Multiciliogenesis: Tricking the cell-cycle machinery to build hundreds of cilia.

Multiciliated cells produce over a hundred motile cilia anchored to the membrane by modified centrioles. Recent work has characterized an alternative cell cycle used by this post-mitotic cell type to generate additional centrioles without undergoing cell division.

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Recent studies have shown that the formation of the primary cilium is associated with a specific cellular organelle known as the midbody remnant (MBR), which is a point-like organelle formed by shedding of the midbody at the end of mitosis. MBRs move along the cell surface close to the center body and regulate it to form primary cilia at the top of the centriole. Primary cilia can act as an organelle to inhibit tumorigenesis, and it is lost in a variety of tumors. Studies have shown that the accumulation of MBRs in tumor cells affects ciliogenesis; in addition, both MBRs and primary cilia are degraded in tumor cells through the autophagy pathway, and MBRs can also transfer tumor signaling pathway factors to primary cilia affecting tumorigenesis. In this article, the basic structure and the formation process of MBR and primary cilia are reviewed and the mechanism of MBRs regulating ciliogenesis is elaborated. The significance of MBR-mediated ciliogenesis in tumorigenesis and its potential as a target for cancer treatment are discussed.

Roles of centrioles in neural attraction of dental pulp stem cells.

BACKGROUND/PURPOSE: Human nerve development is vital, affecting trauma recovery and dental issues. Early embryonic clues link nerves to tooth development via factors like Wnt and Hedgehog pathways. Centrosomes play a role, and centriole issues can disrupt oral development, as in oral facial digital syndrome type 1. This study aimed to delve deeper into the role and influence of centrioles on the development of dental nerves. METHODS: Cell migration assessed by co-culturing mouse neural tissue and human dental pulp stem cells (DPSCs). Centrioles were fluorescently stained, and their positions observed with confocal microscopy. Centrinone was employed to inhibit centriole activity, evaluating its impact on cell mobility under activity inhibition. RESULTS: As the distance between nerve tissue and DPSCs decreased, more DPSCs had centrioles near nerve tissue. Co-culture with nerve tissue increased DPSCs migration toward it. In contrast, DPSCs cultured alone or with fibroblasts showed weaker migration, indicating neural tissue's attractive influence. The addition of 125 nM centrinone halted cell migration and centriole polymerization. After centrinone removal over two days, centrioles returned to normal, suggesting continued motility inhibition. CONCLUSION: Centrioles direct cell movement and polarization. There are two scenarios: centrioles at the cell center with the nucleus moving backward (as in NIH3T3 cells) and both cells and centrioles moving forward (as in DPSCs). DPSCs' attraction to neural tissue may shed light on nerve guidance by tooth germs, aiding embryonic cell differentiation into nerves. However, further in vivo and in vitro studies are needed to confirm the specific mechanism.

Protocols to induce and study ciliogenesis.

Primary cilia (PC) are sensory organelles that function as cellular antennas, transmitting signals between the extracellular and intracellular spaces in many vertebrate tissues. The cell generates and assembles PC through a highly regulated process called ciliogenesis. This complex process is involved in several physiological functions, including embryonic development, locomotion, cell cycle regulation or energetic homeostasis control. In general, when a cell finishes its cell division, the oldest centriole usually migrates to the plasma membrane and becomes a basal body that gives rise to the formation of a cilium. For this reason, the presence of cilia is incompatible with cell division, so when a cell is going to divide, the cilium and the basal body disappear. Ciliogenesis is triggered by various stimuli, all of them related to cell cycle blockade. This cell cycle, and ciliogenesis induction, can be observed by: (1) the influence of growth factors (lack of serum and consequent inability to promote cell cycle exit and increase the proportion of cells in G0); (2) pharmacological cell cycle inhibitors (staurosporine or etoposide); or (3) physiological cell cycle inhibition (excessive contact between neighboring cells). Evaluation of ciliogenesis induction is vitally important for the study of diseases related to ciliary dysfunction, called ciliopathies. That is why the use of correct protocols for inducing cilia formation and an accurate posterior visualization of the cilia after performing said protocols are essential parts in the study of these diseases. To facilitate this task, here we described detailed protocols to induce ciliogenesis in vitro and visualize PC by immunofluorescence microscopy in cultured cells.

