Emerging insights into CP110 removal during early steps of ciliogenesis

Dutch fabric merchant and exemplary microscopy pioneer Antonie van Leeuwenhoek described microorganisms with flagella, otherwise known as motile cilia, as “little animals observed in rain-, well-, sea- and snow-water” in the 1600s (Leeuwenhoek, 1677). However, what later became known as the primary cilium was not identified until 1844 by anatomist Johann Alexander Ecker (Ecker, 1844). Unlike Leeuwenhoek's flagella, primary cilia were not observed “tumbling about and sideways”. Rather, despite their physiological and structural resemblance to flagella, they are non-motile organelles (see Box 1).

For many years, researchers were reluctant to relinquish the notion that the primary cilium somehow plays a role as a motile organelle; indeed, as late as the 20th century the primary cilium was still commonly referred to as the ‘central flagellum’. However, even after electron microscopy studies clearly demonstrated the non-motile nature of the primary cilium, it was again relegated to the ‘backwaters of the cell’ and described as a vestigial organelle with no apparent purpose.

In the past two decades, there has been a massive shift in perception of the primary cilium and a radical enhancement in our understanding of the role of the primary cilium in cellular function. Recent advances have now demonstrated that the primary cilium plays a major role in signal transduction pathways and is required for development in multicellular organisms. Moreover, dozens of known ciliopathies, syndromes that present with kidney disease, obesity, and a variety of developmental and neurodevelopmental disorders, have been described, and hundreds of genes have been implicated in these ciliopathies (for an excellent review, see Reiter and Leroux, 2017). It is notable that some ciliopathies are caused by impaired ciliary signal transduction and/or sensory functions, which might be independent of defects in primary ciliogenesis. Cilia are essential signaling organelles that are enriched in G protein-coupled receptors such as Patched receptor, which transduces Hedgehog signaling (Derderian et al., 2023). Moreover, cilia are a source of extracellular vesicles that can signal by communication with neighboring cells (Ma et al., 2023b).

Several recent publications have provided major insights into early ciliogenesis, particularly regarding mechanisms for the removal of the centriolar capping protein CP110 (also known as CCP110) from the mother centriole. This step represents a key early threshold point regulating the progression of primary ciliogenesis, and several proteins involved in pathways that promote removal and degradation of CP110 from the mother centriole have been implicated in ciliopathies and other pathologies. In this Review, we will focus on describing key early steps of primary ciliogenesis that occur prior to generation of the ciliary vesicle, highlighting recently uncovered mechanisms for the removal of CP110 from the mother centriole. We also provide examples of ciliopathies caused by genetic variants that encode key proteins involved in removal of CP110 and other early steps of ciliogenesis, and conclude by discussing future directions aimed at understanding the complex regulation of mother centriole uncapping in primary ciliogenesis.

Given that primary ciliogenesis occurs when cells are in the G₁/G₀ phases of the cell cycle, and that cells typically cannot divide if they possess a primary cilium, this process is tightly regulated with multiple layers of control (see Fig. 1). As described in Box 1, primary ciliogenesis occurs via one of two known pathways. Given that much less is known about the extracellular pathway, which appears to be prominent primarily in polarized epithelial cells, this Review will focus on the regulation of the intracellular pathway, which occurs in many types of cells.

In most cells, initiation of primary ciliogenesis occurs in response to stress, such as serum starvation. Indeed, even within 10 minutes of removing serum from cell culture, one of the initial steps in ciliogenesis is observed – the recruitment and docking of distal appendage vesicles (DAVs) to the mother centriole (see Fig. 1B). These vesicles contain, among other proteins, RAB11 (herein referring to RAB11A and RAB11B), a member of the Ras-associated binding (RAB) family of small GTPases, and likely originate from the endocytic pathway (Knodler et al., 2010; Westlake et al., 2011). Their recruitment relies on the microtubule- and actin-based motor proteins dynein and myosin Va, respectively (Wu et al., 2018). However, the mechanisms that selectively target such vesicles towards the centrosome remain poorly defined.

