InteractiveFly: GeneBrief

Cep135: Biological Overview | References

BIOLOGICAL OVERVIEW

Cilia and flagella are involved in a variety of processes and human diseases, including ciliopathies and sterility. Their motility is often controlled by a central microtubule (MT) pair localized within the ciliary MT-based skeleton, the axoneme. This study characterized the formation of the motility apparatus in detail in Drosophila spermatogenesis. Assembly of the central MT pair starts prior to the meiotic divisions, with nucleation of a singlet MT within the basal body of a small cilium, and the second MT of the pair only assembles much later, upon flagella formation. BLD10/CEP135, a conserved player in centriole and flagella biogenesis, can bind and stabilize MTs and is required for the early steps of central MT pair formation. This work describes a genetically tractable system to study motile cilia formation and provides an explanation for BLD10/CEP135's role in assembling highly stable MT-based structures, such as motile axonemes and centrioles (Carvalho-Santos, 2012).

Cilia are microtubule (MT)-based organelles involved in a variety of processes, such as cell motility, fluid flow, and sensing mechanical stimuli and signaling molecules. At the base of each cilium there is at least one modified centriole, the basal body (BB), which templates the growth of the axoneme, the MT-based structure of cilia. Centrioles are also essential for the formation of centrosomes, the primary MT organizer of the cell (Bettencourt-Dias, 2011; Nigg, 2009). Cilia can exist as motile or immotile structures. Most motile cilia have a pair of central MTs within the lumen of their axoneme to coordinate motility. Defects in ciliary motility are associated with a variety of human disorders including infertility, respiratory problems, hydrocephalus and situs inversus, commonly found in patients with primary ciliary dyskinesia. Mice mutant for Hydin, a component of the central MT pair apparatus, show motility defects and develop hydrocephalus (Carvalho-Santos, 2012).

Much is known about axonemal components, mostly from work developed in the green algae. In fully assembled axonemes, the central MT pair starts at the most proximal part of the axoneme, a region called the transition zone. MT regulators are likely to control the assembly of the central MT pair: γ-tubulin and heterotrimeric kinesin-2 are required for this process in protozoa and sea urchin, respectively. However, little is known about the molecular mechanisms that govern the switch of centrioles to BBs and, in particular, when and how central MT pair assembly starts and is coordinated with other cellular processes (Carvalho-Santos, 2012).

Drosophila mutants for Bld10, a conserved player in centriole biogenesis, have short centrioles and the majority of their sperm flagella lack the central MT pair (Carvalho-Santos, 2010; Mottier-Pavie, 2009). In Chlamydomonas and Paramecium, BLD10/CEP135 localizes close to the centriolar MTs at the spoke tips of the cartwheel, a nine-fold symmetric structure present at the base of centrioles/BBs that enforces their symmetry (Hiraki, 2007; Jerka-Dziadosz, 2010; Matsuura, 2004). Depletion of BLD10/CEP135 in those organisms severely impairs BB assembly. In human cultured cells, BLD10/CEP135 is required for centriole assembly and localizes to the cartwheel, the centriolar walls, and the lumen of the centriole distal region (Kleylein-Sohn, 2007). Altogether, these data strongly indicate that BLD10/CEP135 has a MT-related function that underlies its role in centriole/BB assembly. Moreover, its localization in the centrioles but not in the axoneme in human and Drosophila suggests a role in the initiation of central MT pair assembly (Carvalho-Santos, 2012).

Drosophila spermatogenesis was used to understand how the central MT pair is assembled and the role played by BLD10/CEP135 in that process. One of the MTs of the pair is assembled prior to the meiotic divisions, within the basal body of a small cilium. This MT is maintained through meiosis, after which the flagellum and the second MT of the pair within it are formed. BLD10/CEP135 directly binds and stabilizes MTs and that in Bld10 mutants the singlet MT does not form and consequently the central MT pair is not assembled (Carvalho-Santos, 2012).

The central MT pair complex is an essential and highly specialized structure present in motile cilia that is required for coordinated motility. The morphological and molecular changes that occur during central MT pair assembly are yet to be characterized. Building on influential morphological work that described spermatogenesis in the fruit fly, this study has established Drosophila spermatogenesis as a genetically tractable system to study central MT pair formation. The assembly of this structure initiates prior to the meiotic divisions, much earlier than previously thought. During that stage, a singlet MT forms within the BB of a small cilium in G2 spermatocytes. This MT is likely very stable as it is present throughout meiotic divisions and until axoneme extension. In early spermatids, the stage at which flagellum assembly takes place, a second MT appears close to the singlet, and completes central MT pair formation. Bld10 emerged as an ideal candidate to regulate central MT pair formation as mutants lack this structure and the protein localizes to both the lumen and distal regions of the centriole/BB (Blachon, 2009; Carvalho-Santos, 2010; Mottier-Pavie, 2009). Accordingly, this study demonstrates that spermatogenesis in Bld10 mutants is carried out without assembly of the singlet MT, thus impacting on central MT pair biogenesis. Bld10 is a MAP whose overexpression leads to cytoplasmic MT stabilization in culture cells. Finally, this study directly links MT stabilizing activity of Bld10 to central MT pair assembly as (1) its N terminus, which binds and stabilizes MTs, contributes to this process, and (2) exposure to colchicine during central MT pair formation accentuates Bld10 mutant phenotype (Carvalho-Santos, 2012).

Morphological findings of central MT pair assembly during Drosophila spermatogenesis are summarized in Schematic Representation of Cilium and Flagellum Structures in Drosophila melanogaster Spermatogenesis (Figure 7). The identification and analysis of different stages of this process using TEM single and serial sections, and tomography, suggest this assembly is much more dynamic than was anticipated. It is proposed that central MT pair biogenesis starts with the formation of a singlet MT within the lumen of the BB. The detailed analysis of this process raises several important questions, including where the first singlet MT is nucleated from, and whether it actively participates in the assembly of the second MT of the pair. The identification of singlet MT or central MT pair markers that do not localize to other BB and axoneme structures will allow further mechanistic studies of central MT pair assembly in a less laborious fashion than as it is with electron microscopy (Carvalho-Santos, 2012).