Early Drosophila Oogenesis: A Tale of Centriolar Asymmetry.

Among the morphological processes that characterize the early stages of Drosophila oogenesis, the dynamic of the centrioles deserves particular attention. We re-examined the architecture and the distribution of the centrioles within the germarium and early stages of the vitellarium. We found that most of the germ cell centrioles diverge from the canonical model and display notable variations in size. Moreover, duplication events were frequently observed within the germarium in the absence of DNA replication. Finally, we report the presence of an unusually long centriole that is first detected in the cystoblast and is always associated with the developing oocyte. This centriole is directly inherited after the asymmetric division of the germline stem cells and persists during the process of oocyte selection, thus already representing a marker for oocyte identification at the beginning of its formation and during the ensuing developmental stages.

The SON RNA splicing factor is required for intracellular trafficking structures that promote centriole assembly and ciliogenesis.

Control of centrosome assembly is critical for cell division, intracellular trafficking, and cilia. Regulation of centrosome number occurs through the precise duplication of centrioles that reside in centrosomes. Here we explored transcriptional control of centriole assembly and find that the RNA splicing factor SON is specifically required for completing procentriole assembly. Whole genome mRNA sequencing identified genes whose splicing and expression are affected by the reduction of SON, with an enrichment in genes involved in the microtubule (MT) cytoskeleton, centrosome, and centriolar satellites. SON is required for the proper splicing and expression of CEP131, which encodes a major centriolar satellite protein and is required to organize the trafficking and MT network around the centrosomes. This study highlights the importance of the distinct MT trafficking network that is intimately associated with nascent centrioles and is responsible for procentriole development and efficient ciliogenesis.

The paternal toolbox for embryo development and health.

The sperm is essential for reconstitution of embryonic diploidy and highly specialized developmental functions. Immediately after gamete fusion, the sperm-borne PLC-zeta triggers activation, generating intracellular free Ca2+ oscillations. Mutations in the PLC-zeta encoding gene are associated with the absence of this factor in mature sperm and inability to achieve fertilization. Sperm play also a role in the greater game of the choreography of fertilization. In the human, the sperm centrioles are introduced into the oocyte environment with gamete fusion. They interact with the oocyte cytoskeletal apparatus to form a functional pair of centrosomes and ultimately regulate pronuclear juxtaposition in preparation for the first cleavage. As a consequence, the fidelity of chromosome segregation during the first cell divisions depends on the function of sperm centrioles. Sperm DNA integrity is essential for embryo development and health. Damaged DNA does not impact on the sperm fertilization ability following ICSI. However, detrimental effects emerge at pre- and post-implantation stages. Sperm-specific epigenetic factors also play an active role in the regulation of embryonic development, as shown by correlations between reduced embryo morphological quality and incorrect chromatin packaging during spermiogenesis or abnormal methylation of sperm CpG islands. This functional landscape demonstrates that the contribution of the sperm to development goes far beyond its well-established role in fertilization. Clinical studies confirm this view and indicate sperm function as a crucial aspect of research to increase the efficacy of assisted reproduction treatments.

A dynamic basal complex modulates mammalian sperm movement.

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.

Mechanical stretch scales centriole number to apical area via Piezo1 in multiciliated cells.

How cells count and regulate organelle number is a fundamental question in cell biology. For example, most cells restrict centrioles to two in number and assemble one cilium; however, multiciliated cells (MCCs) synthesize hundreds of centrioles to assemble multiple cilia. Aberration in centriole/cilia number impairs MCC function and can lead to pathological outcomes. Yet how MCCs control centriole number remains unknown. Using Xenopus, we demonstrate that centriole number scales with apical area over a remarkable 40-fold change in size. We find that tensile forces that shape the apical area also trigger centriole amplification based on both cell stretching experiments and disruption of embryonic elongation. Unexpectedly, Piezo1, a mechanosensitive ion channel, localizes near each centriole suggesting a potential role in centriole amplification. Indeed, depletion of Piezo1 affects centriole amplification and disrupts its correlation with the apical area in a tension-dependent manner. Thus, mechanical forces calibrate cilia/centriole number to the MCC apical area via Piezo1. Our results provide new perspectives to study organelle number control essential for optimal cell function.