As noted above, the distal and subdistal appendages on mother centrioles are crucial for normal ciliogenesis, and protein variants that localize to these structures can lead to ciliopathies. Subdistal appendages comprise proteins such as outer dense fiber of sperm tails 2 (ODF2), ninein, centriolin and centrosomal protein (CEP) CEP170, as well as additional proteins (Ma et al., 2023a). Although several subdistal appendage proteins, such as ODF2 (Ishikawa et al., 2005) and CC2D2A (Veleri et al., 2014), are required for ciliogenesis, subdistal appendages have better defined roles in spindle generation, cell division and differentiation (Hall and Hehnly, 2021). In contrast, numerous proteins that localize to and comprise the distal appendages are crucial for primary ciliogenesis (Schmidt et al., 2012; Tanos et al., 2013), starting with the core protein CEP83, which supports the recruitment of a multitude of proteins to the distal appendages (Tanos et al., 2013). In particular, distal appendages form the docking site for DAVs on the mother centriole. For example, one key distal appendage protein known as CEP164 is required for the docking of the DAVs (Schmidt et al., 2012) and later in ciliogenesis for the recruitment of tau tubulin kinase 2 (TTBK2) (Tanos et al., 2013), which is subsequently necessary for the uncapping of the mother centriole (Goetz et al., 2012), thus allowing DAVs to fuse with one another and form the ciliary vesicle (CV) (Cajanek and Nigg, 2014; Tanos et al., 2013). CEP164 on the distal appendage interacts with RABIN8 (also known as RAB3IP) on DAVs (Cajanek and Nigg, 2014). RABIN8 is a RAB8 (herein referring to RAB8A and RAB8B) GTP-exchange factor that is recruited to the DAVs through its interaction with the GTP-bound form of RAB11 (Knodler et al., 2010; Westlake et al., 2011) and the transport particle II (TRAPPII) complex (Westlake et al., 2011). The central roles played by these proteins in primary ciliogenesis are consistent with their involvement in ciliopathies.

For the fusion of DAVs and their transformation into the CV to occur and allow ciliogenesis to progress, various proteins that localize to the distal cap of the mother centriole must be removed and/or degraded. Key among these proteins is CP110 and its binding partner CEP97, which localize to both mother and daughter centrioles, and are then removed exclusively from the mother centriole during ciliogenesis (Spektor et al., 2007). Notably, despite its primary localization to the distal end of the centriole, CP110 is required to anchor the basal body to the plasma membrane during ciliogenesis and its depletion results in aberrant distribution of subdistal appendage proteins and impaired ciliary vesicle docking (Yadav et al., 2016). Uncapping of the mother centriole or removal of CP110 is promoted by the endocytic regulatory protein Eps15 homology domain protein-1 (EHD1) (Lu et al., 2015) and requires the EHD1-binding partner MICAL-like protein-1 (MICAL-L1) (Xie et al., 2019) and the SNARE protein SNAP29 (Rotem-Yehudar et al., 2001; Xu et al., 2004). Following removal of CP110, SNAP29 mediates SNARE-based fusion of DAVs to generate the CV (Lu et al., 2015). In addition, CP110 interacts with the microtubule-depolymerizing kinesin KIF24, a kinesin-13 homolog that might either recruit or stabilize CP110 at the mother centriole as well as suppress cilia formation through its microtubule-depolymerizing activity (Kobayashi et al., 2011).

Following uncapping of the mother centriole, the axoneme is able to elongate. RAB8 was the first endocytic trafficking protein identified as being involved the process of ciliogenesis (Nachury et al., 2007; Yoshimura et al., 2007), but it acts after CV formation upon activation by RABIN8 (Knodler et al., 2010; Westlake et al., 2011). Although required for growth of the ciliary membrane and axoneme elongation, the actual function of RAB8 in ciliogenesis is not well understood. Studies in zebrafish demonstrate that the well-conserved RAB8 effector Rabaptin-5 interacts with RAB8 and a protein known as Elipsa, which is part of the intraflagellar transport (IFT) complex that is linked to kinesin (Omori et al., 2008). Components of the IFT complex cooperate with the small GTP-binding proteins ARL13B and RAB8 (Nozaki et al., 2017), thus supporting active tubulin transport from the base of the cilium to the tip of the axoneme to promote growth (Bhogaraju et al., 2013), although diffusion of tubulin plays the major role in IFT, at least in Chlamydomonas cilia (Craft Van De Weghe et al., 2020).

