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The centrosome is a cytoplasmic organelle with roles in microtubule organization that has also been proposed to act as a hub for cellular signaling. Some centrosomal components are required for full activation of the DNA damage response. However, whether the centrosome regulates specific DNA repair pathways is not known. Here, we show that centrosome presence is required to fully activate recombination, specifically to completely license its initial step, the so-called DNA end resection. Furthermore, we identify a centriolar structure, the subdistal appendages, and a specific factor, CEP170, as the critical centrosomal component involved in the regulation of recombination and resection. Cells lacking centrosomes or depleted for CEP170 are, consequently, hypersensitive to DNA damaging agents. Moreover, low levels of CEP170 in multiple cancer types correlate with an increase of the mutation burden associated with specific mutational signatures and a better prognosis, suggesting that changes in CEP170 can act as a mutation driver but could also be targeted to improve current oncological treatments., (© 2023 The Authors. Published under the terms of the CC BY NC ND 4.0 license.)

Centrioles are important cellular organelles involved in the formation of both cilia and centrosomes. It is therefore not surprising that their dysfunction may lead to a variety of human pathologies. Studies have identified a conserved pathway of proteins required for centriole formation, and investigations using the embryo of the fruit fly Drosophila melanogaster have been crucial in elucidating their dynamics. However, a full understanding of how these components interact has been hampered by the total absence of centrioles in null mutant backgrounds for any of these core centriole factors. Here, I review our recent work describing a new model for investigating these interactions in the absence of bona fide centrioles. Sas-6 Ana2 Particles (SAPs) form when two core centriole factors, Sas-6 and Ana2, are co-over-expressed in fruit fly eggs. Crucially, they form even in eggs lacking other core centriole proteins. I review our characterisation of SAPs, and provide one example of how they have been used to investigate the role of a core centriole protein in PCM formation. I then consider some of the strengths and weaknesses of the SAP model, and discuss them in the context of other models for centriole study in Drosophila. Similar aggregates have been seen in other systems upon expression of centriole factors, so SAPs may also be a useful approach to study centriole proteins in other organisms., (© 2021 The Author(s).)

Centrosomes, composed of two centrioles and pericentriolar material, organize mitotic spindles during cell division and template cilia during interphase. The first few divisions during mouse development occur without centrioles, which form around embryonic day (E) 3. However, disruption of centriole biogenesis in Sas-4 null mice leads to embryonic arrest around E9. Centriole loss in Sas-4 -/- embryos causes prolonged mitosis and p53-dependent cell death. Studies in vitro discovered a similar USP28-, 53BP1-, and p53-dependent mitotic surveillance pathway that leads to cell cycle arrest. In this study, we show that an analogous pathway is conserved in vivo where 53BP1 and USP28 are upstream of p53 in Sas-4 -/- embryos. The data indicate that the pathway is established around E7 of development, four days after the centrioles appear. Our data suggest that the newly formed centrioles gradually mature to participate in mitosis and cilia formation around the beginning of gastrulation, coinciding with the activation of mitotic surveillance pathway upon centriole loss., (© 2021 The Authors. Published under the terms of the CC BY NC ND 4.0 license.)

Polo-like kinase 4 (Plk4) is a key regulator of centriole biogenesis. Studies have shown that Plk4 undergoes dynamic relocalization from a ring-like pattern around a centriole to a dot-like morphology at the procentriole assembly site and this event is central for inducing centriole biogenesis. However, the detailed mechanisms underlying Plk4's capacity to drive its symmetry-breaking ring-to-dot relocalization remain largely unknown. Here, we showed that Plk4 self-initiates this process in an autophosphorylation-dependent manner and that STIL, its downstream target, is not required for this event. Time-dependent analyses with mEOS-fused photoconvertible Plk4 revealed that a portion of ring-state Plk4 acquires a capacity, presumably through autophosphorylation, to linger around a centriole, ultimately generating a dot-state morphology. Interestingly, Plk4 WT, but not its catalytically inactive mutant, showed the ability to form a nanoscale spherical assembly in the cytosol of human cells or heterologous E. coli , demonstrating its autophosphorylation-dependent self-organizing capacity. At the biochemical level, Plk4 - unlike its N-terminal βTrCP degron motif - robustly autophosphorylated the PC3 SSTT motif within its C-terminal cryptic polo-box, an event critical for inducing its physical clustering. Additional in vivo experiments showed that although STIL was not required for Plk4's initial ring-to-dot conversion, coexpressed STIL greatly enhanced Plk4's ability to generate a spherical condensate and recruit Sas6, a major component of the centriolar cartwheel structure. We propose that Plk4's autophosphorylation-induced clustering is sufficient to induce its ring-to-dot localization conversion and that subsequently recruited STIL potentiates this process to generate a procentriole assembly body critical for Plk4-dependent centriole biogenesis.

Microtubules (MTs) promote important cellular functions including migration, intracellular trafficking, and chromosome segregation. The centrosome, comprised of two centrioles surrounded by the pericentriolar material (PCM), is the cell's central MT-organizing center. Centrosomes in cancer cells are commonly numerically amplified. However, the question of how the amplification of centrosomes alters MT organization capacity is not well studied. We developed a quantitative image-processing and machine learning-aided approach for the semi-automated analysis of MT organization. We designed a convolutional neural network-based approach for detecting centrosomes, and an automated pipeline for analyzing MT organization around centrosomes, encapsulated in a semi-automatic graphical tool. Using this tool, we find that breast cancer cells with supernumerary centrosomes not only have more PCM protein per centrosome, which gradually increases with increasing centriole numbers, but also exhibit expansion in PCM size. Furthermore, cells with amplified centrosomes have more growing MT ends, higher MT density and altered spatial distribution of MTs around amplified centrosomes. Thus, the semi-automated approach developed here enables rapid and quantitative analyses revealing important facets of centrosomal aberrations., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2020. Published by The Company of Biologists Ltd.)

