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Cilia are evolutionarily conserved structures with many sensory and motility-related functions. The ciliary base, composed of the basal body and the transition zone, is critical for cilia assembly and function, but its contribution to cilia diversity remains unknown. Hence, we generated a high-resolution structural and biochemical atlas of the ciliary base of four functionally distinct neuronal and sperm cilia types within an organism, Drosophila melanogaster . We uncovered a common scaffold and diverse structures associated with different localization of 15 evolutionarily conserved components. Furthermore, CEP290 (also known as NPHP6) is involved in the formation of highly diverse transition zone links. In addition, the cartwheel components SAS6 and ANA2 (also known as STIL) have an underappreciated role in basal body elongation, which depends on BLD10 (also known as CEP135). The differential expression of these cartwheel components contributes to diversity in basal body length. Our results offer a plausible explanation to how mutations in conserved ciliary base components lead to tissue-specific diseases. Using electron and three-dimensional structured illumination microscopy methods, Jana et al. characterize the ciliary base in four different cilia types in Drosophila , discovering structural and protein component differences that may be linked to the diversified functions of cilia.

The centrosome linker proteins C-Nap1, rootletin, and CEP68 connect the two centrosomes of a cell during interphase into one microtubule-organizing center. This coupling is important for cell migration, cilia formation, and timing of mitotic spindle formation. Very little is known about the structure of the centrosome linker. Here, we used stimulated emission depletion (STED) microscopy to show that each C-Nap1 ring at the proximal end of the two centrioles organizes a rootletin ring and, in addition, multiple rootletin/CEP68 fibers. Rootletin/CEP68 fibers originating from the two centrosomes form a web-like, interdigitating network, explaining the flexible nature of the centrosome linker. The rootletin/CEP68 filaments are repetitive and highly ordered. Staggered rootletin molecules (N-to-N and C-to-C) within the filaments are 75 nm apart. Rootletin binds CEP68 via its C-terminal spectrin repeat-containing region in 75-nm intervals. The N-to-C distance of two rootletin molecules is ∼35 to 40 nm, leading to an estimated minimal rootletin length of ∼110 nm. CEP68 is important in forming rootletin filaments that branch off centrioles and to modulate the thickness of rootletin fibers. Thus, the centrosome linker consists of a vast network of repeating rootletin units with C-Nap1 as ring organizer and CEP68 as filament modulator.

As daughter centrioles assemble during G2, they recruit conserved Ana3/RTTN followed by its partner Rcd4/PPP1R35. Together, this contributes to the subsequent recruitment of Ana1/CEP295, required for the centriole’s conversion to a centrosome. Here, we show that Rcd4/PPP1R35 is also required to maintain 9-fold centriole symmetry in the Drosophila male germline; its absence causes microtubule triplets to disperse into a reduced number of doublet or singlet microtubules. rcd4 -null mutant spermatocytes display skinny centrioles that elongate normally and localize centriolar components correctly. Mutant spermatocytes also have centrioles of normal girth that splay at their proximal ends when induced to elongate by Ana1 overexpression. Skinny and splayed spermatid centrioles can still recruit a proximal centriole-like (PCL) structure marking a capability to initiate features of centriole duplication in developing sperm. Thus, stable 9-fold symmetry of microtubule triplets is not essential for centriole growth, correct longitudinal association of centriole components, and aspects of centriole duplication. In this study, the authors show that centriole 9-fold microtubule symmetry is not essential for centriole elongation, correct distribution of the centriole’s components along its length, and in initiating aspects of centriole duplication.

Centrioles are microtubule-based cellular structures present in most human cells that build centrosomes and cilia. Proliferating cells have only two centrosomes and this number is stringently maintained through the temporally and spatially controlled processes of centriole assembly and segregation. The assembly of new centrioles begins in early S phase and ends in the third G1 phase from their initiation. This lengthy process of centriole assembly from their initiation to their maturation is characterized by numerous structural and still poorly understood biochemical changes, which occur in synchrony with the progression of cells through three consecutive cell cycles. As a result, proliferating cells contain three structurally, biochemically, and functionally distinct types of centrioles: procentrioles, daughter centrioles, and mother centrioles. This age difference is critical for proper centrosome and cilia function. Here we discuss the centriole assembly process as it occurs in somatic cycling human cells with a focus on the structural, biochemical, and functional characteristics of centrioles of different ages.

Centrioles are cylindrical, ninefold symmetrical structures with peripheral triplet microtubules strictly required to template cilia and flagella. The highly conserved protein SAS-6 constitutes the center of the cartwheel assembly that scaffolds centrioles early in their biogenesis. We determined the x-ray structure of the amino-terminal domain of SAS-6 from zebrafish, and we show that recombinant SAS-6 self-associates in vitro into assemblies that resemble cartwheel centers. Point mutations are consistent with the notion that centriole formation in vivo depends on the interactions that define the self-assemblies observed here. Thus, these interactions are probably essential to the structural organization of cartwheel centers.