The removal of CP110 from the mother centriole (hereafter referred to as CP110 removal), which occurs after distal appendage vesicle docking and before CV assembly (Lu et al., 2015) or upon centriole docking to the plasma membrane (Tanos et al., 2013), is an early and crucial threshold point in the process of ciliogenesis (Spektor et al., 2007). Proteins involved in primary ciliogenesis are often categorized as acting either before or after elimination of the centriolar capping. Levels of CP110 (and the level of its fellow capping and binding partner CEP97), and therefore the process of ciliogenesis, can be controlled at the transcriptional level (Czerny et al., 2022; Lai et al., 2011; Walentek et al., 2016) and by a variety of microRNAs (Cao et al., 2012; Ma et al., 2023c; Song et al., 2014; Wu et al., 2014; and reviewed in Walentek et al., 2014). However, acute degradation of CP110 appears to be a major mode of regulation of ciliogenesis upon serum starvation of cells (summarized in Table 1). Indeed, ubiquitylation of ciliary proteins has been linked to both ciliogenesis and cilia disassembly, and a multitude of ciliary regulatory proteins undergo ubiquitylation at various stages of ciliogenesis (reviewed in Hossain and Tsang, 2019). In recent years, much effort has been made to elucidate the molecular mechanisms that control CP110 removal, and a number of exciting studies have identified distinct but not necessarily mutually exclusive modes for removal of the capping protein, as discussed below.

Both CP110 and its binding partner CEP97 are removed from the mother centriole as a key step leading to axoneme growth (Spektor et al., 2007). Indeed, loss of either protein leads to removal of its partner from the mother centriole, suggesting that targeting of either for degradation might be an effective way to promote primary ciliogenesis (Spektor et al., 2007). One mechanism recently proposed for CP110 removal involves the function of the centrosomal protein CEP78. CEP78 has been previously implicated in ciliogenesis (Goncalves et al., 2021; Hossain et al., 2017) and pathogenic CEP78 gene variants lead to various ciliopathy-like and retinal diseases including cone-rod dystrophy in the retina and hearing loss (CRDHL) (Reiter and Leroux, 2017). CEP78 is recruited to the centrosome by another centrosomal protein known as CEP350, and also binds to the E3 ligase complex EDD1–DYRK2–DDB1^VPRBP (EDD1 is also known as UBR5; hereafter the complex is referred to as DYRK2) (Goncalves et al., 2021). This E3 complex then proceeds to ubiquitylate CP110, leading to its degradation and thus facilitating growth of the axoneme and ciliogenesis (Goncalves et al., 2021) (Fig. 2A).

As noted above, removal of CEP97 leads to destabilization of CP110, its subsequent removal and degradation and the induction of axoneme growth and ciliogenesis (Spektor et al., 2007). One study has identified involvement of the E3 ligase complex known as cullin-3–RBX1–KCTD10 (hereafter refered to as Cullin-KCTD) in the polyubiquitylation of CEP97, leading to removal of both CEP97 and CP110 from the mother centriole and ultimately to ciliogenesis (Nagai et al., 2018) (Fig. 2B). Thus, E3 ligases that ubiquitylate either CP110 or CEP97 can lead to loss of both these centriolar proteins from the mother centriole and promote primary ciliogenesis (Fig. 2), and the possibility of targeting either or both proteins potentially suggests finely tunable regulation of this key early step in ciliogenesis.

A third E3 ligase implicated in CP110 removal is HERC2 (Xie et al., 2023), which had been previously implicated in degradation of CP110 through its interaction with neuralized E3 ubiquitin protein ligase 4 (NEURL4) (Al-Hakim et al., 2012; Loukil et al., 2017). We recently demonstrated that HERC2 is delivered from peripherally localized centriolar satellites to the mother centriole where it interacts with and ubiquitylates CP110, leading to removal of CP110 and subsequent proteasomal degradation (Xie et al., 2023) (Fig. 3A). Both the delivery of HERC2 and the interaction between HERC2 and CP110 require the action of EHD1 (Naslavsky and Caplan, 2005, 2011, 2018). EHD1 has been previously implicated in CP110 removal (Lu et al., 2015) and is recruited to the centrosome by MICAL-L1 (Xie et al., 2019). The mechanism by which EHD1 controls centriolar satellite movement remains unclear. One possibility is that EHD1 modulates microtubule function, as it can interact with β-tubulin (Huang et al., 2020). Thus, EHD1 might regulate the microtubule growth necessary for movement of centriolar satellites to the mother centriole, and it might thereby be required for ubiquitylation of CP110 by HERC2 and subsequent degradation of CP110.

