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Tim Stearns - One of the best experts on this subject based on the ideXlab platform.
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Centrioles are amplified in cycling progenitors of olfactory sensory neurons
PLOS Biology, 2020Co-Authors: Kaitlin Ching, Tim StearnsAbstract: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.
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Centrioles are amplified via rosette formation in cycling progenitors of olfactory sensory neurons
bioRxiv, 2019Co-Authors: Kaitlin Ching, Tim StearnsAbstract:Olfaction in most animals is mediated by sensory neurons bearing receptors on cilia that are accessible to the environment. Within chordates, olfactory sensory neurons (OSNs) usually have multiple cilia, each with a Centriole at its base. OSNs differentiate from stem cells in the olfactory epithelium, and how mature cells with multiple Centrioles are generated during this process is not yet understood. OSNs in the mouse olfactory epithelium have about 15 cilia each, and we show that Centrioles are amplified in precursor cells via formation of Centriole rosette structures both during embryonic development and during turnover of the olfactory epithelium in adults. We also found free Centrioles present in rosette-bearing cells, suggesting that more than one pathway contributes to total Centriole number. Cells with amplified Centrioles can go on to divide, with clustered Centrioles at each pole. Additionally, we found that Centrioles are amplified in early immediate neuronal precursors, coincident with elevation of mRNA for Plk4 and Stil, two key regulators of Centriole duplication. Our findings highlight the importance of accounting for Centriole amplification in the development of olfactory epithelium-derived neuron regeneration therapies.
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Centriole triplet microtubules are required for stable Centriole formation and inheritance in human cells
eLife, 2017Co-Authors: Jennifer T. Wang, Jadranka Lončarek, Dong Kong, Christian R. Hoerner, Tim StearnsAbstract:Centrioles are composed of long-lived microtubules arranged in nine triplets. However, the contribution of triplet microtubules to mammalian Centriole formation and stability is unknown. Little is known of the mechanism of triplet microtubule formation, but experiments in unicellular eukaryotes indicate that delta-tubulin and epsilon-tubulin, two less-studied tubulin family members, are required. Here, we report that Centrioles in delta-tubulin and epsilon-tubulin null mutant human cells lack triplet microtubules and fail to undergo Centriole maturation. These aberrant Centrioles are formed de novo each cell cycle, but are unstable and do not persist to the next cell cycle, leading to a futile cycle of Centriole formation and disintegration. Disintegration can be suppressed by paclitaxel treatment. Delta-tubulin and epsilon-tubulin physically interact, indicating that these tubulins act together to maintain triplet microtubules and that these are necessary for inheritance of Centrioles from one cell cycle to the next.
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Centrioles, in absentia
Science, 2015Co-Authors: Tim StearnsAbstract:The Centriole is one of the organelles that defines eukaryotes. It was present in the last universal common eukaryotic ancestor ( 1 ), and persists in all major branches of the eukaryotic tree. The Centriole nucleates the cilium, which is involved in sensory signaling and in cell motility. In animal cells, the Centriole is also the hub of the centrosome, an accumulation of microtubule-nucleating and -organizing proteins that determine the spatial arrangement of the microtubule cytoskeleton. Duplication and segregation of the Centrioles are strictly controlled such that each cell begins the cell division cycle with a single pair of Centrioles, which duplicate only once and are then segregated on the poles of the mitotic spindle ( 2 ). On page 1155 of this issue, Wong et al. ( 3 ) describe a small-molecule inhibitor of Centriole duplication that allowed them to probe the effects of Centriole loss. Surprisingly, it appears that some cancer cells can proliferate indefinitely without Centrioles, whereas normal cells cannot.