The observation that a singlet MT forms within the BB as a precursor for the biogenesis of the central MT pair of the motile axoneme, implies a broader role for the BB in templating cilia than currently thought. This raises an important question on the significance and conservation of this process. Until now, the study of this process using stills of TEM sections has prevented the discovery and characterization of intermediate steps. Drosophila spermatogenesis proved to be a valuable system to study these fast intermediate stages since central pair assembly takes a few days to be completed. It is proposed that the presence of the singlet MT is an intermediate state until the second MT of the pair is nucleated. In mature sperm both MTs have equal length and therefore is not obvious that one MT was assembled first. In model organisms often used to study flagella assembly, such intermediate stages might have not been observed due to their transitory nature. In Nephrotoma suturalis, an insect that is phylogenetically close to Drosophila melanogaster, a singlet MT has also been observed (LaFountain, 1976). The presence of stages where a singlet MT is easily found can be a consequence of a slower central MT pair assembly in these insects (Carvalho-Santos, 2012).

It is intriguing that the central MT pair starts to assemble so early within the BB. An extensive literature search was conducted for ultrastructural studies on sperm flagella assembly in other species to address whether this was a conserved phenomenon. Surprisingly, in both vertebrate and invertebrate species, central MT pairs have been observed in cilia/flagella that assemble during spermatocyte stages, before meiosis I or II . Why the central MT pair assembles at that stage, and in particular whether spermatocyte cilia motility is needed for spermatogenesis, is an important question that deserves further study. It is still unclear whether in these different species the central MT pairs found in spermatocyte cilia/flagella are precursors of the central MT pair of sperm flagella, as is describe in this study for Drosophila (Carvalho-Santos, 2012).

Although much is known about the components of the central MT pair machinery, little is understood about the molecular regulation of its nucleation. In light of the current findings, BLD10/CEP135 is proposed to regulate the initiation of central MT pair biogenesis through MT stabilization. Bld10 conserved N-terminal domain is proposed to play an important role in this process; however, given that removal of that domain does not completely abolish Bld10 activity, it cannot be excluded that other Bld10 protein regions also stabilize the central MT pair. It is common for MAPs to have different MT binding domains. Since the M- and C-Bld10 truncations localize independently to centrioles, it is possible they also stabilize MTs associated with the centriole and the central MT pair, a hypothesis that should be investigated in the future (Carvalho-Santos, 2012).

How general is BLD10/CEP135 MT-stabilizing function? It is possible that BLD10/CEP135 ancestral function is stabilizing special sets of MTs including the centriole triplets and the singlet MT during axoneme central MT pair assembly. BLD10/CEP135 is present in the genome of most organisms that assemble centrioles and flagella but absent from those that lack these organelles, such as higher plants. While a role for BLD10/CEP135 in central MT pair assembly has not been investigated in vertebrates, TSGA10, a BLD10/CEP135 paralog only present in vertebrates (Carvalho-Santos, 2010), localizes to the sperm flagella and has been linked to male sterility, further corroborating a role for this family of proteins in flagella assembly (Modarressi, 2001). BLD10/CEP135 loss-of-function in several species generates phenotypes associated with centriolar MT defects, including MT triplet loss (Hiraki, 2007; Jerka-Dziadosz, 2010; Matsuura, 2004), and shorter centrioles (Carvalho-Santos, 2010; Mottier-Pavie, 2009). The link between Bld10 and stabilization of these specific MT sets is also supported by its localization to the cartwheel spokes and BB/centriolar MT triplets (in Chlamydomonas, human cells and Drosophila) (Hiraki, 2007; Kleylein-Sohn, 2007; Matsuura, 2004; Mottier-Pavie, 2009). The complete lack of BBs in Chlamydomonas BLD10 mutants might reflect defects both in cartwheel assembly and in the recruitment and/or stabilization of the BB MTs onto the cartwheel (Matsuura, 2004). This is not the case in Drosophila, as centrioles, albeit shorter, are observed in Bld10 mutants (Carvalho-Santos, 2010; Mottier-Pavie, 2009). It is possible that, in the fruit fly, other molecules are redundant with Bld10 in its role of stabilizing cartwheel-associated MTs. In the future, it will be important to understand how Bld10 might stabilize MTs, how its function is regulated in different centriole compartments (cartwheel, lumen, walls) and at different time points, such as during centriole or central MT pair assembly (Carvalho-Santos, 2012).

Centrioles and axonemes are very special organelles, being much more stable than any other MT-based structures (Bettencourt-Dias and Glover, 2007). They not only withstand complex MT remodeling environments but in the case of centrioles they also perdure for several cell generations. Additionally, the assembly and stabilization of centrioles and axonemes involves particular MT regulators and posttranslational modifications (Bettencourt-Dias and Glover, 2007). This study has shown that BLD10/CEP135 is a special MT regulator specifically involved in the formation of highly stable and specialized MTs. The discovery of these specialized MAPs, where SAS4/CPAP could also be included, opens the door to an exciting new biology of MT regulation. Moreover, given the importance of centrioles and cilia in development and homeostasis, this work will allow further contextualization of these cellular structures in human disease (Carvalho-Santos, 2012).

Drosophila Cep135/Bld10 maintains proper centriole structure but is dispensable for cartwheel formation

Cep135/Bld10 is a conserved centriolar protein required for the formation of the central cartwheel, an early intermediate in centriole assembly. Surprisingly, Cep135/Bld10 is not essential for centriole duplication in Drosophila suggesting that either Cep135/Bld10 is not essential for cartwheel formation, or that the cartwheel is not essential for centriole assembly in flies. Using Electron Tomography and superresolution microscopy this study shows that centrioles can form a cartwheel in the absence of Cep135/Bld10, but centriole width is increased and the cartwheel appears to disassemble over time. Using 3D structured illumination microscopy it was shown that Cep135/Bld10 is localised to a region between inner (SAS-6, Ana2) and outer (Asl, DSpd-2 and D-PLP) centriolar components, and the localisation of all these components is subtly perturbed in the absence of Cep135/Bld10, although the 9-fold symmetry of the centriole is maintained. Thus, in flies, Cep135/Bld10 is not essential for cartwheel assembly or for establishing the 9-fold symmetry of centrioles; rather, it appears to stabilise the connection between inner and outer centriole components (Roque, 2012).