Centrosomes, the predominant sites of microtubule nucleation and anchorage, coordinate spindle assembly and cell division in animal cells. At the onset of mitosis, centrioles accumulate microtubule-organizing pericentriolar material (PCM) in a process termed centrosome maturation. To what extent centrosome maturation depends on the continued activity of mitotic regulators or the presence of centrioles has hitherto been unclear. Using the C. elegans early embryo, we show that PCM expansion requires the Polo-like kinase PLK-1 and CEP192 (SPD-2 in C. elegans), but not its upstream regulator Aurora A (AIR-1), while maintenance of the PCM polymer depends exclusively on PLK-1. SPD-2 and PLK-1 are highly concentrated at centrioles. Unexpectedly, laser microsurgery reveals that while centrioles are required for PCM recruitment and centrosome structural integrity they are dispensable for PCM maintenance. We propose a model whereby centrioles promote centrosome maturation by recruiting PLK-1, but subsequent maintenance occurs via PLK-1 acting directly within the PCM., (Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.)

Defects in mitotic spindle orientation (MSO) disrupt the organization of stem cell niches impacting tissue morphogenesis and homeostasis. Mutations in centrosome genes reduce MSO fidelity, leading to tissue dysplasia and causing several diseases such as microcephaly, dwarfism, and cancer. Whether these mutations perturb spindle orientation solely by affecting astral microtubule nucleation or whether centrosome proteins have more direct functions in regulating MSO is unknown. To investigate this question, we analyzed the consequences of deregulating Plk4 (the master centriole duplication kinase) activity in Drosophila asymmetrically dividing neural stem cells. We found that Plk4 functions upstream of MSO control, orchestrating centriole symmetry breaking and consequently centrosome positioning. Mechanistically, we show that Plk4 acts through Spd2 phosphorylation, which induces centriole release from the apical cortex. Overall, this work not only reveals a role for Plk4 in regulating centrosome function but also links the centrosome biogenesis machinery with the MSO apparatus., (Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.)

Centrioles must duplicate as cells progress through the cell cycle but it is unclear how the site of duplication is selected. A recent computational study demonstrates that two separate but interacting feedback mechanisms (autocatalytic activation and substrate depletion) are capable of selecting a single site for centriole biogenesis., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Centrosomes are the major microtubule-organizing centers, consisting of centrioles surrounded by a pericentriolar material (PCM). Centrosomal PCM is spatiotemporally regulated to be minimal during interphase and expands as cells enter mitosis. It is unclear how PCM expansion is initiated at the onset of mitosis. Here, we identify that, in Drosophila, Plk1/Polo kinase phosphorylates the conserved centrosomal protein Sas-4 in vitro. This phosphorylation appears to occur at the onset of mitosis, enabling Sas-4's localization to expand outward from meiotic and mitotic centrosomes. The Plk1/Polo kinase site of Sas-4 is then required for an efficient recruitment of Cnn and γ-tubulin, bona fide PCM proteins that are essential for PCM expansion and centrosome maturation. Point mutations at Plk1/Polo sites of Sas-4 affect neither centrosome structure nor centriole duplication but specifically reduce the affinity to bind Cnn and γ-tubulin. These observations identify Plk1/Polo kinase regulation of Sas-4 as essential for efficient PCM expansion., (Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.)

Throughout biology, specifying cellular events at the correct location and time is necessary for ensuring proper function. The formation of robust microtubule organizing centers (MTOCs) in mitosis is one such event that must be restricted in space to centrosomes to prevent ectopic MTOC formation elsewhere in the cell, a situation that can result in multipolar spindle formation and aneuploidy. The process of reaching maximum centrosome MTOC activity in late G2, known as centrosome maturation, ensures accurate timing of nuclear envelope breakdown and proper chromosome attachment. Although centrosome maturation has been recognized for over a century, the spatial and temporal regulatory mechanisms that direct MTOC activation are poorly understood. Here, we review Sas-4/CPAP, Asterless/Cep152, Spd-2/Cep192, and PLP/Pericentrin, a group of proteins we refer to as 'bridge' proteins that reside at the surface of centrioles, perfectly positioned to serve as the gatekeepers of proper centrosome maturation at the perfect place and time., (© 2018 The Author(s).)

Centrioles are microtubule-based core components of centrosomes and cilia. They are duplicated exactly once during S-phase progression. Central to formation of each new (daughter) centriole is the formation of a nine-fold symmetrical cartwheel structure onto which microtubule triplets are deposited. In recent years, a module comprising the protein kinase polo-like kinase 4 (PLK4) and the two proteins STIL and SAS-6 have been shown to stay at the core of centriole duplication. Depletion of any one of these three proteins blocks centriole duplication and, conversely, overexpression causes centriole amplification. In this short review article, we summarize recent insights into how PLK4, STIL and SAS-6 co-operate in space and time to form a new centriole. These advances begin to shed light on the very first steps of centriole biogenesis., (© 2016 The Author(s).)

Ciliogenesis initiates at the mother centriole through a series of events that include membrane docking, displacement of cilia-inhibitory proteins and axoneme elongation. Centriolar proteins, in particular at distal and subdistal appendages, carry out these functions. Recently, cytoplasmic complexes named centriolar satellites have also been shown to promote ciliogenesis. Little is known about the functional and molecular relationship between appendage proteins, satellites and cilia biogenesis. Here, we identified the WD-repeat protein 8 (WDR8, also known as WRAP73) as a satellite and centriolar component. We show that WDR8 interacts with the satellite proteins SSX2IP and PCM1 as well as the centriolar proximal end component Cep135. Cep135 is required for the recruitment of WDR8 to centrioles. Depletion experiments revealed that WDR8 and Cep135 have strongly overlapping functions in ciliogenesis. Both are indispensable for ciliary vesicle docking to the mother centriole and for unlocking the distal end of the mother centriole from the ciliary inhibitory complex CP110-Cep97. Our data thus point to an important function of centriolar proximal end proteins in ciliary membrane biogenesis, and establish WDR8 and Cep135 as two factors that are essential for the initial steps of ciliation., (© 2016. Published by The Company of Biologists Ltd.)

