Ultrastructural diversity between centrioles of eukaryotes.

Science.gov (United States)

Gupta, Akshari; Kitagawa, Daiju

2018-02-16

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. Here, 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. Additionally, 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.Centriole structure, cartwheel, triplet microtubules, SAS-6, centrosome.

SAS-6 assembly templated by the lumen of cartwheel-less centrioles precedes centriole duplication.

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Fong, Chii Shyang; Kim, Minhee; Yang, T Tony; Liao, Jung-Chi; Tsou, Meng-Fu Bryan

2014-07-28

Centrioles are 9-fold symmetric structures duplicating once per cell cycle. Duplication involves self-oligomerization of the centriolar protein SAS-6, but how the 9-fold symmetry is invariantly established remains unclear. Here, we found that SAS-6 assembly can be shaped by preexisting (or mother) centrioles. During S phase, SAS-6 molecules are first recruited to the proximal lumen of the mother centriole, adopting a cartwheel-like organization through interactions with the luminal wall, rather than via their self-oligomerization activity. The removal or release of luminal SAS-6 requires Plk4 and the cartwheel protein STIL. Abolishing either the recruitment or the removal of luminal SAS-6 hinders SAS-6 (or centriole) assembly at the outside wall of mother centrioles. After duplication, the lumen of engaged mother centrioles becomes inaccessible to SAS-6, correlating with a block for reduplication. These results lead to a proposed model that centrioles may duplicate via a template-based process to preserve their geometry and copy number. Copyright © 2014 Elsevier Inc. All rights reserved.

From centriole biogenesis to cellular function: centrioles are essential for cell division at critical developmental stages.

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Rodrigues-Martins, Ana; Riparbelli, Maria; Callaini, Giuliano; Glover, David M; Bettencourt-Dias, Monica

2008-01-01

Centrioles are essential for the formation of cilia, flagella and centrosome organization. Abnormalities in centrosome structure and number in many cancers can be associated with aberrant cell division and genomic instability.(1,2) Canonical centriole duplication occurs in coordination with the cell division cycle, such that a single new "daughter" centriole arises next to each "mother" centriole. If destroyed, or eliminated during development, centrioles can form de novo.(3-5) Here we discuss our recent data demonstrating a molecular pathway that operates in both de novo and canonical centriole biogenesis involving SAK/PLK4, SAS-6 and SAS-4.(6) We showed that centriole biogenesis is a self-assembly process locally triggered by high SAK/PLK4 activity that may or not be associated with an existing centriole. SAS-6 acts downstream of SAK/PLK4 to organize nine precentriolar units, which we call here enatosomes, fitting together laterally and longitudinally, specifying a tube-like centriole precursor.(7,8) The identification of mutants impaired in centriole biogenesis has permitted the study of the physiological consequences of their absence in the whole organism. In Drosophila, centrioles are not necessary for somatic cell divisions.(9,10) However, we show here that mitotic abnormalities arise in syncytial SAK/PLK4-derived mutant embryos resulting in lethality. Moreover male meiosis fails in both SAK/PLK4 and DSAS-4 mutant spermatids that have no centrioles. These results show diversity in the need for centrioles in cell division. This suggests that tissue specific constraints selected for different contributions of centrosome-independent and dependent mechanisms in spindle function. This heterogeneity should be taken into account both in reaching an understanding of spindle function and when designing drugs that target cell division.

Nonequivalence of maternal centrosomes/centrioles in starfish oocytes: selective casting-off of reproductive centrioles into polar bodies.

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Uetake, Yumi; Kato, Koichi H; Washitani-Nemoto, Setsuko; Nemoto Si, Shin-ichi

2002-07-01

It is believed that in most animals only the paternal centrosome provides the division poles for mitosis in zygotes. This paternal inheritance of the centrosomes depends on the selective loss of the maternal centrosome. In order to understand the mechanism of centrosome inheritance, the behavior of all maternal centrosomes/centrioles was investigated throughout the meiotic and mitotic cycles by using starfish eggs that had polar body (PB) formation suppressed. In starfish oocytes, the centrioles do not duplicate during meiosis II. Hence, each centrosome of the meiosis II spindle has only one centriole, whereas in meiosis I, each has a pair of centrioles. When two pairs of meiosis I centrioles were retained in the cytoplasm of oocytes by complete suppression of PB extrusion, they separated into four single centrioles in meiosis II. However, after completion of the meiotic process, only two of the four single centrioles were found in addition to the pronucleus. When the two single centrioles of a meiosis II spindle were retained in the oocyte cytoplasm by suppressing the extrusion of the second PB, only one centriole was found with the pronucleus after the completion of the meiotic process. When these PB-suppressed eggs were artificially activated to drive the mitotic cycles, all the surviving single centrioles duplicated repeatedly to form pairs of centrioles, which could organize mitotic spindles. These results indicate that the maternal centrioles are not equivalent in their intrinsic stability and reproductive capacity. The centrosomes with the reproductive centrioles are selectively cast off into the PBs, resulting in the mature egg inheriting a nonreproductive centriole, which would degrade shortly after the completion of meiosis. (c) 2002 Elsevier Science (USA).

Centriole Remodeling during Spermiogenesis in Drosophila.

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Khire, Atul; Jo, Kyoung H; Kong, Dong; Akhshi, Tara; Blachon, Stephanie; Cekic, Anthony R; Hynek, Sarah; Ha, Andrew; Loncarek, Jadranka; Mennella, Vito; Avidor-Reiss, Tomer

2016-12-05

The first cell of an animal (zygote) requires centrosomes that are assembled from paternally inherited centrioles and maternally inherited pericentriolar material (PCM) [1]. In some animals, sperm centrioles with typical ultrastructure are the origin of the first centrosomes in the zygote [2-4]. In other animals, however, sperm centrioles lose their proteins and are thought to be degenerated and non-functional during spermiogenesis [5, 6]. Here, we show that the two sperm centrioles (the giant centriole [GC] and the proximal centriole-like structure [PCL]) in Drosophila melanogaster are remodeled during spermiogenesis through protein enrichment and ultrastructure modification in parallel to previously described centrosomal reduction [7]. We found that the ultrastructure of the matured sperm (spermatozoa) centrioles is modified dramatically and that the PCL does not resemble a typical centriole. We also describe a new phenomenon of Poc1 enrichment of the atypical centrioles in the spermatozoa. Using various mutants, protein expression during spermiogenesis, and RNAi knockdown of paternal Poc1, we found that paternal Poc1 enrichment is essential for the formation of centrioles during spermiogenesis and for the formation of centrosomes after fertilization in the zygote. Altogether, these findings demonstrate that the sperm centrioles are remodeled both in their protein composition and in ultrastructure, yet they are functional and are essential for normal embryogenesis in Drosophila. Copyright © 2016 Elsevier Ltd. All rights reserved.

Centrobin-Centrosomal Protein 4.1-associated Protein (CPAP) Interaction Promotes CPAP Localization to the Centrioles during Centriole Duplication*

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Gudi, Radhika; Zou, Chaozhong; Dhar, Jayeeta; Gao, Qingshen; Vasu, Chenthamarakshan

2014-01-01

Centriole duplication is the process by which two new daughter centrioles are generated from the proximal end of preexisting mother centrioles. Accurate centriole duplication is important for many cellular and physiological events, including cell division and ciliogenesis. Centrosomal protein 4.1-associated protein (CPAP), centrosomal protein of 152 kDa (CEP152), and centrobin are known to be essential for centriole duplication. However, the precise mechanism by which they contribute to centriole duplication is not known. In this study, we show that centrobin interacts with CEP152 and CPAP, and the centrobin-CPAP interaction is critical for centriole duplication. Although depletion of centrobin from cells did not have an effect on the centriolar levels of CEP152, it caused the disappearance of CPAP from both the preexisting and newly formed centrioles. Moreover, exogenous expression of the CPAP-binding fragment of centrobin also caused the disappearance of CPAP from both the preexisting and newly synthesized centrioles, possibly in a dominant negative manner, thereby inhibiting centriole duplication and the PLK4 overexpression-mediated centrosome amplification. Interestingly, exogenous overexpression of CPAP in the centrobin-depleted cells did not restore CPAP localization to the centrioles. However, restoration of centrobin expression in the centrobin-depleted cells led to the reappearance of centriolar CPAP. Hence, we conclude that centrobin-CPAP interaction is critical for the recruitment of CPAP to procentrioles to promote the elongation of daughter centrioles and for the persistence of CPAP on preexisting mother centrioles. Our study indicates that regulation of CPAP levels on the centrioles by centrobin is critical for preserving the normal size, shape, and number of centrioles in the cell. PMID:24700465

Centrobin-centrosomal protein 4.1-associated protein (CPAP) interaction promotes CPAP localization to the centrioles during centriole duplication.

Science.gov (United States)

Gudi, Radhika; Zou, Chaozhong; Dhar, Jayeeta; Gao, Qingshen; Vasu, Chenthamarakshan

2014-05-30

Centriole duplication is the process by which two new daughter centrioles are generated from the proximal end of preexisting mother centrioles. Accurate centriole duplication is important for many cellular and physiological events, including cell division and ciliogenesis. Centrosomal protein 4.1-associated protein (CPAP), centrosomal protein of 152 kDa (CEP152), and centrobin are known to be essential for centriole duplication. However, the precise mechanism by which they contribute to centriole duplication is not known. In this study, we show that centrobin interacts with CEP152 and CPAP, and the centrobin-CPAP interaction is critical for centriole duplication. Although depletion of centrobin from cells did not have an effect on the centriolar levels of CEP152, it caused the disappearance of CPAP from both the preexisting and newly formed centrioles. Moreover, exogenous expression of the CPAP-binding fragment of centrobin also caused the disappearance of CPAP from both the preexisting and newly synthesized centrioles, possibly in a dominant negative manner, thereby inhibiting centriole duplication and the PLK4 overexpression-mediated centrosome amplification. Interestingly, exogenous overexpression of CPAP in the centrobin-depleted cells did not restore CPAP localization to the centrioles. However, restoration of centrobin expression in the centrobin-depleted cells led to the reappearance of centriolar CPAP. Hence, we conclude that centrobin-CPAP interaction is critical for the recruitment of CPAP to procentrioles to promote the elongation of daughter centrioles and for the persistence of CPAP on preexisting mother centrioles. Our study indicates that regulation of CPAP levels on the centrioles by centrobin is critical for preserving the normal size, shape, and number of centrioles in the cell. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

What is the function of centrioles?

Science.gov (United States)

Marshall, Wallace F

2007-03-01

The function of centrioles has been controversial and remains incompletely resolved. This is because centrioles, in and of themselves, do not directly perform any physiological activity. Instead, their role is only to act as a jig or breadboard onto which other functional structures can be built. Centrioles are primarily involved in forming two structures-centrosomes and cilia. Centrioles bias the position of spindle pole formation, but because spindle poles can self-organize, the function of the centriole in mitosis is not obligatory. Consequently, lack of centrioles does not generally prevent mitosis, although recent experiments suggest acentriolar spindles have reduced fidelity of chromosome segregation. In contrast, centrioles are absolutely required for the assembly of cilia, including primary cilia that act as cellular antennae. Consistent with this requirement, it is now becoming clear that many ciliary diseases, including nephronophthisis, Bardet-Biedl syndrome, Meckel Syndrome, and Oral-Facial-Digital syndrome, are caused by defects in centriole-associated proteins.

Atypical centrioles are present in Tribolium sperm.

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Fishman, E L; Jo, Kyoung; Ha, Andrew; Royfman, Rachel; Zinn, Ashtyn; Krishnamurthy, Malathi; Avidor-Reiss, Tomer

2017-03-01

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. © 2017 The Authors.

Mode of centriole duplication and distribution

OpenAIRE

1990-01-01

Centriole stability and distribution during the mammalian cell cycle was studied by microinjecting biotinylated tubulin into early G1 cells and analyzing the pattern of incorporation into centrioles. Cells were extracted and cold treated to depolymerize labile microtubules, allowing the fluorescent microscopic visualization of the stable centrioles. The ability to detect single centrioles was confirmed by use of correlative electron microscopy. Indirect hapten and immunofluorescent labeling o...

Centrioles in Symmetric Spaces

OpenAIRE

Quast, Peter

2011-01-01

We describe all centrioles in irreducible simply connected pointed symmetric spaces of compact type in terms of the root system of the ambient space, and we study some geometric properties of centrioles.

PLK4 trans-Autoactivation Controls Centriole Biogenesis in Space.

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Lopes, Carla A M; Jana, Swadhin Chandra; Cunha-Ferreira, Inês; Zitouni, Sihem; Bento, Inês; Duarte, Paulo; Gilberto, Samuel; Freixo, Francisco; Guerrero, Adán; Francia, Maria; Lince-Faria, Mariana; Carneiro, Jorge; Bettencourt-Dias, Mónica

2015-10-26

Centrioles are essential for cilia and centrosome assembly. In centriole-containing cells, centrioles always form juxtaposed to pre-existing ones, motivating a century-old debate on centriole biogenesis control. Here, we show that trans-autoactivation of Polo-like kinase 4 (PLK4), the trigger of centriole biogenesis, is a critical event in the spatial control of that process. We demonstrate that centrioles promote PLK4 activation through its recruitment and local accumulation. Though centriole removal reduces the proportion of active PLK4, this is rescued by concentrating PLK4 to the peroxisome lumen. Moreover, while mild overexpression of PLK4 only triggers centriole amplification at the existing centriole, higher PLK4 levels trigger both centriolar and cytoplasmatic (de novo) biogenesis. Hence, centrioles promote their assembly locally and disfavor de novo synthesis. Similar mechanisms enforcing the local concentration and/or activity of other centriole components are likely to contribute to the spatial control of centriole biogenesis under physiological conditions. Copyright © 2015 Elsevier Inc. All rights reserved.

Isolation and proteomic analysis of Chlamydomonas centrioles.

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Keller, Lani C; Marshall, Wallace F

2008-01-01

Centrioles are barrel-shaped cytoskeletal organelles composed of nine triplet microtubules blades arranged in a pinwheel-shaped array. Centrioles are required for recruitment of pericentriolar material (PCM) during centrosome formation, and they act as basal bodies, which are necessary for the outgrowth of cilia and flagella. Despite being described over a hundred years ago, centrioles are still among the most enigmatic organelles in all of cell biology. To gain molecular insights into the function and assembly of centrioles, we sought to determine the composition of the centriole proteome. Here, we describe a method that allows for the isolation of virtually "naked" centrioles, with little to no obscuring PCM, from the green alga, Chlamydomonas. Proteomic analysis of this material provided evidence that multiple human disease gene products encode protein components of the centriole, including genes involved in Meckel syndrome and Oral-Facial-Digital syndrome. Isolated centrioles can be used in combination with a wide variety of biochemical assays in addition to being utilized as a source for proteomic analysis.

Electron tomography study of isolated human centrioles.

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Ibrahim, Rana; Messaoudi, Cédric; Chichon, Francisco Javier; Celati, Claude; Marco, Sergio

2009-01-01

Centrioles are components of the centrosome, which is present in most eukaryotic cells (from protozoa to mammals). They organize the microtubule skeleton during interphase and the mitotic spindle during cell division. In ciliate cells, centrioles form basal bodies that are involved in cellular motility. Despite their important roles in biology, the detailed structure of centrioles remains obscure. This work contributes to a more complete model of centriole structure. The authors used electron tomography of isolated centrosomes from the human lymphoblast KE37 to explore the details of subdistal appendages and centriole lumen organization in mother centrioles. Their results reveal that each of the nine subdistal appendages is composed of two halves (20 nm diameter each) fused in a 40 nm tip that extends 100 nm from where it anchors to microtubules. The centriole lumen is filled at the distal domain by a 45 nm periodic stack of rings. Each ring has a 30 nm diameter, is 15 nm thick, and appears to be tilted at 53 degrees perpendicular to the centriole axis. The rings are anchored to microtubules by arms. Based on their results, the authors propose a model of the mother centriole distal structure. Copyright 2008 Wiley-Liss, Inc.

Centriole movements in mammalian epithelial cells during cytokinesis

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Tanke Hans J

2010-05-01

Full Text Available Abstract Background In cytokinesis, when the cleavage furrow has been formed, the two centrioles in each daughter cell separate. It has been suggested that the centrioles facilitate and regulate cytokinesis to some extent. It has been postulated that termination of cytokinesis (abscission depends on the migration of a centriole to the intercellular bridge and then back to the cell center. To investigate the involvement of centrioles in cytokinesis, we monitored the movements of centrioles in three mammalian epithelial cell lines, HeLa, MCF 10A, and the p53-deficient mouse mammary tumor cell line KP-7.7, by time-lapse imaging. Centrin1-EGFP and α-Tubulin-mCherry were co-expressed in the cells to visualize respectively the centrioles and microtubules. Results Here we report that separated centrioles that migrate from the cell pole are very mobile during cytokinesis and their movements can be characterized as 1 along the nuclear envelope, 2 irregular, and 3 along microtubules forming the spindle axis. Centriole movement towards the intercellular bridge was only seen occasionally and was highly cell-line dependent. Conclusions These findings show that centrioles are highly mobile during cytokinesis and suggest that the repositioning of a centriole to the intercellular bridge is not essential for controlling abscission. We suggest that centriole movements are microtubule dependent and that abscission is more dependent on other mechanisms than positioning of centrioles.

Conserved molecular interactions in centriole-to-centrosome conversion.

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Fu, Jingyan; Lipinszki, Zoltan; Rangone, Hélène; Min, Mingwei; Mykura, Charlotte; Chao-Chu, Jennifer; Schneider, Sandra; Dzhindzhev, Nikola S; Gottardo, Marco; Riparbelli, Maria Giovanna; Callaini, Giuliano; Glover, David M

2016-01-01

Centrioles are required to assemble centrosomes for cell division and cilia for motility and signalling. New centrioles assemble perpendicularly to pre-existing ones in G1-S and elongate throughout S and G2. Fully elongated daughter centrioles are converted into centrosomes during mitosis to be able to duplicate and organize pericentriolar material in the next cell cycle. Here we show that centriole-to-centrosome conversion requires sequential loading of Cep135, Ana1 (Cep295) and Asterless (Cep152) onto daughter centrioles during mitotic progression in both Drosophila melanogaster and human. This generates a molecular network spanning from the inner- to outermost parts of the centriole. Ana1 forms a molecular strut within the network, and its essential role can be substituted by an engineered fragment providing an alternative linkage between Asterless and Cep135. This conserved architectural framework is essential for loading Asterless or Cep152, the partner of the master regulator of centriole duplication, Plk4. Our study thus uncovers the molecular basis for centriole-to-centrosome conversion that renders daughter centrioles competent for motherhood.

Plk2 regulated centriole duplication is dependent on its localization to the centrioles and a functional polo-box domain.

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Cizmecioglu, Onur; Warnke, Silke; Arnold, Marc; Duensing, Stefan; Hoffmann, Ingrid

2008-11-15

In mammalian cells, the centrosome consists of a pair of centrioles and amorphous pericentriolar material. The centrosome duplicates once per cell cycle. Polo like kinases (Plks) perform crucial functions in cell cycle progression and during mitosis. The polo-like kinase-2, Plk2, is activated near the G(1)/S phase transition, and plays an important role in the reproduction of centrosomes. In this study, we show that the polo-box of Plk2 is required both for association to the centrosome and centriole duplication. Mutation of critical sites in the Plk2 polo-box prevents centrosomal localization and impairs centriole duplication. Plk2 is localized to centrosomes during early G(1) phase where it only associates to the mother centriole and then distributes equally to both mother and daughter centrioles at the onset of S phase. Furthermore, our results imply that Plk2 mediated centriole duplication is dependent on Plk4 function. In addition, we find that siRNA-mediated downregulation of Plk2 leads to the formation of abnormal mitotic spindles confirming that Plk2 may have a function in the reproduction of centrioles.

Centriole, differentiation, and senescence.

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Tkemaladze, J; Chichinadze, K

2010-01-01

Irreversible differentiation (change of morphogenetic status) and programmed death (apoptosis) are observed only in somatic cells, and cell division is the only way by which the morphogenetic status of the offspring cells may be modified. It is known that there is a fixed limit to the number of possible cell divisions, the so-called Hayflick limit. Existing links between cell division, differentiation, and apoptosis make it possible to conclude that all of these processes could be controlled by a single self-reproducing structure. Potential candidates for this replicable structure in a somatic cell are the chromosomes, mitochondria (both contain DNA), and centrioles. Centrioles (a diplosome, or pair of centrioles) are the most likely unit that can fully regulate the processes of irreversible differentiation, determination, and modification of the morphogenetic status. Centrioles may contain differently encoded RNA molecules stacked in a definite order, and during mitosis, these RNA molecules are released one by one into the cytoplasm. In the presence of reverse transcriptase and endonuclease, processing of this RNA presumably changes the status of repressed and potentially active genes and, subsequently, the morphogenetic status of a cell.

Isolation, cryotomography, and three-dimensional reconstruction of centrioles.

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Guichard, Paul; Hamel, Virginie; Neves, Aitana; Gönczy, Pierre

2015-01-01

Centrioles and basal bodies (referred to hereafter as centrioles for simplicity) are microtubule-based cylindrical organelles that are typically ∼450-nm long and ∼250nm in diameter. The centriole is composed of three distinct regions: the distal part characterized by microtubule doublets, the central core that harbors microtubule triplets, which are also present in the proximal part that also contains the cartwheel, a structure crucial for centriole assembly. The cartwheel was initially revealed by conventional electron microscopy of resin-embedded samples and is thought to impart the near universal ninefold symmetry of centrioles. Deciphering the native architecture of the cartwheel has proven challenging owing to its small dimensions and the difficulties in isolating it. Here, we present a method to purify and analyze the structure of the exceptionally long Trichonympha centriole by cryotomography and subtomogram averaging. Using this method, we revealed the native architecture of the proximal cartwheel-containing region at ∼40Å-resolution. This method can be applied as a general strategy for uncovering the structure of centrioles in other species. Copyright © 2015 Elsevier Inc. All rights reserved.