These results suggest that, unlike in Chlamydomonas and Paramecium, Cep135/Bld10 is not essential for cartwheel formation in Drosophila. Instead, Cep135/Bld10 appears to function to stabilise the cartwheel and the interaction between the cartwheel and the more outer regions of the centrioles. In the absence of Cep135/Bld10 cartwheels are initially formed relatively normally but they then appear to deteriorate over time. Cartwheels are often displaced from the centre of the centriole, the diameter of the centriole is increased, and the localisation of both inner and outer centriolar components is subtly perturbed. These phenotypes are in good agreement with localisation studies that show Cep135/Bld10 localising to a region between the inner and outer regions of the centriole (Roque, 2012).

It has previously been shown that Drosophila Cep135/Bld10 is essential for the formation of the central pair of MTs in the flagellar axoneme, and this study shows that it is also essential for the formation of a central tube structure that often extends into the short cilia formed in mature primary spermatocytes. This central tube could be a MT, although 3D-SIM analyses indicates that the cartwheel components DSas-6 and Ana2 localise to this structure. An important caveat to this result is that both of these proteins are overexpressed in these experiments compared to the endogenous proteins, so they could be binding to this structure when they would not normally do so. Nevertheless, it seems reasonable to conclude that this structure is either not a MT (and is in some way related to the central tube of the cartwheel), or is a specialised MT that is capable of binding overexpressed DSas-6 and Ana2 (while other MTs in the centriole do not). How the formation of the central tube in these centrioles and cilia might relate to the formation of the central pair of MTs in the flagellar axoneme is unclear, but it is intriguing that the formation of both structures requires Cep135/Bld10. Perhaps the central tube present in the centrioles is in some way required for the eventual growth of the central pair of MTs in the flagellar axoneme (Roque, 2012).

The centriolar protein Bld10/Cep135 is required to establish centrosome asymmetry in Drosophila neuroblasts

Centrosome asymmetry has been implicated in stem cell fate maintenance in both flies and vertebrates and Drosophila neuroblasts, the neural precursors of the fly's central nervous system, contain molecularly and physically asymmetric centrosomes, established through differences in pericentriolar matrix (PCM) retention. For instance, the daughter centriole maintains PCM and thus microtubule-organizing center (MTOC) activity through Polo-mediated phosphorylation of Centrobin (Cnb). The mother centriole, however, quickly downregulates PCM and moves away from the apical cortex, randomly migrating through the cytoplasm until maturation sets in at prophase. How PCM downregulation is molecularly controlled is currently unknown, but it involves Pericentrin (PCNT)-like protein (plp) to prevent premature Polo localization and thus MTOC activity. This study reports that the centriolar protein bld10, the fly ortholog of Cep135, is required to establish centrosome asymmetry in Drosophila neuroblasts through shedding of Polo from the mother centrosome. bld10 mutants fail to downregulate Polo and PCM, generating two active, improperly positioned MTOCs. Failure to shed Polo and PCM causes spindle alignment and centrosome segregation defects, resulting in neuroblasts incorrectly retaining the older mother centrosome. Since Cep135 is implicated in primary microcephaly, it is speculated that perturbed centrosome asymmetry could contribute to this rare neurodevelopmental disease (Singh, 2014).

In a gene candidate approach to identify molecules required for centrosome asymmetry in Drosophila neuroblasts, this study identified bld10/Cep135 as a potential centrosome dematuration regulator. bld10 is a ubiquitous centriolar protein, localizing to centrioles in Drosophila larval neuroblasts and other cell types. To investigate centrosome asymmetry, live imaging experiments were performed in intact third-instar larval brains, labeling centrosomes with the centriolar markers DSas6::GFP or DSas4::GFP and mCherry::Jupiter. jupiter encodes for a microtubule binding protein, sharing properties with several structural microtubule-associated proteins (MAPs), and is ideally suited to visualize microtubule dynamics and microtubule-organizing center (MTOC) activity. In agreement with previous findings, it was found that wild-type (WT) interphase neuroblasts contained one apical MTOC only. The second MTOC appeared during prophase in close proximity to the basal cortex. By prometaphase, both MTOCs reached maximal activity and intensity. However, in bld10 mutant interphase neuroblasts (bld10^c04199/Df(3L)Brd15, two centrosomes of similar size and MTOC activity were observed close together on the apical cortex. The two centrosomes progressively separated from each other until they reached their respective positions on the apical and basal cortex by prometaphase. Thus, in contrast to the wild-type, bld10 mutant neuroblasts show symmetric centrosome behavior. bld10's centrosome asymmetry defect could be rescued with bld10::GFP, and immunohistochemistry experiments confirmed the live imaging results (Singh, 2014).

The bld10^c04199 allele is predicted to produce a truncated protein, retaining bld10's N terminus. A new N-terminal deletion allele (bld10ΔN was generated that showed the same centrosome asymmetry phenotype. In addition, neuroblasts were found containing monopolar and multipolar spindles, which are not observed with the bld10^c04199 allele. This suggests that bld10^c04199 is a separation-of-function allele, specifically disrupting centrosome asymmetry. Unless otherwise noted, all of the experiments described in the following sections were performed with the bld10^c04199 allele (Singh, 2014).

The lack of centrosome asymmetry in bld10 mutant neuroblasts could be due to aberrant centriole migration. For example, the mother centriole could either fail to migrate through the cytoplasm or migrates back to the apical cortex to mature. This hypothesis was tested, measuring centriole migration as a function of time, and it was observed that centriolar migration in wild-type and bld10 mutant neuroblasts occur in two distinct phases: (1) centrioles steadily separated from each other, followed by (2) a sudden increase in intercentriolar distance, which peaked when centrioles reached a separation distance of ~4-6 μm in the wild-type and bld10 mutants. Centrioles in bld10 mutants did not require more time to reach this threshold distance and did not return to the apical cortex to mature. It is concluded that bld10's centrosome asymmetry defect is not due to aberrant centriole migration (Singh, 2014).