Centrosomes are dominant microtubule organizing centers in animal cells with a pair of centrioles at their core. They template cilia during interphase and help organize the mitotic spindle for a more efficient cell division. Here, we review the roles of centrosomes in the early developing mouse and during organ formation. Mammalian cells respond to centrosome loss‐of‐function by activating the mitotic surveillance pathway, a timing mechanism that, when a defined mitotic duration is exceeded, leads to p53‐dependent cell death in the descendants. Mouse embryos without centrioles are highly susceptible to this pathway and undergo embryonic arrest at mid‐gestation. The complete loss of the centriolar core results in earlier and more severe phenotypes than that of other centrosomal proteins. Finally, different developing tissues possess varying thresholds and mount graded responses to the loss of centrioles that go beyond the germ layer of origin. [ABSTRACT FROM AUTHOR]

Two mother centrioles in an animal cell are linked by intercentriolar fibers that have CROCC/rootletin as their main building block. Here, we investigated the regulatory role of intercentriolar/rootlet fibers in cilia assembly. The cilia formation rates were significantly reduced in the CEP250/C-NAP1 and CROCC/rootletin knockout (KO) cells, irrespective of the departure of the young mother centrioles from the basal bodies. In addition, centriolar satellites were dispersed throughout the cytoplasm in the CEP250 and CROCC KO cells. We observed that PCM1 directly binds to CROCC. Their interaction is critical not only for the accumulation of centriolar satellites near the centrosomes/basal bodies but also for cilia formation. Finally, we observed that the centriolar satellite proteins are localized at the intercentriolar/rootlet fibers in the kidney epithelial cells. Based on these findings, we propose that the intercentriolar/rootlet fibers function as docking sites for centriolar satellites near the centrosomes/basal bodies and facilitate the cilia assembly process. [ABSTRACT FROM AUTHOR]

Centrosomes are the primary microtubule organizer in eukaryotic cells. In addition to shaping the intracellular microtubule network and the mitotic spindle, centrosomes are responsible for positioning cilia and flagella. To fulfill these diverse functions, centrosomes must be properly located within cells, which requires that they undergo intracellular transport. Importantly, centrosome mispositioning has been linked to ciliopathies, cancer, and infertility. The mechanisms by which centrosomes migrate are diverse and context dependent. In many cells, centrosomes move via indirect motor transport, whereby centrosomal microtubules engage anchored motor proteins that exert forces on those microtubules, resulting in centrosome movement. However, in some cases, centrosomes move via direct motor transport, whereby the centrosome or centriole functions as cargo that directly binds molecular motors which then walk on stationary microtubules. In this review, we summarize the mechanisms of centrosome motility and the consequences of centrosome mispositioning and identify key questions that remain to be addressed. [ABSTRACT FROM AUTHOR]

The triplet microtubule, a core structure of centrioles crucial for the organization of centrosomes, cilia, and flagella, consists of unclosed incomplete microtubules. The mechanisms of its assembly represent a fundamental open question in biology. Here, we discover that the ciliopathy protein HYLS1 and the β-tubulin isotype TUBB promote centriole triplet microtubule assembly. HYLS1 or a C-terminal tail truncated version of TUBB generates tubulin-based superstructures composed of centriole-like incomplete microtubule chains when overexpressed in human cells. AlphaFold-based structural models and mutagenesis analyses further suggest that the ciliopathy-related residue D211 of HYLS1 physically traps the wobbling C-terminal tail of TUBB, thereby suppressing its inhibitory role in the initiation of the incomplete microtubule assembly. Overall, our findings provide molecular insights into the biogenesis of atypical microtubule architectures conserved for over a billion years. Centrioles are characterized by an atypical triplet microtubule structure. Here, the authors discover that the ciliopathy protein HYLS1 promotes the assembly of triplet microtubules within human centrioles. [ABSTRACT FROM AUTHOR]

The centrosome is a non-membrane-bound organelle that is conserved across most animal cells and serves various functions throughout the cell cycle. In dividing cells, the centrosome is known as the spindle pole and nucleates a robust microtubule spindle to separate genetic material equally into two daughter cells. In nondividing cells, the mother centriole, a substructure of the centrosome, matures into a basal body and nucleates cilia, which acts as a signaltransducing antenna. The functions of centrosomes and their substructures are important for embryonic development and have been studied extensively using in vitro mammalian cell culture or in vivo using invertebrate models. However, there are considerable differences in the composition and functions of centrosomes during different aspects of vertebrate development, and these are less studied. In this Review, we discuss the roles played by centrosomes, highlighting conserved and divergent features across species, particularly during fertilization and embryonic development. [ABSTRACT FROM AUTHOR]

Tardigrades are microscopic ecdysozoans that can withstand extreme environmental conditions. Several tardigrade species undergo reversible morphological transformations and enter into cryptobiosis, which helps them to survive periods of unfavorable environmental conditions. However, the underlying molecular mechanisms of cryptobiosis are mostly unknown. Tubulins are evolutionarily conserved components of the microtubule cytoskeleton that are crucial in many cellular processes. We hypothesize that microtubules are necessary for the morphological changes associated with successful cryptobiosis. The molecular composition of the microtubule cytoskeleton in tardigrades is unknown. Therefore, we analyzed and characterized tardigrade tubulins and identified 79 tardigrade tubulin sequences in eight taxa. We found three α-, seven β-, one γ-, and one ε-tubulin isoform. To verify in silico identified tardigrade tubulins, we also isolated and sequenced nine out of ten predicted Hypsibius exemplaris tubulins. All tardigrade tubulins were localized as expected when overexpressed in mammalian cultured cells: to the microtubules or to the centrosomes. The presence of a functional ε-tubulin, clearly localized to centrioles, is attractive from a phylogenetic point of view. Although the phylogenetically close Nematoda lost their δ- and ε-tubulins, some groups of Arthropoda still possess them. Thus, our data support the current placement of tardigrades into the Panarthropoda clade. [ABSTRACT FROM AUTHOR]