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The Centriole duplication cycle
Philosophical Transactions of the Royal Society B: Biological Sciences, 2014Co-Authors: Elif Nur Firat-karalar, Tim StearnsAbstract:Centrosomes are the main microtubule-organizing centre of animal cells and are important for many critical cellular and developmental processes from cell polarization to cell division. At the core of the centrosome are Centrioles, which recruit pericentriolar material to form the centrosome and act as basal bodies to nucleate formation of cilia and flagella. Defects in Centriole structure, function and number are associated with a variety of human diseases, including cancer, brain diseases and ciliopathies. In this review, we discuss recent advances in our understanding of how new Centrioles are assembled and how Centriole number is controlled. We propose a general model for Centriole duplication control in which cooperative binding of duplication factors defines a Centriole ‘origin of duplication’ that initiates duplication, and passage through mitosis effects changes that license the Centriole for a new round of duplication in the next cell cycle. We also focus on variations on the general theme in which many Centrioles are created in a single cell cycle, including the specialized structures associated with these variations, the deuterosome in animal cells and the blepharoplast in lower plant cells.
Mengfu Bryan Tsou - One of the best experts on this subject based on the ideXlab platform.
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PPP1R35 ensures Centriole homeostasis by promoting Centriole-to-centrosome conversion.
Molecular Biology of the Cell, 2018Co-Authors: Chii Shyang Fong, Kanako Ozaki, Mengfu Bryan TsouAbstract:Centriole-to-centrosome conversion (CCC) safeguards Centriole homeostasis by coupling Centriole duplication with segregation, and is essential for stabilization of mature vertebrate Centrioles naturally devoid of the geometric scaffold or the cartwheel. Here we identified PPP1R35, a putative regulator of the protein phosphatase PP1, as a novel centriolar protein required for CCC. We found that PPP1R35 is enriched at newborn daughter Centrioles in S or G2 phase. In the absence of PPP1R35, Centriole assembly initiates normally in S phase, but none of the nascent Centrioles can form active centrosomes or recruit CEP295, an essential factor for CCC. Instead, all PPP1R35-null Centrioles, although stable during their birth in interphase, become disintegrated after mitosis upon cartwheel removal. Surprisingly, we found that neither the centriolar localization nor the function of PPP1R35 in CCC requires the putative PP1-interacting motif. PPP1R35 is thus acting upstream of CEP295 to induce CCC for proper Centriole maintenance.
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A Cell-Free System for Real-Time Analyses of Centriole Disengagement and Centriole-to-Centrosome Conversion.
Methods in Molecular Biology, 2016Co-Authors: Rajesh K Soni, Mengfu Bryan TsouAbstract:Centriole or centrosome number in cycling cells is strictly maintained through coordinated duplication and segregation. Duplication is limited to once only per cell cycle by separating the assembly event that occurs in S/G2 phase from the two licensing events, Centriole disengagement and Centriole-to-centrosome conversion, both of which occurs in mitosis. In addition to duplication licensing, Centriole-to-centrosome conversion also enables Centrioles to associate with spindle poles and thereby to segregate equally during cell division. Centriole disengagement and Centriole-to-centrosome conversion thus constitute the major regulatory module ensuring centrosome homeostasis in cycling cells. Using Xenopus egg extracts and purified engaged Centrioles, we here describe an in vitro assay allowing us to synchronously induce the initiation of Centriole disengagement and centrosome formation, pause the reaction anytime during the process, and more importantly, preserve "reaction intermediates" for further analyses.
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Promotion and Suppression of Centriole Duplication Are Catalytically Coupled through PLK4 to Ensure Centriole Homeostasis.
Cell Reports, 2016Co-Authors: Minhee Kim, Rajesh K Soni, Brian P. O'rourke, Prasad V. Jallepalli, Ronald C. Hendrickson, Mengfu Bryan TsouAbstract:PLK4 is the major kinase driving Centriole duplication. Duplication occurs only once per cell cycle, forming one new (or daughter) Centriole that is tightly engaged to the preexisting (or mother) Centriole. Centriole engagement is known to block the reduplication of mother Centrioles, but the molecular identity responsible for the block remains unclear. Here, we show that the centriolar cartwheel, the geometric scaffold for Centriole assembly, forms the identity of daughter Centrioles essential for the block, ceasing further duplication of the mother Centriole to which it is engaged. To ensure a steady block, we found that the cartwheel requires constant maintenance by PLK4 through phosphorylation of the same substrate that drives Centriole assembly, revealing a parsimonious control in which "assembly" and "block for new assembly" are linked through the same catalytic reaction to achieve homeostasis. Our results support a recently deduced model that the cartwheel-bound PLK4 directly suppresses Centriole reduplication.
