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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.

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.

Primary cilia play critical roles in development and disease. Their assembly is triggered by mature centrioles (basal bodies) and requires centrosomal protein 164kDa (Cep164), a component of distal appendages. Here we show that loss of Cep164 leads to early defects in ciliogenesis, reminiscent of the phenotypic consequences of mutations in TTBK2 (Tau tubulin kinase 2). We identify Cep164 as a likely physiological substrate of TTBK2 and demonstrate that Cep164 and TTBK2 form a complex. We map the interaction domains and demonstrate that complex formation is crucial for the recruitment of TTBK2 to basal bodies. Remarkably, ciliogenesis can be restored in Cep164-depleted cells by expression of chimeric proteins in which TTBK2 is fused to the C-terminal centriole-targeting domain of Cep164. These findings indicate that one of the major functions of Cep164 in ciliogenesis is to recruit active TTBK2 to centrioles. Once positioned, TTBK2 then triggers key events required for ciliogenesis, including removal of CP110 and recruitment of intraflagellar transport proteins. In addition, our data suggest that TTBK2 also acts upstream of Cep164, contributing to the assembly of distal appendages.

Centrosomes are microtubule-organizing centres of animal cells. They influence the morphology of the microtubule cytoskeleton, function as the base for the primary cilium and serve as a nexus for important signalling pathways. At the core of a typical centrosome are two cylindrical microtubule-based structures termed centrioles, which recruit a matrix of associated pericentriolar material. Cells begin the cell cycle with exactly one centrosome, and the duplication of centrioles is constrained such that it occurs only once per cell cycle and at a specific site in the cell. As a result of this duplication mechanism, the two centrioles differ in age and maturity, and thus have different functions; for example, the older of the two centrioles can initiate the formation of a ciliary axoneme. We discuss spatial aspects of the centrosome duplication cycle, the mechanism of centriole assembly and the possible consequences of the inherent asymmetry of centrioles and centrosomes.

Centrioles play a key role in the development of the fly. They are needed for the correct formation of centrosomes, the organelles at the poles of the spindle that can persist as microtubule organizing centers (MTOCs) into interphase. The ability to nucleate cytoplasmic microtubules (MTs) is a property of the surrounding pericentriolar material (PCM). The centriole has a dual life, existing not only as the core of the centrosome but also as the basal body, the structure that templates the formation of cilia and flagellae. Thus the structure and functions of the centriole, the centrosome, and the basal body have an impact upon many aspects of development and physiology that can readily be modeled in Drosophila. Centrosomes are essential to give organization to the rapidly increasing numbers of nuclei in the syncytial embryo and for the spatially precise execution of cell division in numerous tissues, particularly during male meiosis. Although mitotic cell cycles can take place in the absence of centrosomes, this is an error-prone process that opens up the fly to developmental defects and the potential of tumor formation. Here, we review the structure and functions of the centriole, the centrosome, and the basal body in different tissues and cultured cells of Drosophila melanogaster, highlighting their contributions to different aspects of development and cell division.

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.

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.

Centrosomes, composed of centrioles and pericentriolar matrix proteins, are traditionally viewed as essential microtubule-organizing centers (MTOCs) that facilitate bipolar spindle formation and chromosome segregation during spermatogenesis. In this study, we investigated the role of centrioles in male germ cell development by using a murine conditional knockout (cKO) of Sas4 , a critical component of centriole biogenesis. We found that while centriole duplication was impaired in Sas4 cKO spermatocytes, these cells were still capable of progressing through meiosis I and II. Chromosome segregation was able to proceed through the formation of a non-centrosomal MTOC, indicating that centrioles are not required for meiotic divisions. However, spermatids that inherited fewer than two centrioles exhibited severe defects in spermiogenesis, including improper manchette formation, constricted perinuclear rings, disrupted acrosome morphology, and failure to form flagella. Consequently, Sas4 cKO males were infertile due to the absence of functional spermatozoa. Our findings demonstrate that while centrioles are dispensable for meiosis in male germ cells, they are essential for spermiogenesis and sperm maturation. This work provides key insights into the role of centrosomes in male fertility and may have implications for understanding certain conditions of male infertility associated with centriole defects.

Centrioles are microtubule-based organelles critical for signaling, motility and division. The microtubule-binding protein SAS-1 is homologous to the human ciliopathy component C2CD3 and contributes to centriole integrity in C. elegans , but how this function is exerted is incompletely understood. Here, through the generation of a null allele and analysis with U-Ex-STED, we establish that SAS-1 is dispensable for the onset of centriole assembly, but essential for organelle integrity during oogenesis, spermatogenesis and in the early embryo. Additionally, we uncover that SAS-1 is present at the transition zone of sensory neurons, and contributes in a partially redundant manner to ciliary function. Furthermore, we investigate the relationship between SAS-1 and the C. elegans Sjögren’s Syndrome Nuclear Antigen 1 protein SSNA-1, establishing that SSNA-1 localizes next to the SAS-1 C-terminus in the centriole architecture. Moreover, through molecular epistasis experiments with null alleles of both components, we reveal that SAS-1 is essential for SSNA-1 localization to centrioles during oogenesis and to the transition zone during ciliogenesis. Moreover, using a heterologous human cell assay, we establish that SAS-1 recruits SSNA-1 to microtubules. Overall, our findings help clarify how SAS-1, together with SSNA-1, ensures centriole integrity, and reveal that it contributes to cilium function.

Chromosomal instability (CIN) is associated with many human diseases, including neurodevelopmental or neurodegenerative conditions, age-related disorders and cancer, and is a key driver for disease initiation and progression. A major source of structural chromosome instability (s-CIN) leading to structural chromosome aberrations is “replication stress”, a condition in which stalled or slowly progressing replication forks interfere with timely and error-free completion of the S phase. On the other hand, mitotic errors that result in chromosome mis-segregation are the cause of numerical chromosome instability (n-CIN) and aneuploidy. In this review, we will discuss recent evidence showing that these two forms of chromosomal instability can be mechanistically interlinked. We first summarize how replication stress causes structural and numerical CIN, focusing on mechanisms such as mitotic rescue of replication stress (MRRS) and centriole disengagement, which prevent or contribute to specific types of structural chromosome aberrations and segregation errors. We describe the main outcomes of segregation errors and how micronucleation and aneuploidy can be the key stimuli promoting inflammation, senescence, or chromothripsis. At the end, we discuss how CIN can reduce cellular fitness and may behave as an anticancer barrier in noncancerous cells or precancerous lesions, whereas it fuels genomic instability in the context of cancer, and how our current knowledge may be exploited for developing cancer therapies.