Centrosomes are orbited by centriolar satellites, dynamic multiprotein assemblies nucleated by Pericentriolar material 1 (PCM1). To study the requirement for centriolar satellites, we generated mice lacking PCM1, a crucial component of satellites. Pcm1-/- mice display partially penetrant perinatal lethality with survivors exhibiting hydrocephalus, oligospermia, and cerebellar hypoplasia, and variably expressive phenotypes such as hydronephrosis. As many of these phenotypes have been observed in human ciliopathies and satellites are implicated in cilia biology, we investigated whether cilia were affected. PCM1 was dispensable for ciliogenesis in many cell types, whereas Pcm1-/- multiciliated ependymal cells and human PCM1-/- retinal pigmented epithelial 1 (RPE1) cells showed reduced ciliogenesis. PCM1-/- RPE1 cells displayed reduced docking of the mother centriole to the ciliary vesicle and removal of CP110 and CEP97 from the distal mother centriole, indicating compromised early ciliogenesis. Similarly, Pcm1-/- ependymal cells exhibited reduced removal of CP110 from basal bodies in vivo. We propose that PCM1 and centriolar satellites facilitate efficient trafficking of proteins to and from centrioles, including the departure of CP110 and CEP97 to initiate ciliogenesis, and that the threshold to trigger ciliogenesis differs between cell types. [ABSTRACT FROM AUTHOR]

Organization of microtubule arrays requires spatio-temporal regulation of the microtubule nucleator γ-tubulin ring complex (γTuRC) at microtubule organizing centers (MTOCs). MTOC-localized adapter proteins are thought to recruit and activate γTuRC, but the molecular underpinnings remain obscure. Here we show that at interphase centrosomes, rather than adapters, the microtubule polymerase ch-TOG (also named chTOG or CKAP5) ultimately controls γTuRC recruitment and activation. ch-TOG co-assembles with γTuRC to stimulate nucleation around centrioles. In the absence of ch-TOG, γTuRC fails to localize to these sites, but not the centriole lumen. However, whereas some ch-TOG is stably bound at subdistal appendages, it only transiently associates with PCM. ch-TOG's dynamic behavior requires its tubulin-binding TOG domains and a C-terminal region involved in localization. In addition, ch-TOG also promotes nucleation from the Golgi. Thus, at interphase centrosomes stimulation of nucleation and γTuRC attachment are mechanistically coupled through transient recruitment of ch-TOG, and ch-TOG's nucleation-promoting activity is not restricted to centrosomes. The molecular mechanisms underpinning the organization of microtubule arrays remain unclear. Here the authors show that in human cells, the microtubule polymerase ch-TOG promotes nucleation of microtubules at the interphase centrosome and the Golgi through a mechanism that involves transient interaction with the microtubule nucleator γTuRC. [ABSTRACT FROM AUTHOR]

Piezo1 and 2 are evolutionarily conserved mechanosensory cation channels known to function on the cell surface by responding to external pressure and transducing a mechanically activated Ca2+ current. Here we show that both Piezo1 and 2 also exhibit concentrated intracellular localization at centrosomes. Both Piezo1 and 2 loss-of-function and Piezo1 activation by the small molecule Yoda1 result in supernumerary centrosomes, premature centriole disengagement, multi-polar spindles, and mitotic delay. By using a GFP, Calmodulin and M13 Protein fusion (GCaMP) Ca2+-sensitive reporter, we show that perturbations in Piezo modulate Ca2+ flux at centrosomes. Moreover, the inhibition of Polo-like-kinase 1 eliminates Yoda1-induced centriole disengagement. Because previous studies have implicated force generation by microtubules as essential for maintaining centrosomal integrity, we propose that mechanotransduction by Piezo maintains pericentrosomal Ca2+ within a defined range, possibly through sensing cell intrinsic forces from microtubules. [ABSTRACT FROM AUTHOR]

The centriole is the microtubule-based backbone that ensures integrity, function, and cell cycle-dependent duplication of centrosomes. Mostly unclear mechanisms control structural integrity of centrioles. Here, we show that the centrosome protein CEP350 functions as scaffold that coordinates distal-end properties of centrioles such as length, stability, and formation of distal and subdistal appendages. CEP350 fulfills these diverse functions by ensuring centriolar localization of WDR90, recruiting the proteins CEP78 and OFD1 to the distal end of centrioles and promoting the assembly of subdistal appendages that have a role in removing the daughter-specific protein Centrobin. The CEP350-FOP complex in association with CEP78 or OFD1 controls centriole microtubule length. Centrobin safeguards centriole distal end stability, especially in the compromised CEP350-/- cells, while the CEP350-FOP-WDR90 axis secures centriole integrity. This study identifies CEP350 as a guardian of the distal-end region of centrioles without having an impact on the proximal PCM part. [ABSTRACT FROM AUTHOR]

During its division the cell must ensure the equal distribution of its genetic material in the two newly created cells, but it must also distribute organelles such as the Golgi apparatus, the mitochondria and the centrosome. DNA, the carrier of heredity, located in the nucleus of the cell, has made it possible to define the main principles that regulate the progression of the cell cycle. The cell cycle, which includes interphase and mitosis, is essentially a nuclear cycle, or a DNA cycle, since the interphase stages names (G1, S, G2) phases are based on processes that occur exclusively with DNA. However, centrosome duplication and segregation are two equally important events for the two new cells that must inherit a single centrosome. The centrosome, long considered the center of the cell, is made up of two small cylinders, the centrioles, made up of microtubules modified to acquire a very high stability. It is the main nucleation center of microtubules in the cell. Apart from a few exceptions, each cell in G1 phase has only one centrosome, consisting in of two centrioles and pericentriolar materials (PCM), which must be duplicated before the cell divides so that the two new cells formed inherit a single centrosome. The centriole is also the origin of the primary cilia, motile cilia and flagella of some cells. [ABSTRACT FROM AUTHOR]