CP110 exhibits novel regulatory activities during centriole assembly in Drosophila

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Franz, Anna; Roque, Hélio; Saurya, Saroj; Dobbelaere, Jeroen

2013-01-01

CP110 is a conserved centriole protein implicated in the regulation of cell division, centriole duplication, and centriole length and in the suppression of ciliogenesis. Surprisingly, we report that mutant flies lacking CP110 (CP110Δ) were viable and fertile and had no obvious defects in cell division, centriole duplication, or cilia formation. We show that CP110 has at least three functions in flies. First, it subtly influences centriole length by counteracting the centriole-elongating activity of several centriole duplication proteins. Specifically, we report that centrioles are ∼10% longer than normal in CP110Δ mutants and ∼20% shorter when CP110 is overexpressed. Second, CP110 ensures that the centriolar microtubules do not extend beyond the distal end of the centriole, as some centriolar microtubules can be more than 50 times longer than the centriole in the absence of CP110. Finally, and unexpectedly, CP110 suppresses centriole overduplication induced by the overexpression of centriole duplication proteins. These studies identify novel and surprising functions for CP110 in vivo in flies. PMID:24297749

Asterless is required for centriole length control and sperm development

Science.gov (United States)

Galletta, Brian J.; Jacobs, Katherine C.; Fagerstrom, Carey J.

2016-01-01

Centrioles are the foundation of two organelles, centrosomes and cilia. Centriole numbers and functions are tightly controlled, and mutations in centriole proteins are linked to a variety of diseases, including microcephaly. Loss of the centriole protein Asterless (Asl), the Drosophila melanogaster orthologue of Cep152, prevents centriole duplication, which has limited the study of its nonduplication functions. Here, we identify populations of cells with Asl-free centrioles in developing Drosophila tissues, allowing us to assess its duplication-independent function. We show a role for Asl in controlling centriole length in germline and somatic tissue, functioning via the centriole protein Cep97. We also find that Asl is not essential for pericentriolar material recruitment or centrosome function in organizing mitotic spindles. Lastly, we show that Asl is required for proper basal body function and spermatid axoneme formation. Insights into the role of Asl/Cep152 beyond centriole duplication could help shed light on how Cep152 mutations lead to the development of microcephaly. PMID:27185836

Pix proteins and the evolution of centrioles.

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Woodland, Hugh R; Fry, Andrew M

2008-01-01

We have made a wide phylogenetic survey of Pix proteins, which are constituents of vertebrate centrioles in most eukaryotes. We have also surveyed the presence and structure of flagella or cilia and centrioles in these organisms, as far as is possible from published information. We find that Pix proteins are present in a vast range of eukaryotes, but not all. Where centrioles are absent so are Pix proteins. If one considers the maintenance of Pix proteins over evolutionary time scales, our analysis would suggest that their key function is to make cilia and flagella, and the same is true of centrioles. Moreover, this survey raises the possibility that Pix proteins are only maintained to make cilia and flagella that undulate, and even then only when they are constructed by transporting ciliary constituents up the cilium using the intraflagellar transport (IFT) system. We also find that Pix proteins have become generally divergent within Ecdysozoa and between this group and other taxa. This correlates with a simplification of centrioles within Ecdysozoa and a loss or divergence of cilia/flagella. Thus Pix proteins act as a weathervane to indicate changes in centriole function, whose core activity is to make cilia and flagella.

Centrobin–tubulin interaction is required for centriole elongation and stability

Science.gov (United States)

Gudi, Radhika; Zou, Chaozhong; Li, Jun

2011-01-01

Centrobin is a daughter centriole protein that is essential for centrosome duplication. However, the molecular mechanism by which centrobin functions during centriole duplication remains undefined. In this study, we show that centrobin interacts with tubulin directly, and centrobin–tubulin interaction is pivotal for the function of centrobin during centriole duplication. We found that centrobin is recruited to the centriole biogenesis site via its interaction with tubulins during the early stage of centriole biogenesis, and its recruitment is dependent on hSAS-6 but not centrosomal P4.1–associated protein (CPAP) and CP110. The function of centrobin is also required for the elongation of centrioles, which is likely mediated by its interaction with tubulin. Furthermore, disruption of centrobin–tubulin interaction led to destabilization of existing centrioles and the preformed procentriole-like structures induced by CPAP expression, indicating that centrobin–tubulin interaction is critical for the stability of centrioles. Together, our study demonstrates that centrobin facilitates the elongation and stability of centrioles via its interaction with tubulins. PMID:21576394

The Origin of the Second Centriole in the Zygote of Drosophila melanogaster

Science.gov (United States)

Blachon, Stephanie; Khire, Atul; Avidor-Reiss, Tomer

2014-01-01

Centrosomes are composed of two centrioles surrounded by pericentriolar material (PCM). However, the sperm and the oocyte modify or lose their centrosomes. Consequently, how the zygote establishes its first centrosome, and in particular, the origin of the second zygotic centriole, is uncertain. Drosophila melanogaster spermatids contain a single centriole called the Giant Centriole (GC) and a Proximal centriole-like (PCL) structure whose function is unknown. We found that, like the centriole, the PCL loses its protein markers at the end of spermiogenesis. After fertilization, the first two centrioles are observed via the recruitment of the zygotic PCM proteins and are seen in asterless mutant embryos that cannot form centrioles. The zygote’s centriolar proteins label only the daughter centrioles of the first two centrioles. These observations demonstrate that the PCL is the origin for the second centriole in the Drosophila zygote and that a paternal centriole precursor, without centriolar proteins, is transmitted to the egg during fertilization. PMID:24532732

p53 protects against genome instability following centriole duplication failure

Science.gov (United States)

Lambrus, Bramwell G.; Uetake, Yumi; Clutario, Kevin M.; Daggubati, Vikas; Snyder, Michael; Sluder, Greenfield

2015-01-01

Centriole function has been difficult to study because of a lack of specific tools that allow persistent and reversible centriole depletion. Here we combined gene targeting with an auxin-inducible degradation system to achieve rapid, titratable, and reversible control of Polo-like kinase 4 (Plk4), a master regulator of centriole biogenesis. Depletion of Plk4 led to a failure of centriole duplication that produced an irreversible cell cycle arrest within a few divisions. This arrest was not a result of a prolonged mitosis, chromosome segregation errors, or cytokinesis failure. Depleting p53 allowed cells that fail centriole duplication to proliferate indefinitely. Washout of auxin and restoration of endogenous Plk4 levels in cells that lack centrioles led to the penetrant formation of de novo centrioles that gained the ability to organize microtubules and duplicate. In summary, we uncover a p53-dependent surveillance mechanism that protects against genome instability by preventing cell growth after centriole duplication failure. PMID:26150389

The presence of centrioles and centrosomes in ovarian mature cystic teratoma cells suggests human parthenotes developed in vitro can differentiate into mature cells without a sperm centriole

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Lee, Bo Yon, E-mail: boyonlee@gmail.com [Department of Obstetrics and Gynecology, Kyung Hee University Hospital, Kyung Hee University, School of Medicine, Seoul (Korea, Republic of); Shim, Sang Woo; Kim, Young Sun; Kim, Seung Bo [Department of Obstetrics and Gynecology, Kyung Hee University Hospital, Kyung Hee University, School of Medicine, Seoul (Korea, Republic of)

2011-11-18

Highlights: Black-Right-Pointing-Pointer The sperm centriole is the progenitor of centrosomes in all somatic cells. Black-Right-Pointing-Pointer Centrioles and centrosomes exist in parthenogenetic ovarian teratoma cells. Black-Right-Pointing-Pointer Without a sperm centriole, parthenogenetic oocytes produce centrioles and centrosomes. Black-Right-Pointing-Pointer Parthenogenetic human oocytes can develop and differentiate into mature cells. -- Abstract: In most animals, somatic cell centrosomes are inherited from the centriole of the fertilizing spermatozoa. The oocyte centriole degenerates during oogenesis, and completely disappears in metaphase II. Therefore, the embryos generated by in vitro parthenogenesis are supposed to develop without any centrioles. Exceptional acentriolar and/or acentrosomal developments are possible in mice and in some experimental cells; however, in most animals, the full developmental potential of parthenogenetic cells in vitro and the fate of their centrioles/centrosomes are not clearly understood. To predict the future of in vitro human parthenogenesis, we explored the centrioles/centrosomes in ovarian mature cystic teratoma cells by immunofluorescent staining and transmission electron microscopy. We confirmed the presence of centrioles and centrosomes in these well-known parthenogenetic ovarian tumor cells. Our findings clearly demonstrate that, even without a sperm centriole, parthenotes that develop from activated oocytes can produce their own centrioles/centrosomes, and can even develop into the well-differentiated mature tissue.

The presence of centrioles and centrosomes in ovarian mature cystic teratoma cells suggests human parthenotes developed in vitro can differentiate into mature cells without a sperm centriole

International Nuclear Information System (INIS)

Lee, Bo Yon; Shim, Sang Woo; Kim, Young Sun; Kim, Seung Bo

2011-01-01

Highlights: ► The sperm centriole is the progenitor of centrosomes in all somatic cells. ► Centrioles and centrosomes exist in parthenogenetic ovarian teratoma cells. ► Without a sperm centriole, parthenogenetic oocytes produce centrioles and centrosomes. ► Parthenogenetic human oocytes can develop and differentiate into mature cells. -- Abstract: In most animals, somatic cell centrosomes are inherited from the centriole of the fertilizing spermatozoa. The oocyte centriole degenerates during oogenesis, and completely disappears in metaphase II. Therefore, the embryos generated by in vitro parthenogenesis are supposed to develop without any centrioles. Exceptional acentriolar and/or acentrosomal developments are possible in mice and in some experimental cells; however, in most animals, the full developmental potential of parthenogenetic cells in vitro and the fate of their centrioles/centrosomes are not clearly understood. To predict the future of in vitro human parthenogenesis, we explored the centrioles/centrosomes in ovarian mature cystic teratoma cells by immunofluorescent staining and transmission electron microscopy. We confirmed the presence of centrioles and centrosomes in these well-known parthenogenetic ovarian tumor cells. Our findings clearly demonstrate that, even without a sperm centriole, parthenotes that develop from activated oocytes can produce their own centrioles/centrosomes, and can even develop into the well-differentiated mature tissue.

Pix proteins and the evolution of centrioles.

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Hugh R Woodland

Full Text Available We have made a wide phylogenetic survey of Pix proteins, which are constituents of vertebrate centrioles in most eukaryotes. We have also surveyed the presence and structure of flagella or cilia and centrioles in these organisms, as far as is possible from published information. We find that Pix proteins are present in a vast range of eukaryotes, but not all. Where centrioles are absent so are Pix proteins. If one considers the maintenance of Pix proteins over evolutionary time scales, our analysis would suggest that their key function is to make cilia and flagella, and the same is true of centrioles. Moreover, this survey raises the possibility that Pix proteins are only maintained to make cilia and flagella that undulate, and even then only when they are constructed by transporting ciliary constituents up the cilium using the intraflagellar transport (IFT system. We also find that Pix proteins have become generally divergent within Ecdysozoa and between this group and other taxa. This correlates with a simplification of centrioles within Ecdysozoa and a loss or divergence of cilia/flagella. Thus Pix proteins act as a weathervane to indicate changes in centriole function, whose core activity is to make cilia and flagella.

Male gametogenesis without centrioles.

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Riparbelli, Maria Giovanna; Callaini, Giuliano

2011-01-15

The orientation of the mitotic spindle plays a central role in specifying stem cell-renewal by enabling interaction of the daughter cells with external cues: the daughter cell closest to the hub region is instructed to self-renew, whereas the distal one starts to differentiate. Here, we have analyzed male gametogenesis in DSas-4 Drosophila mutants and we have reported that spindle alignment and asymmetric divisions are properly executed in male germline stem cells that lack centrioles. Spermatogonial divisions also correctly proceed in the absence of centrioles, giving rise to cysts of 16 primary spermatocytes. By contrast, abnormal meiotic spindles assemble in primary spermatocytes. These results point to different requirements for centrioles during male gametogenesis of Drosophila. Spindle formation during germ cell mitosis may be successfully supported by an acentrosomal pathway that is inadequate to warrant the proper execution of meiosis. Copyright © 2010 Elsevier Inc. All rights reserved.

Centrioles in the beginning of human development.

OpenAIRE

Sathananthan, A H; Kola, I; Osborne, J; Trounson, A; Ng, S C; Bongso, A; Ratnam, S S

1991-01-01

We demonstrate the presence of centrioles in fertilized human oocytes at syngamy. Single or double centrioles within centrosomes were detected by transmission electron microscopy at one pole of the first cleavage spindle in normal and dispermic embryos (25-26 hr after insemination). Sperm centrioles were also closely associated with the male pronucleus (16-20 hr after insemination) in pronuclear stage embryos. A tripolar spindle derived from a tripronuclear embryo is also demonstrated with tw...

A homeostatic clock sets daughter centriole size in flies

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Aydogan, Mustafa G.; Steinacker, Thomas L.; Novak, Zsofia A.; Baumbach, Janina; Muschalik, Nadine

2018-01-01

Centrioles are highly structured organelles whose size is remarkably consistent within any given cell type. New centrioles are born when Polo-like kinase 4 (Plk4) recruits Ana2/STIL and Sas-6 to the side of an existing “mother” centriole. These two proteins then assemble into a cartwheel, which grows outwards to form the structural core of a new daughter. Here, we show that in early Drosophila melanogaster embryos, daughter centrioles grow at a linear rate during early S-phase and abruptly stop growing when they reach their correct size in mid- to late S-phase. Unexpectedly, the cartwheel grows from its proximal end, and Plk4 determines both the rate and period of centriole growth: the more active the centriolar Plk4, the faster centrioles grow, but the faster centriolar Plk4 is inactivated and growth ceases. Thus, Plk4 functions as a homeostatic clock, establishing an inverse relationship between growth rate and period to ensure that daughter centrioles grow to the correct size. PMID:29500190

The conversion of centrioles to centrosomes: essential coupling of duplication with segregation.

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Wang, Won-Jing; Soni, Rajesh Kumar; Uryu, Kunihiro; Tsou, Meng-Fu Bryan

2011-05-16

Centrioles are self-reproducing organelles that form the core structure of centrosomes or microtubule-organizing centers (MTOCs). However, whether duplication and MTOC organization reflect innate activities of centrioles or activities acquired conditionally is unclear. In this paper, we show that newly formed full-length centrioles had no inherent capacity to duplicate or to organize pericentriolar material (PCM) but acquired both after mitosis through a Plk1-dependent modification that occurred in early mitosis. Modified centrioles initiated PCM recruitment in G1 and segregated equally in mitosis through association with spindle poles. Conversely, unmodified centrioles segregated randomly unless passively tethered to modified centrioles. Strikingly, duplication occurred only in centrioles that were both modified and disengaged, whereas unmodified centrioles, engaged or not, were "infertile," indicating that engagement specifically blocks modified centrioles from reduplication. These two requirements, centriole modification and disengagement, fully exclude unlimited duplication in one cell cycle. We thus uncovered a Plk1-dependent mechanism whereby duplication and segregation are coupled to maintain centriole homeostasis.

Mother centrioles do a cartwheel to produce just one daughter.

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Chen, Jieyan V; Megraw, Timothy L

2014-07-28

In this issue of Developmental Cell, Fong et al. (2014) present evidence for a model of centriole duplication whereby the cartwheel-the starting building block in centriole biogenesis-assembles within the lumen of the mother centriole before templating the daughter centriole to ensure a single duplication event per cell cycle. Copyright © 2014 Elsevier Inc. All rights reserved.

The interrelationship between APC/C and Plk1 activities in centriole disengagement

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Toshiyuki Hatano

2012-09-01

Mother–daughter centriole disengagement, the necessary first step in centriole duplication, involves Plk1 activity in early mitosis and separase activity after APC/C activity mediates securin degradation. Plk1 activity is thought to be essential and sufficient for centriole disengagement with separase activity playing a supporting but non-essential role. In separase null cells, however, centriole disengagement is substantially delayed. The ability of APC/C activity alone to mediate centriole disengagement has not been directly tested. We investigate the interrelationship between Plk1 and APC/C activities in disengaging centrioles in S or G2 HeLa and RPE1 cells, cell types that do not reduplicate centrioles when arrested in S phase. Knockdown of the interphase APC/C inhibitor Emi1 leads to centriole disengagement and reduplication of the mother centrioles, though this is slow. Strong inhibition of Plk1 activity, if any, during S does not block centriole disengagement and mother centriole reduplication in Emi1 depleted cells. Centriole disengagement depends on APC/C–Cdh1 activity, not APC/C–Cdc20 activity. Also, Plk1 and APC/C–Cdh1 activities can independently promote centriole disengagement in G2 arrested cells. Thus, Plk1 and APC/C–Cdh1 activities are independent but slow pathways for centriole disengagement. By having two slow mechanisms for disengagement working together, the cell ensures that centrioles will not prematurely separate in late G2 or early mitosis, thereby risking multipolar spindle assembly, but rather disengage in a timely fashion only late in mitosis.

CDK1 Prevents Unscheduled PLK4-STIL Complex Assembly in Centriole Biogenesis.

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Zitouni, Sihem; Francia, Maria E; Leal, Filipe; Montenegro Gouveia, Susana; Nabais, Catarina; Duarte, Paulo; Gilberto, Samuel; Brito, Daniela; Moyer, Tyler; Kandels-Lewis, Steffi; Ohta, Midori; Kitagawa, Daiju; Holland, Andrew J; Karsenti, Eric; Lorca, Thierry; Lince-Faria, Mariana; Bettencourt-Dias, Mónica

2016-05-09

Centrioles are essential for the assembly of both centrosomes and cilia. Centriole biogenesis occurs once and only once per cell cycle and is temporally coordinated with cell-cycle progression, ensuring the formation of the right number of centrioles at the right time. The formation of new daughter centrioles is guided by a pre-existing, mother centriole. The proximity between mother and daughter centrioles was proposed to restrict new centriole formation until they separate beyond a critical distance. Paradoxically, mother and daughter centrioles overcome this distance in early mitosis, at a time when triggers for centriole biogenesis Polo-like kinase 4 (PLK4) and its substrate STIL are abundant. Here we show that in mitosis, the mitotic kinase CDK1-CyclinB binds STIL and prevents formation of the PLK4-STIL complex and STIL phosphorylation by PLK4, thus inhibiting untimely onset of centriole biogenesis. After CDK1-CyclinB inactivation upon mitotic exit, PLK4 can bind and phosphorylate STIL in G1, allowing pro-centriole assembly in the subsequent S phase. Our work shows that complementary mechanisms, such as mother-daughter centriole proximity and CDK1-CyclinB interaction with centriolar components, ensure that centriole biogenesis occurs once and only once per cell cycle, raising parallels to the cell-cycle regulation of DNA replication and centromere formation. Copyright © 2016 Elsevier Ltd. All rights reserved.

Double life of centrioles: CP110 in the spotlight.

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Bettencourt-Dias, Mónica; Carvalho-Santos, Zita

2008-01-01

Centrioles lead an important double life: they can give rise to the centrosome or convert to basal bodies and template cilia. Little is known about the control of centriole fate. Spektor and colleagues have now identified a centriolar complex, composed of CP110 and CEP97, which inhibits centriole to basal body conversion, preventing cilia formation. This work paves the way to understanding centriole and cilia biogenesis, which are two processes misregulated in human diseases, such as cancer and polycystic kidney disease.

Drosophila Ana1 is required for centrosome assembly and centriole elongation.

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Saurya, Saroj; Roque, Hélio; Novak, Zsofia A; Wainman, Alan; Aydogan, Mustafa G; Volanakis, Adam; Sieber, Boris; Pinto, David Miguel Susano; Raff, Jordan W

2016-07-01

Centrioles organise centrosomes and cilia, and these organelles have an important role in many cell processes. In flies, the centriole protein Ana1 is required for the assembly of functional centrosomes and cilia. It has recently been shown that Cep135 (also known as Bld10) initially recruits Ana1 to newly formed centrioles, and that Ana1 then recruits Asl (known as Cep152 in mammals) to promote the conversion of these centrioles into centrosomes. Here, we show that ana1 mutants lack detectable centrosomes in vivo, that Ana1 is irreversibly incorporated into centrioles during their assembly and appears to play a more important role in maintaining Asl at centrioles than in initially recruiting Asl to centrioles. Unexpectedly, we also find that Ana1 promotes centriole elongation in a dose-dependent manner: centrioles are shorter when Ana1 dosage is reduced and are longer when Ana1 is overexpressed. This latter function of Ana1 appears to be distinct from its role in centrosome and cilium function, as a GFP-Ana1 fusion lacking the N-terminal 639 amino acids of the protein can support centrosome assembly and cilium function but cannot promote centriole over-elongation when overexpressed. © 2016. Published by The Company of Biologists Ltd.