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stabilization of cartwheel less Centrioles for duplication requires cep295 mediated Centriole to centrosome conversion
Cell Reports, 2014Co-Authors: Denisse Izquierdo, Wonjing Wang, Kunihiro Uryu, Mengfu Bryan TsouAbstract:Summary Vertebrate Centrioles lose their geometric scaffold, the cartwheel, during mitosis, concurrently with gaining the ability to recruit the pericentriolar material (PCM) and thereby function as the centrosome. Cartwheel removal has recently been implicated in Centriole duplication, but whether "cartwheel-less" Centrioles are intrinsically stable or must be maintained through other modifications remains unclear. Here, we identify a newborn Centriole-enriched protein, KIAA1731/CEP295, specifically mediating Centriole-to-centrosome conversion but dispensable for cartwheel removal. In the absence of CEP295, Centrioles form in the S/G2 phase and lose their associated cartwheel in mitosis but cannot be converted to centrosomes, uncoupling the two events. Strikingly, Centrioles devoid of both the PCM and the cartwheel progressively lose centriolar components, whereas Centrioles associating with either the cartwheel or PCM alone can exist stably. Thus, cartwheel removal can have grave repercussions to Centriole stability, and Centriole-to-centrosome conversion mediated by CEP295 must occur in parallel to maintain cartwheel-less Centrioles for duplication.
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stabilization of cartwheel less Centrioles for duplication requires cep295 mediated Centriole to centrosome conversion
Cell Reports, 2014Co-Authors: Denisse Izquierdo, Wonjing Wang, Kunihiro Uryu, Mengfu Bryan TsouAbstract:Summary Vertebrate Centrioles lose their geometric scaffold, the cartwheel, during mitosis, concurrently with gaining the ability to recruit the pericentriolar material (PCM) and thereby function as the centrosome. Cartwheel removal has recently been implicated in Centriole duplication, but whether "cartwheel-less" Centrioles are intrinsically stable or must be maintained through other modifications remains unclear. Here, we identify a newborn Centriole-enriched protein, KIAA1731/CEP295, specifically mediating Centriole-to-centrosome conversion but dispensable for cartwheel removal. In the absence of CEP295, Centrioles form in the S/G2 phase and lose their associated cartwheel in mitosis but cannot be converted to centrosomes, uncoupling the two events. Strikingly, Centrioles devoid of both the PCM and the cartwheel progressively lose centriolar components, whereas Centrioles associating with either the cartwheel or PCM alone can exist stably. Thus, cartwheel removal can have grave repercussions to Centriole stability, and Centriole-to-centrosome conversion mediated by CEP295 must occur in parallel to maintain cartwheel-less Centrioles for duplication.
Tomer Avidor-reiss - One of the best experts on this subject based on the ideXlab platform.
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The Evolution of Centriole Structure: Heterochrony, Neoteny, and Hypermorphosis.
Results and Problems in Cell Differentiation, 2019Co-Authors: Tomer Avidor-reiss, Katerina A. TurnerAbstract:Centrioles are subcellular organelles that were present in the last eukaryotic common ancestor, where the Centriole’s ancestral role was to form cilia. Centrioles have maintained a remarkably conserved structure in eukaryotes that have cilia, while groups that lack cilia have lost their Centrioles, highlighting the structure–function relationship that exists between the Centriole and the cilium. In contrast, animal sperm cells, a ciliated cell, exhibit remarkable structural diversity in the Centriole. Understanding how this structural diversity evolved may provide insight into Centriole assembly and function, as well as their unique role in sperm. Here, we apply concepts used in the study of the evolution of animal morphology to gain insight into the evolution of Centriole structure. We propose that Centrioles with an atypical structure form because of changes in the timing of Centriole assembly events, which can be described as centriolar “heterochrony.” Atypical Centrioles of insects and mammals appear to have evolved through different types of heterochrony. Here, we discuss two particular types of heterochrony: neoteny and hypermorphosis. The Centriole assembly of insect sperm cells exhibits the retention of “juvenile” Centriole structure, which can be described as centriolar “neoteny.” Mammalian sperm cells have an extended Centriole assembly program through the addition of novel steps such as centrosome reduction and Centriole remodeling to form atypical Centrioles, a form of Centriole “hypermorphosis.” Overall, Centriole heterochrony appears to be a common mechanism for the development of the atypical Centriole during the evolution of Centriole assembly of various animals’ sperm.