In addition to delivery of HERC2 to the mother centriole, centriolar satellites potentially regulate ciliogenesis via additional pathways. Centriolar satellites were first described and implicated in ciliogenesis almost 25 years ago (Kubo et al., 1999). Although a multitude of studies have linked centriolar satellites to the process of primary ciliogenesis (reviewed in Odabasi et al., 2020), the precise role of satellites has remained poorly defined. Among other potential mechanisms, centriolar satellites control the activity of Aurora kinase A, a known regulator of both ciliary disassembly and ciliogenesis (Arslanhan et al., 2021). Recent studies have highlighted the cell type-specific role of centriolar satellites in ciliogenesis. Pericentriolar material 1 protein (PCM1) is an essential component of centriolar satellites. PCM1^−/− retinal pigmented epithelial 1 cells, which lack centriolar satellites, fail to generate primary cilia (Hall et al., 2023; Xie et al., 2023). However, the situation is more complex in vivo. Mouse embryonic fibroblast (MEF) cells from Pcm1^−/− mice fail to remove CP110 from the mother centriole, but nonetheless are capable of generating primary cilia, suggesting that at least in these cells CP110 removal is not a prerequisite for primary ciliogenesis (Hall et al., 2023). Further support for the role of centriolar satellites in the regulation of ciliogenesis comes from a study demonstrating that the E3 ligase component N-recognin 5 (UBR5) interacts with and ubiquitylates centrosome and spindle pole associated protein 1 (CSPP1) on centriolar satellites to maintain satellite stability (Shearer et al., 2018).

Another ubiquitin-dependent mechanism for CP110 removal comes from a recent study by Shen and colleagues, who have demonstrated the involvement of the linear ubiquitin assembly chain complex (LUBAC) in primary ciliogenesis through its ubiquitylation of CP110 (Shen et al., 2022). LUBAC is a unique E3 enzyme complex that generates linear polyubiquitin chains by linking a ubiquitin protein, via its C-terminal carboxyl group, to the N-terminal methionine on the preceding ubiquitin (Iwai, 2021). Such a mechanism for linear ubiquitylation is not mutually exclusive with the function of other E3 ligases. For example, EDD1–DYRK2–DDB1^VPRBP, Cullin-KCTD and HERC2 could function in concert with LUBAC, as heterotypic ubiquitin chains, comprising both lysine-linked and methionine-linked ubiquitin moieties, have been identified in multiple pathways (Haakonsen and Rape, 2019).

Additional mechanisms have been described for removal of CP110, which focus on elimination of proteins that either recruit or maintain CP110 at the mother centriole. For example, a recent study has shown that KIF24 (Kobayashi et al., 2011) associates with the mother centriole and serves to recruit M-phase phosphoprotein 9 (MPP9; also known as MPHOSPH9). MPP9 binds to the CP110 and CEP97 centriolar capping proteins, thus recruiting and maintaining them on the mother centriole and preventing ciliogenesis from proceeding (Huang et al., 2018; Kobayashi et al., 2011) (Fig. 3B). In the early stages of ciliogenesis, MPP9 undergoes phosphorylation by TTBK2, causing MPP9 to undergo proteasomal degradation and triggering destabilization and subsequent removal of the CP110–CEP97 complex from the mother centriole (Huang et al., 2018) (Fig. 3B). Phosphorylation of another substrate, CEP83, by TTBK2, has also been linked to removal of CP110 and ciliogenesis (Lo et al., 2019; Tanos et al., 2013). In addition, the recruitment of TTBK2 to the basal body is regulated by lipid kinases and phosphatases. Phosphatidylinositol-4-phosphate interferes with the interaction of TTBK2 and its recruiting protein, CEP164, and prevents recruitment of TTBK2 to the basal body, removal of CP110 and ciliogenesis (Xu et al., 2016). CEP164 itself is recruited by the centrin-binding protein SFI1, which is also required for removal of CP110 and ciliogenesis (Laporte et al., 2001). Also of note, a form of Parkinson's disease results from an arginine-to-cysteine mutation at residue 1441 in the leucine-rich repeat kinase 2 (LRRK2). LRRK2 phosphorylates RAB10 and enhances its binding to the Rab-interacting lysosomal protein RILPL1, causing a failure in TTBK2 recruitment to the basal body; this leads to impaired removal of CP110 and inhibits ciliogenesis (Sobu et al., 2021).