Asymmetric Centriole Numbers at Spindle Poles Cause Chromosome Missegregation in Cancer

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Marco R. Cosenza

2017-08-01

Full Text Available Chromosomal instability is a hallmark of cancer and correlates with the presence of extra centrosomes, which originate from centriole overduplication. Overduplicated centrioles lead to the formation of centriole rosettes, which mature into supernumerary centrosomes in the subsequent cell cycle. While extra centrosomes promote chromosome missegregation by clustering into pseudo-bipolar spindles, the contribution of centriole rosettes to chromosome missegregation is unknown. We used multi-modal imaging of cells with conditional centriole overduplication to show that mitotic rosettes in bipolar spindles frequently harbor unequal centriole numbers, leading to biased chromosome capture that favors binding to the prominent pole. This results in chromosome missegregation and aneuploidy. Rosette mitoses lead to viable offspring and significantly contribute to progeny production. We further show that centrosome abnormalities in primary human malignancies frequently consist of centriole rosettes. As asymmetric centriole rosettes generate mitotic errors that can be propagated, rosette mitoses are sufficient to cause chromosome missegregation in cancer.

The presence of centrioles and centrosomes in ovarian mature cystic teratoma cells suggests human parthenotes developed in vitro can differentiate into mature cells without a sperm centriole.

Science.gov (United States)

Lee, Bo Yon; Shim, Sang Woo; Kim, Young Sun; Kim, Seung Bo

2011-11-18

In most animals, somatic cell centrosomes are inherited from the centriole of the fertilizing spermatozoa. The oocyte centriole degenerates during oogenesis, and completely disappears in metaphase II. Therefore, the embryos generated by in vitro parthenogenesis are supposed to develop without any centrioles. Exceptional acentriolar and/or acentrosomal developments are possible in mice and in some experimental cells; however, in most animals, the full developmental potential of parthenogenetic cells in vitro and the fate of their centrioles/centrosomes are not clearly understood. To predict the future of in vitro human parthenogenesis, we explored the centrioles/centrosomes in ovarian mature cystic teratoma cells by immunofluorescent staining and transmission electron microscopy. We confirmed the presence of centrioles and centrosomes in these well-known parthenogenetic ovarian tumor cells. Our findings clearly demonstrate that, even without a sperm centriole, parthenotes that develop from activated oocytes can produce their own centrioles/centrosomes, and can even develop into the well-differentiated mature tissue. Copyright © 2011 Elsevier Inc. All rights reserved.

The mother centriole plays an instructive role in defining cell geometry.

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Jessica L Feldman

2007-06-01

Full Text Available 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.

Lack of centrioles and primary cilia in STIL(-/-) mouse embryos.

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David, Ahuvit; Liu, Fengying; Tibelius, Alexandra; Vulprecht, Julia; Wald, Diana; Rothermel, Ulrike; Ohana, Reut; Seitel, Alexander; Metzger, Jasmin; Ashery-Padan, Ruth; Meinzer, Hans-Peter; Gröne, Hermann-Josef; Izraeli, Shai; Krämer, Alwin

2014-01-01

Although most animal cells contain centrosomes, consisting of a pair of centrioles, their precise contribution to cell division and embryonic development is unclear. Genetic ablation of STIL, an essential component of the centriole replication machinery in mammalian cells, causes embryonic lethality in mice around mid gestation associated with defective Hedgehog signaling. Here, we describe, by focused ion beam scanning electron microscopy, that STIL(-/-) mouse embryos do not contain centrioles or primary cilia, suggesting that these organelles are not essential for mammalian development until mid gestation. We further show that the lack of primary cilia explains the absence of Hedgehog signaling in STIL(-/-) cells. Exogenous re-expression of STIL or STIL microcephaly mutants compatible with human survival, induced non-templated, de novo generation of centrioles in STIL(-/-) cells. Thus, while the abscence of centrioles is compatible with mammalian gastrulation, lack of centrioles and primary cilia impairs Hedgehog signaling and further embryonic development.

Cortical neurogenesis in the absence of centrioles.

Science.gov (United States)

Insolera, Ryan; Bazzi, Hisham; Shao, Wei; Anderson, Kathryn V; Shi, Song-Hai

2014-11-01

Neuronal production in the mammalian cortex depends on extensive mitoses of radial glial progenitors (RGPs) residing in the ventricular zone (VZ). We examined the function of centrioles in RGPs during cortical neurogenesis in mice by conditional removal of SAS-4, a protein that is required for centriole biogenesis. SAS-4 deletion led to a progressive loss of centrioles, accompanied by RGP detachment from the VZ. Delocalized RGPs did not become outer subventricular zone RGPs (oRGs). Although they remained proliferative, ectopic RGPs, as well as those in the VZ, with a centrosomal deficit exhibited prolonged mitosis, p53 upregulation and apoptosis, resulting in neuronal loss and microcephaly. Simultaneous removal of p53 fully rescued RGP death and microcephaly, but not RGP delocalization and randomized mitotic spindle orientation. Our findings define the functions of centrioles in anchoring RGPs in the VZ and ensuring their efficient mitoses, and reveal the robust adaptability of RGPs in the developing cortex.

Centrioles: active players or passengers during mitosis?

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Debec, Alain; Sullivan, William; Bettencourt-Dias, Monica

2010-07-01

Centrioles are cylinders made of nine microtubule (MT) triplets present in many eukaryotes. Early studies, where centrosomes were seen at the poles of the mitotic spindle led to their coining as "the organ for cell division". However, a variety of subsequent observational and functional studies showed that centrosomes might not always be essential for mitosis. Here we review the arguments in this debate. We describe the centriole structure and its distribution in the eukaryotic tree of life and clarify its role in the organization of the centrosome and cilia, with an historical perspective. An important aspect of the debate addressed in this review is how centrioles are inherited and the role of the spindle in this process. In particular, germline inheritance of centrosomes, such as their de novo formation in parthenogenetic species, poses many interesting questions. We finish by discussing the most likely functions of centrioles and laying out new research avenues.

Asterless licenses daughter centrioles to duplicate for the first time in Drosophila embryos.

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Novak, Zsofia A; Conduit, Paul T; Wainman, Alan; Raff, Jordan W

2014-06-02

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. Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.

The daughter centriole controls ciliogenesis by regulating Neurl-4 localization at the centrosome

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Loukil, Abdelhalim; Tormanen, Kati

2017-01-01

The two centrioles of the centrosome differ in age and function. Although the mother centriole mediates most centrosome-dependent processes, the role of the daughter remains poorly understood. A recent study has implicated the daughter centriole in centriole amplification in multiciliated cells, but its contribution to primary ciliogenesis is unclear. We found that manipulations that prevent daughter centriole formation or induce its separation from the mother abolish ciliogenesis. This defect was caused by stabilization of the negative ciliogenesis regulator CP110 and was corrected by CP110 depletion. CP110 dysregulation may be caused by effects on Neurl-4, a daughter centriole–associated ubiquitin ligase cofactor, which was required for ciliogenesis. Centrosome-targeted Neurl-4 was sufficient to restore ciliogenesis in cells with manipulated daughter centrioles. Interestingly, early during ciliogenesis, Neurl-4 transiently associated with the mother centriole in a process that required mother–daughter centriole proximity. Our data support a model in which the daughter centriole promotes ciliogenesis through Neurl-4–dependent regulation of CP110 levels at the mother centriole. PMID:28385950

PLK1 regulation of PCNT cleavage ensures fidelity of centriole separation during mitotic exit.

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Kim, Jaeyoun; Lee, Kwanwoo; Rhee, Kunsoo

2015-12-09

Centrioles are duplicated and segregated in close link to the cell cycle. During mitosis, daughter centrioles are disengaged and eventually separated from mother centrioles. New daughter centrioles may be generated only after centriole separation. Therefore, centriole separation is considered a licensing step for centriole duplication. It was previously known that separase specifically cleaves pericentrin (PCNT) during mitotic exit. Here we report that PCNT has to be phosphorylated by PLK1 to be a suitable substrate of separase. Phospho-resistant mutants of PCNT are not cleaved by separase and eventually inhibit centriole separation. Furthermore, phospho-mimetic PCNT mutants rescue centriole separation even in the presence of a PLK1 inhibitor. On the basis on these results, we propose that PLK1 phosphorylation is a priming step for separase-mediated cleavage of PCNT and eventually for centriole separation. PLK1 phosphorylation of PCNT provides an additional layer of regulatory mechanism to ensure the fidelity of centriole separation during mitotic exit.

Centriole distribution during tripolar mitosis in Chinese hamster ovary cells

Science.gov (United States)

1984-01-01

During bipolar mitosis a pair of centrioles is distributed to each cell but the activities of the two centrioles within the pair are not equivalent. The parent is normally surrounded by a cloud of pericentriolar material that serves as a microtubule-organizing center. The daughter does not become associated with pericentriolar material until it becomes a parent in the next cell cycle (Rieder, C.L., and G. G. Borisy , 1982, Biol. Cell., 44:117-132). We asked whether the microtubule-organizing activity associated with a centriole was dependent on its becoming a parent. We induced multipolar mitosis in Chinese hamster ovary cells by treatment with 0.04 micrograms/ml colcemid for 4 h. After recovery from this colcemid block, the majority of cells divided into two, but 40% divided into three and 2% divided into four. The tripolar mitotic cells were examined by antitubulin immunofluorescence and by high voltage electron microscopy of serial thick (0.25-micron) sections. The electron microscope analysis showed that centriole number was conserved and that the centrioles were distributed among the three spindle poles, generally in a 2:1:1 or 2:2:0 pattern. The first pattern shows that centriole parenting is not prerequisite for association with pole function; the second pattern indicates that centrioles per se are not required at all. However, the frequency of midbody formation and successful division was higher when centrioles were present in the 2:1:1 pattern. We suggest that the centrioles may help the proper distribution and organization of the pericentriolar cloud, which is needed for the formation of a functional spindle pole. PMID:6373793

C-NAP1 and rootletin restrain DNA damage-induced centriole splitting and facilitate ciliogenesis.

Science.gov (United States)

Conroy, Pauline C; Saladino, Chiara; Dantas, Tiago J; Lalor, Pierce; Dockery, Peter; Morrison, Ciaran G

2012-10-15

Cilia are found on most human cells and exist as motile cilia or non-motile primary cilia. Primary cilia play sensory roles in transducing various extracellular signals, and defective ciliary functions are involved in a wide range of human diseases. Centrosomes are the principal microtubule-organizing centers of animal cells and contain two centrioles. We observed that DNA damage causes centriole splitting in non-transformed human cells, with isolated centrioles carrying the mother centriole markers CEP170 and ninein but not kizuna or cenexin. Loss of centriole cohesion through siRNA depletion of C-NAP1 or rootletin increased radiation-induced centriole splitting, with C-NAP1-depleted isolated centrioles losing mother markers. As the mother centriole forms the basal body in primary cilia, we tested whether centriole splitting affected ciliogenesis. While irradiated cells formed apparently normal primary cilia, most cilia arose from centriolar clusters, not from isolated centrioles. Furthermore, C-NAP1 or rootletin knockdown reduced primary cilium formation. Therefore, the centriole cohesion apparatus at the proximal end of centrioles may provide a target that can affect primary cilium formation as part of the DNA damage response.

Structural basis of the 9-fold symmetry of centrioles.

Science.gov (United States)

Kitagawa, Daiju; Vakonakis, Ioannis; Olieric, Natacha; Hilbert, Manuel; Keller, Debora; Olieric, Vincent; Bortfeld, Miriam; Erat, Michèle C; Flückiger, Isabelle; Gönczy, Pierre; Steinmetz, Michel O

2011-02-04

The centriole, and the related basal body, is an ancient organelle characterized by a universal 9-fold radial symmetry and is critical for generating cilia, flagella, and centrosomes. The mechanisms directing centriole formation are incompletely understood and represent a fundamental open question in biology. Here, we demonstrate that the centriolar protein SAS-6 forms rod-shaped homodimers that interact through their N-terminal domains to form oligomers. We establish that such oligomerization is essential for centriole formation in C. elegans and human cells. We further generate a structural model of the related protein Bld12p from C. reinhardtii, in which nine homodimers assemble into a ring from which nine coiled-coil rods radiate outward. Moreover, we demonstrate that recombinant Bld12p self-assembles into structures akin to the central hub of the cartwheel, which serves as a scaffold for centriole formation. Overall, our findings establish a structural basis for the universal 9-fold symmetry of centrioles. Copyright © 2011 Elsevier Inc. All rights reserved.

Proteomic analysis of isolated chlamydomonas centrioles reveals orthologs of ciliary-disease genes.

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Keller, Lani C; Romijn, Edwin P; Zamora, Ivan; Yates, John R; Marshall, Wallace F

2005-06-21

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. 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. By analyzing isolated Chlamydomonas basal bodies, we have been able to obtain the first reported proteomic analysis of the centriole.

Distinct mechanisms eliminate mother and daughter centrioles in meiosis of starfish oocytes.

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Borrego-Pinto, Joana; Somogyi, Kálmán; Karreman, Matthia A; König, Julia; Müller-Reichert, Thomas; Bettencourt-Dias, Mónica; Gönczy, Pierre; Schwab, Yannick; Lénárt, Péter

2016-03-28

Centriole elimination is an essential process that occurs in female meiosis of metazoa to reset centriole number in the zygote at fertilization. How centrioles are eliminated remains poorly understood. Here we visualize the entire elimination process live in starfish oocytes. Using specific fluorescent markers, we demonstrate that the two older, mother centrioles are selectively removed from the oocyte by extrusion into polar bodies. We show that this requires specific positioning of the second meiotic spindle, achieved by dynein-driven transport, and anchorage of the mother centriole to the plasma membrane via mother-specific appendages. In contrast, the single daughter centriole remaining in the egg is eliminated before the first embryonic cleavage. We demonstrate that these distinct elimination mechanisms are necessary because if mother centrioles are artificially retained, they cannot be inactivated, resulting in multipolar zygotic spindles. Thus, our findings reveal a dual mechanism to eliminate centrioles: mothers are physically removed, whereas daughters are eliminated in the cytoplasm, preparing the egg for fertilization. © 2016 Borrego-Pinto et al.

Integrity of the Pericentriolar Material Is Essential for Maintaining Centriole Association during M Phase

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Rhee, Kunsoo

2015-01-01

A procentriole is assembled next to the mother centriole during S phase and remains associated until M phase. After functioning as a spindle pole during mitosis, the mother centriole and procentriole are separated at the end of mitosis. A close association of the centriole pair is regarded as an intrinsic block to the centriole reduplication. Therefore, deregulation of this process may cause a problem in the centriole number control, resulting in increased genomic instability. Despite its importance for faithful centriole duplication, the mechanism of centriole separation is not fully understood yet. Here, we report that centriole pairs are prematurely separated in cells whose cell cycle is arrested at M phase by STLC. Dispersal of the pericentriolar material (PCM) was accompanied. This phenomenon was independent of the separase activity but needed the PLK1 activity. Nocodazole effectively inhibited centriole scattering in STLC-treated cells, possibly by reducing the microtubule pulling force around centrosomes. Inhibition of PLK1 also reduced the premature separation of centrioles and the PCM dispersal as well. These results revealed the importance of PCM integrity in centriole association. Therefore, we propose that PCM disassembly is one of the driving forces for centriole separation during mitotic exit. PMID:26407333

Integrity of the Pericentriolar Material Is Essential for Maintaining Centriole Association during M Phase.

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Mi Young Seo

Full Text Available A procentriole is assembled next to the mother centriole during S phase and remains associated until M phase. After functioning as a spindle pole during mitosis, the mother centriole and procentriole are separated at the end of mitosis. A close association of the centriole pair is regarded as an intrinsic block to the centriole reduplication. Therefore, deregulation of this process may cause a problem in the centriole number control, resulting in increased genomic instability. Despite its importance for faithful centriole duplication, the mechanism of centriole separation is not fully understood yet. Here, we report that centriole pairs are prematurely separated in cells whose cell cycle is arrested at M phase by STLC. Dispersal of the pericentriolar material (PCM was accompanied. This phenomenon was independent of the separase activity but needed the PLK1 activity. Nocodazole effectively inhibited centriole scattering in STLC-treated cells, possibly by reducing the microtubule pulling force around centrosomes. Inhibition of PLK1 also reduced the premature separation of centrioles and the PCM dispersal as well. These results revealed the importance of PCM integrity in centriole association. Therefore, we propose that PCM disassembly is one of the driving forces for centriole separation during mitotic exit.

Life without centrioles: cilia in the spotlight.

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Badano, Jose L; Katsanis, Nicholas

2006-06-30

Centrioles are critical cellular components that form the architectural core of both centrosomes and basal bodies, the nucleating structures of cilia. New work, including a study in this issue (), highlights the unexpected finding that lack of centrioles does not impede development in the fruit fly. Rather, flies reach maturity but then die because their sensory neurons lack cilia.

Centriole maturation requires regulated Plk1 activity during two consecutive cell cycles.

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Kong, Dong; Farmer, Veronica; Shukla, Anil; James, Jana; Gruskin, Richard; Kiriyama, Shigeo; Loncarek, Jadranka

2014-09-29

Newly formed centrioles in cycling cells undergo a maturation process that is almost two cell cycles long before they become competent to function as microtubule-organizing centers and basal bodies. As a result, each cell contains three generations of centrioles, only one of which is able to form cilia. It is not known how this long and complex process is regulated. We show that controlled Plk1 activity is required for gradual biochemical and structural maturation of the centrioles and timely appendage assembly. Inhibition of Plk1 impeded accumulation of appendage proteins and appendage formation. Unscheduled Plk1 activity, either in cycling or interphase-arrested cells, accelerated centriole maturation and appendage and cilia formation on the nascent centrioles, erasing the age difference between centrioles in one cell. These findings provide a new understanding of how the centriole cycle is regulated and how proper cilia and centrosome numbers are maintained in the cells.

Protein Phosphatase 1 Down Regulates ZYG-1 Levels to Limit Centriole Duplication.

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Nina Peel

2017-01-01

Full Text Available In humans perturbations of centriole number are associated with tumorigenesis and microcephaly, therefore appropriate regulation of centriole duplication is critical. The C. elegans homolog of Plk4, ZYG-1, is required for centriole duplication, but our understanding of how ZYG-1 levels are regulated remains incomplete. We have identified the two PP1 orthologs, GSP-1 and GSP-2, and their regulators I-2SZY-2 and SDS-22 as key regulators of ZYG-1 protein levels. We find that down-regulation of PP1 activity either directly, or by mutation of szy-2 or sds-22 can rescue the loss of centriole duplication associated with a zyg-1 hypomorphic allele. Suppression is achieved through an increase in ZYG-1 levels, and our data indicate that PP1 normally regulates ZYG-1 through a post-translational mechanism. While moderate inhibition of PP1 activity can restore centriole duplication to a zyg-1 mutant, strong inhibition of PP1 in a wild-type background leads to centriole amplification via the production of more than one daughter centriole. Our results thus define a new pathway that limits the number of daughter centrioles produced each cycle.

Lack of centrioles and primary cilia in STIL−/− mouse embryos

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David, Ahuvit; Liu, Fengying; Tibelius, Alexandra; Vulprecht, Julia; Wald, Diana; Rothermel, Ulrike; Ohana, Reut; Seitel, Alexander; Metzger, Jasmin; Ashery-Padan, Ruth; Meinzer, Hans-Peter; Gröne, Hermann-Josef; Izraeli, Shai; Krämer, Alwin

2014-01-01

Although most animal cells contain centrosomes, consisting of a pair of centrioles, their precise contribution to cell division and embryonic development is unclear. Genetic ablation of STIL, an essential component of the centriole replication machinery in mammalian cells, causes embryonic lethality in mice around mid gestation associated with defective Hedgehog signaling. Here, we describe, by focused ion beam scanning electron microscopy, that STIL−/− mouse embryos do not contain centrioles or primary cilia, suggesting that these organelles are not essential for mammalian development until mid gestation. We further show that the lack of primary cilia explains the absence of Hedgehog signaling in STIL−/− cells. Exogenous re-expression of STIL or STIL microcephaly mutants compatible with human survival, induced non-templated, de novo generation of centrioles in STIL−/− cells. Thus, while the abscence of centrioles is compatible with mammalian gastrulation, lack of centrioles and primary cilia impairs Hedgehog signaling and further embryonic development. PMID:25486474

Cep63 and cep152 cooperate to ensure centriole duplication.

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Nicola J Brown

Full Text Available Centrosomes consist of two centrioles embedded in pericentriolar material and function as the main microtubule organising centres in dividing animal cells. They ensure proper formation and orientation of the mitotic spindle and are therefore essential for the maintenance of genome stability. Centrosome function is crucial during embryonic development, highlighted by the discovery of mutations in genes encoding centrosome or spindle pole proteins that cause autosomal recessive primary microcephaly, including Cep63 and Cep152. In this study we show that Cep63 functions to ensure that centriole duplication occurs reliably in dividing mammalian cells. We show that the interaction between Cep63 and Cep152 can occur independently of centrosome localisation and that the two proteins are dependent on one another for centrosomal localisation. Further, both mouse and human Cep63 and Cep152 cooperate to ensure efficient centriole duplication by promoting the accumulation of essential centriole duplication factors upstream of SAS-6 recruitment and procentriole formation. These observations describe the requirement for Cep63 in maintaining centriole number in dividing mammalian cells and further establish the order of events in centriole formation.

Ofd1, a human disease gene, regulates the length and distal structure of centrioles.

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Singla, Veena; Romaguera-Ros, Miriam; Garcia-Verdugo, Jose Manuel; Reiter, Jeremy F

2010-03-16

Centrosomes and their component centrioles represent the principal microtubule organizing centers of animal cells. Here, we show that the gene underlying orofaciodigital syndrome 1, Ofd1, is a component of the distal centriole that controls centriole length. In the absence of Ofd1, distal regions of centrioles, but not procentrioles, elongate abnormally. These long centrioles are structurally similar to normal centrioles but contain destabilized microtubules with abnormal posttranslational modifications. Ofd1 is also important for centriole distal appendage formation and centriolar recruitment of the intraflagellar transport protein Ift88. To model OFD1 syndrome in embryonic stem cells, we replaced the Ofd1 gene with missense alleles from human OFD1 patients. Distinct disease-associated mutations cause different degrees of excessive or decreased centriole elongation, all of which are associated with diminished ciliogenesis. Our results indicate that Ofd1 acts at the distal centriole to build distal appendages, recruit Ift88, and stabilize centriolar microtubules at a defined length. Copyright 2010 Elsevier Inc. All rights reserved.