Super-Resolution Microscopy and FIB-SEM Imaging Reveal Parental Centriole-Derived, Hybrid Cilium in Mammalian Multiciliated Cells.

Motile cilia are cellular beating machines that play a critical role in mucociliary clearance, cerebrospinal fluid movement, and fertility. In the airways, hundreds of motile cilia present on the surface of a multiciliated epithelia cell beat coordinately to protect the epithelium from bacteria, viruses, and harmful particulates. During multiciliated cell differentiation, motile cilia are templated from basal bodies, each extending a basal foot-an appendage linking motile cilia together to ensure coordinated beating. Here, we demonstrate that among the many motile cilia of a multiciliated cell, a hybrid cilium with structural features of both primary and motile cilia is harbored. The hybrid cilium is conserved in mammalian multiciliated cells, originates from parental centrioles, and its cellular position is biased and dependent on ciliary beating. Furthermore, we show that the hybrid cilium emerges independently of other motile cilia and functions in regulating basal body alignment.

Architecture of the centriole cartwheel-containing region revealed by cryo-electron tomography.

Centrioles are evolutionarily conserved barrels of microtubule triplets that form the core of the centrosome and the base of the cilium. While the crucial role of the proximal region in centriole biogenesis has been well documented, its native architecture and evolutionary conservation remain relatively unexplored. Here, using cryo-electron tomography of centrioles from four evolutionarily distant species, we report on the architectural diversity of the centriole's proximal cartwheel-bearing region. Our work reveals that the cartwheel central hub is constructed from a stack of paired rings with cartwheel inner densities inside. In both Paramecium and Chlamydomonas, the repeating structural unit of the cartwheel has a periodicity of 25 nm and consists of three ring pairs, with 6 radial spokes emanating and merging into a single bundle that connects to the microtubule triplet via the D2-rod and the pinhead. Finally, we identified that the cartwheel is indirectly connected to the A-C linker through the triplet base structure extending from the pinhead. Together, our work provides unprecedented evolutionary insights into the architecture of the centriole proximal region, which underlies centriole biogenesis.

Growth disadvantage associated with centrosome amplification drives population-level centriole number homeostasis.

The centriole duplication cycle normally ensures that centriole number is maintained at two centrioles per G1 cell. However, some circumstances can result in an aberrant increase in centriole number-a phenotype that is particularly prevalent in several types of cancer. Following an artificial increase in centriole number without tetraploidization due to transient overexpression of the kinase PLK4, human cells return to a normal centriole number during the proliferation of the population. We examine the mechanisms responsible for this return to normal centriole number at the population level in human retinal pigment epithelial cells. We find that the return to normal centriole number in the population of induced cells cannot be explained by limited duplication of centrioles, instability of extra centrioles, or by grossly asymmetric segregation of extra centrioles in mitosis. However, cells with extra centrioles display heterogenous phenotypes including extended cell cycle arrest, longer interphase durations, and death, which overall results in a proliferative disadvantage relative to normal cells in the population. Although about half of cells with extra centrioles in a population were able to divide, the extent of the disadvantages conferred by other fates is sufficient to account for the observed rate of return to normal centriole number. These results suggest that only under conditions of positive selection for cells with extra centrioles, continuous generation of such centrioles, or alleviation of the disadvantageous growth phenotypes would they be maintained in a population.

Centrioles are amplified in cycling progenitors of olfactory sensory neurons.

Olfaction in most animals is mediated by neurons bearing cilia that are accessible to the environment. Olfactory sensory neurons (OSNs) in chordates usually have multiple cilia, each with a centriole at its base. OSNs differentiate from stem cells in the olfactory epithelium, and how the epithelium generates cells with many centrioles is not yet understood. We show that centrioles are amplified via centriole rosette formation in both embryonic development and turnover of the olfactory epithelium in adult mice, and rosette-bearing cells often have free centrioles in addition. Cells with amplified centrioles can go on to divide, with centrioles clustered at each pole. Additionally, we found that centrioles are amplified in immediate neuronal precursors (INPs) concomitant with elevation of mRNA for polo-like kinase 4 (Plk4) and SCL/Tal1-interrupting locus gene (Stil), key regulators of centriole duplication. These results support a model in which centriole amplification occurs during a transient state characterized by elevated Plk4 and Stil in early INP cells. These cells then go on to divide at least once to become OSNs, demonstrating that cell division with amplified centrioles, known to be tolerated in disease states, can occur as part of a normal developmental program.