Centrioles have a unique, conserved architecture formed by three linked, ‘triplet’, microtubules arranged in ninefold symmetry. The mechanisms by which these triplet microtubules are formed remain unclear but likely involve the noncanonical tubulins delta-tubulin and epsilon-tubulin. Previously, we found that human cells lacking delta-tubulin or epsilon-tubulin form abnormal centrioles, characterized by an absence of triplet microtubules, lack of central core protein POC5, and a futile cycle of centriole formation and disintegration (Wang et al., 2017). Here, we show that human cells lacking either TEDC1 or TEDC2 have similar abnormalities. Using ultrastructure expansion microscopy, we observed that mutant centrioles elongate to the same length as control centrioles in G2 phase and fail to recruit central core scaffold proteins. Remarkably, mutant centrioles also have an expanded proximal region. During mitosis, these mutant centrioles further elongate before fragmenting and disintegrating. All four proteins physically interact and TEDC1 and TEDC2 can form a subcomplex in the absence of the tubulins, supporting an AlphaFold Multimer model of the tetramer. TEDC1 and TEDC2 localize to centrosomes and are mutually dependent on each other and on delta-tubulin and epsilon-tubulin for localization. Our results demonstrate that delta-tubulin, epsilon-tubulin, TEDC1, and TEDC2 function together to promote robust centriole architecture, laying the foundation for future studies on the mechanisms underlying the assembly of triplet microtubules and their interactions with centriole structure.

Centriole duplication occurs once per cell cycle to ensure robust formation of bipolar spindles and chromosome segregation. Each newly-formed daughter centriole remains connected to its mother centriole until late mitosis. The disengagement of the centriole pair is required for centriole duplication. However, the mechanisms underlying centriole engagement remain poorly understood. Here, we show that Cep57 is required for pericentriolar material (PCM) organization that regulates centriole engagement. Depletion of Cep57 causes PCM disorganization and precocious centriole disengagement during mitosis. The disengaged daughter centrioles acquire ectopic microtubule-organizing-center activity, which results in chromosome mis-segregation. Similar defects are observed in mosaic variegated aneuploidy syndrome patient cells with cep57 mutations. We also find that Cep57 binds to the well-conserved PACT domain of pericentrin. Microcephaly osteodysplastic primordial dwarfism disease pericentrin mutations impair the Cep57-pericentrin interaction and lead to PCM disorganization. Together, our work demonstrates that Cep57 provides a critical interface between the centriole core and PCM. Centriole disengagement occurs towards mitotic exit and involves cleavage of pericentrin, a component of the pericentriolar material. Here the authors show that depletion of the centrosomal protein Cep57 leads to precocious centriole disengagement, and that Cep57 binds pericentrin.

Reproductive success depends on efficient sperm movement driven by axonemal dynein-mediated microtubule sliding. Models predict sliding at the base of the tail – the centriole – but such sliding has never been observed. Centrioles are ancient organelles with a conserved architecture; their rigidity is thought to restrict microtubule sliding. Here, we show that, in mammalian sperm, the atypical distal centriole (DC) and its surrounding atypical pericentriolar matrix form a dynamic basal complex (DBC) that facilitates a cascade of internal sliding deformations, coupling tail beating with asymmetric head kinking. During asymmetric tail beating, the DC’s right side and its surroundings slide ~300 nm rostrally relative to the left side. The deformation throughout the DBC is transmitted to the head-tail junction; thus, the head tilts to the left, generating a kinking motion. These findings suggest that the DBC evolved as a dynamic linker coupling sperm head and tail into a single self-coordinated system. Centrioles are ancient organelles with a conserved architecture and their rigidity is thought to restrict microtubule sliding. Here authors show that, in mammalian sperm, the atypical distal centriole and its surrounding atypical pericentriolar matrix form a dynamic basal complex that facilitates a cascade of internal sliding deformations, coupling tail beating with asymmetric head kinking.

Mutations in many centriolar protein-encoding genes cause primary microcephaly. Using super-resolution and electron microscopy, we find that the human microcephaly protein, RTTN, is recruited to the proximal end of the procentriole at early S phase, and is located at the inner luminal walls of centrioles. Further studies demonstrate that RTTN directly interacts with STIL and acts downstream of STIL-mediated centriole assembly. CRISPR/Cas9-mediated RTTN gene knockout in p53-deficient cells induce amplification of primitive procentriole bodies that lack the distal-half centriolar proteins, POC5 and POC1B. Additional analyses show that RTTN serves as an upstream effector of CEP295, which mediates the loading of POC1B and POC5 to the distal-half centrioles. Interestingly, the naturally occurring microcephaly-associated mutant, RTTN (A578P), shows a low affinity for STIL binding and blocks centriole assembly. These findings reveal that RTTN contributes to building full-length centrioles and illuminate the molecular mechanism through which the RTTN (A578P) mutation causes primary microcephaly. Mutations in many centriolar protein-encoding genes cause primary microcephaly. Here the authors show that human microcephaly protein RTTN directly interacts with STIL and acts downstream of STIL-mediated centriole assembly, contributing to building full-length centrioles

Centrosomes play an important role in various cellular processes, including spindle formation and chromosome segregation. They are composed of two orthogonally arranged centrioles, whose duplication occurs only once per cell cycle. Accurate control of centriole numbers is essential for the maintenance of genomic integrity. Although it is well appreciated that polo-like kinase 4 (Plk4) plays a central role in centriole biogenesis, how it is recruited to centrosomes and whether this step is necessary for centriole biogenesis remain largely elusive. Here we showed that Plk4 localizes to distinct subcentrosomal regions in a temporally and spatially regulated manner, and that Cep192 and Cep152 serve as two distinct scaffolds that recruit Plk4 to centrosomes in a hierarchical order. Interestingly, Cep192 and Cep152 competitively interacted with the cryptic polo box of Plk4 through their homologous N-terminal sequences containing acidic-α-helix and N/Q-rich motifs. Consistent with these observations, the expression of either one of these N-terminal fragments was sufficient to delocalize Plk4 from centrosomes. Furthermore, loss of the Cep192- or Cep152-dependent interaction with Plk4 resulted in impaired centriole duplication that led to delayed cell proliferation. Thus, the spatiotemporal regulation of Plk4 localization by two hierarchical scaffolds, Cep192 and Cep152, is critical for centriole biogenesis.