Another potential mechanism for the removal and degradation of CP110 involves the protein enkurin domain containing 1 (ENKD1). ENKD1 competes with CEP97 for interaction with CP110 at the mother centriole (Song et al., 2022) (Fig. 4A). Given that the interaction between CEP97 and CP110 leads to a stable complex at the mother centriole, the competition between ENKD1 and CEP97 for CP110 binding destabilizes CP110, prompting its removal from the mother centriole (Song et al., 2022) (Fig. 4A). It is notable that both of these mechanisms that rely on destabilization of CP110 are not mutually exclusive with the mechanisms dependent on E3 ligase ubiquitylation and degradation of CP110. The process of destabilization of CP110 might in fact temporally precede ubiquitylation. A recent study, which demonstrated the cytosolic carboxypeptidase family members CCP5 and CCP6 as stabilizers of CP110, showed that CCP5 and CCP6 levels are transiently downregulated to facilitate removal of CP110 during the initiation of ciliogenesis (Wang et al., 2023). ELMOD2 is another stabilizing protein that acts upstream of CP110 to prevent its spurious removal from the mother centriole (Turn et al., 2021). Additional CP110 interactors are the centriolar protein centrin 2 (Prosser and Morrison, 2015) and centrobin (Ogungbenro et al., 2018). In serum-starved retinal pigmented epithelial cells, depletion of either centrin 2 or centrobin led to a failure to remove CP110 from the mother centriole and impaired the formation of primary cilia (Prosser and Morrison, 2015). A number of additional proteins have been implicated in regulation of CP110 at the mother centriole, including Gap junction protein α1 (GJA1) (Jang et al., 2022), WD-repeat protein 8 (WDR8; also known as WRAP73) (Kurtulmus et al., 2016), EHD4 (Jones et al., 2022), the retromer complex (Xie et al., 2022) and microtubule affinity regulating kinase 4 (MARK4) (Kuhns et al., 2013), although it is unknown whether these proteins interact with CP110 or how they participate in regulating removal of CP110 (see Table 1).

A fascinating mechanism has recently been elucidated in which autophagosomes are recruited to the mother centriole to degrade CP110. This process depends upon the NudC-like protein 2 (NUDC2) autophagy receptor (Liu et al., 2021) (Fig. 4B). In this mechanism (demonstrated in MEF cells), NUDC2 interacts with CP110 on the mother centriole, and then serves as a receptor for LC3-positive autophagic vesicles that subsequently engulf CP110 and target it for autolysosomal degradation. Notably, during centriole amplification, NUDC2 interacts with and stabilizes HERC2, and knockdown of either protein produces centriole over-duplication effects (Li et al., 2019).

A more complete understanding of the early steps that regulate ciliogenesis might be key to resolving the molecular basis of many ciliopathies. Indeed, genetic variants that code for a number of key proteins involved in the early steps of primary ciliogenesis have been implicated in ciliopathies. Below we discuss examples of ciliopathies caused by genetic variants of several of these key proteins, which include centriolar proteins, distal appendage proteins and subdistal appendage proteins.

Ciliopathies can result from defects in either primary or motile cilia, and in some cases, from dysfunction in both types of cilia. Primary cilia dysfunction has wide-ranging effects on the brain and development in addition to the kidneys and liver. Such dysfunction can also impact sensory organs including the eyes, ears and nose. Indeed, primary cilia dysfunction and/or perturbed ciliogenesis has been implicated in anosmia induced by the SARS-CoV-2 (COVID-19) virus (Li et al., 2020). This potentially results from interactions of the viral Nsp13 protein with various centrosomal proteins (Gordon et al., 2020). Defective ciliary proteins that ordinarily function at the axoneme or ciliary tip can lead to ciliopathies, but ciliopathies can also result from variants of protein that regulate early steps in the process of ciliogenesis. We will next highlight select examples of inherited ciliopathies that result from defective centriolar proteins and both distal and subdistal appendage proteins.