Centrioles want to move out and make cilia.

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Pearson, Chad G; Culver, Brady P; Winey, Mark

2007-09-01

Cilia formation in mammalian cells requires basal bodies that are either derived from centrioles that transition from their cytoplasmic role in centrosome organization or that form en masse in multiciliated cells. Several recent studies have begun to uncover the links between centriole duplication and their transformation to basal bodies.

CEP295 interacts with microtubules and is required for centriole elongation.

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Chang, Ching-Wen; Hsu, Wen-Bin; Tsai, Jhih-Jie; Tang, Chieh-Ju C; Tang, Tang K

2016-07-01

Centriole duplication is a tightly ordered process during which procentrioles are assembled in G1-S and elongate during S and G2. Here, we show that human CEP295 (Drosophila Ana1) is not essential for initial cartwheel assembly, but is required to build distal half centrioles during S and G2. Using super-resolution and immunogold electron microscopy, we demonstrate that CEP295 is recruited to the proximal end of procentrioles in early S phase, when it is also localized at the centriolar microtubule wall that surrounds the human SAS6 cartwheel hub. Interestingly, depletion of CEP295 not only inhibits the recruitments of POC5 and POC1B to the distal half centrioles in G2, resulting in shorter centrioles, it also blocks the post-translational modification of centriolar microtubules (e.g. acetylation and glutamylation). Importantly, our results indicate that CEP295 directly interacts with microtubules, and that excess CEP295 could induce the assembly of overly long centrioles. Furthermore, exogenous expression of the N-terminal domain of CEP295 exerts a dominant-negative effect on centriole elongation. Collectively, these findings suggest that CEP295 is essential for building the distal half centrioles and for post-translational modification of centriolar microtubules. © 2016. Published by The Company of Biologists Ltd.

Klp10A modulates the localization of centriole-associated proteins during Drosophila male gametogenesis.

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Gottardo, Marco; Callaini, Giuliano; Riparbelli, Maria Giovanna

2016-12-16

Mutations in Klp10A, a microtubule-depolymerising Kinesin-13, lead to overly long centrioles in Drosophila male germ cells. We demonstrated that the loss of Klp10A modifies the distribution of typical proteins involved in centriole assembly and function. In the absence of Klp10A the distribution of Drosophila pericentrin-like protein (Dplp), Sas-4 and Sak/Plk4 that are restricted in control testes to the proximal end of the centriole increase along the centriole length. Remarkably, the cartwheel is lacking or it appears abnormal in mutant centrioles, suggesting that this structure may spatially delimit protein localization. Moreover, the parent centrioles that in control cells have the same dimensions grow at different rates in mutant testes with the mother centrioles longer than the daughters. Daughter centrioles have often an ectopic position with respect to the proximal end of the mothers and failed to recruit Dplp.

Rapid centriole assembly in Naegleria reveals conserved roles for both de novo and mentored assembly.

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Fritz-Laylin, Lillian K; Levy, Yaron Y; Levitan, Edward; Chen, Sean; Cande, W Zacheus; Lai, Elaine Y; Fulton, Chandler

2016-03-01

Centrioles are eukaryotic organelles whose number and position are critical for cilia formation and mitosis. Many cell types assemble new centrioles next to existing ones ("templated" or mentored assembly). Under certain conditions, centrioles also form without pre-existing centrioles (de novo). The synchronous differentiation of Naegleria amoebae to flagellates represents a unique opportunity to study centriole assembly, as nearly 100% of the population transitions from having no centrioles to having two within minutes. Here, we find that Naegleria forms its first centriole de novo, immediately followed by mentored assembly of the second. We also find both de novo and mentored assembly distributed among all major eukaryote lineages. We therefore propose that both modes are ancestral and have been conserved because they serve complementary roles, with de novo assembly as the default when no pre-existing centriole is available, and mentored assembly allowing precise regulation of number, timing, and location of centriole assembly. © 2016 Wiley Periodicals, Inc.

Plk4-dependent phosphorylation of STIL is required for centriole duplication

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Anne-Sophie Kratz

2015-02-01

Full Text Available Duplication of centrioles, namely the formation of a procentriole next to the parental centriole, is regulated by the polo-like kinase Plk4. Only a few other proteins, including STIL (SCL/TAL1 interrupting locus, SIL and Sas-6, are required for the early step of centriole biogenesis. Following Plk4 activation, STIL and Sas-6 accumulate at the cartwheel structure at the initial stage of the centriole assembly process. Here, we show that STIL interacts with Plk4 in vivo. A STIL fragment harboring both the coiled-coil domain and the STAN motif shows the strongest binding affinity to Plk4. Furthermore, we find that STIL is phosphorylated by Plk4. We identified Plk4-specific phosphorylation sites within the C-terminal domain of STIL and show that phosphorylation of STIL by Plk4 is required to trigger centriole duplication.

Destruction of maternal centrioles during fertilization of the brown alga, Scytosiphon lomentaria (Scytosiphonales, Phaeophyceae).

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Nagasato, Chikako; Motomura, Taizo

2004-10-01

In brown algal fertilization, a pair of centrioles is derived from the male gamete, irrespective of the sexual reproduction pattern, i.e., isogamy, anisogamy, or oogamy. In this study, the manner in which the maternal centriole structure is destroyed in early zygotes of the isogamous brown alga Scytosiphon lomentaria was examined by electron microscopy. At fertilization, the zygote had two pairs of centrioles (flagellar basal bodies) derived from motile male and female gametes, and there was no morphological difference between the two pairs. The flagellar basal plate and the axonemal microtubules were still connected with the distal end of centrioles. Ultrastructural observations showed that the integrity of maternal-derived centrioles began to degenerate even in the 1-h-old zygote. At that time, the cylinder of triplet microtubules of the maternal centrioles became shorter from the distal end, and a section passing through the centrioles indicated that a part of the nine triplets of microtubules changed into doublet or singlet microtubules by degeneration of B and/or C tubules. In 2-h-old zygote, there was no trace of maternal centrioles ultrastructurally, and only the paternal centrioles remained. Further, reduction of centrin accompanying destruction of the maternal centrioles was examined in immunofluorescence microscopy. Centrin localized at the paternal and the maternal centrioles had the same fluorescence intensity in the early zygotes. At 4-6 h after fertilization, two spots indicating centrin localization showed different fluorescence intensity. Later, the weaker spot disappeared completely. These results showed that there is a difference in time between the destruction of the centriolar cylinders and the reduction of centrin molecules around them. Copyright 2004 Wiley-Liss, Inc.

The E2F-DP1 Transcription Factor Complex Regulates Centriole Duplication in Caenorhabditis elegans

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Jacqueline G. Miller

2016-03-01

Full Text Available Centrioles play critical roles in the organization of microtubule-based structures, from the mitotic spindle to cilia and flagella. In order to properly execute their various functions, centrioles are subjected to stringent copy number control. Central to this control mechanism is a precise duplication event that takes place during S phase of the cell cycle and involves the assembly of a single daughter centriole in association with each mother centriole . Recent studies have revealed that posttranslational control of the master regulator Plk4/ZYG-1 kinase and its downstream effector SAS-6 is key to ensuring production of a single daughter centriole. In contrast, relatively little is known about how centriole duplication is regulated at a transcriptional level. Here we show that the transcription factor complex EFL-1-DPL-1 both positively and negatively controls centriole duplication in the Caenorhabditis elegans embryo. Specifically, we find that down regulation of EFL-1-DPL-1 can restore centriole duplication in a zyg-1 hypomorphic mutant and that suppression of the zyg-1 mutant phenotype is accompanied by an increase in SAS-6 protein levels. Further, we find evidence that EFL-1-DPL-1 promotes the transcription of zyg-1 and other centriole duplication genes. Our results provide evidence that in a single tissue type, EFL-1-DPL-1 sets the balance between positive and negative regulators of centriole assembly and thus may be part of a homeostatic mechanism that governs centriole assembly.

Maintaining the proper connection between the centrioles and the pericentriolar matrix requires Drosophila centrosomin.

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Lucas, Eliana P; Raff, Jordan W

2007-08-27

Centrosomes consist of two centrioles surrounded by an amorphous pericentriolar matrix (PCM), but it is unknown how centrioles and PCM are connected. We show that the centrioles in Drosophila embryos that lack the centrosomal protein Centrosomin (Cnn) can recruit PCM components but cannot maintain a proper attachment to the PCM. As a result, the centrioles "rocket" around in the embryo and often lose their connection to the nucleus in interphase and to the spindle poles in mitosis. This leads to severe mitotic defects in embryos and to errors in centriole segregation in somatic cells. The Cnn-related protein CDK5RAP2 is linked to microcephaly in humans, but cnn mutant brains are of normal size, and we observe only subtle defects in the asymmetric divisions of mutant neuroblasts. We conclude that Cnn maintains the proper connection between the centrioles and the PCM; this connection is required for accurate centriole segregation in somatic cells but is not essential for the asymmetric division of neuroblasts.

Cleavage of cohesin rings coordinates the separation of centrioles and chromatids.

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Schöckel, Laura; Möckel, Martin; Mayer, Bernd; Boos, Dominik; Stemmann, Olaf

2011-07-10

Cohesin pairs sister chromatids by forming a tripartite Scc1-Smc1-Smc3 ring around them. In mitosis, cohesin is removed from chromosome arms by the phosphorylation-dependent prophase pathway. Centromeric cohesin is protected by shugoshin 1 and protein phosphatase 2A (Sgo1-PP2A) and opened only in anaphase by separase-dependent cleavage of Scc1 (refs 4-6). Following chromosome segregation, centrioles loosen their tight orthogonal arrangement, which licenses later centrosome duplication in S phase. Although a role of separase in centriole disengagement has been reported, the molecular details of this process remain enigmatic. Here, we identify cohesin as a centriole-engagement factor. Both premature sister-chromatid separation and centriole disengagement are induced by ectopic activation of separase or depletion of Sgo1. These unscheduled events are suppressed by expression of non-cleavable Scc1 or inhibition of the prophase pathway. When endogenous Scc1 is replaced by artificially cleavable Scc1, the corresponding site-specific protease triggers centriole disengagement. Separation of centrioles can alternatively be induced by ectopic cleavage of an engineered Smc3. Thus, the chromosome and centrosome cycles exhibit extensive parallels and are coordinated with each other by dual use of the cohesin ring complex.

Structures of SAS-6 suggest its organization in centrioles.

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van Breugel, Mark; Hirono, Masafumi; Andreeva, Antonina; Yanagisawa, Haru-aki; Yamaguchi, Shoko; Nakazawa, Yuki; Morgner, Nina; Petrovich, Miriana; Ebong, Ima-Obong; Robinson, Carol V; Johnson, Christopher M; Veprintsev, Dmitry; Zuber, Benoît

2011-03-04

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

Positioning of centrioles is a conserved readout of Frizzled planar cell polarity signalling.

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Carvajal-Gonzalez, Jose Maria; Roman, Angel-Carlos; Mlodzik, Marek

2016-03-29

Planar cell polarity (PCP) signalling is a well-conserved developmental pathway regulating cellular orientation during development. An evolutionarily conserved pathway readout is not established and, moreover, it is thought that PCP mediated cellular responses are tissue-specific. A key PCP function in vertebrates is to regulate coordinated centriole/cilia positioning, a function that has not been associated with PCP in Drosophila. Here we report instructive input of Frizzled-PCP (Fz/PCP) signalling into polarized centriole positioning in Drosophila wings. We show that centrioles are polarized in pupal wing cells as a readout of PCP signalling, with both gain and loss-of-function Fz/PCP signalling affecting centriole polarization. Importantly, loss or gain of centrioles does not affect Fz/PCP establishment, implicating centriolar positioning as a conserved PCP-readout, likely downstream of PCP-regulated actin polymerization. Together with vertebrate data, these results suggest a unifying model of centriole/cilia positioning as a common downstream effect of PCP signalling from flies to mammals.

Behavior and function of paternally inherited centrioles in brown algal zygotes.

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Nagasato, Chikako

2005-12-01

In brown algal cells, the centrosome, consisting of a pair of centrioles and the pericentriolar material, is primarily involved in the organization of microtubules (MTs) throughout the cell cycle. In motile cells, the centrioles participate in the formation of flagellar axoneme as flagellar basal bodies, and in somatic cells they play a crucial role in many cellular activities as a part of the centrosome. With respect to the role of the centrosome as a microtubule organizing center (MTOC), brown algal cells resemble animal cells. In most animal fertilization processes, the sperm cell introduces centrioles, the core of the centrosome, into the egg cytoplasm. In this study, the behavior of centrioles from gametogenesis and fertilization to the first cell division of the zygote was examined in the three sexual reproduction patterns occurring in brown algae, i.e., oogamy, anisogamy and isogamy, by electron- and immunofluorescence-microscopy. The pair of centrioles contained in somatic cells was shown to be derived from the male gamete, irrespective of the sexual reproductive pattern. The paternally derived centrioles were duplicated before mitosis and were involved in spindle pole formation. Moreover, MTs from the centrosome play a crucial role in the process of cytokinesis, as the position of centrosomes accompanying daughter nuclei seems to determine the cytokinetic plane. A new approach to clarifying the mode of cytokinesis in brown algae is presented in this study.

Cdk1 Phosphorylates Drosophila Sas-4 to Recruit Polo to Daughter Centrioles and Convert Them to Centrosomes.

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Novak, Zsofia A; Wainman, Alan; Gartenmann, Lisa; Raff, Jordan W

2016-06-20

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. Copyright © 2016 The Author(s). Published by Elsevier Inc. All rights reserved.

Link between DNA damage and centriole disengagement/reduplication in untransformed human cells.

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Douthwright, Stephen; Sluder, Greenfield

2014-10-01

The radiation and radiomimetic drugs used to treat human tumors damage DNA in both cancer cells and normal proliferating cells. Centrosome amplification after DNA damage is well established for transformed cell types but is sparsely reported and not fully understood in untransformed cells. We characterize centriole behavior after DNA damage in synchronized untransformed human cells. One hour treatment of S phase cells with the radiomimetic drug, Doxorubicin, prolongs G2 by at least 72 h, though 14% of the cells eventually go through mitosis in that time. By 72 h after DNA damage we observe a 52% incidence of centriole disengagement plus a 10% incidence of extra centrioles. We find that either APC/C or Plk activities can disengage centrioles after DNA damage, though they normally work in concert. All disengaged centrioles are associated with γ-tubulin and maturation markers and thus, should in principle be capable of reduplicating and organizing spindle poles. The low incidence of reduplication of disengaged centrioles during G2 is due to the p53-dependent expression of p21 and the consequent loss of Cdk2 activity. We find that 26% of the cells going through mitosis after DNA damage contain disengaged or extra centrioles. This could produce genomic instability through transient or persistent spindle multipolarity. Thus, for cancer patients the use of DNA damaging therapies raises the chances of genomic instability and evolution of transformed characteristics in proliferating normal cell populations. © 2014 Wiley Periodicals, Inc.

The conversion of centrioles to centrosomes: essential coupling of duplication with segregation

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Wang, Won-Jing; Soni, Rajesh Kumar; Uryu, Kunihiro; Bryan Tsou, Meng-Fu

2011-01-01

Centrioles are self-reproducing organelles that form the core structure of centrosomes or microtubule-organizing centers (MTOCs). However, whether duplication and MTOC organization reflect innate activities of centrioles or activities acquired conditionally is unclear. In this paper, we show that newly formed full-length centrioles had no inherent capacity to duplicate or to organize pericentriolar material (PCM) but acquired both after mitosis through a Plk1-dependent modification that occur...

Loss of centrioles causes chromosomal instability in vertebrate somatic cells.

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Sir, Joo-Hee; Pütz, Monika; Daly, Owen; Morrison, Ciaran G; Dunning, Mark; Kilmartin, John V; Gergely, Fanni

2013-12-09

Most animal cells contain a centrosome, which comprises a pair of centrioles surrounded by an ordered pericentriolar matrix (PCM). Although the role of this organelle in organizing the mitotic spindle poles is well established, its precise contribution to cell division and cell survival remains a subject of debate. By genetically ablating key components of centriole biogenesis in chicken DT40 B cells, we generated multiple cell lines that lack centrioles. PCM components accumulated in acentriolar microtubule (MT)-organizing centers but failed to adopt a higher-order structure, as shown by three-dimensional structured illumination microscopy. Cells without centrioles exhibited both a delay in bipolar spindle assembly and a high rate of chromosomal instability. Collectively, our results expose a vital role for centrosomes in establishing a mitotic spindle geometry that facilitates correct kinetochore-MT attachments. We propose that centrosomes are essential in organisms in which rapid segregation of a large number of chromosomes needs to be attained with fidelity.

Structured illumination of the interface between centriole and peri-centriolar material

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Fu, Jingyan; Glover, David M.

2012-01-01

The increase in centrosome size in mitosis was described over a century ago, and yet it is poorly understood how centrioles, which lie at the core of centrosomes, organize the pericentriolar material (PCM) in this process. Now, structured illumination microscopy reveals in Drosophila that, before clouds of PCM appear, its proteins are closely associated with interphase centrioles in two tube-like layers: an inner layer occupied by centriolar microtubules, Sas-4, Spd-2 and Polo kinase; and an outer layer comprising Pericentrin-like protein (Dplp), Asterless (Asl) and Plk4 kinase. Centrosomin (Cnn) and γ-tubulin associate with this outer tube in G2 cells and, upon mitotic entry, Polo activity is required to recruit them together with Spd-2 into PCM clouds. Cnn is required for Spd-2 to expand into the PCM during this maturation process but can itself contribute to PCM independently of Spd-2. By contrast, the centrioles of spermatocytes elongate from a pre-existing proximal unit during the G2 preceding meiosis. Sas-4 is restricted to the microtubule-associated, inner cylinder and Dplp and Cnn to the outer cylinder of this proximal part. γ-Tubulin and Asl associate with the outer cylinder and Spd-2 with the inner cylinder throughout the entire G2 centriole. Although they occupy different spatial compartments on the G2 centriole, Cnn, Spd-2 and γ-tubulin become diminished at the centriole upon entry into meiosis to become part of PCM clouds. PMID:22977736

Centrioles initiate cilia assembly but are dispensable for maturation and maintenance in C. elegans.

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Serwas, Daniel; Su, Tiffany Y; Roessler, Max; Wang, Shaohe; Dammermann, Alexander

2017-06-05

Cilia are cellular projections that assemble on centriole-derived basal bodies. While cilia assembly is absolutely dependent on centrioles, it is not known to what extent they contribute to downstream events. The nematode C. elegans provides a unique opportunity to address this question, as centrioles do not persist at the base of mature cilia. Using fluorescence microscopy and electron tomography, we find that centrioles degenerate early during ciliogenesis. The transition zone and axoneme are not completely formed at this time, indicating that cilia maturation does not depend on intact centrioles. The hydrolethalus syndrome protein HYLS-1 is the only centriolar protein known to remain at the base of mature cilia and is required for intraflagellar transport trafficking. Surprisingly, targeted degradation of HYLS-1 after initiation of ciliogenesis does not affect ciliary structures. Taken together, our results indicate that while centrioles are essential to initiate cilia formation, they are dispensable for cilia maturation and maintenance. © 2017 Serwas et al.

Loss of Centrobin Enables Daughter Centrioles to Form Sensory Cilia in Drosophila.

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Gottardo, Marco; Pollarolo, Giulia; Llamazares, Salud; Reina, Jose; Riparbelli, Maria G; Callaini, Giuliano; Gonzalez, Cayetano

2015-08-31

Sensory cilia are organelles that convey information to the cell from the extracellular environment. In vertebrates, ciliary dysfunction results in ciliopathies that in humans comprise a wide spectrum of developmental disorders. In Drosophila, sensory cilia are found only in the neurons of type I sensory organs, but ciliary dysfunction also has dramatic consequences in this organism because it impairs the mechanosensory properties of bristles and chaetae and leads to uncoordination, a crippling condition that causes lethality shortly after eclosion. The cilium is defined by the ciliary membrane, a protrusion of the cell membrane that envelops the core structure known as the axoneme, a microtubule array that extends along the cilium from the basal body. In vertebrates, basal body function requires centriolar distal and subdistal appendages and satellites. Because these structures are acquired through centriole maturation, only mother centrioles can serve as basal bodies. Here, we show that although centriole maturity traits are lacking in Drosophila, basal body fate is reserved to mother centrioles in Drosophila type I neurons. Moreover, we show that depletion of the daughter-centriole-specific protein Centrobin (CNB) enables daughter centrioles to dock on the cell membrane and to template an ectopic axoneme that, although structurally defective, protrudes out of the cell and is enveloped by a ciliary membrane. Conversely, basal body capability is inhibited in mother centrioles modified to carry CNB. These results reveal the crucial role of CNB in regulating basal body function in Drosophila ciliated sensory organs. Copyright © 2015 Elsevier Ltd. All rights reserved.

TSKS concentrates in spermatid centrioles during flagellogenesis.