To get mechanistic insight into the bld10 phenotype, live imaging was used to measure the dynamic localization of three GFP-tagged pericentriolar matrix (PCM) markers: γ-tubulin (γ-Tub), Mini spindles (Msps; CKAP5 in vertebrates) and centrosomin (Cnn; CDK5Rap2 in vertebrates). Wild-type neuroblasts showed robust localization of γ-Tub, Msps, and Cnn to the apical centrosome during interphase. After centrosome splitting, all three PCM markers were downregulated from the basal centrosome (shedding phase) but reaccumulated during prophase (maturation phase. bld10 mutant neuroblasts also correctly localized γ-Tub, Msps, and Cnn to the apical interphase centrosome. However, similar to the MTOC marker Jupiter, γ-Tub, Msps, and Cnn were not downregulated from the separating centriole. Centrosome size was measured and a centrosome asymmetry index was plotted, starting at centrosome splitting until metaphase. Wild-type centrosomes developed a clear size asymmetry during the shedding phase and reduced it during the maturation phase. bld10 mutant centrosomes stayed similar in size, manifested in an asymmetry index below 1.5. Centrosome size and intensity measurements also revealed that in most wild-type neuroblasts, γ-Tub, Msps, and Cnn were removed from the basal centrosome ~15 min after centrosome splitting. Basal wild-type centrosomes were essentially devoid of γ-Tub after that time, whereas bld10 mutants contained equal amounts of this PCM marker. Changes in centrosome size were further compared, and it was found that wild-type apical centrosomes predominantly grew, whereas basal centrosomes increased (maturation phase) and decreased (shedding phase) their size to almost the same extent. bld10 mutant centrosomes were able to enlarge but showed very little size reduction, comparable to apical wild-type centrosomes. It is concluded that bld10 mutant centrosomes are able to mature but fail to downregulate the PCM markers γ-Tub, Msps, and Cnn (Singh, 2014).

The results suggest two possible mechanisms for centrosome asymmetry: (1) bld10 could prevent premature mother centrosome maturation by blocking the precocious accumulation of PCM proteins. (2) Alternatively, bld10 could promote PCM shedding right after centrosomes separate, thereby preventing the basal mother centrosome to prematurely become an MTOC. An in vivo pulse-chase labeling experiment was devised to distinguish between these two possibilities. To this end, Cnn at its endogenous locus was tagged with the photoconvertable fluorescent protein mDendra2. If bld10 blocks premature PCM accumulation, mother centrioles should quickly shed photoconverted Cnn and prematurely reaccumulate unconverted Cnn in bld10 mutants. Vice versa, if PCM shedding is compromised, it should be possible to follow the photoconverted centrosomes from the moment they separate until telophase. It was found that apical wild-type daughter centrioles retained the majority of photoconverted Cnn::mDendra2 from early interphase until prophase (possibly longer), indicating that very little Cnn protein gets exchanged. The basal mother centriole, however, lost photoconverted Cnn::mDendra2 within approximately 10-15 min after centriole separation, confirming that Cnn is shed quickly. Interestingly, bld10 centrioles retained photoconverted Cnn::mDendra2 for at least 45 min after separation. In some cases, one of the centrioles decorated with photoconverted Cnn::mDendra2 was even inherited by the newly formed GMC. It is conclude that (1) on the apical centrosome, Cnn protein turnover is absent or significantly reduced during interphase, that (2) on the basal centrosome, Cnn is shed quickly and replaced with new Cnn when maturation sets in, and that (3) bld10's centrosome asymmetry defect is not due to premature centrosome maturation. Instead, separating basal centrioles fail to shed Cnn in particular and possibly PCM proteins in general (Singh, 2014).

To elucidate the molecular mechanism underlying PCM shedding, the relationship was analyzed between bld10, Centrobin (Cnb), and Pericentrin (PCNT)-like protein (plp). Recently, it was shown that Cnb is necessary and sufficient for PCM retention on the apical daughter neuroblast centrosome. However, gain- and loss-of-function experiments with Cnb did not perturb bld10's localization. Similarly, as in the wild-type, Cnb was localized asymmetrically in bld10 mutants. plp mutants fail to downregulate γ-Tub on the mother centrosome. In plp mutant neuroblasts, the basal centrosome retained Cnn and MTOC activity during interphase. Interestingly, photoconversion experiments showed that similar to bld10, Cnn shedding from the basal centrosome was compromised in plp mutants. However, plp localization is not perturbed in bld10 mutant neuroblasts, and bld10 was normally localized in plp mutants. Knockdown of plp in bld10 mutants did not enhance bld10 PCM shedding phenotype, but due to the occurrence of additional phenotypes (fragmented or multiple centrosomes), the shedding phenotype could also be partially masked. In sum, it is concluded that bld10 is regulating centrosome asymmetry independently of Cnb and that plp is also required to shed Cnn (Singh, 2014).

Since the mitotic kinase Polo has been implicated in PCM retention during interphase, Polo localization dynamics were analyzed in wild-type and bld10 mutant neuroblasts. Recently, it was reported that Polo localizes to the apical centrosome during interphase and is only detectable at the basal centrosome during prophase, when maturation sets in. A Polo::GFP protein trap line was used and it was confirmed that Polo is stably localized to the apical interphase centrosome. Surprisingly, weak Polo was also found on the separating mother centrosome. Subsequently, Polo disappeared from the basal mother centriole within 10 min, comparable to Cnn, γ-Tub, and Msps shedding times. With a genomic Polo::GFP transgene, showing lower fluorescence intensity, Polo was found to be localized on both centrosomes in bld10 mutants. These data suggest that in wild-type neuroblasts, Polo is not just recruited onto the basal mother centrosome by prophase as previously reported, but is also subject to shedding during interphase. Polo is required for PCM retention since in bld10 mutant neuroblasts treated with the Polo inhibitor BI2536, both centrosomes lose MTOC activity. Thus, it is concluded that shedding of Polo is a requirement for the subsequent shedding of Cnn, γ-Tub, and Msps, enabling basal mother centrosome dematuration and the establishment of centrosome asymmetry (Singh, 2014).

Finally, the consequences were analyzed of disrupted centrosome asymmetry. The daughter centriole was labeled with Cnb::YFP ] and centrosome segregation was assayed. It was confirmed that wild-type neuroblasts faithfully retain the Cnb* daughter centriole, whereas the Cnb- centrosome segregates into the GMC. bld10 mutants showed correct asymmetric Cnb localization, but ~45% of bld10 mutant neuroblasts wrongly retained the mother centriole and segregated the daughter centriole into the GMC. Cnb+ centrosomes are usually bigger in wild-type and bld10 mutant neuroblasts, but centrosome segregation is independent of MTOC activity and size since bld10 mutant neuroblasts often retained the smaller centrosome. It is concluded that centrosome asymmetry is required for faithful centrosome segregation (Singh, 2014).