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It takes two (Centrioles) to tango.
Reproduction, 2019Co-Authors: Tomer Avidor-reiss, Emily L. FishmanAbstract:Cells that divide during embryo development require precisely two Centrioles during interphase and four Centrioles during mitosis. This precise number is maintained by allowing each Centriole to nucleate only one Centriole per cell cycle (i.e. Centriole duplication). Yet, how the first cell of the embryo, the zygote, obtains two Centrioles has remained a mystery in most mammals and insects. The mystery arose because the female gamete (oocyte) is thought to have no functional Centrioles and the male gamete (spermatozoon) is thought to have only one functional Centriole, resulting in a zygote with a single Centriole. However, recent studies in fruit flies, beetles and mammals, including humans, suggest an alternative explanation: spermatozoa have a typical Centriole and an atypical Centriole. The sperm typical Centriole has a normal structure but distinct protein composition, whereas the sperm atypical Centriole is distinct in both. During fertilization, the atypical Centriole is released into the zygote, nucleates a new Centriole and participates in spindle pole formation. Thus, the spermatozoa's atypical Centriole acts as a second Centriole in the zygote. Here, we review Centriole biology in general and especially in reproduction, we describe the discovery of the spermatozoon atypical Centriole, and we provide an updated model for Centriole inherence during sexual reproduction. While we focus on humans and other non-rodent mammals, we also provide a broader evolutionary perspective.
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Rapid Evolution of Sperm Produces Diverse Centriole Structures that Reveal the Most Rudimentary Structure Needed for Function
MDPI AG, 2018Co-Authors: Tomer Avidor-reissAbstract:Centrioles are ancient subcellular protein-based organelles that maintain a conserved number and structure across many groups of eukaryotes. Centriole number (two per cells) is tightly regulated; each pre-existing Centriole nucleates only one Centriole as the cell prepares for division. The structure of Centrioles is barrel-shaped, with a nine-fold symmetry of microtubules. This organization of microtubules is essential for the ancestral function of Centriole–cilium nucleation. In animal cells, Centrioles have gained an additional role: recruiting pericentriolar material (PCM) to form a centrosome. Therefore, it is striking that in animal spermatozoa, the Centrioles have a remarkable diversity of structures, where some are so anomalous that they are referred to as atypical Centrioles and are barely recognizable. The atypical Centriole maintains the ability to form a centrosome and nucleate a new Centriole, and therefore reveals the most rudimentary structure that is needed for Centriole function. However, the atypical Centriole appears to be incapable of forming a cilium. Here, we propose that the diversity in sperm Centriole structure is due to rapid evolution in the shape of the spermatozoa head and neck. The enhanced diversity may be driven by a combination of direct selection for novel Centriole functions and pleiotropy, which eliminates Centriole properties that are dispensable in the spermatozoa function
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Atypical Centrioles are present in Tribolium sperm.