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Xu, Bingfang; Hao, Zhonglin; Jha, Kula N; Zhang, Zhibing; Urekar, Craig; Digilio, Laura; Pulido, Silvia; Strauss, Jerome F; Flickinger, Charles J; Herr, John C

2008-07-15

Centrosomal coiled-coil proteins paired with kinases play critical roles in centrosomal functions within somatic cells, however knowledge regarding gamete centriolar proteins is limited. In this study, the substrate of TSSK1 and 2, TSKS, was localized during spermiogenesis to the centrioles of post-meiotic spermatids, where it reached its greatest concentration during the period of flagellogenesis. This centriolar localization persisted in ejaculated human spermatozoa, while centriolar TSKS diminished in mouse sperm, where centrioles are known to undergo complete degeneration. In addition to the centriolar localization during flagellogenesis, mouse TSKS and the TSSK2 kinase localized in the tail and acrosomal regions of mouse epididymal sperm, while TSSK2 was found in the equatorial segment, neck and the midpiece of human spermatozoa. TSSK2/TSKS is the first kinase/substrate pair localized to the centrioles of spermatids and spermatozoa. Coupled with the infertility due to haploinsufficiency noted in chimeric mice with deletion of Tssk1 and 2 (companion paper) this centriolar kinase/substrate pair is predicted to play an indispensable role during spermiogenesis.

De novo centriole formation in human cells is error-prone and does not require SAS-6 self-assembly.

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Wang, Won-Jing; Acehan, Devrim; Kao, Chien-Han; Jane, Wann-Neng; Uryu, Kunihiro; Tsou, Meng-Fu Bryan

2015-11-26

Vertebrate centrioles normally propagate through duplication, but in the absence of preexisting centrioles, de novo synthesis can occur. Consistently, centriole formation is thought to strictly rely on self-assembly, involving self-oligomerization of the centriolar protein SAS-6. Here, through reconstitution of de novo synthesis in human cells, we surprisingly found that normal looking centrioles capable of duplication and ciliation can arise in the absence of SAS-6 self-oligomerization. Moreover, whereas canonically duplicated centrioles always form correctly, de novo centrioles are prone to structural errors, even in the presence of SAS-6 self-oligomerization. These results indicate that centriole biogenesis does not strictly depend on SAS-6 self-assembly, and may require preexisting centrioles to ensure structural accuracy, fundamentally deviating from the current paradigm.

Centriolar SAS-7 acts upstream of SPD-2 to regulate centriole assembly and pericentriolar material formation

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Sugioka, Kenji; Hamill, Danielle R; Lowry, Joshua B; McNeely, Marie E; Enrick, Molly; Richter, Alyssa C; Kiebler, Lauren E; Priess, James R; Bowerman, Bruce

2017-01-01

The centriole/basal body is a eukaryotic organelle that plays essential roles in cell division and signaling. Among five known core centriole proteins, SPD-2/Cep192 is the first recruited to the site of daughter centriole formation and regulates the centriolar localization of the other components in C. elegans and in humans. However, the molecular basis for SPD-2 centriolar localization remains unknown. Here, we describe a new centriole component, the coiled-coil protein SAS-7, as a regulator of centriole duplication, assembly and elongation. Intriguingly, our genetic data suggest that SAS-7 is required for daughter centrioles to become competent for duplication, and for mother centrioles to maintain this competence. We also show that SAS-7 binds SPD-2 and regulates SPD-2 centriolar recruitment, while SAS-7 centriolar localization is SPD-2-independent. Furthermore, pericentriolar material (PCM) formation is abnormal in sas-7 mutants, and the PCM-dependent induction of cell polarity that defines the anterior-posterior body axis frequently fails. We conclude that SAS-7 functions at the earliest step in centriole duplication yet identified and plays important roles in the orchestration of centriole and PCM assembly. DOI: http://dx.doi.org/10.7554/eLife.20353.001 PMID:28092264

Ana3 is a conserved protein required for the structural integrity of centrioles and basal bodies.

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Stevens, Naomi R; Dobbelaere, Jeroen; Wainman, Alan; Gergely, Fanni; Raff, Jordan W

2009-11-02

Recent studies have identified a conserved "core" of proteins that are required for centriole duplication. A small number of additional proteins have recently been identified as potential duplication factors, but it is unclear whether any of these proteins are components of the core duplication machinery. In this study, we investigate the function of one of these proteins, Drosophila melanogaster Ana3. We show that Ana3 is present in centrioles and basal bodies, but its behavior is distinct from that of the core duplication proteins. Most importantly, we find that Ana3 is required for the structural integrity of both centrioles and basal bodies and for centriole cohesion, but it is not essential for centriole duplication. We show that Ana3 has a mammalian homologue, Rotatin, that also localizes to centrioles and basal bodies and appears to be essential for cilia function. Thus, Ana3 defines a conserved family of centriolar proteins and plays an important part in ensuring the structural integrity of centrioles and basal bodies.

Determination of Mother Centriole Maturation in CPAP-Depleted Cells Using the Ninein Antibody

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Miseon Lee

2015-03-01

Full Text Available BackgroundMutations in centrosomal protein genes have been identified in a number of genetic diseases in brain development, including microcephaly. Centrosomal P4.1-associated protein (CPAP is one of the causal genes implicated in primary microcephaly. We previously proposed that CPAP is essential for mother centriole maturation during mitosis.MethodsWe immunostained CPAP-depleted cells using the ninein antibody, which selectively detects subdistal appendages in mature mother centrioles.ResultsNinein signals were significantly impaired in CPAP-depleted cells.ConclusionThe results suggest that CPAP is required for mother centriole maturation in mammalian cells. The selective absence of centriolar appendages in young mother centrioles may be responsible for asymmetric spindle pole formation in CPAP-depleted cells.

Do centrioles generate a polar ejection force?

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Wells, Jonathan

2005-01-01

A microtubule-dependent polar ejection force that pushes chromosomes away from spindle poles during prometaphase is observed in animal cells but not in the cells of higher plants. Elongating microtubules and kinesin-like motor molecules have been proposed as possible causes, but neither accounts for all the data. In the hypothesis proposed here a polar ejection force is generated by centrioles, which are found in animals but not in higher plants. Centrioles consist of nine microtubule triplets arranged like the blades of a tiny turbine. Instead of viewing centrioles through the spectacles of molecular reductionism and neo-Darwinism, this hypothesis assumes that they are holistically designed to be turbines. Orthogonally oriented centriolar turbines could generate oscillations in spindle microtubules that resemble the motion produced by a laboratory vortexer. The result would be a microtubule-mediated ejection force tending to move chromosomes away from the spindle axis and the poles. A rise in intracellular calcium at the onset of anaphase could regulate the polar ejection force by shutting down the centriolar turbines, but defective regulation could result in an excessive force that contributes to the chromosomal instability characteristic of most cancer cells.

Centrioles, centrosomes, and cilia in health and disease.

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Nigg, Erich A; Raff, Jordan W

2009-11-13

Centrioles are barrel-shaped structures that are essential for the formation of centrosomes, cilia, and flagella. Here we review recent advances in our understanding of the function and biogenesis of these organelles, and we emphasize their connection to human disease. Deregulation of centrosome numbers has long been proposed to contribute to genome instability and tumor formation, whereas mutations in centrosomal proteins have recently been genetically linked to microcephaly and dwarfism. Finally, structural or functional centriole aberrations contribute to ciliopathies, a variety of complex diseases that stem from the absence or dysfunction of cilia.

Human microcephaly protein RTTN interacts with STIL and is required to build full-length centrioles.

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Chen, Hsin-Yi; Wu, Chien-Ting; Tang, Chieh-Ju C; Lin, Yi-Nan; Wang, Won-Jing; Tang, Tang K

2017-08-15

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

Mother centrioles are kicked out so that starfish zygote can grow.

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Verlhac, Marie-Hélène

2016-03-28

Most oocytes eliminate their centrioles during meiotic divisions through unclear mechanisms. In this issue, Borrego-Pinto et al. (2016. J Cell. Biol. http://dx.doi.org/10.1083/jcb.201510083) show that mother centrioles need to be eliminated from starfish oocytes by extrusion into the polar bodies for successful embryo development. © 2016 Verlhac.

Centrioles regulate centrosome size by controlling the rate of Cnn incorporation into the PCM.

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Conduit, Paul T; Brunk, Kathrin; Dobbelaere, Jeroen; Dix, Carly I; Lucas, Eliana P; Raff, Jordan W

2010-12-21

centrosomes are major microtubule organizing centers in animal cells, and they comprise a pair of centrioles surrounded by an amorphous pericentriolar material (PCM). Centrosome size is tightly regulated during the cell cycle, and it has recently been shown that the two centrosomes in certain stem cells are often asymmetric in size. There is compelling evidence that centrioles influence centrosome size, but how centrosome size is set remains mysterious. we show that the conserved Drosophila PCM protein Cnn exhibits an unusual dynamic behavior, because Cnn molecules only incorporate into the PCM closest to the centrioles and then spread outward through the rest of the PCM. Cnn incorporation into the PCM is driven by an interaction with the conserved centriolar proteins Asl (Cep152 in humans) and DSpd-2 (Cep192 in humans). The rate of Cnn incorporation into the PCM is tightly regulated during the cell cycle, and this rate influences the amount of Cnn in the PCM, which in turn is an important determinant of overall centrosome size. Intriguingly, daughter centrioles in syncytial embryos only start to incorporate Cnn as they disengage from their mothers; this generates a centrosome size asymmetry, with mother centrioles always initially organizing more Cnn than their daughters. centrioles can control the amount of PCM they organize by regulating the rate of Cnn incorporation into the PCM. This mechanism can explain how centrosome size is regulated during the cell cycle and also allows mother and daughter centrioles to set centrosome size independently of one another.

Crystal structures of the CPAP/STIL complex reveal its role in centriole assembly and human microcephaly

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Cottee, Matthew A; Muschalik, Nadine; Wong, Yao Liang; Johnson, Christopher M; Johnson, Steven; Andreeva, Antonina; Oegema, Karen; Lea, Susan M; Raff, Jordan W; van Breugel, Mark

2013-01-01

Centrioles organise centrosomes and template cilia and flagella. Several centriole and centrosome proteins have been linked to microcephaly (MCPH), a neuro-developmental disease associated with small brain size. CPAP (MCPH6) and STIL (MCPH7) are required for centriole assembly, but it is unclear how mutations in them lead to microcephaly. We show that the TCP domain of CPAP constitutes a novel proline recognition domain that forms a 1:1 complex with a short, highly conserved target motif in STIL. Crystal structures of this complex reveal an unusual, all-β structure adopted by the TCP domain and explain how a microcephaly mutation in CPAP compromises complex formation. Through point mutations, we demonstrate that complex formation is essential for centriole duplication in vivo. Our studies provide the first structural insight into how the malfunction of centriole proteins results in human disease and also reveal that the CPAP–STIL interaction constitutes a conserved key step in centriole biogenesis. DOI: http://dx.doi.org/10.7554/eLife.01071.001 PMID:24052813

SAS-1 Is a C2 Domain Protein Critical for Centriole Integrity in C. elegans

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Delattre, Marie; Balestra, Fernando R.; Blanchoud, Simon; Finger, Susanne; Knott, Graham; Müller-Reichert, Thomas; Gönczy, Pierre

2014-01-01

Centrioles are microtubule-based organelles important for the formation of cilia, flagella and centrosomes. Despite progress in understanding the underlying assembly mechanisms, how centriole integrity is ensured is incompletely understood, including in sperm cells, where such integrity is particularly critical. We identified C. elegans sas-1 in a genetic screen as a locus required for bipolar spindle assembly in the early embryo. Our analysis reveals that sperm-derived sas-1 mutant centrioles lose their integrity shortly after fertilization, and that a related defect occurs when maternal sas-1 function is lacking. We establish that sas-1 encodes a C2 domain containing protein that localizes to centrioles in C. elegans, and which can bind and stabilize microtubules when expressed in human cells. Moreover, we uncover that SAS-1 is related to C2CD3, a protein required for complete centriole formation in human cells and affected in a type of oral-facial-digital (OFD) syndrome. PMID:25412110

SAS-1 is a C2 domain protein critical for centriole integrity in C. elegans.

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Lukas von Tobel

2014-11-01

Full Text Available Centrioles are microtubule-based organelles important for the formation of cilia, flagella and centrosomes. Despite progress in understanding the underlying assembly mechanisms, how centriole integrity is ensured is incompletely understood, including in sperm cells, where such integrity is particularly critical. We identified C. elegans sas-1 in a genetic screen as a locus required for bipolar spindle assembly in the early embryo. Our analysis reveals that sperm-derived sas-1 mutant centrioles lose their integrity shortly after fertilization, and that a related defect occurs when maternal sas-1 function is lacking. We establish that sas-1 encodes a C2 domain containing protein that localizes to centrioles in C. elegans, and which can bind and stabilize microtubules when expressed in human cells. Moreover, we uncover that SAS-1 is related to C2CD3, a protein required for complete centriole formation in human cells and affected in a type of oral-facial-digital (OFD syndrome.

The HPV-16 E7 oncoprotein induces centriole multiplication through deregulation of Polo-like kinase 4 expression

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Duensing Stefan

2011-05-01

Full Text Available Abstract Background Infection with high-risk human papillomaviruses (HPVs such as HPV-16 is intimately associated with squamous cell carcinomas (SCCs of the anogenital tract and a subset of oropharyngeal carcinomas. Such lesions, including pre-invasive precursors, frequently show multipolar mitoses and aneuploidy. The high-risk HPV-16-encoded E7 oncoprotein has been shown to rapidly induce centrosome abnormalities thereby causing the formation of supernumerary mitotic spindle poles and increasing the risk for chromosome missegregation. HPV-16 E7 has been found to rapidly induce centriole overduplication, in part, through the simultaneous formation of more than one daughter centriole at single maternal centrioles (centriole multiplication. The precise molecular mechanism that underlies HPV-16 E7-induced centriole multiplication, however, remains poorly understood. Findings Here, we show that human keratinocytes engineered to stably express the HPV-16 E7 oncoprotein exhibit aberrant Polo-like kinase 4 (PLK4 protein expression at maternal centrioles. Real-time quantitative reverse transcriptase (qRT-PCR analysis of these cells revealed an increase of PLK4 mRNA levels compared to control cells. Importantly, the ability of the HPV-16 E7 oncoprotein to induce centriole multiplication was found to correlate with its ability to activate the PLK4 promoter and to up-regulate PLK4 mRNA. Conclusions These results highlight the critical role of PLK4 transcriptional deregulation in centriole multiplication in HPV-16 E7-expressing cells. Our findings encourage further experiments to test transcriptional inhibitors or small molecules targeting PLK4 to prevent centriole abnormalities, mitotic infidelity and malignant progression in HPV-associated neoplasms and other tumors in which PLK4 regulation is disrupted.

The Centrioles, Centrosomes, Basal Bodies, and Cilia of Drosophila melanogaster.

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Lattao, Ramona; Kovács, Levente; Glover, David M

2017-05-01

Centrioles play a key role in the development of the fly. They are needed for the correct formation of centrosomes, the organelles at the poles of the spindle that can persist as microtubule organizing centers (MTOCs) into interphase. The ability to nucleate cytoplasmic microtubules (MTs) is a property of the surrounding pericentriolar material (PCM). The centriole has a dual life, existing not only as the core of the centrosome but also as the basal body, the structure that templates the formation of cilia and flagellae. Thus the structure and functions of the centriole, the centrosome, and the basal body have an impact upon many aspects of development and physiology that can readily be modeled in Drosophila Centrosomes are essential to give organization to the rapidly increasing numbers of nuclei in the syncytial embryo and for the spatially precise execution of cell division in numerous tissues, particularly during male meiosis. Although mitotic cell cycles can take place in the absence of centrosomes, this is an error-prone process that opens up the fly to developmental defects and the potential of tumor formation. Here, we review the structure and functions of the centriole, the centrosome, and the basal body in different tissues and cultured cells of Drosophila melanogaster , highlighting their contributions to different aspects of development and cell division. Copyright © 2017 Lattao et al.

Apparent diffusive motion of centrin foci in living cells: implications for diffusion-based motion in centriole duplication

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Rafelski, Susanne M.; Keller, Lani C.; Alberts, Jonathan B.; Marshall, Wallace F.

2011-04-01

The degree to which diffusion contributes to positioning cellular structures is an open question. Here we investigate the question of whether diffusive motion of centrin granules would allow them to interact with the mother centriole. The role of centrin granules in centriole duplication remains unclear, but some proposed functions of these granules, for example, in providing pre-assembled centriole subunits, or by acting as unstable 'pre-centrioles' that need to be captured by the mother centriole (La Terra et al 2005 J. Cell Biol. 168 713-22), require the centrin foci to reach the mother. To test whether diffusive motion could permit such interactions in the necessary time scale, we measured the motion of centrin-containing foci in living human U2OS cells. We found that these centrin foci display apparently diffusive undirected motion. Using the apparent diffusion constant obtained from these measurements, we calculated the time scale required for diffusion to capture by the mother centrioles and found that it would greatly exceed the time available in the cell cycle. We conclude that mechanisms invoking centrin foci capture by the mother, whether as a pre-centriole or as a source of components to support later assembly, would require a form of directed motility of centrin foci that has not yet been observed.

Intracellular Position of Centrioles and the Direction of Homeostatic Epithelial Cell Movements in the Mouse Cornea.

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Silverman, Erika; Zhao, Jin; Merriam, John C; Nagasaki, Takayuki

2017-02-01

Corneal epithelial cells exhibit continuous centripetal movements at a rate of about 30 µm per day, but neither the driving force nor the mechanism that determines the direction of movements is known. To facilitate the investigation of homeostatic cell movement, we examined if the intracellular position of a centriole can be used as a directional marker of epithelial cell movements in the mouse cornea. A direction of cell movements was estimated in fixed specimens from a pattern of underlying subepithelial nerve fibers. Intracellular position of centrioles was determined by gamma-tubulin immunohistology and plotted in a narrow strip along the entire diameter of a cornea from limbus to limbus. When we determined the position of centrioles in the peripheral cornea where cell movements proceed generally along a radial path, about 55% of basal epithelial cells contained a centriole in the front half of a cell. However, in the central cornea where cells exhibit a spiral pattern of movements, centrioles were distributed randomly. These results suggest that centrioles tend to be positioned toward the direction of movement in corneal basal epithelial cells when they are moving centripetally at a steady rate.

Centrioles to basal bodies in the spermiogenesis of Mastotermes darwiniensis (Insecta, Isoptera).

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Riparbelli, Maria Giovanna; Callaini, Giuliano; Mercati, David; Hertel, Horst; Dallai, Romano

2009-05-01

In addition to their role in centrosome organization, the centrioles have another distinct function as basal bodies for the formation of cilia and flagella. Centriole duplication has been reported to require two alternate assembly pathways: template or de novo. Since spermiogenesis in the termite Mastotermes darwiniensis lead to the formation of multiflagellate sperm, this process represents a useful model system in which to follow basal body formation and flagella assembly. We present evidence of a possible de novo pathway for basal body formation in the differentiating germ cell. This cell also contains typical centrosomal proteins, such as centrosomin, pericentrin-like protein, gamma-tubulin, that undergo redistribution as spermatid differentiation proceeds. The spermatid centrioles are long structures formed by nine doublet rather than triplet microtubules provided with short projections extending towards the surrounding cytoplasm and with links between doublets. The sperm basal bodies are aligned in parallel beneath the nucleus. They consist of long regions close to the nucleus showing nine doublets in a cartwheel array devoid of any projections; on the contrary, the short region close to the plasma membrane, where the sperm flagella emerge, is characterized by projections similar to those observed in the centrioles linking the basal body to the plasma membrane. It is hypothesized that this appearance is in connection with the centriole elongation and further with the flagellar axonemal organization. Microtubule doublets of sperm flagellar axonemes are provided with outer dynein arms, while inner arms are rarely visible. (c) 2009 Wiley-Liss, Inc.

A mutation in the centriole-associated protein centrin causes genomic instability via increased chromosome loss in Chlamydomonas reinhardtii

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Marshall Wallace F

2005-05-01

Full Text Available Abstract Background The role of centrioles in mitotic spindle function remains unclear. One approach to investigate mitotic centriole function is to ask whether mutation of centriole-associated proteins can cause genomic instability. Results We addressed the role of the centriole-associated EF-hand protein centrin in genomic stability using a Chlamydomonas reinhardtii centrin mutant that forms acentriolar bipolar spindles and lacks the centrin-based rhizoplast structures that join centrioles to the nucleus. Using a genetic assay for loss of heterozygosity, we found that this centrin mutant showed increased genomic instability compared to wild-type cells, and we determined that the increase in genomic instability was due to a 100-fold increase in chromosome loss rates compared to wild type. Live cell imaging reveals an increased rate in cell death during G1 in haploid cells that is consistent with an elevated rate of chromosome loss, and analysis of cell death versus centriole copy number argues against a role for multipolar spindles in this process. Conclusion The increased chromosome loss rates observed in a centrin mutant that forms acentriolar spindles suggests a role for centrin protein, and possibly centrioles, in mitotic fidelity.

Auditory hair cell centrioles undergo confined Brownian motion throughout the developmental migration of the kinocilium.