Since bld10 mutant neuroblasts have mispositioned MTOCs in relation to the apical-basal division axis, spindle orientation was analyzed. Immunohistochemistry experiments showed that bld10 mutant neuroblast spindles deviate from the regular orientation range, with isolated cases of extreme misalignment. Metaphase spindles, aligned orthogonally to the apical-basal polarity axis, can induce symmetric neuroblast divisions, resulting in an increase of the neural stem cell pool. However, neuroblast numbers were unchanged in bld10 mutants compared to control brains, and symmetric neuroblast divisions were not found with live imaging. Instead, time-lapse experiments showed that bld10 mutant centrosomes prematurely formed misaligned bipolar spindles. Spindle rotation during metaphase corrected this misalignment. Apical-basal polarity is a prerequisite for correct spindle orientation, but apical and basal polarity markers localized normally in bld10 mutants. Similarly, the spindle orientation regulators, Partner of Inscuteable (Pins; LGN/AGS3 in vertebrates) and the NuMA ortholog Mud, were also correctly localized. It is concluded that controlled PCM shedding and maturation is required for correct centrosome positioning but backup mechanisms exist, correcting for misaligned metaphase spindles (Singh, 2014).

Many cell types, including stem and progenitor cells, contain asymmetric centrosomes and segregate them nonrandomly, suggesting a connection between centrosome asymmetry and cell fate. How centrosome asymmetry is regulated is currently not understood, but centrosome dematuration is a critical step in establishing centrosome asymmetry. It was found that the centriolar protein bld10/Cep135, known as a centriole duplication and elongation factor, is required to establish centrosome asymmetry. On the basis of these data, it is proposed that plp and bld10 induce Polo's removal from the mother centriole, triggering the shedding of PCM proteins such as Cnn, γ-Tub, and Msps. Polo has been reported to be closely associated with centrioles, ideally positioned to phosphorylate both centriolar and PCM proteins. Thus, it is proposed that Polo-mediated phosphorylation of PCM proteins maintains a stable interaction between the centriole and surrounding PCM (Singh, 2014).

How Polo localization is regulated is currently not known, and no direct molecular interaction was detected between bld10 and Polo. Although no centriolar markers were found to be mislocalized inbld10 mutants (at the resolution level of confocal microscopy), it is possible that structural centriole defects, as detected in bld10 mutant spermatocytes and wing disc cells, could affect PCM turnover rates. However, since bld10 is not asymmetrically localized, it is difficult to conceive how such defects specifically compromise the behavior of the mother but not the daughter centrosome (Singh, 2014).

Although perturbed centrosome asymmetry does not seem to undermine neuroblast polarity, the cell cycle, or physical and molecular asymmetric cell division, the possibility cannot be excluded that centrosome asymmetry could have long-term consequences currently beyond the ability to detect. Interestingly, defects in centrosome maturation or mutations in Cep135 can cause neurodevelopmental disorders such as primary microcephaly. It will be interesting to address the question whether lack of Cep135 is causing microcephaly due to compromised centrosome asymmetry and dematuration (Singh, 2014).

Superresolution characterization of core centriole architecture

The centrosome is the main microtubule-organizing center in animal cells. It comprises of two centrioles and the surrounding pericentriolar material. Protein organization at the outer layer of the centriole and outward has been studied extensively; however, an overall picture of the protein architecture at the centriole core has been missing. This paper reports a direct view of Drosophila centriolar proteins at ~50-nm resolution. This reveals a Sas6 ring at the C-terminus, where it overlaps with the C-terminus of Cep135. The ninefold symmetrical pattern of Cep135 is further conveyed through Ana1-Asterless axes that extend past the microtubule wall from between the blades. Ana3 and Rcd4, whose termini are close to Cep135, are arranged in ninefold symmetry that does not match the above axes. During centriole biogenesis, Ana3 and Rcd4 are sequentially loaded on the newly formed centriole and are required for centriole-to-centrosome conversion through recruiting the Cep135-Ana1-Asterless complex. Together, these results provide a spatiotemporal map of the centriole core and implications of how the structure might be built (Tian, 2021).

The centrosome has multiple crucial functions, including the assembly of the mitotic spindle and establishing the axis of cell division. It comprises two principal components: a pair of orthogonally arranged centrioles and the surrounding pericentriolar material (PCM). Centrioles are stable cylindrical structures comprising nine microtubule blades arranged at the end of nine spokes that radiate from a central hub. During each cell cycle, the centriole pair disengages at the mitotic exit, allowing the new centrioles (or daughter centrioles) to gradually assemble next to each preexisting centriole (the mother centriole). A mother centriole serves as a recruitment and assembly scaffold for the PCM proteins to form spindle poles in mitosis; in many cell types, it also provides a template for cilium or flagellum assembly during cell quiescence, forming a crucial organelle for chemical sensation, signal transduction, locomotion, and so forth. Centrosome defects have been related to a wide range of human diseases, including cancer, microcephaly, and a group of disorders collectively known as the 'ciliopathies' (Tian, 2021).

Understanding how the centrosome functions requires knowledge of its protein composition and organization. The centrosome is composed of >100 different proteins. Their architectural arrangement has begun to be systematically examined since the application of superresolution microscopy. Using 3D structured illumination microscopy (3D-SIM), distinct concentric domains within a centrosome have been documented (e.g., zones I-V of the Drosophila centrosome) and that the PCM has a conserved, ordered structure. Protein organization at several compartments of the centrosome, such as the distal and subdistal appendages, the transition zone, the centrosome linker, and the longitudinal axis of the centriole, has also been studied via 3D-SIM, stimulated emission depletion (STED) microscopy, or stochastic optical reconstruction microscopy. Meanwhile, proteins at the core of the centriole remain largely unresolved. This cartwheel region, revealed as zone I by 3D-SIM, contains the central hub of ~22-nm diameter and the nine spokes that determine the ninefold symmetrical feature of the centriole (Tian, 2021).