The sperm centrioles.

Centrioles are eukaryotic subcellular structures that produce and regulate massive cytoskeleton superstructures. They form centrosomes and cilia, regulate new centriole formation, anchor cilia to the cell, and regulate cilia function. These basic centriolar functions are executed in sperm cells during their amplification from spermatogonial stem cells during their differentiation to spermatozoa, and finally, after fertilization, when the sperm fuses with the egg. However, sperm centrioles exhibit many unique characteristics not commonly observed in other cell types, including structural remodeling, centriole-flagellum transition zone migration, and cell membrane association during meiosis. Here, we discuss five roles of sperm centrioles: orchestrating early spermatogenic cell divisions, forming the spermatozoon flagella, linking the spermatozoon head and tail, controlling sperm tail beating, and organizing the cytoskeleton of the zygote post-fertilization. We present the historic discovery of the centriole as a sperm factor that initiates embryogenesis, and recent genetic studies in humans and other mammals evaluating the current evidence for the five functions of sperm centrioles. We also examine information connecting the various sperm centriole functions to distinct clinical phenotypes. The emerging picture is that centrioles are essential sperm components with remarkable functional diversity and specialization that will require extensive and in-depth future studies.

Cep97 Is Required for Centriole Structural Integrity and Cilia Formation in Drosophila.

Centrioles are highly elaborate microtubule-based structures responsible for the formation of centrosomes and cilia. Despite considerable variation across species and tissues within any given tissue, their size is essentially constant [1, 2]. While the diameter of the centriole cylinder is set by the dimensions of the inner scaffolding structure of the cartwheel [3], how centriole length is set so precisely and stably maintained over many cell divisions is not well understood. Cep97 and CP110 are conserved proteins that localize to the distal end of centrioles and have been reported to limit centriole elongation in vertebrates [4, 5]. Here, we examine Cep97 function in Drosophila melanogaster. We show that Cep97 is essential for formation of full-length centrioles in multiple tissues of the fly. We further identify the microtubule deacetylase Sirt2 as a Cep97 interactor. Deletion of Sirt2 likewise affects centriole size. Interestingly, so does deletion of the acetylase Atat1, indicating that loss of stabilizing acetyl marks impairs centriole integrity. Cep97 and CP110 were originally identified as inhibitors of cilia formation in vertebrate cultured cells [6], and loss of CP110 is a widely used marker of basal body maturation. In contrast, in Drosophila, Cep97 appears to be only transiently removed from basal bodies and loss of Cep97 strongly impairs ciliogenesis. Collectively, our results support a model whereby Cep97 functions as part of a protective cap that acts together with the microtubule acetylation machinery to maintain centriole stability, essential for proper function in cilium biogenesis.

Tissue specific requirement of Drosophila Rcd4 for centriole duplication and ciliogenesis.

Rcd4 is a poorly characterized Drosophila centriole component whose mammalian counterpart, PPP1R35, is suggested to function in centriole elongation and conversion to centrosomes. Here, we show that rcd4 mutants exhibit fewer centrioles, aberrant mitoses, and reduced basal bodies in sensory organs. Rcd4 interacts with the C-terminal part of Ana3, which loads onto the procentriole during interphase, ahead of Rcd4 and before mitosis. Accordingly, depletion of Ana3 prevents Rcd4 recruitment but not vice versa. We find that neither Ana3 nor Rcd4 participates directly in the mitotic conversion of centrioles to centrosomes, but both are required to load Ana1, which is essential for such conversion. Whereas ana3 mutants are male sterile, reflecting a requirement for Ana3 for centriole development in the male germ line, rcd4 mutants are fertile and have male germ line centrioles of normal length. Thus, Rcd4 is essential in somatic cells but is not absolutely required in spermatogenesis, indicating tissue-specific roles in centriole and basal body formation.

Evolution of centriole assembly.

In this Primer, Nabais et al. discuss the evolution of the structure and function of centrioles and basal bodies, describe conserved centriole assembly features and the diversity in centriole architecture across eukaryotes, and highlight important outstanding evolutionary questions concerning centriole assembly.