Open Biology, 2017Co-Authors: Emily L. Fishman, Rachel Royfman, Ashtyn Zinn, Malathi Krishnamurthy, Tomer Avidor-reissAbstract:Typical Centrioles are made of microtubules organized in ninefold symmetry. Most animal somatic cells have two Centrioles for normal cell division and function. These Centrioles originate from the zygote, but because the oocyte does not provide any Centrioles, it is surprising that the zygotes of many animals are thought to inherit only one Centriole from the sperm. Recently, in the sperm of Drosophila melanogaster, we discovered a second centriolar structure, the proximal Centriole-like structure (PCL), which functions in the zygote. Whether the sperm of other insects has a second centriolar structure is unknown. Here, we characterized spermiogenesis in the red flour beetle, Tribolium castaneum Electron microscopy suggests that Tribolium has one microtubule-based Centriole at the tip of the axoneme and a structure similar to the PCL, which lacks microtubules and lies in a cytoplasmic invagination of the nucleus. Immunostaining against the orthologue of the Centriole/PCL protein, Ana1, also recognizes two Centrioles near the nucleus during spermiogenesis: one that is microtubule-based at the tip of the axoneme, suggesting it is the Centriole; and another that is more proximal and appears during early spermiogenesis, suggesting it is the PCL. Together, these findings suggest that Tribolium sperm has one microtubule-based Centriole and one microtubule-lacking Centriole.
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Atypical Centrioles during sexual reproduction.
Frontiers in Cell and Developmental Biology, 2015Co-Authors: Tomer Avidor-reiss, Atul Khire, Emily L. FishmanAbstract:Centrioles are conserved, self-replicating, microtubule-based 9-fold symmetric subcellular organelles that are essential for proper cell division and function. Most cells have two Centrioles and maintaining this number of Centrioles is important for animal development and physiology. However, how animals gain their first two Centrioles during reproduction is only partially understood. It is well established that in most animals, the Centrioles are contributed to the zygote by the sperm. However, in humans and many animals, the sperm Centrioles are modified in their structure and protein composition, or they appear to be missing altogether. In these animals, the origin of the first Centrioles is not clear. Here, we review various hypotheses on how Centrioles are gained during reproduction and describe specialized functions of the zygotic Centrioles. In particular, we discuss a new and atypical Centriole found in sperm and zygote, the proximal Centriole-like structure (PCL). We also discuss another type of atypical Centriole, the “zombie” Centriole, which is degenerated but functional. Together, the presence of Centrioles, PCL, and zombie Centrioles suggests a universal mechanism of Centriole inheritance among animals and new causes of infertility. Since the atypical Centrioles of sperm and zygote share similar functions with typical Centrioles in somatic cells, they can provide unmatched insight into Centriole biology.
Jordan W Raff - One of the best experts on this subject based on the ideXlab platform.
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cdk1 phosphorylates drosophila sas 4 to recruit polo to daughter Centrioles and convert them to centrosomes
Developmental Cell, 2016Co-Authors: Zsofia A Novak, Alan Wainman, Lisa Gartenmann, Jordan W RaffAbstract:Summary Centrosomes and cilia are organized by a Centriole pair comprising an older mother and a younger daughter. Centriole numbers are tightly regulated, and daughter Centrioles (which assemble in S phase) cannot themselves duplicate or organize centrosomes until they have passed through mitosis. It is unclear how this mitotic "Centriole conversion" is regulated, but it requires Plk1/Polo kinase. Here we show that in flies, Cdk1 phosphorylates the conserved Centriole protein Sas-4 during mitosis. This creates a Polo-docking site that helps recruit Polo to daughter Centrioles and is required for the subsequent recruitment of Asterless (Asl), a protein essential for Centriole duplication and mitotic centrosome assembly. Point mutations in Sas-4 that prevent Cdk1 phosphorylation or Polo docking do not block Centriole disengagement during mitosis, but block efficient Centriole conversion and lead to embryonic lethality. These observations can explain why daughter Centrioles have to pass through mitosis before they can duplicate and organize a centrosome.