Mitotic centrosomes are formed when centrioles start to recruit large amounts of pericentriolar material (PCM) around themselves in preparation for mitosis. This centrosome "maturation" requires the centrioles and also Polo/PLK1 protein kinase. The PCM comprises several hundred proteins and, in Drosophila, Polo cooperates with the conserved centrosome proteins Spd‐2/CEP192 and Cnn/CDK5RAP2 to assemble a PCM scaffold around the mother centriole that then recruits other PCM client proteins. We show here that in Drosophila syncytial blastoderm embryos, centrosomal Polo levels rise and fall during the assembly process—peaking, and then starting to decline, even as levels of the PCM scaffold continue to rise and plateau. Experiments and mathematical modelling indicate that a centriolar pulse of Polo activity, potentially generated by the interaction between Polo and its centriole receptor Ana1 (CEP295 in humans), could explain these unexpected scaffold assembly dynamics. We propose that centrioles generate a local pulse of Polo activity prior to mitotic entry to initiate centrosome maturation, explaining why centrioles and Polo/PLK1 are normally essential for this process. Synopsis: As cells prepare to enter mitosis, the centrioles recruit pericentriolar material (PCM) to form mitotic centrosomes. Here, experiments and modelling show centrioles to generate a pulse of Polo activity prior to mitosis, to stimulate the assembly of a Spd‐2/Polo/Cnn scaffold that can recruit other PCM components. As Drosophila embryos prepare to enter mitosis, Ana1 is activated at centrioles, allowing it to recruit and activate Polo prior to the entry into mitosis.The Polo recruited by Ana1 phosphorylates Spd‐2, allowing Spd‐2 to form a scaffold that fluxes outwards and recruits and activates more Polo, and also Cnn.Polo phosphorylates Cnn, allowing it to assemble into a stronger scaffold that helps to support the weaker Spd‐2 scaffold.Polo also inactivates Ana1, so centrosomal Polo and Spd‐2 levels decline as embryos enter mitosis; the Cnn scaffold is maintained as Polo is globally activated in the mitotic cytoplasm. [ABSTRACT FROM AUTHOR]

Centrioles are central structural elements of centrosomes and cilia. In human cells, daughter centrioles are assembled adjacent to existing centrioles in S-phase and reach their full functionality with the formation of distal and subdistal appendages one-and-a-half cell cycles later, as they exit their second mitosis. Current models postulate that the centriolar protein centrobin acts as placeholder for distal appendage proteins that must be removed to complete distal appendage formation. Here, we investigated, in non-transformed human epithelial RPE1 cells, the mechanisms controlling centrobin removal and its effect on distal appendage formation. Our data are consistent with a speculative model in which centrobin is removed from older centrioles due to a higher affinity for the newly born daughter centrioles, under the control of the centrosomal kinase PLK1. This removal also depends on the presence of subdistal appendage proteins on the oldest centriole. Removing centrobin, however, is not required for the recruitment of distal appendage proteins, even though this process is equally dependent on PLK1. We conclude that PLK1 kinase regulates centrobin removal and distal appendage formation during centriole maturation via separate pathways. [ABSTRACT FROM AUTHOR]

Centrosomes are the main microtubule-organizing centres, playing essential roles in the organization of the cytoskeleton during interphase, and in the mitotic spindle, which controls chromosome segregation, during cell division. Centrosomes also act as the basal body of cilia, regulating cilium length and affecting extracellular signal reception as well as the integration of intracellular signalling pathways. Centrosomes are self-replicative and duplicate once every cell cycle to generate two centrosomes. The core support structure of the centrosome consists of two molecularly distinct centrioles. The mother (mature) centriole exhibits accessory appendages and is surrounded by both pericentriolar material and centriolar satellites, structures that the daughter (immature) centriole lacks. In this Review, we discuss what is currently known about centrosome duplication, its dialogue with the cell cycle and the sequential acquisition of specific components during centriole maturation. We also describe our current understanding of the mature centriolar structures that are required to build a cilium. Altogether, the built-in centrosome asymmetries that stem from the two centrosomes inheriting molecularly different centrioles sets the foundation for cell division being an intrinsically asymmetric process. [ABSTRACT FROM AUTHOR]

Background: BRCA1‐associated protein (BAP1) is a tumor suppressor whose loss is associated with various malignancies. The primary cilium is an organelle involved in signal transduction and cell cycle progression. Primary cilia have been shown to be absent in melanoma but retained to some extent in melanocytic nevi, and the severity of dysplasia influences the degree of cilia loss. Additionally, studies have revealed roles for BAP1 in centrosome and mitotic spindle formation. Because the primary cilium is nucleated on the mother centriole, we examined the connection between the presence of primary cilia and the formation of centrosomes in BAP1‐inactivated melanocytic tumors (BIMTs). Methods: We evaluated the cilia and centrosomes in 11 BIMTs and five conventional melanocytic nevi using immunofluorescence staining of acetylated alpha‐tubulin and gamma‐tubulin. Results: We found that, compared to nevi, BIMTs show loss of primary cilia and amplification of centrosomes. Occasional nevi also showed increased centrioles; however, these foci of amplification were more likely to be ciliated than those in BIMTs. Conclusions: Although centrosome amplification does not absolutely correlate with loss of primary cilia in melanocytic neoplasms, absence of BAP1 exacerbates the phenotype. Moreover, aberrant centrosome and cilia formation are likely critical in the pathogenesis of other BAP1‐inactivated tumors. [ABSTRACT FROM AUTHOR]

Centriole biogenesis and maintenance are crucial for cells to generate cilia and assemble centrosomes that function as microtubule organizing centers (MTOCs). Centriole biogenesis and MTOC function both require the microtubule nucleator γ-tubulin ring complex (γTuRC). It is widely accepted that γTuRC nucleates microtubules from the pericentriolar material that is associated with the proximal part of centrioles. However, γTuRC also localizes more distally and in the centriole lumen, but the significance of these findings is unclear. Here we identify spatially and functionally distinct subpopulations of centrosomal γTuRC. Luminal localization is mediated by augmin, which is linked to the centriole inner scaffold through POC5. Disruption of luminal localization impairs centriole integrity and interferes with cilium assembly. Defective ciliogenesis is also observed in γTuRC mutant fibroblasts from a patient suffering from microcephaly with chorioretinopathy. These results identify a non-canonical role of augmin-γTuRC in the centriole lumen that is linked to human disease. The γ-tubulin ring complex (γTuRC) nucleates microtubules at the centrosome, but how this function is related to γTuRC subcentrosomal distribution is unclear. Here the authors show that γTuRC in the centriole lumen has a nucleation-independent role in centriole integrity and cilium assembly. [ABSTRACT FROM AUTHOR]