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Lepelletier, Léa; de Monvel, Jacques Boutet; Buisson, Johanna; Desdouets, Chantal; Petit, Christine

2013-07-02

Planar polarization of the forming hair bundle, the mechanosensory antenna of auditory hair cells, depends on the poorly characterized center-to-edge displacement of a primary cilium, the kinocilium, at their apical surface. Taking advantage of the gradient of hair cell differentiation along the cochlea, we reconstituted a map of the kinocilia displacements in the mouse embryonic cochlea. We then developed a cochlear organotypic culture and video-microscopy approach to monitor the movements of the kinocilium basal body (mother centriole) and its daughter centriole, which we analyzed using particle tracking and modeling. We found that both hair cell centrioles undergo confined Brownian movements around their equilibrium positions, under the apparent constraint of a radial restoring force of ∼0.1 pN. This magnitude depended little on centriole position, suggesting nonlinear interactions with constraining, presumably cytoskeletal elements. The only dynamic change observed during the period of kinocilium migration was a doubling of the centrioles' confinement area taking place early in the process. It emerges from these static and dynamic observations that kinocilia migrate gradually in parallel with the organization of hair cells into rows during cochlear neuroepithelium extension. Analysis of the confined motion of hair cell centrioles under normal and pathological conditions should help determine which structures contribute to the restoring force exerting on them. Copyright © 2013 Biophysical Society. Published by Elsevier Inc. All rights reserved.

Overly long centrioles and defective cell division upon excess of the SAS-4-related protein CPAP.

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Kohlmaier, Gregor; Loncarek, Jadranka; Meng, Xing; McEwen, Bruce F; Mogensen, Mette M; Spektor, Alexander; Dynlacht, Brian D; Khodjakov, Alexey; Gönczy, Pierre

2009-06-23

The centrosome is the principal microtubule organizing center (MTOC) of animal cells. Accurate centrosome duplication is fundamental for genome integrity and entails the formation of one procentriole next to each existing centriole, once per cell cycle. The procentriole then elongates to eventually reach the same size as the centriole. The mechanisms that govern elongation of the centriolar cylinder and their potential relevance for cell division are not known. Here, we show that the SAS-4-related protein CPAP is required for centrosome duplication in cycling human cells. Furthermore, we demonstrate that CPAP overexpression results in the formation of abnormally long centrioles. This also promotes formation of more than one procentriole in the vicinity of such overly long centrioles, eventually resulting in the presence of supernumerary MTOCs. This in turn leads to multipolar spindle assembly and cytokinesis defects. Overall, our findings suggest that centriole length must be carefully regulated to restrict procentriole number and thus ensure accurate cell division.

Apparent diffusive motion of centrin foci in living cells: implications for diffusion-based motion in centriole duplication

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Rafelski, Susanne M; Keller, Lani C; Marshall, Wallace F; Alberts, Jonathan B

2011-01-01

The degree to which diffusion contributes to positioning cellular structures is an open question. Here we investigate the question of whether diffusive motion of centrin granules would allow them to interact with the mother centriole. The role of centrin granules in centriole duplication remains unclear, but some proposed functions of these granules, for example, in providing pre-assembled centriole subunits, or by acting as unstable 'pre-centrioles' that need to be captured by the mother centriole (La Terra et al 2005 J. Cell Biol. 168 713–22), require the centrin foci to reach the mother. To test whether diffusive motion could permit such interactions in the necessary time scale, we measured the motion of centrin-containing foci in living human U2OS cells. We found that these centrin foci display apparently diffusive undirected motion. Using the apparent diffusion constant obtained from these measurements, we calculated the time scale required for diffusion to capture by the mother centrioles and found that it would greatly exceed the time available in the cell cycle. We conclude that mechanisms invoking centrin foci capture by the mother, whether as a pre-centriole or as a source of components to support later assembly, would require a form of directed motility of centrin foci that has not yet been observed

SAS-6 engineering reveals interdependence between cartwheel and microtubules in determining centriole architecture.

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Hilbert, Manuel; Noga, Akira; Frey, Daniel; Hamel, Virginie; Guichard, Paul; Kraatz, Sebastian H W; Pfreundschuh, Moritz; Hosner, Sarah; Flückiger, Isabelle; Jaussi, Rolf; Wieser, Mara M; Thieltges, Katherine M; Deupi, Xavier; Müller, Daniel J; Kammerer, Richard A; Gönczy, Pierre; Hirono, Masafumi; Steinmetz, Michel O

2016-04-01

Centrioles are critical for the formation of centrosomes, cilia and flagella in eukaryotes. They are thought to assemble around a nine-fold symmetric cartwheel structure established by SAS-6 proteins. Here, we have engineered Chlamydomonas reinhardtii SAS-6-based oligomers with symmetries ranging from five- to ten-fold. Expression of a SAS-6 mutant that forms six-fold symmetric cartwheel structures in vitro resulted in cartwheels and centrioles with eight- or nine-fold symmetries in vivo. In combination with Bld10 mutants that weaken cartwheel-microtubule interactions, this SAS-6 mutant produced six- to eight-fold symmetric cartwheels. Concurrently, the microtubule wall maintained eight- and nine-fold symmetries. Expressing SAS-6 with analogous mutations in human cells resulted in nine-fold symmetric centrioles that exhibited impaired length and organization. Together, our data suggest that the self-assembly properties of SAS-6 instruct cartwheel symmetry, and lead us to propose a model in which the cartwheel and the microtubule wall assemble in an interdependent manner to establish the native architecture of centrioles.

Centrioles in flies: the exception to the rule?

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Gogendeau, Delphine; Basto, Renata

2010-04-01

Centrioles and basal bodies are MT based structures that present a highly conserved ninefold symmetry. Centrioles can be found at the core of the centrosome where they participate in PCM recruitment and organization, contributing to cytoplasmic MT nucleation. Basal bodies are normally located closely to the plasma membrane where they are responsible for axoneme assembly to form structures such as cilia or flagella. While it is well accepted that these organelles have important roles in cell and tissue organization, their contribution to certain phases of animal development is still not entirely established. Here we review the role of centrosomes and cilia in Drosophila melanogaster and briefly discuss the implications of these findings to other model organisms. Copyright 2009 Elsevier Ltd. All rights reserved.

Spindle formation in the mouse embryo requires Plk4 in the absence of centrioles.

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Coelho, Paula A; Bury, Leah; Sharif, Bedra; Riparbelli, Maria G; Fu, Jingyan; Callaini, Giuliano; Glover, David M; Zernicka-Goetz, Magdalena

2013-12-09

During the first five rounds of cell division in the mouse embryo, spindles assemble in the absence of centrioles. Spindle formation initiates around chromosomes, but the microtubule nucleating process remains unclear. Here we demonstrate that Plk4, a protein kinase known as a master regulator of centriole formation, is also essential for spindle assembly in the absence of centrioles. Depletion of maternal Plk4 prevents nucleation and growth of microtubules and results in monopolar spindle formation. This leads to cytokinesis failure and, consequently, developmental arrest. We show that Plk4 function depends on its kinase activity and its partner protein, Cep152. Moreover, tethering Cep152 to cellular membranes sequesters Plk4 and is sufficient to trigger spindle assembly from ectopic membranous sites. Thus, the Plk4-Cep152 complex has an unexpected role in promoting microtubule nucleation in the vicinity of chromosomes to mediate bipolar spindle formation in the absence of centrioles. Copyright © 2013 The Authors. Published by Elsevier Inc. All rights reserved.

Taking Centrioles to the Elimination Round.

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Schoborg, Todd A; Rusan, Nasser M

2016-07-11

Two recent papers published in The Journal of Cell Biology (Borrego-Pinto et al., 2016) and Science (Pimenta-Marques et al., 2016) have begun to shed light on the mechanism of centriole elimination during female oogenesis, highlighting a protective role for Polo kinase and the pericentriolar material. Published by Elsevier Inc.

Centrobin-mediated Regulation of the Centrosomal Protein 4.1-associated Protein (CPAP) Level Limits Centriole Length during Elongation Stage*

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Gudi, Radhika; Haycraft, Courtney J.; Bell, P. Darwin; Li, Zihai; Vasu, Chenthamarakshan

2015-01-01

Microtubule-based centrioles in the centrosome mediate accurate bipolar cell division, spindle orientation, and primary cilia formation. Cellular checkpoints ensure that the centrioles duplicate only once in every cell cycle and achieve precise dimensions, dysregulation of which results in genetic instability and neuro- and ciliopathies. The normal cellular level of centrosomal protein 4.1-associated protein (CPAP), achieved by its degradation at mitosis, is considered as one of the major mechanisms that limits centriole growth at a predetermined length. Here we show that CPAP levels and centriole elongation are regulated by centrobin. Exogenous expression of centrobin causes abnormal elongation of centrioles due to massive accumulation of CPAP in the cell. Conversely, CPAP was undetectable in centrobin-depleted cells, suggesting that it undergoes degradation in the absence of centrobin. Only the reintroduction of full-length centrobin, but not its mutant form that lacks the CPAP binding site, could restore cellular CPAP levels in centrobin-depleted cells, indicating that persistence of CPAP requires its interaction with centrobin. Interestingly, inhibition of the proteasome in centrobin-depleted cells restored the cellular and centriolar CPAP expression, suggesting its ubiquitination and proteasome-mediated degradation when centrobin is absent. Intriguingly, however, centrobin-overexpressing cells also showed proteasome-independent accumulation of ubiquitinated CPAP and abnormal, ubiquitin-positive, elongated centrioles. Overall, our results show that centrobin interacts with ubiquitinated CPAP and prevents its degradation for normal centriole elongation function. Therefore, it appears that loss of centrobin expression destabilizes CPAP and triggers its degradation to restrict the centriole length during biogenesis. PMID:25616662

Determination of Mother Centriole Maturation in CPAP-Depleted Cells Using the Ninein Antibody

OpenAIRE

Lee, Miseon; Rhee, Kunsoo

2015-01-01

Background Mutations in centrosomal protein genes have been identified in a number of genetic diseases in brain development, including microcephaly. Centrosomal P4.1-associated protein (CPAP) is one of the causal genes implicated in primary microcephaly. We previously proposed that CPAP is essential for mother centriole maturation during mitosis. Methods We immunostained CPAP-depleted cells using the ninein antibody, which selectively detects subdistal appendages in mature mother centrioles. ...

Role of CAP350 in centriolar tubule stability and centriole assembly.

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Mikael Le Clech

Full Text Available BACKGROUND: Centrioles are microtubule-based cylindrical structures composed of nine triplet tubules and are required for the formation of the centrosome, flagella and cilia. Despite theirs importance, centriole biogenesis is poorly understood. Centrosome duplication is initiated at the G1/S transition by the sequential recruitment of a set of conserved proteins under the control of the kinase Plk4. Subsequently, the procentriole is assembled by the polymerization of centriolar tubules via an unknown mechanism involving several tubulin paralogs. METHODOLOGY/PRINCIPAL FINDINGS: Here, we developed a cellular assay to study centrosome duplication and procentriole stability based on its sensitivity to the microtubule-depolymerizing drug nocodazole. By using RNA interference experiments, we show that the stability of growing procentrioles is regulated by the microtubule-stabilizing protein CAP350, independently of hSAS-6 and CPAP which initiate procentriole growth. Furthermore, our analysis reveals the critical role of centriolar tubule stability for an efficient procentriole growth. CONCLUSIONS/SIGNIFICANCE: CAP350 belongs to a new class of proteins which associate and stabilize centriolar tubules to control centriole duplication.

Rootletin interacts with C-Nap1 and may function as a physical linker between the pair of centrioles/basal bodies in cells.

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Yang, Jun; Adamian, Michael; Li, Tiansen

2006-02-01

Rootletin, a major structural component of the ciliary rootlet, is located at the basal bodies and centrosomes in ciliated and nonciliated cells, respectively. Here we investigated its potential role in the linkage of basal bodies/centrioles and the mechanism involved in such linkages. We show that rootletin interacts with C-Nap1, a protein restricted at the ends of centrioles and functioning in centrosome cohesion in interphase cells. Their interaction in vivo is supported by their colocalization at the basal bodies/centrioles and coordinated association with the centrioles during the cell cycle. Ultrastructural examinations demonstrate that rootletin fibers connect the basal bodies in ciliated cells and are present both at the ends of and in between the pair of centrioles in nonciliated cells. The latter finding stands in contrast with C-Nap1, which is present only at the ends of the centrioles. Transient expression of C-Nap1 fragments dissociated rootletin fibers from the centrioles, resulting in centrosome separation in interphase. Overexpression of rootletin in cells caused multinucleation, micronucleation, and irregularity of nuclear shape and size, indicative of defects in chromosome separation. These data suggest that rootletin may function as a physical linker between the pair of basal bodies/centrioles by binding to C-Nap1.

Talpid3-binding centrosomal protein Cep120 is required for centriole duplication and proliferation of cerebellar granule neuron progenitors.

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Chuanqing Wu

Full Text Available Granule neuron progenitors (GNPs are the most abundant neuronal type in the cerebellum. GNP proliferation and thus cerebellar development require Sonic hedgehog (Shh secreted from Purkinje cells. Shh signaling occurs in primary cilia originating from the mother centriole. Centrioles replicate only once during a typical cell cycle and are responsible for mitotic spindle assembly and organization. Recent studies have linked cilia function to cerebellar morphogenesis, but the role of centriole duplication in cerebellar development is not known. Here we show that centrosomal protein Cep120 is asymmetrically localized to the daughter centriole through its interaction with Talpid3 (Ta3, another centrosomal protein. Cep120 null mutant mice die in early gestation with abnormal heart looping. Inactivation of Cep120 in the central nervous system leads to both hydrocephalus, due to the loss of cilia on ependymal cells, and severe cerebellar hypoplasia, due to the failed proliferation of GNPs. The mutant GNPs lack Hedgehog pathway activity. Cell biological studies show that the loss of Cep120 results in failed centriole duplication and consequently ciliogenesis, which together underlie Cep120 mutant cerebellar hypoplasia. Thus, our study for the first time links a centrosomal protein necessary for centriole duplication to cerebellar morphogenesis.

A new theory of the origin of cancer: quantum coherent entanglement, centrioles, mitosis, and differentiation.

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Hameroff, Stuart R

2004-11-01

Malignant cells are characterized by abnormal segregation of chromosomes during mitosis ("aneuploidy"), generally considered a result of malignancy originating in genetic mutations. However, recent evidence supports a century-old concept that maldistribution of chromosomes (and resultant genomic instability) due to abnormalities in mitosis itself is the primary cause of malignancy rather than a mere byproduct. In normal mitosis chromosomes replicate into sister chromatids which are then precisely separated and transported into mirror-like sets by structural protein assemblies called mitotic spindles and centrioles, both composed of microtubules. The elegant yet poorly understood ballet-like movements and geometric organization occurring in mitosis have suggested guidance by some type of organizing field, however neither electromagnetic nor chemical gradient fields have been demonstrated or shown to be sufficient. It is proposed here that normal mirror-like mitosis is organized by quantum coherence and quantum entanglement among microtubule-based centrioles and mitotic spindles which ensure precise, complementary duplication of daughter cell genomes and recognition of daughter cell boundaries. Evidence and theory supporting organized quantum states in cytoplasm/nucleoplasm (and quantum optical properties of centrioles in particular) at physiological temperature are presented. Impairment of quantum coherence and/or entanglement among microtubule-based mitotic spindles and centrioles can result in abnormal distribution of chromosomes, abnormal differentiation and uncontrolled growth, and account for all aspects of malignancy. New approaches to cancer therapy and stem cell production are suggested via non-thermal laser-mediated effects aimed at quantum optical states of centrioles.

Simulations of centriole of polarized centrosome as a monopole antenna in immune and viral synapses.

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Dvorak, Josef; Melichar, Bohuslav; Filipova, Alzbeta; Grimova, Jana; Grimova, Nela; Rozsypalova, Aneta; Buka, David; Voboril, Rene; Zapletal, Radek; Buchler, Tomas; Richter, Igor; Buka, David

2018-01-01

The immune synapse (IS) is a temporary interface between an antigen-presenting cell and an effector lymphocyte. Viral synapse is a molecularly organized cellular junction that is structurally similar to the IS. Primary cilium is considered as a functional homologue of the IS due to the morphological and functional similarities in architecture between both micotubule structures. It has been hypothesized that endogenous electromagnetic field in the cell is generated by a unique cooperating system between mitochondria and microtubules. We are extending this prior hypothesis of the endogenous electromagnetic field in the cell postulating that polarized centriole in immune and viral synapse could serve as a monopole antenna. This is an addition to our hypothesis that primary cilium could serve as a monopole antenna. We simulated the distribution of electric field of centriole of polarized centrosome as a monopole antenna in immune and viral synapse. Very weak electromagnetic field of polarized centriole of CD8+ T lymphocyte in IS can contribute to the transport of cytolytic granules into the attacked (cancer) cell. Analogically, very weak electromagnetic field of polarized centriole in viral synapse of infected CD4 cells can aid the transport of viruses (human immunodeficiency virus) to non-infected CD4 cells. We hypothesized that healthy organisms need these monopole antennas. If, during the neoplastic transformation, healthy cells lose monopole antennas in form of primary cilia, the IS aims to replace them by monopole antennas of polarized centrioles in IS to restore homeostasis.

Stress-induced localization of HSPA6 (HSP70B') and HSPA1A (HSP70-1) proteins to centrioles in human neuronal cells.

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Khalouei, Sam; Chow, Ari M; Brown, Ian R

2014-05-01

The localization of yellow fluorescent protein (YFP)-tagged HSP70 proteins was employed to identify stress-sensitive sites in human neurons following temperature elevation. Stable lines of human SH-SY5Y neuronal cells were established that expressed YFP-tagged protein products of the human inducible HSP70 genes HSPA6 (HSP70B') and HSPA1A (HSP70-1). Following a brief period of thermal stress, YFP-tagged HSPA6 and HSPA1A rapidly appeared at centrioles in the cytoplasm of human neuronal cells, with HSPA6 demonstrating a more prolonged signal compared to HSPA1A. Each centriole is composed of a distal end and a proximal end, the latter linking the centriole doublet. The YFP-tagged HSP70 proteins targeted the proximal end of centrioles (identified by γ-tubulin marker) rather than the distal end (centrin marker). Centrioles play key roles in cellular polarity and migration during neuronal differentiation. The proximal end of the centriole, which is involved in centriole stabilization, may be stress-sensitive in post-mitotic, differentiating human neurons.

SAS-4 is recruited to a dynamic structure in newly forming centrioles that is stabilized by the gamma-tubulin-mediated addition of centriolar microtubules.

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Dammermann, Alexander; Maddox, Paul S; Desai, Arshad; Oegema, Karen

2008-02-25

Centrioles are surrounded by pericentriolar material (PCM), which is proposed to promote new centriole assembly by concentrating gamma-tubulin. Here, we quantitatively monitor new centriole assembly in living Caenorhabditis elegans embryos, focusing on the conserved components SAS-4 and SAS-6. We show that SAS-4 and SAS-6 are coordinately recruited to the site of new centriole assembly and reach their maximum levels during S phase. Centriolar SAS-6 is subsequently reduced by a mechanism intrinsic to the early assembly pathway that does not require progression into mitosis. Centriolar SAS-4 remains in dynamic equilibrium with the cytoplasmic pool until late prophase, when it is stably incorporated in a step that requires gamma-tubulin and microtubule assembly. These results indicate that gamma-tubulin in the PCM stabilizes the nascent daughter centriole by promoting microtubule addition to its outer wall. Such a mechanism may help restrict new centriole assembly to the vicinity of preexisting parent centrioles that recruit PCM.

[New hypothesis on the replication of centrioles and basal bodies].

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Mignot, J P

1996-12-01

Certain morphological data, obtained in studies of the ultrastructure of centrioles and basal bodies in cells of metazoa and protists, lead us to think that the cartwheel represents of the most appropriate organization for a self-reproducing and transmissible centriolar organizer. Centrioles and basal bodies might then not be simply the centres of replication of those organizers, but also reservoirs containing several superposed centriolar organizers, which are released depending on the requirements of the cell. As an isolated cartwheel is extremely unlikely to be detected, either in conventional electron microscopy or in immunocytochemistry, it is thus the reservoir which has so far been under consideration. Such a hypothesis would permit the explanation that biogenesis de novo and biogenesis in proximity to preexisting organelles may differ only in terms of the number of morphogenetic units involved.

CAND1 Promotes PLK4-Mediated Centriole Overduplication and Is Frequently Disrupted in Prostate Cancer

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Nina Korzeniewski

2012-09-01

Full Text Available Centrosomes play a crucial role in the maintenance of genome stability by orchestrating bipolar mitotic spindle formation. The centrosome normally duplicates precisely once before mitosis in a process that is extensively regulated by protein degradation including SKP1-Cullin 1 (CUL1-F-box (SCF E3 ubiquitin ligase activity. The core SCF component CUL1 has recently been found to be required to suppress the formation of supernumerary centrosomes and centrioles, the core-forming units of centrosomes. Here, we identify the CUL1-interacting protein cullin-associated and neddylation-dissociated 1 (CAND1 as a novel centrosomal protein with a role in centriole duplication control. CAND1 was found to synergize with Polo-like kinase 4 (PLK4, a master regulator of centriole biogenesis, in the induction of centriole overduplication. We provide evidence that CAND1 functions in this process by increasing PLK4 protein stability. Furthermore, mutants of CUL1 that lack the ability to interact with CAND1 and are unable to assemble functional E3 ubiquitin ligase complexes were impaired in their ability to restrain aberrant daughter centriole synthesis. To corroborate a role of CAND1 in human carcinogenesis, we analyzed a series of prostate adenocarcinomas and found altered expression of CAND1 on the mRNA or protein level in 52.9% and 40.8%, respectively, of the tumor samples analyzed. These results highlight the role of altered SCF components in cancer in general and encourage further studies to explore the SCF-CAND1 axis for the development of novel predictive biomarkers and therapeutic approaches in prostate cancer.