CEP290 is a key centriolar protein that is required for ciliogenesis (Tsang et al., 2008), and over 100 CEP290 protein variants have been identified that lead to a range of different ciliopathies, potentially through altered interaction with other centriolar proteins (Coppieters et al., 2010). For example, human tectonic family member 1 (TCTN1) localizes to the transition zone and interacts with CEP290, and TCTN1 variants can cause Joubert syndrome, a ciliopathy which affects the cerebellum and causes impaired balance and coordination (Garcia-Gonzalo et al., 2011). Moreover, variants in other proteins that form a complex with TCTN1 have also been implicated in Joubert syndrome as well as Meckel syndrome, the latter of which presents with skull defects and brain protrusion through the skull, kidney cysts, extra digits and multiple abnormalities in the head, face and various internal organs (Garcia-Gonzalo et al., 2011). Another example is the outer centriolar wall protein hydrolethalus syndrome protein 1 (HYLS-1), which acts early in the ciliogenesis pathway to organize the basal body at the plasma membrane and promote ciliary axoneme elongation (Dammermann et al., 2009). A missense mutation in the HYLS1 gene (D211G) leads to hydrolethalus syndrome (and prenatal mortality) (Mee et al., 2005), whereas homozygous variants that extend the C-terminal end of the protein 11 amino acids beyond the stop codon can lead to a milder defect, with individuals surviving and presenting with Joubert syndrome (Oka et al., 2016).

In the 1960s, a subset of individuals with oral, facial and digital malformations (known as oral-facial-digital syndrome type 1) were first identified as also having polycystic kidney disease (Tucker et al., 1966). Eventually, a centriolar protein known as OFD1 was implicated in this disease as well as in other types of ciliopathies (Ferrante et al., 2001). Another gene responsible for an oral-facial-digital syndrome ciliopathy is C2CD3 (Ye et al., 2014). The C2CD3 protein binds to OFD1 at the distal end of centrioles and has been implicated in controlling centriole length (Thauvin-Robinet et al., 2014). Additionally, the human homolog of the chicken TALPID3 protein (also known as KIAA0586) localizes to the basal body, and gene variants in TALPID3 can lead to Joubert syndrome and other ciliopathies (Alby et al., 2015; Bachmann-Gagescu et al., 2015; Roosing et al., 2015).

Distal and subdistal appendages are key to ciliogenesis, as they exist exclusively on the mother centriole that forms the basal body and thus distinguish the mother centriole from the daughter centriole. Unsurprisingly, many subdistal and distal appendage proteins are required for ciliogenesis, and pathogenic gene variants lead to various ciliopathies. For example, pathogenic variants coding for the subdistal appendage protein CC2D2A lead to developmental disorders and various ciliopathies, including Meckel syndrome and Joubert syndrome (Gorden et al., 2008; Noor et al., 2008; Tallila et al., 2008). The kinesins KIF2A and KIF3A, microtubule motor proteins, localize to subdistal appendages, and variations in either protein can cause impaired ciliogenesis and lead to ciliopathies and/or developmental disorders (Broix et al., 2018; Cogne et al., 2020; Gomez Garcia and Knoers, 2009). CEP89 (also known as CEP123 and CCDC123) is a subdistal and distal appendage protein required for ciliogenesis, and it has also been implicated in autosomal dominant polycystic kidney disease (Sillibourne et al., 2013; Skalicka et al., 2018; Tanos et al., 2013).

Proteins that localize selectively to the distal (but not subdistal) appendages are also crucial for ciliogenesis and many have been implicated in ciliopathies. CEP83 (also known as CCDC41), is required for mother centriole docking at the plasma membrane to promote ciliogenesis (Tanos et al., 2013), as well for the docking of DAVs on the distal appendages (Joo et al., 2013). Homozygous variants of the CEP83 gene have been identified in multiple families afflicted with renal ciliopathies (nephronophthisis), learning disabilities and hydrocephalus (Failler et al., 2014). CEP83 acts at the top of a hierarchy for the recruitment of other distal appendage proteins to centrioles, including sodium channel and clathrin linker 1 (SCLT1) and TTBK2 (Tanos et al., 2013), which was later found to be directly recruited by CEP164 (Cajanek and Nigg, 2014; Graser et al., 2007). Consistent with a role for distal appendage proteins in ciliogenesis, pathogenic CEP164 gene variants also lead to nephronophthisis (Chaki et al., 2012; Slaats et al., 2014) and SCLT1 variants have been identified in individuals with oral-facial-digital syndrome type IX (Adly et al., 2014). Interestingly, early ciliogenesis requires phosphorylation of CEP83 by TTBK2 (Lo et al., 2019), and pathogenic TTBK2 gene variants have been directly implicated in spinocerebellar ataxia type 11, which causes atrophy of Purkinje neurons in the cerebellum, loss of balance and slurred speech (Bowie et al., 2018).