Polo kinase (PLK1 in mammals) is a master cell cycle regulator that is recruited to various subcellular structures, often by its polo-box domain (PBD), which binds to phosphorylated S-pS/pT motifs. Polo/PLK1 kinases have multiple functions at centrioles and centrosomes, and we have previously shown that in Drosophila phosphorylated Sas-4 initiates Polo recruitment to newly formed centrioles, while phosphorylated Spd-2 recruits Polo to the pericentriolar material (PCM) that assembles around mother centrioles in mitosis. Here, we show that Ana1 (Cep295 in humans) also helps to recruit Polo to mother centrioles in Drosophila. If Ana1-dependent Polo recruitment is impaired, mother centrioles can still duplicate, disengage from their daughters and form functional cilia, but they can no longer efficiently assemble mitotic PCM or elongate during G2. We conclude that Ana1 helps recruit Polo to mother centrioles to specifically promote mitotic centrosome assembly and centriole elongation in G2, but not centriole duplication, centriole disengagement or cilia assembly. [ABSTRACT FROM AUTHOR]

The centrosome influences the shape, orientation and activity of the microtubule cytoskeleton. The pericentriolar material (PCM), determines this functionality by providing a dynamic platform for nucleating microtubules and acts as a nexus for molecular signaling. Although great strides have been made in understanding PCM activity, its diffraction-limited size and amorphous appearance on electron microscopy (EM) have limited analysis of its high-order organization. Here, we outline current knowledge of PCM architecture and assembly, emphasizing recent super-resolution imaging studies that revealed the PCM has a layered structure made of fibers and matrices conserved from flies to humans. Notably, these studies debunk the long-standing view of an amorphous PCM and provide a paradigm to dissect the supramolecular organization of organelles in cells.

Centrioles are microtubule-based organelles that are embedded within pericentriolar material (PCM). Together, they comprise the centrosome, a microtubule-organizing center. PCM can sometimes exist in the absence of centrioles, but a new example of acentriolar PCM in neurons offers deeper insight into the relationship between these two entities. [ABSTRACT FROM AUTHOR]

Centrioles duplicate in interphase only once per cell cycle. Newly formed centrioles remain associated with their mother centrioles. The two centrioles disengage at the end of mitosis, which licenses centriole duplication in the next cell cycle. Therefore, timely centriole disengagement is critical for the proper centriole duplication cycle. However, the mechanisms underlying centriole engagement during interphase are poorly understood. Here, we show that Cep57 and Cep57L1 cooperatively maintain centriole engagement during interphase. Codepletion of Cep57 and Cep57L1 induces precocious centriole disengagement in interphase without compromising cell cycle progression. The disengaged daughter centrioles convert into centrosomes during interphase in a Plk1-dependent manner. Furthermore, the centrioles reduplicate and the centriole number increases, which results in chromosome segregation errors. Overall, these findings demonstrate that the maintenance of centriole engagement by Cep57 and Cep57L1 during interphase is crucial for the tight control of centriole copy number and thus for proper chromosome segregation. [ABSTRACT FROM AUTHOR]

Exon junction complexes (EJCs) mark untranslated spliced mRNAs and are crucial for the mRNA lifecycle. An imbalance in EJC dosage alters mouse neural stem cell (mNSC) division and is linked to human neurodevelopmental disorders. In quiescent mNSC and immortalized human retinal pigment epithelial (RPE1) cells, centrioles form a basal body for ciliogenesis. Here, we report that EJCs accumulate at basal bodies of mNSC or RPE1 cells and decline when these cells differentiate or resume growth. A high-throughput smFISH screen identifies two transcripts accumulating at centrosomes in quiescent cells, NIN and BICD2. In contrast to BICD2, the localization of NIN transcripts is EJC-dependent. NIN mRNA encodes a core component of centrosomes required for microtubule nucleation and anchoring. We find that EJC down-regulation impairs both pericentriolar material organization and ciliogenesis. An EJC-dependent mRNA trafficking towards centrosome and basal bodies might contribute to proper mNSC division and brain development. Exon junction complexes (EJCs) that mark untranslated mRNA are involved in transport, translation and nonsense-mediated mRNA decay. Here the authors show centrosomal localization of EJCs which appears to be required for both the localization of NIN mRNA around centrosomes and ciliogenesis. [ABSTRACT FROM AUTHOR]

Centriole copy number is tightly maintained by the once‐per‐cycle duplication of these organelles. Centrioles constitute the core of centrosomes, which organize the microtubule cytoskeleton and form the poles of the mitotic spindle. Centrosome amplification is frequently observed in tumors, where it promotes aneuploidy and contributes to invasive phenotypes. In non‐transformed cells, centrosome amplification triggers PIDDosome activation as a protective response to inhibit cell proliferation, but how extra centrosomes activate the PIDDosome remains unclear. Using a genome‐wide screen, we identify centriole distal appendages as critical for PIDDosome activation in cells with extra centrosomes. The distal appendage protein ANKRD26 is found to interact with and recruit the PIDDosome component PIDD1 to centriole distal appendages, and this interaction is required for PIDDosome activation following centrosome amplification. Furthermore, a recurrent ANKRD26 mutation found in human tumors disrupts PIDD1 localization and PIDDosome activation in cells with extra centrosomes. Our data support a model in which ANKRD26 initiates a centriole‐derived signal to limit cell proliferation in response to centrosome amplification. SYNOPSIS: Centrosome amplification contributes to tumorigenesis, but in non‐transformed cells activates the PIDDosome to inhibit cell proliferation. A genome‐wide screen identifies the centriolar distal appendage protein ANKRD26 as PIDD1 recruiter and PIDDosome activator in such situations. ANKRD26 recruits the PIDDosome component PIDD1 to centriole distal appendages.Centriolar recruitment of PIDD1 is required for PIDDosome activation following centrosome amplification.A cancer‐linked ANKRD26 mutation disrupts PIDD1 centriolar recruitment and PIDDosome activation in cells with extra centrosomes.ANKRD26 initiates a centriole‐derived signal to limit cell proliferation following centrosome amplification. [ABSTRACT FROM AUTHOR]