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asterless licenses daughter Centrioles to duplicate for the first time in drosophila embryos
Current Biology, 2014Co-Authors: Zsofia A Novak, Paul T Conduit, Alan Wainman, Jordan W RaffAbstract:Centrioles form centrosomes and cilia, and defects in any of these three organelles are associated with human disease [1]. Centrioles duplicate once per cell cycle, when a mother Centriole assembles an adjacent daughter during S phase. Daughter Centrioles cannot support the assembly of another daughter until they mature into mothers during the next cell cycle [2–5]. The molecular nature of this daughter-to-mother transition remains mysterious. Pioneering studies in C. elegans identified a set of core proteins essential for Centriole duplication [6–12], and a similar set have now been identified in other species [10, 13–18]. The protein kinase ZYG-1/Sak/Plk4 recruits the inner Centriole cartwheel components SAS-6 and SAS-5/Ana2/STIL, which then recruit SAS-4/CPAP, which in turn helps assemble the outer Centriole microtubules [19, 20]. In flies and humans, the Asterless/Cep152 protein interacts with Sak/Plk4 and Sas-4/CPAP and is required for Centriole duplication, although its precise role in the assembly pathway is unclear [21–24]. Here, we show that Asl is not incorporated into daughter Centrioles as they assemble during S phase but is only incorporated once mother and daughter separate at the end of mitosis. The initial incorporation of Asterless (Asl) is irreversible, requires DSas-4, and, crucially, is essential for daughter Centrioles to mature into mothers that can support Centriole duplication. We therefore propose a “dual-licensing” model of Centriole duplication, in which Asl incorporation provides a permanent primary license to allow new Centrioles to duplicate for the first time, while Centriole disengagement provides a reduplication license to allow mother Centrioles to duplicate again.
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cnn dynamics drive centrosome size asymmetry to ensure daughter Centriole retention in drosophila neuroblasts
Current Biology, 2010Co-Authors: Paul T Conduit, Jordan W RaffAbstract:Centrosomes comprise a pair of Centrioles surrounded by an amorphous network of pericentriolar material (PCM). In certain stem cells, the two centrosomes differ in size, and this appears to be important for asymmetric cell division [1, 2]. In some cases, centrosome asymmetry is linked to Centriole age because the older, mother Centriole always organizes more PCM than the daughter Centriole, thus ensuring that the mother Centriole is always retained in the stem cell after cell division [3]. This has raised the possibility that an "immortal" mother Centriole may help maintain stem cell fate [4, 5]. It is unclear, however, how centrosome size asymmetry is generated in stem cells. Here we provide compelling evidence that centrosome size asymmetry in Drosophila neuroblasts is generated by the differential regulation of Cnn incorporation into the PCM at mother and daughter Centrioles. Shortly after Centriole separation, mother and daughter Centrioles organize similar amounts of PCM, but Cnn incorporation is then rapidly downregulated at the mother Centriole, while it is maintained at the daughter Centriole. This ensures that the daughter Centriole maintains its PCM and so its position at the apical cortex. Thus, the daughter Centriole, rather than an "immortal" mother Centriole, is ultimately retained in these stem cells.
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Overexpressing Centriole-Replication Proteins In Vivo Induces Centriole Overduplication and De Novo Formation
Current Biology, 2007Co-Authors: Nina Peel, Naomi R. Stevens, Renata Basto, Jordan W RaffAbstract:Summary Background: Centrosomes have important roles in many aspects of cell organization, and aberrations in their number and function are associated with various diseases, including cancer. Centrosomes consist of a pair of Centrioles surrounded by a pericentriolar matrix (PCM), and their replication is tightly regulated. Here, we investigate the effects of overexpressing the three proteins known to be required for Centriole replication in Drosophila—DSas-6, DSas-4, and Sak. Results: By directly observing Centriole replication in living Drosophila embryos, we show that the overexpression of GFP-DSas-6 can drive extra rounds of Centriole replication within a single cell cycle. Extra Centriole-like structures also accumulate in brain cells that overexpress either GFP-DSas-6 or GFP-Sak, but not DSas-4-GFP. No extra Centrioles accumulate in spermatocytes that overexpress any of these three proteins. Most remarkably, the overexpression of any one of these three proteins results in the rapid de novo formation of many hundreds of Centriole-like structures in unfertilized eggs, which normally do not contain Centrioles. Conclusions: Our data suggest that the levels of centriolar DSas-6 determine the number of daughter Centrioles formed during Centriole replication. Overexpression of either DSas-6 or Sak can induce the formation of extra Centrioles in some tissues but not others, suggesting that Centriole replication is regulated differently in different tissues. The finding that the overexpression of DSas-4, DSas-6, or Sak can rapidly induce the de novo formation of Centriole-like structures in Drosophila eggs suggests that this process results from the stabilization of Centriole-precursors that are normally present in the egg.