Drosophila cultured cells present a consistent model for the study of the centriole core because, contrary to the vertebrate centrosome, the cartwheel persists in the mature centriole. The centriole is composed of doublet microtubules arranged in a ninefold symmetrical cylinder, which is ∼200 nm wide and long and has a cartwheel formation along the entire length. This study first determined which proteins known to be required for Drosophila centriole duplication are the components of the centriole core. A direct view of these proteins is presented at ~50-nm resolution, and a timing order of their assembly is presented using several superresolution techniques. These revealed a ninefold radial scaffold comprising Spindle assembly abnormal 6 (Sas6), Centrosomal protein 135kDa (Cep135), Anastral spindle 1 (Ana1), and Asterless (Asl), as well as concentric toroids formed by Anastral spindle 3 (Ana3) and Reduction in Cnn dots 4 (Rcd4), two novel core centriolar components that are also organized in ninefold symmetry. During centriole biogenesis, Ana3 is recruited to the newly formed daughter centriole later than Sas6 but before Rcd4 and Cep135. These findings thus provide a spatiotemporal map of the centriole core and a model of how the proteins might interact to build the structure (Tian, 2021).

These data reveal the spatiotemporal organization of the proteins at the core region of the Drosophila centriole (see Schematics depicting the lateral organization of centriole core). By superimposing the current measurements to the electron cryotomography data of the Trichonympha, Chlamydomonas, and Drosophila centrioles, this study found that Cep135 overlaps with the C-terminus of Sas6 on the spokes via its C-terminus and extends to the pinheads via the N-terminus. Ana1 localizes from the pinheads to the outer edge of the doublet microtubules. Asl slightly overlaps with the doublet microtubules and extends into PCM in a ninefold manner. It is proposed that the core region of the centriole is composed of two dimensions. One is the ninefold radial dimension that is established by elongated molecules overlapping through their adjacent termini: Sas6, Cep135, Ana1, and Asl. They likely constitute the spoke-pinhead axes and further transmit the ninefold symmetrical geometry to the microtubule wall and into the core PCM. The other is a circular dimension established by a group of compact proteins that are also arranged in ninefold symmetry: Ana3, Rcd4, and possibly Ana2. They likely decorate the radial axes and provide the physical support for the ninefold configuration (Tian, 2021).

Previous work has shown that Cep135, Ana1, and Asl form a complex that is responsible for the centriole-to-centrosome conversion), the final stage in the assembly of the daughter centriole that converts it into a mother centriole able to duplicate. With improved spatial resolution, this study shows that the three proteins are each organized in ninefold manner, reinforcing the idea they are the bona fide components of the spoke-pinhead scaffold. The ninefold radial axes then extend past the centriole microtubule wall via the C-terminus of Ana1, which is positioned between the microtubule blades. Recently, an electron cryotomography study showed that, between adjacent microtubule blades, there are ninefold amorphous brushlike structures in the Drosophila S2 centriole. This study suggests that it could contain Ana1 and Asl, both of which exhibit ninefold symmetry at this region (Tian, 2021).

These findings allocate a role to Drosophila Ana3 and Rcd4, previously known from genome-wide RNAi screens to be required for centriole duplication. Ana3 was later reported to be responsible for the structural integrity of centrioles and basal bodies and for centriole cohesion in the Drosophila testes. This study now provides evidence that both Ana3 and Rcd4 are core centriolar components, localizing to the region where Cep135 is. The N-terminus of Ana3 localizes closest to the center of the centriole, followed by the C-termini of Ana3 and Rcd4 and the N-terminus of Rcd4. Both Ana3 and Rcd4 are organized in ninefold symmetry but seem to be positioned in axes that are not in line with the Cep135-Ana1-Asl complex. Spatial overlapping of Ana3 and Rcd4 indicates these two proteins might interact, which has recently been reported (Panda, 2020) and is conserved to their human counterparts, RTTN and PPP1R35. Depletion of either Ana3 or Rcd4 leads to failure in loading the Cep135-Ana1-Asl complex during centriole biogenesis and thus causes defects in centriole-to-centrosome conversion and the reduction of the centrosome number. This pathway is also conserved in human cells, where PPP1R35 was reported to promote centriole-to-centrosome conversion upstream of Cep295 (human homologue of Ana1) and RTTN and PPP1R35 serve as upstream effectors of Cep295 in mediating centriole elongation (Tian, 2021).

Taken together, these data provide an overall picture of the protein architecture at the centriole core and implications of how the ninefold symmetrical structure might be built. Knowing the spatiotemporal restraints of individual centriolar components will guide the immediate study of the molecular interaction partners and understanding of their functions. Meanwhile, it would also provide information for a higher-resolution approach, including cryo-EM, to eventually obtain a 3D map of the centriole (Tian, 2021).

A proximal centriole-like structure is present in Drosophila spermatids and can serve as a model to study centriole duplication

Most animals have two centrioles in spermatids (the distal and proximal centrioles), but insect spermatids seem to contain only one centriole, which functionally resembles the distal centriole. Using fluorescent centriolar markers, this study has identified a structure near the fly distal centriole that is reminiscent of a proximal centriole (i.e., proximal centriole-like, or PCL). The PCL exhibits several features of daughter centrioles. First, a single PCL forms near the proximal segment of the older centriole. Second, the centriolar proteins SAS-6, Ana1, and Bld10p/Cep135 are in the PCL. Third, PCL formation depends on SAK/PLK4 and SAS-6. Using a genetic screen for PCL defect, a mutation was identifed in the gene encoding the conserved centriolar protein POC1, which is part of the daughter centriole initiation site in Tetrahymena. It is conclude that the PCL resembles an early intermediate structure of a forming centriole, which may explain why no typical centriolar structure is observed under electron microscopy. It is proposed that, during the evolution of insects, the proximal centriole was simplified by eliminating the later steps in centriole assembly. The PCL may provide a unique model to study early steps of centriole formation (Blachon, 2009).