Centrosomes are composed of two orthogonally arranged centrioles surrounded by an electron-dense matrix called the pericentriolar material (PCM). Centrioles are cylinders with diameters of ~250 nm, are several hundred nanometres in length and consist of 9-fold symmetrically arranged microtubules (MT). In dividing animal cells, centrosomes act as the principal MT-organising centres and they also organise actin, which tunes cytoplasmic MT nucleation. In some specialised cells, the centrosome acquires additional critical structures and converts into the base of a cilium with diverse functions including signalling and motility. These structures are found in most eukaryotes and are essential for development and homoeostasis at both cellular and organism levels. The ultrastructure of centrosomes and their derived organelles have been known for more than half a century. However, recent advances in a number of techniques have revealed the high-resolution structures (at Å-to-nm scale resolution) of centrioles and have begun to uncover the molecular principles underlying their properties, including: protein components; structural elements; and biogenesis in various model organisms. This review covers advances in our understanding of the features and processes that are critical for the biogenesis of the evolutionarily conserved structures of the centrosomes. Furthermore, it discusses how variations of these aspects can generate diversity in centrosome structure and function among different species and even between cell types within a multicellular organism. ga1 [ABSTRACT FROM AUTHOR]

• Organization of microtubules by centrosomes for cell division may vary from cell to cell and in different phases of cell life. • Mitotic PCM might act as a phase-separated condensate involved in microtubule aster formation. • Mitotic spindle orientation is regulated through Plk1 signaling. • Centromere dysfunction may cause PCM dispersion and mispositioning of centrioles. The centrosome is present in most animal cells and functions as the major microtubule-organizing center to ensure faithful chromosome segregation during cell division. As cells transition from interphase to mitosis, the duplicated centrosomes separate and move to opposite sides of the cell where the spindle assembles. Centrosomes not only nucleate but also organize microtubules of the mitotic spindle. The mitotic spindle is anchored to the cell cortex by the astral microtubules emanating from the centrosomes. Proper orientation of the mitotic spindle is essential for correct cell division. Centrosome-localized polo-like kinase Plk1 has been linked to regulation of proper spindle orientation. A number of proteins including MISP and NuMA have been implicated in the Plk1-mediated spindle orientation pathway. [ABSTRACT FROM AUTHOR]

Centrosomes comprise two centrioles, the mother and daughter, embedded within a multi-layered proteinaceous matrix known as the pericentriolar material. In proliferating cells, centrosomes duplicate once per cell cycle and organise interphase and mitotic microtubule arrays, whereas in quiescent cells, the mother centriole templates primary cilium formation. Centrosomes have acquired various accessory structures to facilitate these disparate functions. In some eukaryotic lineages, mother centrioles can be distinguished from their daughter by the presence of appendages at their distal end, which anchor microtubule minus ends and tether Golgi-derived vesicles involved in ciliogenesis. Moreover, in vertebrate cells, centrosomes are surrounded by a system of cytoplasmic granules known as centriolar satellites. In this review, we will discuss these centriolar accessories and outline recent findings pertaining to their composition, assembly and regulation. [ABSTRACT FROM AUTHOR]

The centrosome, which consists of centrioles and pericentriolar material (PCM), becomes mature and assembles mitotic spindles by increasing the number of microtubules (MTs) emanating from the PCM. Among the molecules involved in centrosome maturation, Cep192 and Aurora A (AurA, also known as AURKA) are primarily responsible for recruitment of γ-tubulin and MT nucleators, whereas pericentrin (PCNT) is required for PCM organization. However, the role of Cep215 (also known as CDK5RAP2) in centrosome maturation remains elusive. Cep215 possesses binding domains for γ-tubulin, PCNT and MT motors that transport acentrosomal MTs towards the centrosome. We identify a mitosis-specific centrosome-targeting domain of Cep215 (215N) that interacts with Cep192 and phosphorylated AurA (pAurA). Cep192 is essential for targeting 215N to centrosomes, and centrosomal localization of 215N and pAurA is mutually dependent. Cep215 has a relatively minor role in γ-tubulin recruitment to the mitotic centrosome. However, it has been shown previously that this protein is important for connecting mitotic centrosomes to spindle poles. Based on the results of rescue experiments using versions of Cep215 with different domain deletions, we conclude that Cep215 plays a role in maintaining the structural integrity of the spindle pole by providing a platform for the molecules involved in centrosome maturation. [ABSTRACT FROM AUTHOR]

NUP188 encodes a scaffold component of the nuclear pore complex (NPC) and has been implicated as a congenital heart disease gene through an ill-defined function at centrioles. Here, we explore the mechanisms that physically and functionally segregate Nup188 between the pericentriolar material (PCM) and NPCs. Pulse-chase fluorescent labeling indicates that Nup188 populates centrosomes with newly synthesized protein that does not exchange with NPCs even after mitotic NPC breakdown. In addition, the steady-state levels of Nup188 are controlled by the sensitivity of the PCM pool, but not the NPC pool, to proteasomal degradation. Proximity-labeling and super-resolution microscopy show that Nup188 is vicinal to the inner core of the interphase centrosome. Consistent with this, we demonstrate direct binding between Nup188 and Cep152. We further show that Nup188 functions in centriole duplication at or upstream of Sas6 loading. Together, our data establish Nup188 as a component of PCM needed to duplicate the centriole with implications for congenital heart disease mechanisms. [ABSTRACT FROM AUTHOR]