Wallace F. Marshall - One of the best experts on this subject based on the ideXlab platform.
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influence of Centriole number on mitotic spindle length and symmetry
Cytoskeleton, 2010Co-Authors: Lani C. Keller, Kimberly A Wemmer, Wallace F. MarshallAbstract:The functional role of Centrioles or basal bodies in mitotic spindle assembly and function is currently unclear. Although supernumerary Centrioles have been associated with multipolar spindles in cancer cells, suggesting Centriole number might dictate spindle polarity, bipolar spindles are able to assemble in the complete absence of Centrioles, suggesting a level of Centriole-independence in the spindle assembly pathway. In this report we perturb Centriole number using mutations in Chlamydomonas reinhardtii, and measure the response of the mitotic spindle to these perturbations in Centriole number. Although altered Centriole number increased the frequency of monopolar and multipolar spindles, the majority of spindles remained bipolar regardless of the Centriole number. But even when spindles were bipolar, abnormal Centriole numbers led to asymmetries in tubulin distribution, half-spindle length and spindle pole focus. Half spindle length correlated directly with number of Centrioles at a pole, such that an imbalance in Centriole number between the two poles of a bipolar spindle correlated with increased asymmetry between half spindle lengths. These results are consistent with Centrioles playing an active role in regulating mitotic spindle length. Mutants with Centriole number alteration also show increased cytokinesis defects, but these do not correlate with Centriole number in the dividing cell and may therefore reflect downstream consequences of defects in preceding cell divisions. © 2010 Wiley-Liss, Inc.
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Molecular Architecture of the Centriole Proteome: The Conserved WD40 Domain Protein POC1 Is Required for Centriole Duplication and Length Control
Molecular Biology of the Cell, 2009Co-Authors: Lani C. Keller, Stefan Geimer, Edwin P. Romijn, John R. Yates, Ivan Zamora, Wallace F. MarshallAbstract:Centrioles are intriguing cylindrical organelles composed of triplet microtubules. Proteomic data suggest that a large number of proteins besides tubulin are necessary for the formation and maintenance of a Centriole's complex structure. Expansion of the preexisting Centriole proteome from the green alga Chlamydomonas reinhardtii revealed additional human disease genes, emphasizing the significance of Centrioles in normal human tissue homeostasis. We found that two classes of ciliary disease genes were highly represented among the basal body proteome: cystic kidney disease (especially nephronophthisis) syndromes, including Meckel/Joubert-like and oral-facial-digital syndrome, caused by mutations in CEP290, MKS1, OFD1, and AHI1/Jouberin proteins and cone-rod dystrophy syndrome genes, including UNC-119/HRG4, NPHP4, and RPGR1. We further characterized proteome of the Centriole (POC) 1, a highly abundant WD40 domain-containing Centriole protein. We found that POC1 is recruited to nascent proCentrioles and localizes in a highly asymmetrical pattern in mature Centrioles corresponding to sites of basal-body fiber attachment. Knockdown of POC1 in human cells caused a reduction in Centriole duplication, whereas overexpression caused the appearance of elongated Centriole-like structures. Together, these data suggest that POC1 is involved in early steps of Centriole duplication as well as in the later steps of Centriole length control.
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The mother Centriole plays an instructive role in defining cell geometry.