Stepwise evolution of the centriole-assembly pathway

The centriole and basal body (CBB) structure nucleates cilia and flagella, and is an essential component of the centrosome, underlying eukaryotic microtubule-based motility, cell division and polarity. In recent years, components of the CBB-assembly machinery have been identified, but little is known about their regulation and evolution. Given the diversity of cellular contexts encountered in eukaryotes, but the remarkable conservation of CBB morphology, it was asked whether general mechanistic principles could explain CBB assembly. The distribution of each component of the human CBB-assembly machinery was analyzed across eukaryotes as a strategy to generate testable hypotheses. It was found an evolutionarily cohesive and ancestral module, which was termed UNIMOD, is defined by three components (SAS6, SAS4/CPAP and BLD10/CEP135), that correlates with the occurrence of CBBs. Unexpectedly, other players (SAK/PLK4, SPD2/CEP192 and CP110) emerged in a taxon-specific manner. Gene duplication plays an important role in the evolution of CBB components, and, in the case of BLD10/CEP135, this is a source of tissue specificity in CBB and flagella biogenesis. Moreover, extreme protein divergence was observed among CBB components, and it was shown experimentally that there is loss of cross-species complementation among SAK/PLK4 family members, suggesting species-specific adaptations in CBB assembly. It is proposed that the UNIMOD theory explains the conservation of CBB architecture and that taxon- and tissue-specific molecular innovations, gained through emergence, duplication and divergence, play important roles in coordinating CBB biogenesis and function in different cellular contexts (Carvalho-Santos, 2010).

The conservation of the morphology of the CBB structure contrasts with the diversity of contexts in which it assembles and operates in eukaryotic life. Focusing on the phylogenetic distribution of six proteins essential for centriole assembly in humans, it was found that, in contrast to the previously observed conservation of ciliary and flagella components, CBB-assembly mechanisms evolved in a stepwise fashion. It is proposed that a subset of these proteins, which belong to what is called the universal module (UNIMOD), are necessary to define the CBB structure: the ninefold symmetry and the recruitment and tethering of centriolar microtubules. These proteins have a similar phylogenetic distribution to that previously observed for ciliary and flagella components, and it is likely that new centriole components, such as POC1, will also fall into this subset. Furthermore, the set of proteins needed to form a centriole is likely to be larger than the UNIMOD, including proteins that also have non-centriolar functions and are present in organisms that do not have CBBs, such as α- and β-tubulins and centrin. Mechanisms such as duplication with subfunctionalization of ancestral components (e.g., PLK and the BLD10/CEP135 families), divergence (e.g., SAK/PLK4) and the emergence of new genes (e.g., SPD2/CEP192 and CP110) play important roles in the evolution of CBB biogenesis. It was have shown experimentally that subfunctionalization might have played a role in CBB evolution at least twice. In the case of BLD10/CEP135, duplication and subfunctionalization with the generation of TSGA10 is likely to be important in the development of tissue-specific mechanisms of CBB assembly and flagella formation. In the case of the PLK family, the appearance of SAK/PLK4 with subfunctionalization is likely to play a role in uncoupling the regulation of CBB biogenesis from other cell-cycle events performed by PLKs. It was also shown experimentally that divergence in the PLK4 family leads to loss of cross-species complementation, which might create conditions for further development of species-specific regulation of CBB-assembly mechanisms. Finally, the emergence of novel molecules might have allowed adaptation to new contexts of assembly and new functions of the structure. The appearance in unikonts of SPD2/CEP192, a molecule whose ancestral function is thought to be in PCM recruitment, might have permitted, in animals, CBB biogenesis in contexts in which there is less PCM, such as duplication of the basal body upon fertilization. In animals, CP110 might have coupled the assembly of CBBs to the acquisition of new functions, such as cilia assembly and cytokinesis. Overall, these results strongly support the notion that the molecular machinery that defines the CBB structure is an innovation that emerged in the last eukaryotic common ancestor. This structure evolved through the emergence and divergence of new components that adapted CBB biogenesis and function to the diversification of subcellular contexts and tissue types in which they assemble and function (Carvalho-Santos, 2010).

In its evolutionary mechanisms, the CBB machinery is similar to multiprotein complexes and protein-trafficking pathways. In the former, a conserved core that presumably defines the basic function of the complex can acquire tissue- and organism-specific functions by duplication and specialization of specific components, as well as recruitment of novel interactions. The observation of heterogeneous phylogenetic distributions revealed extensive species-specific adaptations, which suggests that this study has uncovered an approach to identify novel CBB biogenesis players and functions using phylogenetic profiling. It was shown, for example, that both CP110 and CEP97, which are biochemical partners, appeared in animals. This study reveals that it is possible to extend the predictive power of evolutionary-based approaches by considering phylogenetic distributions of genes together with biological structures, and that this will be helpful in predicting both protein functions and interactions. In the future, it will be important to increase the repertoire of species whose genome is sequenced and to thoroughly describe the morphology and function of their CBBs (Carvalho-Santos, 2010).

It was surprising to observe species in which CBBs have not been described, but whose genomes contain SAS6 and SAS4: the algae Ostreococcus and the microsporidiae Encephalitozoon cuniculi and Enterocytozoon bienusi. The Ostreococcus genome also encodes orthologs of axonemal dyneins and other centriolar proteins, such as POC1. However, many flagella components are missing from the Ostreococcus genome. It is proposed that this organism might have an elusive CBB remnant, with no associated flagella, such as that described in the non-flagellated, non-sequenced green algae Kirchneriella. The significance of the presence of these proteins, although severely truncated, in the highly reduced genomes of microsporidial intracellular parasites remains to be determined. Further cell biology research in these enigmatic organisms should reveal mechanisms coupling the loss of cellular structures to the evolution of their molecular assembly machinery or alternatively unveil other functions exhibited by these proteins (Carvalho-Santos, 2010).

Drosophila bld10 is a centriolar protein that regulates centriole, basal body, and motile cilium assembly

Cilia and flagella play multiple essential roles in animal development and cell physiology. Defective cilium assembly or motility represents the etiological basis for a growing number of human diseases. Therefore, how cilia and flagella assemble and the processes that drive motility are essential for understanding these diseases. This study shows that Drosophila Bld10, the ortholog of Chlamydomonas reinhardtii Bld10p and human Cep135, is a ubiquitous centriolar protein that also localizes to the spermatid basal body. Mutants that lack Bld10 assemble centrioles and form functional centrosomes, but centrioles and spermatid basal bodies are short in length. bld10 mutant flies are viable but male sterile, producing immotile sperm whose axonemes are deficient in the central pair of microtubules. These results show that Drosophila Bld10 is required for centriole and axoneme assembly to confer cilium motility (Mottier-Pavie, 2009).