Finally, in light of the recent findings demonstrating that centriolar satellites are required for removal of the centriolar capping protein CP110 and thus the promotion of ciliogenesis (Hall et al., 2023; Xie et al., 2023), it is intriguing to note that these centriolar satellites concentrate and assemble many proteins involved in ciliopathies (Lopes et al., 2011).

Primary ciliogenesis is an exquisitely complex process that involves hundreds of proteins and requires tight and highly nuanced regulation. Although there are many points at which control of ciliogenesis occurs, a key early step is the removal of the capping protein CP110 along with its interaction partner CEP97 from the mother centriole. As highlighted in this Review, in recent years, a number of new studies have provided potential mechanisms to explain how removal of CP110 is mediated. For the most part, such mechanisms, although diverse, are neither mutually exclusive nor incompatible with one another. At least three different E3 ligases have been implicated in ubiquitylation of the CP110–CEP97 capping complex: EDD1–DYRK2–DDB1^VPRBP, Cullin-KCTD and HERC2. These might function in concert to promote ubiquitylation and degradation of CP110 and CEP97. In addition, each or all of these E3 ubiquitin ligases could function in tandem with LUBAC, whereby CP110 and/or CEP97 might undergo K63 or linear mixed-chain ubiquitylation. The role of these various E3 ligases and the degree to which CP110 and CEP97 undergo linear versus K63 (or K48) ubiquitylation remains an important question in the field. Additional studies suggest that phosphorylation and removal of MPP9 by TTBK2 or competition between CEP97 and ENKD1 for binding to CP110 can destabilize the CP110–CEP97 complex, leading to removal of CP110. Importantly, the mechanisms involving ubiquitylation are not mutually exclusive with mechanisms that cause destabilization of CP110 and/or CEP97. Finally, although the recruitment of autophagosomes for removal and degradation of CP110 might appear incongruent with ubiquitin-based proteasomal degradation, it has been demonstrated that linear-ubiquitylated proteins can undergo both proteasomal and autophagic degradation (Dittmar and Winklhofer, 2019), further suggesting a potential connection between the various proposed mechanisms for removal of CP110. Whichever mechanism(s) are ultimately primarily responsible for removal of CP110 and its degradation, it is clear that ubiquitylation of CP110 is key to the process. Accordingly, an important future goal will be to elucidate how ubiquitylated CP110 is extracted from the mother centriole. An intriguing possibility is that the valosin-containing protein (VCP, also known as p97) AAA ATPase machinery might be required to extract CP110 from the mother centriole and unfold it prior to degradation by the proteasome. Initial support for involvement of VCP comes from a recent study implicating UBXN10, a ubiquitin-binding VCP adaptor protein, in ciliogenesis (Raman et al., 2015).

Overall, the regulation of removal of CP110 might require tightly coordinated control by multiple E3 ligases, some of which might be delivered by centriolar satellite trafficking. Moreover, proteins that are involved in CP110 destabilization on the mother centriole might similarly be delivered by centriolar satellites. Given the significance of CP110 in the progression of ciliogenesis, it would not be surprising to discover ciliopathies associated with CP110 variants, or variants in additional proteins that regulate removal of CP110. Indeed, it has been recently demonstrated that an amino acid substitution in EHD1, which is required for removal of CP110, causes developmental disorders, hearing loss and polycystic kidney disease, consistent with impaired ciliogenesis (Issler et al., 2022). Future studies will need to delineate the relationship between proteasomal degradation of CP110 at the mother centriole and autophagic removal of CP110. Finally, elucidating how all of these proposed mechanisms selectively target CP110 only at the mother centriole remains a crucial question to resolve.

We apologize to colleagues whose research was not cited due to space constraints. Figures were created using partial templates from BioRender.com (2023).