zone? One speculation is that γTuRCs are recruited from the cytoplasm during assembly of this compartment. Alternatively, they could be derived from the γTuRC population that is present in the centriole lumen. It is worth noting that luminal augmin–γTuRC has been mapped to the POC5-containing central core of the centriole cylinder (Le Guennec et al., 2020; Schweizer et al., 2020 preprint; Steib et al., 2020), whereas the transition zone in ciliated cells is located more distally in the axoneme (Figs 2 and 3). However, a previous large-scale analysis of the centrosome/cilia interactome identified all augmin subunits and NEDD1 as proximity-interactors of CEP162 (Gupta et al., 2015; https://prohits-web.lunenfeld.ca/). CEP162 binds to the distal tip of centrioles and promotes assembly of the transition zone (Wang et al., 2013), suggesting that some augmin–γTuRC might localize more distally than anticipated. The nucleation of central pair microtubules by luminal γ-TuRC thus remains an attractive hypothesis that awaits further investigation.

Centrosomal γTuRC is widely regarded as a bona fide PCM component that nucleates centrosome-associated microtubules. However, it is now clear that centrosomal γTuRC comprises multiple spatially and functionally distinct populations, along the outer wall of the centriole cylinder, inside the centriole lumen and at the subdistal appendages (Table 1). The identification of γTuRC populations with distinct, sub-centrosomal distribution and function warrants a more precise and differentiated terminology when referring to centrosomal γTuRC. Moreover, a more detailed analysis of γTuRC subpopulations is required in experiments where centrosomal γTuRC localization and activity are used as readouts, including the study of mutations in γTuRC subunits and regulators that have been linked to human disorders (Baldwin et al., 2021; Bond et al., 2005; Da Palma et al., 2020; Griffith et al., 2008; Gungor et al., 2021; Hassan et al., 2007; Hull et al., 2019; Issa et al., 2013; Martin et al., 2014; Maver et al., 2019; Mitani et al., 2019; Pagnamenta et al., 2012, 2016; Puffenberger et al., 2012; Rafi and Butler, 2020; Scheidecker et al., 2015; Tan et al., 2014). In the absence of such analysis, the findings have to be interpreted with caution. For example, the puzzling observation that centrosomal targeting and activity of γTuRC are affected by a surprisingly wide range of proteins (Paz and Lüders, 2018) may in part be explained by their effects on distinct γTuRC subpopulations. Exciting future challenges include the identification of γTuRC-targeting factors and regulators that mediate its localization and function at distinct sub-centrosomal sites. Their discovery and manipulation may uncover surprising new ways by which γTuRC contributes to the intricate biology of the centrosome.

We thank Fabian Zimmermann for critical reading and feedback on the manuscript.

Funding

N.S. was supported by an European Molecular Biology Organization (EMBO) long-term fellowship (ALTF 820-2015) and by funding from the European Union's Horizon 2020 research and innovation programme under the Marie Sklodowska-Curie grant agreement No 703907. J.L. acknowledges support by Ministerio de Ciencia e Innovación (MICINN) grants PGC2018-099562-B-I00, Agència de Gestió d'Ajuts Universitaris i de Recerca (AGAUR) 2017 SGR 1089 and by intramural funds of Institute for Research in Biomedicine (IRB) Barcelona, which is a recipient of a Severo Ochoa Centre of Excellence Award from the Spanish Ministry of Science and Innovation and supported by Centres de Recerca de Catalunya (CERCA) (Generalitat de Catalunya).

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Competing interests

The authors declare no competing or financial interests.