Drosophila PLP assembles pericentriolar clouds that promote centriole stability, cohesion and MT nucleation

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Pratt, Metta B.; Johnson, Errin

2018-01-01

Pericentrin is a conserved centrosomal protein whose dysfunction has been linked to several human diseases. It has been implicated in many aspects of centrosome and cilia function, but its precise role is unclear. Here, we examine Drosophila Pericentrin-like-protein (PLP) function in vivo in tissues that form both centrosomes and cilia. Plp mutant centrioles exhibit four major defects: (1) They are short and have subtle structural abnormalities; (2) They disengage prematurely, and so overduplicate; (3) They organise fewer cytoplasmic MTs during interphase; (4) When forming cilia, they fail to establish and/or maintain a proper connection to the plasma membrane—although, surprisingly, they can still form an axoneme-like structure that can recruit transition zone (TZ) proteins. We show that PLP helps assemble “pericentriolar clouds” of electron-dense material that emanate from the central cartwheel spokes and spread outward to surround the mother centriole. We propose that the partial loss of these structures may largely explain the complex centriole, centrosome and cilium defects we observe in Plp mutant cells. PMID:29425198

Drosophila PLP assembles pericentriolar clouds that promote centriole stability, cohesion and MT nucleation.

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Helio Roque

2018-02-01

Full Text Available Pericentrin is a conserved centrosomal protein whose dysfunction has been linked to several human diseases. It has been implicated in many aspects of centrosome and cilia function, but its precise role is unclear. Here, we examine Drosophila Pericentrin-like-protein (PLP function in vivo in tissues that form both centrosomes and cilia. Plp mutant centrioles exhibit four major defects: (1 They are short and have subtle structural abnormalities; (2 They disengage prematurely, and so overduplicate; (3 They organise fewer cytoplasmic MTs during interphase; (4 When forming cilia, they fail to establish and/or maintain a proper connection to the plasma membrane-although, surprisingly, they can still form an axoneme-like structure that can recruit transition zone (TZ proteins. We show that PLP helps assemble "pericentriolar clouds" of electron-dense material that emanate from the central cartwheel spokes and spread outward to surround the mother centriole. We propose that the partial loss of these structures may largely explain the complex centriole, centrosome and cilium defects we observe in Plp mutant cells.

A genome-wide RNAi screen to dissect centriole duplication and centrosome maturation in Drosophila.

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Jeroen Dobbelaere

2008-09-01

Full Text Available Centrosomes comprise a pair of centrioles surrounded by an amorphous pericentriolar material (PCM. Here, we have performed a microscopy-based genome-wide RNA interference (RNAi screen in Drosophila cells to identify proteins required for centriole duplication and mitotic PCM recruitment. We analysed 92% of the Drosophila genome (13,059 genes and identified 32 genes involved in centrosome function. An extensive series of secondary screens classified these genes into four categories: (1 nine are required for centriole duplication, (2 11 are required for centrosome maturation, (3 nine are required for both functions, and (4 three genes regulate centrosome separation. These 32 hits include several new centrosomal components, some of which have human homologs. In addition, we find that the individual depletion of only two proteins, Polo and Centrosomin (Cnn can completely block centrosome maturation. Cnn is phosphorylated during mitosis in a Polo-dependent manner, suggesting that the Polo-dependent phosphorylation of Cnn initiates centrosome maturation in flies.

From stem cell to embryo without centrioles.

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Stevens, Naomi R; Raposo, Alexandre A S F; Basto, Renata; St Johnston, Daniel; Raff, Jordan W

2007-09-04

Centrosome asymmetry plays a key role in ensuring the asymmetric division of Drosophila neural stem cells (neuroblasts [NBs]) and male germline stem cells (GSCs) [1-3]. In both cases, one centrosome is anchored close to a specific cortical region during interphase, thus defining the orientation of the spindle during the ensuing mitosis. To test whether asymmetric centrosome behavior is a general feature of stem cells, we have studied female GSCs, which divide asymmetrically, producing another GSC and a cystoblast. The cystoblast then divides and matures into an oocyte, a process in which centrosomes exhibit a series of complex behaviors proposed to play a crucial role in oogenesis [4-6]. We show that the interphase centrosome does not define spindle orientation in female GSCs and that DSas-4 mutant GSCs [7], lacking centrioles and centrosomes, invariably divide asymmetrically to produce cystoblasts that proceed normally through oogenesis-remarkably, oocyte specification, microtubule organization, and mRNA localization are all unperturbed. Mature oocytes can be fertilized, but embryos that cannot support centriole replication arrest very early in development. Thus, centrosomes are dispensable for oogenesis but essential for early embryogenesis. These results reveal that asymmetric centrosome behavior is not an essential feature of stem cell divisions.

Polo-like kinase 2-dependent phosphorylation of NPM/B23 on serine 4 triggers centriole duplication.

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Annekatrin Krause

Full Text Available Duplication of the centrosome is well controlled during faithful cell division while deregulation of this process leads to supernumary centrosomes, chromosome missegregation and aneuploidy, a hallmark of many cancer cells. We previously reported that Polo-like kinase 2 (Plk2 is activated near the G1/S phase transition, and regulates the reproduction of centrosomes. In search for Plk2 interacting proteins we have identified NPM/B23 (Nucleophosmin as a novel Plk2 binding partner. We find that Plk2 and NPM/B23 interact in vitro in a Polo-box dependent manner. An association between both proteins was also observed in vivo. Moreover, we show that Plk2 phosphorylates NPM/B23 on serine 4 in vivo in S-phase. Notably, expression of a non-phosphorylatable NPM/B23 S4A mutant interferes with centriole reduplication in S-phase arrested cells and leads to a dilution of centriole numbers in unperturbed U2OS cells. The corresponding phospho-mimicking mutants have the opposite effect and their expression leads to the accumulation of centrioles. These findings suggest that NPM/B23 is a direct target of Plk2 in the regulation of centriole duplication and that phosphorylation on serine 4 can trigger this process.

Identification of BCAP, a new protein associated with basal bodies and centrioles.

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Ponsard, Cecile; Seltzer, Virginie; Perret, Eric; Tournier, Frederic; Middendorp, Sandrine

2007-05-01

Cilia exert critical functions in numerous organisms, including that of cell motility, fluid transport and protozoan locomotion. Defects in this organelle can lead to lethal pathologies in humans, including primary ciliary dyskinesia. An understanding of the cilia formation process would lead to better characterization of defects involved in such pathologies. In the present study, we identified a gene encoding a novel human protein, BCAP for Basal body Centriole-Associated Protein, which shares homologies with a previously described protein, Outer Dense Fiber 2 (ODF2). ODF2, a major component of the sperm tail cytoskeleton, is required for the formation of mother centriole distal/subdistal appendages and the generation of primary cilia. Here, we show that the bcap gene contains 18 alternatively spliced exons and encodes five different isoforms, three long and two short ones. BCAP is preferentially expressed in cilia/flagella containing tissues. Moreover, its expression is correlated with cilia formation during mucociliary differentiation of human nasal epithelial cells. Using immunofluorescence analyses, BCAP was localized within basal bodies of ciliated cells and within centrioles of proliferating cells. In light of the several spliced isoforms of BCAP and the particular localization of the protein, BCAP isoforms could play distinct roles in cilia and in centrosomes.

CEP128 Localizes to the Subdistal Appendages of the Mother Centriole and Regulates TGF-β/BMP Signaling at the Primary Cilium

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Mönnich, Maren; Borgeskov, Louise; Breslin, Loretta

2018-01-01

The centrosome is the main microtubule-organizing center in animal cells and comprises a mother and daughter centriole surrounded by pericentriolar material. During formation of primary cilia, the mother centriole transforms into a basal body that templates the ciliary axoneme. Ciliogenesis depends...... on mother centriole-specific distal appendages, whereas the role of subdistal appendages in ciliary function is unclear. Here, we identify CEP128 as a centriole subdistal appendage protein required for regulating ciliary signaling. Loss of CEP128 did not grossly affect centrosomal or ciliary structure...... but caused impaired transforming growth factor-β/bone morphogenetic protein (TGF-β/BMP) signaling in zebrafish and at the primary cilium in cultured mammalian cells. This phenotype is likely the result of defective vesicle trafficking at the cilium as ciliary localization of RAB11 was impaired upon loss...

A key centriole assembly interaction interface between human PLK4 and STIL appears to not be conserved in flies

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Matthew A. Cottee

2017-03-01

Full Text Available A small number of proteins form a conserved pathway of centriole duplication. In humans and flies, the binding of PLK4/Sak to STIL/Ana2 initiates daughter centriole assembly. In humans, this interaction is mediated by an interaction between the Polo-Box-3 (PB3 domain of PLK4 and the coiled-coil domain of STIL (HsCCD. We showed previously that the Drosophila Ana2 coiled-coil domain (DmCCD is essential for centriole assembly, but it forms a tight parallel tetramer in vitro that likely precludes an interaction with PB3. Here, we show that the isolated HsCCD and HsPB3 domains form a mixture of homo-multimers in vitro, but these readily dissociate when mixed to form the previously described 1:1 HsCCD:HsPB3 complex. In contrast, although Drosophila PB3 (DmPB3 adopts a canonical polo-box fold, it does not detectably interact with DmCCD in vitro. Thus, surprisingly, a key centriole assembly interaction interface appears to differ between humans and flies.

hPOC5 is a centrin-binding protein required for assembly of full-length centrioles.

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Azimzadeh, Juliette; Hergert, Polla; Delouvée, Annie; Euteneuer, Ursula; Formstecher, Etienne; Khodjakov, Alexey; Bornens, Michel

2009-04-06

Centrin has been shown to be involved in centrosome biogenesis in a variety of eukaryotes. In this study, we characterize hPOC5, a conserved centrin-binding protein that contains Sfi1p-like repeats. hPOC5 is localized, like centrin, in the distal portion of human centrioles. hPOC5 recruitment to procentrioles occurs during G2/M, a process that continues up to the full maturation of the centriole during the next cell cycle and is correlated with hyperphosphorylation of the protein. In the absence of hPOC5, RPE1 cells arrest in G1 phase, whereas HeLa cells show an extended S phase followed by cell death. We show that hPOC5 is not required for the initiation of procentriole assembly but is essential for building the distal half of centrioles. Interestingly, the hPOC5 family reveals an evolutionary divergence between vertebrates and organisms like Drosophila melanogaster or Caenorhabditis elegans, in which the loss of hPOC5 may correlate with the conspicuous differences in centriolar structure.

Centrosomes are autocatalytic droplets of pericentriolar material organized by centrioles

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Zwicker, David; Decker, Markus; Jaensch, Steffen; Hyman, Anthony A.; Jülicher, Frank

2014-03-01

We propose a physical description of the centrosome, a membrane-less organelle involved in cell division. In our model, centrosome material occurs in a soluble form in the cytosol and a form that tends to undergo phase separation from the cytosol. We find that an autocatalytic chemical transition between these forms accounts for the temporal evolution observed in experiments. Interestingly, the nucleation of centrosomes can be controlled by an enzymatic activity of the centrioles, which are present at the core of all centrosomes. This non-equilibrium feature also allows for multiple stable centrosomes, a situation which is unstable in equilibrium phase separation. Our theory explains the growth dynamics of centrosomes for all cell sizes down to the eight-cell stage of the C. elegans embryo. It also accounts for data acquired in experiments with aberrant numbers of centrosomes and altered cell volumes. Furthermore, our model can describe unequal centrosome sizes observed in cells with disturbed centrioles. Our example suggests a general picture of the organization of membrane-less organelles.

Laser irradiation of centrosomes in newt eosinophils: evidence of centriole role in motility

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Koonce, M.P.; Cloney, R.A.; Berns, M.W.

1984-06-01

Newt eosinophils are motile granulated leukocytes that uniquely display a highly visible centrosomal area. Electron microscope and tubulin antibody fluorescence confirms the presence of centrioles, pericentriolar material, and radiating microtubules within this visible area. Actin antibodies intensely stain the advancing cell edges and tail but only weakly stain pseudopods being withdrawn into the cell. Randomly activated eosinophils follow a roughly consistent direction with an average rate of 22.5 ..mu..m/min. The position of the centrosome is always located between the trailing cell nucleus and advancing cell edge. If the cell extends more than one pseudopod, the one closest to or containing the centrosome is always the one in which motility continues. Laser irradiation of the visible centrosomal area resulted in rapid cell rounding. After several minutes following irradiation, most cells flattened and movement continued. However, postirradiation motility was uncoordinated and directionless, and the rate decreased to an average of 14.5 ..mu..m/min. Electron microscopy and tubulin immunofluorescence indicated that an initial disorganization of microtubules resulted from the laser microirradiations. After several minutes, organized microtubules reappeared, but the centrioles appeared increasingly damaged. The irregularities in motility due to irradiation are probably related to the damaged centrioles. The results presented in this paper suggest that the centrosome is an important structure in controlling the rate and direction of newt eosinophil motility.

Evolution: Tracing the origins of centrioles, cilia, and flagella.

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Carvalho-Santos, Zita; Azimzadeh, Juliette; Pereira-Leal, José B; Bettencourt-Dias, Mónica

2011-07-25

Centrioles/basal bodies (CBBs) are microtubule-based cylindrical organelles that nucleate the formation of centrosomes, cilia, and flagella. CBBs, cilia, and flagella are ancestral structures; they are present in all major eukaryotic groups. Despite the conservation of their core structure, there is variability in their architecture, function, and biogenesis. Recent genomic and functional studies have provided insight into the evolution of the structure and function of these organelles.

Odf2-deficient mother centrioles lack distal/subdistal appendages and the ability to generate primary cilia.

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Ishikawa, Hiroaki; Kubo, Akiharu; Tsukita, Shoichiro; Tsukita, Sachiko

2005-05-01

Outer dense fibre 2 (Odf2; also known as cenexin) was initially identified as a main component of the sperm tail cytoskeleton, but was later shown to be a general scaffold protein that is specifically localized at the distal/subdistal appendages of mother centrioles. Here we show that Odf2 expression is suppressed in mouse F9 cells when both alleles of Odf2 genes are deleted. Unexpectedly, the cell cycle of Odf2(-/-) cells does not seem to be affected. Immunofluorescence and ultrathin-section electron microscopy reveals that in Odf2(-/-) cells, distal/subdistal appendages disappear from mother centrioles, making it difficult to distinguish mother from daughter centrioles. In Odf2(-/-) cells, however, the formation of primary cilia is completely suppressed, although approximately 25% of wild-type F9 cells are ciliated under the steady-state cell cycle. The loss of primary cilia in Odf2(-/-) F9 cells can be rescued by exogenous Odf2 expression. These findings indicate that Odf2 is indispensable for the formation of distal/subdistal appendages and the generation of primary cilia, but not for other cell-cycle-related centriolar functions.

A Wnt/beta-catenin pathway antagonist Chibby binds Cenexin at the distal end of mother centrioles and functions in primary cilia formation.

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Nathan Steere

Full Text Available The mother centriole of the centrosome is distinguished from immature daughter centrioles by the presence of accessory structures (distal and subdistal appendages, which play an important role in the organization of the primary cilium in quiescent cells. Primary cilia serve as sensory organelles, thus have been implicated in mediating intracellular signal transduction pathways. Here we report that Chibby (Cby, a highly conserved antagonist of the Wnt/β-catenin pathway, is a centriolar component specifically located at the distal end of the mother centriole and essential for assembly of the primary cilium. Cby appeared as a discrete dot in the middle of a ring-like structure revealed by staining with a distal appendage component of Cep164. Cby interacted with one of the appendage components, Cenexin (Cnx, which thereby abrogated the inhibitory effect of Cby on β-catenin-mediated transcriptional activation in a dose-dependent manner. Cby and Cnx did not precisely align, as Cby was detected at a more distal position than Cnx. Cnx emerged earlier than Cby during the cell cycle and was required for recruitment of Cby to the mother centriole. However, Cby was dispensable for Cnx localization to the centriole. During massive centriogenesis in in vitro cultured mouse tracheal epithelial cells, Cby and Cnx were expressed in a similar pattern, which was coincident with the expression of Foxj1. Our results suggest that Cby plays an important role in organization of both primary and motile cilia in collaboration with Cnx.

A Wnt/beta-catenin pathway antagonist Chibby binds Cenexin at the distal end of mother centrioles and functions in primary cilia formation.

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Steere, Nathan; Chae, Vanessa; Burke, Michael; Li, Feng-Qian; Takemaru, Ken-ichi; Kuriyama, Ryoko

2012-01-01

The mother centriole of the centrosome is distinguished from immature daughter centrioles by the presence of accessory structures (distal and subdistal appendages), which play an important role in the organization of the primary cilium in quiescent cells. Primary cilia serve as sensory organelles, thus have been implicated in mediating intracellular signal transduction pathways. Here we report that Chibby (Cby), a highly conserved antagonist of the Wnt/β-catenin pathway, is a centriolar component specifically located at the distal end of the mother centriole and essential for assembly of the primary cilium. Cby appeared as a discrete dot in the middle of a ring-like structure revealed by staining with a distal appendage component of Cep164. Cby interacted with one of the appendage components, Cenexin (Cnx), which thereby abrogated the inhibitory effect of Cby on β-catenin-mediated transcriptional activation in a dose-dependent manner. Cby and Cnx did not precisely align, as Cby was detected at a more distal position than Cnx. Cnx emerged earlier than Cby during the cell cycle and was required for recruitment of Cby to the mother centriole. However, Cby was dispensable for Cnx localization to the centriole. During massive centriogenesis in in vitro cultured mouse tracheal epithelial cells, Cby and Cnx were expressed in a similar pattern, which was coincident with the expression of Foxj1. Our results suggest that Cby plays an important role in organization of both primary and motile cilia in collaboration with Cnx.

Mother Centriole Distal Appendages Mediate Centrosome Docking at the Immunological Synapse and Reveal Mechanistic Parallels with Ciliogenesis.

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Stinchcombe, Jane C; Randzavola, Lyra O; Angus, Karen L; Mantell, Judith M; Verkade, Paul; Griffiths, Gillian M

2015-12-21

Cytotoxic T lymphocytes (CTLs) are highly effective serial killers capable of destroying virally infected and cancerous targets by polarized release from secretory lysosomes. Upon target contact, the CTL centrosome rapidly moves to the immunological synapse, focusing microtubule-directed release at this point [1-3]. Striking similarities have been noted between centrosome polarization at the synapse and basal body docking during ciliogenesis [1, 4-8], suggesting that CTL centrosomes might dock with the plasma membrane during killing, in a manner analogous to primary cilia formation [1, 4]. However, questions remain regarding the extent and function of centrosome polarization at the synapse, and recent reports have challenged its role [9, 10]. Here, we use high-resolution transmission electron microscopy (TEM) tomography analysis to show that, as in ciliogenesis, the distal appendages of the CTL mother centriole contact the plasma membrane directly during synapse formation. This is functionally important as small interfering RNA (siRNA) targeting of the distal appendage protein, Cep83, required for membrane contact during ciliogenesis [11], impairs CTL secretion. Furthermore, the regulatory proteins CP110 and Cep97, which must dissociate from the mother centriole to allow cilia formation [12], remain associated with the mother centriole in CTLs, and neither axoneme nor transition zone ciliary structures form. Moreover, complete centrosome docking can occur in proliferating CTLs with multiple centriole pairs. Thus, in CTLs, centrosomes dock transiently with the membrane, within the cell cycle and without progression into ciliogenesis. We propose that this transient centrosome docking without cilia formation is important for CTLs to deliver rapid, repeated polarized secretion directed by the centrosome. Copyright © 2015 The Authors. Published by Elsevier Ltd.. All rights reserved.

Potential role of centrioles in determining the morphogenetic status of animal somatic cells.

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Tkemaladze, J; Chichinadze, K

2005-05-01

Irreversible differentiation (change of morphogenetic status) and programmed death (apoptosis) are observed only in somatic cells. Cell division is the only way by which the morphogenetic status of the offspring cells may be modified. It is known that there is a fixed limit to the number of possible cell divisions, the so-called 'Hayflick limit'. Existing links between cell division, differentiation and apoptosis make it possible to conclude that all these processes could be controlled by a single self-reproducing structure. Potential candidates for this replicable structure in a somatic cell are chromosomes, mitochondria (both contain DNA), and centrioles. Centrioles (diplosome) are the most likely unit that can fully regulate the processes of irreversible differentiation, determination and modification of the morphogenetic status. It may contain differently encoded RNA molecules stacked in a definite order. During mitosis, these RNA molecules are released one by one into the cytoplasm. In the presence of reverse transcriptase and endonuclease, RNA can be embedded in nuclear DNA. This process presumably changes the status of repressed and potentially active genes and, subsequently, the morphogenetic status of a cell.

Gamma-tubulin-containing abnormal centrioles are induced by insufficient Plk4 in human HCT116 colorectal cancer cells.

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Kuriyama, Ryoko; Bettencourt-Dias, Monica; Hoffmann, Ingrid; Arnold, Marc; Sandvig, Lisa

2009-06-15

Cancer cells frequently induce aberrant centrosomes, which have been implicated in cancer initiation and progression. Human colorectal cancer cells, HCT116, contain aberrant centrioles composed of disorganized cylindrical microtubules and displaced appendages. These cells also express unique centrosome-related structures associated with a subset of centrosomal components, including gamma-tubulin, centrin and PCM1. During hydroxyurea treatment, these abnormal structures become more abundant and undergo a change in shape from small dots to elongated fibers. Although gamma-tubulin seems to exist as a ring complex, the abnormal structures do not support microtubule nucleation. Several lines of evidence suggest that the fibers correspond to a disorganized form of centriolar microtubules. Plk4, a mammalian homolog of ZYG-1 essential for initiation of centriole biogenesis, is not associated with the gamma-tubulin-specific abnormal centrosomes. The amount of Plk4 at each centrosome was less in cells with abnormal centrosomes than cells without gamma-tubulin-specific abnormal centrosomes. In addition, the formation of abnormal structures was abolished by expression of exogenous Plk4, but not SAS6 and Cep135/Bld10p, which are downstream regulators required for the organization of nine-triplet microtubules. These results suggest that HCT116 cells fail to organize the ninefold symmetry of centrioles due to insufficient Plk4.