Bld10/Cep135 stabilizes basal bodies to resist cilia-generated forces

Basal bodies nucleate, anchor, and organize cilia. As the anchor for motile cilia, basal bodies must be resistant to the forces directed toward the cell as a consequence of ciliary beating. The molecules and generalized mechanisms that contribute to the maintenance of basal bodies remain to be discovered. Bld10/Cep135 is a basal body outer cartwheel domain protein that has established roles in the assembly of nascent basal bodies. This study showed that Tetrahymena Bld10 protein first incorporates stably at basal bodies early during new assembly. Bld10 protein continues to accumulate at basal bodies after assembly, and it is hypothesized that the full complement of Bld10 is required to stabilize basal bodies. A novel mechanism was identified for Bld10/Cep135 in basal body maintenance so that basal bodies can withstand the forces produced by motile cilia. Bld10 stabilizes basal bodies by promoting the stability of the A- and C-tubules of the basal body triplet microtubules and by properly positioning the triplet microtubule blades. The forces generated by ciliary beating promote basal body disassembly in bld10Delta cells. Thus Bld10/Cep135 acts to maintain the structural integrity of basal bodies against the forces of ciliary beating in addition to its separable role in basal body assembly (Bayless, 2012).

Search PubMed for articles about Drosophila Cep135

Bayless, B. A., Giddings, T. H., Jr., Winey, M. and Pearson, C. G. (2012). Bld10/Cep135 stabilizes basal bodies to resist cilia-generated forces. Mol Biol Cell 23: 4820-4832. PubMed ID: 23115304

Bettencourt-Dias, M. and Glover, D. M. (2007). Centrosome biogenesis and function: centrosomics brings new understanding. Nat Rev Mol Cell Biol 8: 451-463. PubMed ID: 17505520

Bettencourt-Dias, M., Hildebrandt, F., Pellman, D., Woods, G. and Godinho, S. A. (2011). Centrosomes and cilia in human disease. Trends Genet 27: 307-315. PubMed ID: 21680046

Blachon, S., Cai, X., Roberts, K. A., Yang, K., Polyanovsky, A., Church, A. and Avidor-Reiss, T. (2009). A proximal centriole-like structure is present in Drosophila spermatids and can serve as a model to study centriole duplication. Genetics 182: 133-144. PubMed ID: 19293139

Carvalho-Santos, Z., et al. (2010). Stepwise evolution of the centriole-assembly pathway. J. Cell Sci. 123(Pt 9): 1414-26. PubMed ID: 20392737

Carvalho-Santos, Z., Azimzadeh, J., Pereira-Leal, J. B. and Bettencourt-Dias, M. (2011). Evolution: Tracing the origins of centrioles, cilia, and flagella. J Cell Biol 194: 165-175. PubMed ID: 21788366

Carvalho-Santos, Z., Machado, P., Alvarez-Martins, I., Gouveia, S. M., Jana, S. C., Duarte, P., Amado, T., Branco, P., Freitas, M. C., Silva, S. T., Antony, C., Bandeiras, T. M. and Bettencourt-Dias, M. (2012). BLD10/CEP135 is a microtubule-associated protein that controls the formation of the flagellum central microtubule pair. Dev Cell 23: 412-424. PubMed ID: 22898782

Hiraki, M., Nakazawa, Y., Kamiya, R. and Hirono, M. (2007). Bld10p constitutes the cartwheel-spoke tip and stabilizes the 9-fold symmetry of the centriole. Curr Biol 17: 1778-1783. PubMed ID: 17900905

Jerka-Dziadosz, M., Gogendeau, D., Klotz, C., Cohen, J., Beisson, J. and Koll, F. (2010). Basal body duplication in Paramecium: the key role of Bld10 in assembly and stability of the cartwheel. Cytoskeleton (Hoboken) 67: 161-171. PubMed ID: 20217679

Kleylein-Sohn, J., Westendorf, J., Le Clech, M., Habedanck, R., Stierhof, Y. D. and Nigg, E. A. (2007). Plk4-induced centriole biogenesis in human cells. Dev Cell 13: 190-202. PubMed ID: 17681131

LaFountain, J. R., Jr. (1976). Analysis of birefringence and ultrastructure of spindles in primary spermatocytes of Nephrotoma suturalis during anaphase. J Ultrastruct Res 54: 333-346. PubMed ID: 943566

Matsuura, K., Lefebvre, P. A., Kamiya, R. and Hirono, M. (2004). Bld10p, a novel protein essential for basal body assembly in Chlamydomonas: localization to the cartwheel, the first ninefold symmetrical structure appearing during assembly. J Cell Biol 165: 663-671. PubMed ID: 15173189

Modarressi, M. H., Cameron, J., Taylor, K. E. and Wolfe, J. (2001). Identification and characterisation of a novel gene, TSGA10, expressed in testis. Gene 262: 249-255. PubMed ID: 11179690

Mottier-Pavie, V. and Megraw, T. L. (2009). Drosophila bld10 is a centriolar protein that regulates centriole, basal body, and motile cilium assembly. Mol Biol Cell 20: 2605-2614. PubMed ID: 19321663

Nigg, E. A. and Raff, J. W. (2009). Centrioles, centrosomes, and cilia in health and disease. Cell 139: 663-678. PubMed ID: 19914163

Panda, P., Kovacs, L., Dzhindzhev, N., Fatalska, A., Persico, V., Geymonat, M., Riparbelli, M. G., Callaini, G. and Glover, D. M. (2020). Tissue specific requirement of Drosophila Rcd4 for centriole duplication and ciliogenesis. J Cell Biol 219(8). PubMed ID: 32543652

Roque, H., Wainman, A., Richens, J., Kozyrska, K., Franz, A. and Raff, J. W. (2012). Drosophila Cep135/Bld10 maintains proper centriole structure but is dispensable for cartwheel formation. J Cell Sci. PubMed ID: 22976301

Singh, P., Ramdas Nair, A. and Cabernard, C. (2014). The centriolar protein Bld10/Cep135 is required to establish centrosome asymmetry in Drosophila neuroblasts. Curr Biol 24: 1548-1555. PubMed ID: 24954048

Tian, Y., Wei, C., He, J., Yan, Y., Pang, N., Fang, X., Liang, X. and Fu, J. (2021). Superresolution characterization of core centriole architecture. J Cell Biol 220(4). PubMed ID: 33533934

date revised: 5 November 2023

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