PLoS biology, 2007Co-Authors: Jessica L. Feldman, Stefan Geimer, Wallace F. MarshallAbstract:Centriole positioning is a key step in establishment and propagation of cell geometry, but the mechanism of this positioning is unknown. The ability of pre-existing Centrioles to induce formation of new Centrioles at a defined angle relative to themselves suggests they may have the capacity to transmit spatial information to their daughters. Using three-dimensional computer-aided analysis of cell morphology in Chlamydomonas, we identify six genes required for Centriole positioning relative to overall cell polarity, four of which have known sequences. We show that the distal portion of the Centriole is critical for positioning, and that the Centriole positions the nucleus rather than vice versa. We obtain evidence that the daughter Centriole is unable to respond to normal positioning cues and relies on the mother for positional information. Our results represent a clear example of “cytotaxis” as defined by Sonneborn, and suggest that Centrioles can play a key function in propagation of cellular geometry from one generation to the next. The genes documented here that are required for proper Centriole positioning may represent a new class of ciliary disease genes, defects in which would be expected to cause disorganized ciliary position and impaired function.
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proteomic analysis of isolated chlamydomonas Centrioles reveals orthologs of ciliary disease genes
Current Biology, 2005Co-Authors: Lani C. Keller, Edwin P. Romijn, John R. Yates, Ivan Zamora, Wallace F. MarshallAbstract:Summary Background: The Centriole is one of the most enigmatic organelles in the cell. Centrioles are cylindrical, microtubule-based barrels found in the core of the centrosome. Centrioles also act as basal bodies during interphase to nucleate the assembly of cilia and flagella. There are currently only a handful of known Centriole proteins. Results: We used mass-spectrometry-based MudPIT (multidimensional protein identification technology) to identify the protein composition of basal bodies (Centrioles) isolated from the green alga Chlamydomonas reinhardtii . This analysis detected the majority of known Centriole proteins, including centrin, epsilon tubulin, and the cartwheel protein BLD10p. By combining proteomic data with information about gene expression and comparative genomics, we identified 45 cross-validated Centriole candidate proteins in two classes. Members of the first class of proteins (BUG1–BUG27) are encoded by genes whose expression correlates with flagellar assembly and which therefore may play a role in ciliogenesis-related functions of basal bodies. Members of the second class (POC1–POC18) are implicated by comparative-genomics and -proteomics studies to be conserved components of the Centriole. We confirmed centriolar localization for the human homologs of four candidate proteins. Three of the cross-validated Centriole candidate proteins are encoded by orthologs of genes ( OFD1 , NPHP-4 , and PACRG ) implicated in mammalian ciliary function and disease, suggesting that oral-facial-digital syndrome and nephronophthisis may involve a dysfunction of Centrioles and/or basal bodies. Conclusions: By analyzing isolated Chlamydomonas basal bodies, we have been able to obtain the first reported proteomic analysis of the Centriole.
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Research papersKinetics and regulation of de novo Centriole assembly: Implications for the mechanism of Centriole duplication
2001Co-Authors: Wallace F. Marshall, Yvonne Vucica, Joel L. RosenbaumAbstract:Background: Centriole duplication is a key step in the cell cycle whose mechanism is completely unknown. Why new Centrioles always form next to preexisting ones is a fundamental question. The simplest model is that preexisting Centrioles nucleate the assembly of new Centrioles, and that although Centrioles can in some cases form de novo without this nucleation, the de novo assembly mechanism should be too slow to compete with normal duplication in order to maintain fidelity of Centriole duplication. Results: We have measured the rate of de novo Centriole assembly in vegetatively dividing cells that normally always contain Centrioles. By using mutants of Chlamydomonas that are defective in Centriole segregation, we obtained viable Centrioleless cells that continue to divide, and find that within a single generation, 50% of these cells reacquire new Centrioles by de novo assembly. This suggests that the rate of de novo assembly is approximately half the rate of templated duplication. A mutation in the VFL3 gene causes a complete loss of the templated assembly pathway without eliminating de novo assembly. A mutation in the centrin gene also reduced the rate of templated assembly. Conclusions: These results suggest that there are two pathways for Centriole assembly, namely a templated pathway that requires preexisting Centrioles to nucleate new Centriole assembly, and a de novo assembly pathway that is normally turned off when Centrioles are present.