The Drosophila Nonspecific Lethal (NSL) complex is a major transcriptional regulator of housekeeping genes. It contains at least seven subunits that are conserved in the human KANSL complex: Nsl1/Wah (KANSL1), Dgt1/Nsl2 (KANSL2), Rcd1/Nsl3 (KANSL3), Rcd5 (MCRS1), MBD-R2 (PHF20), Wds (WDR5) and Mof (MOF/KAT8). Previous studies have shown that Dgt1, Rcd1 and Rcd5 are implicated in centrosome maintenance. Here, we analyzed the mitotic phenotypes caused by RNAi-mediated depletion of Rcd1, Rcd5, MBD-R2 or Wds in greater detail. Depletion of any of these proteins in Drosophila S2 cells led to defects in chromosome segregation. Consistent with these findings, Rcd1, Rcd5 and MBD-R2 RNAi cells showed reduced levels of both Cid/CENP-A and the kinetochore component Ndc80. In addition, RNAi against any of the four genes negatively affected centriole duplication. In Wds-depleted cells, the mitotic phenotypes were similar but milder than those observed in Rcd1-, Rcd5- or MBD-R2-deficient cells. RT-qPCR experiments and interrogation of published datasets revealed that transcription of many genes encoding centromere/kinetochore proteins (e.g., cid, Mis12 and Nnf1b), or involved in centriole duplication (e.g., Sas-6, Sas-4 and asl) is substantially reduced in Rcd1, Rcd5 and MBD-R2 RNAi cells, and to a lesser extent in wds RNAi cells. During mitosis, both Rcd1-GFP and Rcd5-GFP accumulate at the centrosomes and the telophase midbody, MBD-R2-GFP is enriched only at the chromosomes, while Wds-GFP accumulates at the centrosomes, the kinetochores, the midbody, and on a specific chromosome region. Collectively, our results suggest that the mitotic phenotypes caused by Rcd1, Rcd5, MBD-R2 or Wds depletion are primarily due to reduced transcription of genes involved in kinetochore assembly and centriole duplication. The differences in the subcellular localizations of the NSL components may reflect direct mitotic functions that are difficult to detect at the phenotypic level, because they are masked by the transcription-dependent deficiency of kinetochore and centriolar proteins. [ABSTRACT FROM AUTHOR]

The primary cilium is a cellular antenna found on the surface of many eukaryotic cells, whose main role is to sense and transduce signals that regulate growth, development, and differentiation. Although once believed to be a vestigial organelle without important function, it has become clear that defects in primary cilium are responsible for a wide variety of genetic diseases affecting many organs and tissues, including the brain, eyes, heart, kidneys, liver, and pancreas. The primary cilium is mainly present in quiescent and differentiated cells, and controls must exist to ensure that this organelle is assembled or disassembled at the right time. Although many protein components required for building the cilium have been identified, mechanistic details of how these proteins are spatially and temporally regulated and how these regulations are connected to external cues are beginning to emerge. This review article highlights the role of ubiquitination and in particular, E3 ubiquitin ligases and deubiquitinases, in the control of primary cilia assembly and disassembly. [ABSTRACT FROM AUTHOR]

The centrosome is a small but important organelle that participates in centriole duplication, spindle formation, and ciliogenesis. Each event is regulated by key enzymatic reactions, but how these processes are integrated remains unknown. Recent studies have reported that ciliogenesis is controlled by distal appendage proteins such as FBF1, also known as Albatross. However, the precise role of Albatross in the centrosome cycle, including centriole duplication and centrosome separation, remains to be determined. Here, we report a novel function for Albatross at the proximal ends of centrioles. Using Albatross monospecific antibodies, full‐length constructs, and siRNAs for rescue experiments, we found that Albatross mediates centriole duplication by recruiting HsSAS‐6, a cartwheel protein of centrioles. Moreover, Albatross participates in centrosome separation during mitosis by recruiting Plk1 to residue S348 of Albatross after its phosphorylation. Taken together, our results show that Albatross is a novel protein that spatiotemporally integrates different aspects of centrosome function, namely ciliogenesis, centriole duplication, and centrosome separation. Protein–protein interactions involved in the centrosome cycle (Adapted from "Regulating the transition from centriole to basal body", The Journal of Cell Biology, Vol. 193, No. 3, p436). Full‐length Albatross was confirmed to contribute to ciliation (1), and the C‐terminus and the N‐terminus of Albatross were found to interact with the Plk4‐STIL‐HsSAS‐6 complex and Plk1 for centriole duplication (2) and centrosome separation (3), respectively. [ABSTRACT FROM AUTHOR]

An inherent asymmetry exists between the two centrosomes of a dividing cell. One centrosome is structurally more mature (mother centrosome) than the other (daughter centrosome). Post division, one daughter cell inherits the mother centrosome while the other daughter cell inherits the daughter centrosome. Remarkably, the kind of centrosome inherited is associated with cell fate in several developmental contexts such as in radial glial progenitors in the developing mouse cortex, Drosophila neuroblast divisions and in Drosophila male germline stem cells. However, the role of centrosome inheritance in granule neuron progenitors in the developing cerebellum has not been investigated. Here, we show that mother and daughter centrosomes do exist in these progenitors, and the amount of pericentriolar material (PCM) each centrosome possesses is different. However, we failed to observe any correlation between the fate adopted by the daughter cell and the nature of centrosome it inherited. [ABSTRACT FROM AUTHOR]

Several decades of centriole research have revealed the beautiful symmetry present in these microtubule-based organelles, which are required to form centrosomes, cilia and flagella in many eukaryotes. Centriole architecture is largely conserved across most organisms; however, individual centriolar features such as the central cartwheel or microtubule walls exhibit considerable variability when examined with finer resolution. In this paper, we review the ultrastructural characteristics of centrioles in commonly studied organisms, highlighting the subtle and not-so-subtle differences between specific structural components of these centrioles. In addition, we survey some non-canonical centriole structures that have been discovered in various species, from the coaxial bicentrioles of protists and lower land plants to the giant irregular centrioles of the fungus gnat Sciara. Finally, we speculate on the functional significance of these differences between centrioles, and the contribution of individual structural elements such as the cartwheel or microtubules towards the stability of centrioles. [ABSTRACT FROM AUTHOR]

Abstract: The centrosome executes diverse functions including the nucleation and organization of microtubules. A subset of centrosomal proteins is found to be involved in regulating the nucleation, stability, and dynamics of microtubules. Literature is flooded with reports of centrosomal proteins regulating microtubule nucleation. However, the centrosomal proteins that regulate microtubule stability are underexplored. Here, we review the centrosomal proteins, which either enhance or reduce the stability of microtubules and thereby regulate microtubule dynamics. We also discuss unexplored aspects of the centrosomal proteins that influence microtubule stability. © 2018 IUBMB Life, 70(7):602–611, 2018 [ABSTRACT FROM AUTHOR]