SAS-6 Association with γ-Tubulin Ring Complex Is Required for Centriole Duplication in Human Cells.

Centrioles are essential components of centrosome, the main microtubule-organizing center of animal cells required for robust spindle bipolarity [1, 2]. They are duplicated once during the cell cycle [3], and the duplication involves assembly of a cartwheel on the pre-existing centriole followed by assembly of triplet microtubules around the cartwheel [4, 5]. Although the molecular details of cartwheel formation are understood [6-13], the mechanisms initiating the formation of centriolar microtubules are not known. Here, we show that the central component of cartwheel, HsSAS-6 plays a crucial role in the formation of centriolar microtubules by interacting with the microtubule nucleation machinery, γ-tubulin ring complex (γ-TuRC) in human cells. The globular N terminus and the central coiled-coil domain of SAS-6 are required for formation of the cartwheel [7, 14], whereas the function of its C-terminal outer cartwheel region in centriole duplication remains unclear. We find that deletion of HsSAS-6 C terminus disrupts microtubule formation in daughter centriole, and as a result, cells fail to form the new centriole. Consequently, this results in mitotic cells having only two centrioles localized at a single site. Detailed molecular analyses showed that HsSAS-6 interacts with the γ-TuRC proteins and associates with the γ-TuRC at the centrosome, and furthermore, the C terminus is essential for this association. High-resolution microscopy revealed localization of the γ-TuRC protein, γ-tubulin as multiple lobes surrounding the HsSAS-6-containing central hub in the centriole. Together, the results indicate that HsSAS-6 regulates centriolar microtubule assembly by anchoring γ-TuRCs to the pro-centriole at the onset of daughter centriole formation.

Unraveling the mysteries of centriolar satellites: time to rewrite the textbooks about the centrosome/cilium complex.

Centriolar satellites are membraneless granules that localize and move around centrosomes and cilia. Once referred to as structures with no obvious function, research in the past decade has identified satellites as key regulators of a wide range of cellular and organismal processes. Importantly, these studies have revealed a substantial overlap between functions, proteomes, and disease links of satellites with centrosomes and cilia. Therefore, satellites are now accepted as the "third component" of the vertebrate centrosome/cilium complex, which profoundly changes the way we think about the assembly, maintenance, and remodeling of the complex at the cellular and organismal levels. In this perspective, we first provide an overview of the cellular and structural complexities of centriolar satellites. We then describe the progress in the identification of the satellite interactome, which have paved the way to a molecular understanding of their mechanism of action and assembly mechanisms. After exploring current insights into their functions as recently described by loss-of-function studies and comparative evolutionary approaches, we discuss major unanswered questions regarding their functional and compositional diversity and their functions outside centrosomes and cilia.

Centriole Number and the Accumulation of Microtubules Modulate the Timing of Apical Insertion during Radial Intercalation.

Centrioles are microtubule (MT)-based structures that provide important functions during cell migration, cell division, and cell signaling [1]. Modulating centriole number in 3D cell cultures has been shown to influence protrusive behavior [2-5]. Here, we address in vivo the role of centrioles and the accumulation of MTs on the protrusive behavior required during the initiation of radial intercalation. Radial intercalation is an important developmental process whereby cells undergo polarized movements and interdigitate into a more superficial layer [6, 7]. It is commonly employed during metamorphic events, such as the tissue thinning coupled with expansion or during the introduction of different cell types into an epithelium. During radial intercalation, cells emerge from a basal layer by undergoing a process of apical migration, apical insertion, and expansion [8]. In Xenopus skin, multiciliated cells (MCCs), which contain ∼150 centrioles, and ionocytes (ICs), which contain two centrioles, differentiate during the same developmental window, but MCCs complete intercalation prior to ICs. Here, we utilize this difference in timing to create a quantifiable assay for insertion and find that the timing of insertion is modulated by changes in centriole number and the accumulation of acetylated MTs. Additionally, centrioles align between the nucleus and the leading edge creating an axis of migration with apically oriented (+) ends. Using the MT (-) end protein CAMSAP1 fused to the apically positioned Par6 protein, we have artificially reversed the orientation of MTs and find that the accumulation of MTs in either orientation is sufficient to promote apical insertion.