Two appendages homologous between basal bodies and centrioles are formed using distinct Odf2 domains.

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Tateishi, Kazuhiro; Yamazaki, Yuji; Nishida, Tomoki; Watanabe, Shin; Kunimoto, Koshi; Ishikawa, Hiroaki; Tsukita, Sachiko

2013-11-11

Ciliogenesis is regulated by context-dependent cellular cues, including some transduced through appendage-like structures on ciliary basal bodies called transition fibers and basal feet. However, the molecular basis for this regulation is not fully understood. The Odf2 gene product, ODF2/cenexin, is essential for both ciliogenesis and the formation of the distal and subdistal appendages on centrioles, which become basal bodies. We examined the effects of Odf2 deletion constructs on ciliogenesis in Odf2-knockout F9 cells. Electron microscopy revealed that ciliogenesis and transition fiber formation required the ODF2/cenexin fragment containing amino acids (aa) 188-806, whereas basal foot formation required aa 1-59 and 188-806. These sequences also formed distal and subdistal appendages, respectively, indicating that the centriole appendages are molecularly analogous to those on basal bodies. We used the differential formation of appendages by Odf2 deletion constructs to study the incorporation and function of molecules associated with each appendage type. We found that transition fibers and distal appendages were required for ciliogenesis and subdistal appendages stabilized the centrosomal microtubules.

Mutation in PLK4, encoding a master regulator of centriole formation, defines a novel locus for primordial dwarfism.

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Shaheen, Ranad; Al Tala, Saeed; Almoisheer, Agaadir; Alkuraya, Fowzan S

2014-12-01

Primordial dwarfism (PD) is a heterogeneous clinical entity characterised by severe prenatal and postnatal growth deficiency. Despite the recent wave of disease gene discovery, the causal mutations in many PD patients remain unknown. To describe a PD family that maps to a novel locus. Clinical, imaging and laboratory phenotyping of a new family with PD followed by autozygosity mapping, linkage analysis and candidate gene sequencing. We describe a multiplex consanguineous Saudi family in which two full siblings and one half-sibling presented with classical features of Seckel syndrome in addition to optic nerve hypoplasia. We were able to map the phenotype to a single novel locus on 4q25-q28.2, in which we identified a five base-pair deletion in PLK4, which encodes a master regulator of centriole duplication. Our discovery further confirms the role of genes involved in centriole biology in the pathogenesis of PD. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://group.bmj.com/group/rights-licensing/permissions.

Novel POC1A mutation in primordial dwarfism reveals new insights for centriole biogenesis.

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Koparir, Asuman; Karatas, Omer F; Yuceturk, Betul; Yuksel, Bayram; Bayrak, Ali O; Gerdan, Omer F; Sagiroglu, Mahmut S; Gezdirici, Alper; Kirimtay, Koray; Selcuk, Ece; Karabay, Arzu; Creighton, Chad J; Yuksel, Adnan; Ozen, Mustafa

2015-10-01

POC1A encodes a WD repeat protein localizing to centrioles and spindle poles and is associated with short stature, onychodysplasia, facial dysmorphism and hypotrichosis (SOFT) syndrome. These main features are related to the defect in cell proliferation of chondrocytes in growth plate. In the current study, we aimed at identifying the molecular basis of two patients with primordial dwarfism (PD) in a single family through utilization of whole-exome sequencing. A novel homozygous p.T120A missense mutation was detected in POC1A in both patients, a known causative gene of SOFT syndrome, and confirmed using Sanger sequencing. To test the pathogenicity of the detected mutation, primary fibroblast cultures obtained from the patients and a control individual were used. For evaluating the global gene expression profile of cells carrying p.T120A mutation in POC1A, we performed the gene expression array and compared their expression profiles to those of control fibroblast cells. The gene expression array analysis showed that 4800 transcript probes were significantly deregulated in cells with p.T120A mutation in comparison to the control. GO term association results showed that deregulated genes are mostly involved in the extracellular matrix and cytoskeleton. Furthermore, the p.T120A missense mutation in POC1A caused the formation of abnormal mitotic spindle structure, including supernumerary centrosomes, and changes in POC1A were accompanied by alterations in another centrosome-associated WD repeat protein p80-katanin. As a result, we identified a novel mutation in POC1A of patients with PD and showed that this mutation causes the formation of multiple numbers of centrioles and multipolar spindles with abnormal chromosome arrangement. © The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Epstein-Barr virus thymidine kinase is a centrosomal resident precisely localized to the periphery of centrioles.

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Gill, Michael B; Kutok, Jeffery L; Fingeroth, Joyce D

2007-06-01

The thymidine kinase (TK) encoded by Epstein-Barr virus (EBV) differs not only from that of the alphaherpesviruses but also from that of the gamma-2 herpesvirus subfamily. Because cellular location is frequently a determinant of regulatory function, to gain insight into additional role(s) of EBV TK and to uncover how the lymphocryptovirus and rhadinovirus enzymes differ, the subcellular localizations of EBV TK and the related cercopithecine herpesvirus-15 TK were investigated. We show that in contrast to those of the other family members, the gamma-1 herpesvirus TKs localize to the centrosome and even more precisely to the periphery of the centriole, tightly encircling the tubulin-rich centrioles in a microtubule-independent fashion. Centrosomal localization is observed in diverse cell types and occurs whether the protein is expressed independently or in the context of lytic EBV infection. Surprisingly, analysis of mutants revealed that the unique N-terminal domain was not critical for targeting to the centrosome, but rather, peptide sequences located C terminal to this domain were key. This is the first herpesvirus protein documented to reside in the centrosome, or microtubule-organizing center, an amembranous organelle that regulates the structural biology of the cell cycle through control of chromosome separation and cytokinesis. More recently, proteasome-mediated degradation of cell cycle regulatory proteins, production and loading of antigenic peptides onto HLA molecules, and transient homing of diverse virion proteins required for entry and/or egress have been shown to be coordinated at the centrosome. Potential implications of centrosomal localization for EBV TK function are discussed.

A centrosome-autonomous signal that involves centriole disengagement permits centrosome duplication in G2 phase after DNA damage.

LENUS (Irish Health Repository)

2010-11-15

DNA damage can induce centrosome overduplication in a manner that requires G2-to-M checkpoint function, suggesting that genotoxic stress can decouple the centrosome and chromosome cycles. How this happens is unclear. Using live-cell imaging of cells that express fluorescently tagged NEDD1\\/GCP-WD and proliferating cell nuclear antigen, we found that ionizing radiation (IR)-induced centrosome amplification can occur outside S phase. Analysis of synchronized populations showed that significantly more centrosome amplification occurred after irradiation of G2-enriched populations compared with G1-enriched or asynchronous cells, consistent with G2 phase centrosome amplification. Irradiated and control populations of G2 cells were then fused to test whether centrosome overduplication is allowed through a diffusible stimulatory signal, or the loss of a duplication-inhibiting signal. Irradiated G2\\/irradiated G2 cell fusions showed significantly higher centrosome amplification levels than irradiated G2\\/unirradiated G2 fusions. Chicken-human cell fusions demonstrated that centrosome amplification was limited to the irradiated partner. Our finding that only the irradiated centrosome can duplicate supports a model where a centrosome-autonomous inhibitory signal is lost upon irradiation of G2 cells. We observed centriole disengagement after irradiation. Although overexpression of dominant-negative securin did not affect IR-induced centrosome amplification, Plk1 inhibition reduced radiation-induced amplification. Together, our data support centriole disengagement as a licensing signal for DNA damage-induced centrosome amplification.

A primary microcephaly protein complex forms a ring around parental centrioles.

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Sir, Joo-Hee; Barr, Alexis R; Nicholas, Adeline K; Carvalho, Ofelia P; Khurshid, Maryam; Sossick, Alex; Reichelt, Stefanie; D'Santos, Clive; Woods, C Geoffrey; Gergely, Fanni

2011-10-09

Autosomal recessive primary microcephaly (MCPH) is characterized by a substantial reduction in prenatal human brain growth without alteration of the cerebral architecture and is caused by biallelic mutations in genes coding for a subset of centrosomal proteins. Although at least three of these proteins have been implicated in centrosome duplication, the nature of the centrosome dysfunction that underlies the neurodevelopmental defect in MCPH is unclear. Here we report a homozygous MCPH-causing mutation in human CEP63. CEP63 forms a complex with another MCPH protein, CEP152, a conserved centrosome duplication factor. Together, these two proteins are essential for maintaining normal centrosome numbers in cells. Using super-resolution microscopy, we found that CEP63 and CEP152 co-localize in a discrete ring around the proximal end of the parental centriole, a pattern specifically disrupted in CEP63-deficient cells derived from patients with MCPH. This work suggests that the CEP152-CEP63 ring-like structure ensures normal neurodevelopment and that its impairment particularly affects human cerebral cortex growth.

Epitope of titin A-band-specific monoclonal antibody Tit1 5 H1.1 is highly conserved in several Fn3 domains of the titin molecule. Centriole staining in human, mouse and zebrafish cells

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Mikelsaar Aavo-Valdur

2012-09-01

Full Text Available Abstract Background Previously we have reported on the development of a new mouse anti-titin monoclonal antibody, named MAb Titl 5 H1.1, using the synthetic peptide N-AVNKYGIGEPLESDSVVAK-C which corresponds to an amino acid sequence in the A-region of the titin molecule as immunogen. In the human skeletal muscles, MAb Titl 5 H1.1 reacts specifically with titin in the A-band of the sarcomere and in different non-muscle cell types with nucleus and cytoplasm, including centrioles. In this report we have studied the evolutionary aspects of the binding of MAb Tit1 5 H1.1 with its target antigen (titin. Results We have specified the epitope area of MAb Tit1 5 H1.1 by subpeptide mapping to the hexapeptide N-AVNKYG-C. According to protein databases this amino acid sequence is located in the COOH-terminus of several different Fn3 domains of the A-region of titin molecule in many organisms, such as human being, mouse, rabbit, zebrafish (Danio rerio, and even in sea squirt (Ciona intestinalis. Our immunohisto- and cytochemical studies with MAb Tit1 5 H1.1 in human, mouse and zebrafish tissues and cell cultures showed a striated staining pattern in muscle cells and also staining of centrioles, cytoplasm and nuclei in non-muscle cells. Conclusions The data confirm that titin can play, in addition to the known roles in striated muscle cells also an important role in non-muscle cells as a centriole associated protein. This phenomenon is highly conserved in the evolution and is related to Fn3 domains of the titin molecule. Using titin A-band-specific monoclonal antibody MAb Tit1 5 H1.1 it was possible to locate titin in the sarcomeres of skeletal muscle cells and in the centrioles, cytoplasm and nuclei of non-muscle cells in phylogenetically so distant organisms as Homo sapiens, Mus musculus and zebrafish (Danio rerio.

Centrioles are freed from cilia by severing prior to mitosis.

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Parker, Jeremy D K; Hilton, Laura K; Diener, Dennis R; Rasi, M Qasim; Mahjoub, Moe R; Rosenbaum, Joel L; Quarmby, Lynne M

2010-07-01

Cilia are necessary for normal tissue development and homeostasis and are generally present during interphase, but not in mitosis. The precise mechanism of premitotic ciliary loss has been controversial, with data supporting either sequential disassembly through the transition zone or, alternatively, a severing event at the base of the cilia. Here we show by live cell imaging and immunofluorescence microscopy that resorbing flagella of Chlamydomonas leave remnants associated with the mother cell wall. We postulated that the remnants are the product of severing of doublet microtubules between the basal bodies and the flagellar transition zone, thereby freeing the centrioles to participate in spindle organization. We show via TEM that flagellar remnants are indeed flagellar transition zones encased in vesicles derived from the flagellar membrane. This transition zone vesicle can be lodged within the cell wall or it can be expelled into the environment. This process is observable in Chlamydomonas, first because the released flagellar remnants can remain associated with the cell by virtue of attachments to the cell wall, and second because the Chlamydomonas transition zone is particularly rich with electron-dense structure. However, release of basal bodies for spindle-associated function is likely to be conserved among the eukaryotes. 2010 Wiley-Liss, Inc.

Live Imaging of Centriole Dynamics by Fluorescently Tagged Proteins in Starfish Oocyte Meiosis.

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Borrego-Pinto, Joana; Somogyi, Kálmán; Lénárt, Péter

2016-01-01

High throughput DNA sequencing, the decreasing costs of DNA synthesis, and universal techniques for genetic manipulation have made it much easier and quicker to establish molecular tools for any organism than it has been 5 years ago. This opens a great opportunity for reviving "nonconventional" model organisms, which are particularly suited to study a specific biological process and many of which have already been established before the era of molecular biology. By taking advantage of transcriptomics, in particular, these systems can now be easily turned into full fetched models for molecular cell biology.As an example, here we describe how we established molecular tools in the starfish Patiria miniata, which has been a popular model for cell and developmental biology due to the synchronous and rapid development, transparency, and easy handling of oocytes, eggs, and embryos. Here, we detail how we used a de novo assembled transcriptome to produce molecular markers and established conditions for live imaging to investigate the molecular mechanisms underlying centriole elimination-a poorly understood process essential for sexual reproduction of animal species.

Disruption of mouse Cenpj, a regulator of centriole biogenesis, phenocopies Seckel syndrome.

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McIntyre, Rebecca E; Lakshminarasimhan Chavali, Pavithra; Ismail, Ozama; Carragher, Damian M; Sanchez-Andrade, Gabriela; Forment, Josep V; Fu, Beiyuan; Del Castillo Velasco-Herrera, Martin; Edwards, Andrew; van der Weyden, Louise; Yang, Fengtang; Ramirez-Solis, Ramiro; Estabel, Jeanne; Gallagher, Ferdia A; Logan, Darren W; Arends, Mark J; Tsang, Stephen H; Mahajan, Vinit B; Scudamore, Cheryl L; White, Jacqueline K; Jackson, Stephen P; Gergely, Fanni; Adams, David J

2012-01-01

Disruption of the centromere protein J gene, CENPJ (CPAP, MCPH6, SCKL4), which is a highly conserved and ubiquitiously expressed centrosomal protein, has been associated with primary microcephaly and the microcephalic primordial dwarfism disorder Seckel syndrome. The mechanism by which disruption of CENPJ causes the proportionate, primordial growth failure that is characteristic of Seckel syndrome is unknown. By generating a hypomorphic allele of Cenpj, we have developed a mouse (Cenpj(tm/tm)) that recapitulates many of the clinical features of Seckel syndrome, including intrauterine dwarfism, microcephaly with memory impairment, ossification defects, and ocular and skeletal abnormalities, thus providing clear confirmation that specific mutations of CENPJ can cause Seckel syndrome. Immunohistochemistry revealed increased levels of DNA damage and apoptosis throughout Cenpj(tm/tm) embryos and adult mice showed an elevated frequency of micronucleus induction, suggesting that Cenpj-deficiency results in genomic instability. Notably, however, genomic instability was not the result of defective ATR-dependent DNA damage signaling, as is the case for the majority of genes associated with Seckel syndrome. Instead, Cenpj(tm/tm) embryonic fibroblasts exhibited irregular centriole and centrosome numbers and mono- and multipolar spindles, and many were near-tetraploid with numerical and structural chromosomal abnormalities when compared to passage-matched wild-type cells. Increased cell death due to mitotic failure during embryonic development is likely to contribute to the proportionate dwarfism that is associated with CENPJ-Seckel syndrome.

Disruption of Mouse Cenpj, a Regulator of Centriole Biogenesis, Phenocopies Seckel Syndrome

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McIntyre, Rebecca E.; Lakshminarasimhan Chavali, Pavithra; Forment, Josep V.; Fu, Beiyuan; Del Castillo Velasco-Herrera, Martin; Edwards, Andrew; van der Weyden, Louise; Yang, Fengtang; Ramirez-Solis, Ramiro; Estabel, Jeanne; Gallagher, Ferdia A.; Logan, Darren W.; Arends, Mark J.; Tsang, Stephen H.; Mahajan, Vinit B.; Scudamore, Cheryl L.; White, Jacqueline K.; Jackson, Stephen P.; Gergely, Fanni; Adams, David J.

2012-01-01

Disruption of the centromere protein J gene, CENPJ (CPAP, MCPH6, SCKL4), which is a highly conserved and ubiquitiously expressed centrosomal protein, has been associated with primary microcephaly and the microcephalic primordial dwarfism disorder Seckel syndrome. The mechanism by which disruption of CENPJ causes the proportionate, primordial growth failure that is characteristic of Seckel syndrome is unknown. By generating a hypomorphic allele of Cenpj, we have developed a mouse (Cenpjtm/tm) that recapitulates many of the clinical features of Seckel syndrome, including intrauterine dwarfism, microcephaly with memory impairment, ossification defects, and ocular and skeletal abnormalities, thus providing clear confirmation that specific mutations of CENPJ can cause Seckel syndrome. Immunohistochemistry revealed increased levels of DNA damage and apoptosis throughout Cenpjtm/tm embryos and adult mice showed an elevated frequency of micronucleus induction, suggesting that Cenpj-deficiency results in genomic instability. Notably, however, genomic instability was not the result of defective ATR-dependent DNA damage signaling, as is the case for the majority of genes associated with Seckel syndrome. Instead, Cenpjtm/tm embryonic fibroblasts exhibited irregular centriole and centrosome numbers and mono- and multipolar spindles, and many were near-tetraploid with numerical and structural chromosomal abnormalities when compared to passage-matched wild-type cells. Increased cell death due to mitotic failure during embryonic development is likely to contribute to the proportionate dwarfism that is associated with CENPJ-Seckel syndrome. PMID:23166506

Disruption of mouse Cenpj, a regulator of centriole biogenesis, phenocopies Seckel syndrome.

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Rebecca E McIntyre

Full Text Available Disruption of the centromere protein J gene, CENPJ (CPAP, MCPH6, SCKL4, which is a highly conserved and ubiquitiously expressed centrosomal protein, has been associated with primary microcephaly and the microcephalic primordial dwarfism disorder Seckel syndrome. The mechanism by which disruption of CENPJ causes the proportionate, primordial growth failure that is characteristic of Seckel syndrome is unknown. By generating a hypomorphic allele of Cenpj, we have developed a mouse (Cenpj(tm/tm that recapitulates many of the clinical features of Seckel syndrome, including intrauterine dwarfism, microcephaly with memory impairment, ossification defects, and ocular and skeletal abnormalities, thus providing clear confirmation that specific mutations of CENPJ can cause Seckel syndrome. Immunohistochemistry revealed increased levels of DNA damage and apoptosis throughout Cenpj(tm/tm embryos and adult mice showed an elevated frequency of micronucleus induction, suggesting that Cenpj-deficiency results in genomic instability. Notably, however, genomic instability was not the result of defective ATR-dependent DNA damage signaling, as is the case for the majority of genes associated with Seckel syndrome. Instead, Cenpj(tm/tm embryonic fibroblasts exhibited irregular centriole and centrosome numbers and mono- and multipolar spindles, and many were near-tetraploid with numerical and structural chromosomal abnormalities when compared to passage-matched wild-type cells. Increased cell death due to mitotic failure during embryonic development is likely to contribute to the proportionate dwarfism that is associated with CENPJ-Seckel syndrome.

3D-structured illumination microscopy provides novel insight into architecture of human centrosomes

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Katharina F. Sonnen

2012-08-01

Centrioles are essential for the formation of cilia and flagella. They also form the core of the centrosome, which organizes microtubule arrays important for cell shape, polarity, motility and division. Here, we have used super-resolution 3D-structured illumination microscopy to analyse the spatial relationship of 18 centriole and pericentriolar matrix (PCM components of human centrosomes at different cell cycle stages. During mitosis, PCM proteins formed extended networks with interspersed γ-Tubulin. During interphase, most proteins were arranged at specific distances from the walls of centrioles, resulting in ring staining, often with discernible density masses. Through use of site-specific antibodies, we found the C-terminus of Cep152 to be closer to centrioles than the N-terminus, illustrating the power of 3D-SIM to study protein disposition. Appendage proteins showed rings with multiple density masses, and the number of these masses was strongly reduced during mitosis. At the proximal end of centrioles, Sas-6 formed a dot at the site of daughter centriole assembly, consistent with its role in cartwheel formation. Plk4 and STIL co-localized with Sas-6, but Cep135 was associated mostly with mother centrioles. Remarkably, Plk4 formed a dot on the surface of the mother centriole before Sas-6 staining became detectable, indicating that Plk4 constitutes an early marker for the site of nascent centriole formation. Our study provides novel insights into the architecture of human centrosomes and illustrates the power of super-resolution microscopy in revealing the relative localization of centriole and PCM proteins in unprecedented detail.

Production of Basal Bodies in bulk for dense multicilia formation [version 1; referees: 2 approved

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Xiumin Yan

2016-06-01

Full Text Available Centriole number is normally under tight control and is directly linked to ciliogenesis. In cells that use centrosomes as mitotic spindle poles, one pre-existing mother centriole is allowed to duplicate only one daughter centriole per cell cycle. In multiciliated cells, however, many centrioles are generated to serve as basal bodies of the cilia. Although deuterosomes were observed more than 40 years ago using electron microscopy and are believed to produce most of the basal bodies in a mother centriole-independent manner, the underlying molecular mechanisms have remained unknown until recently. From these findings arise more questions and a call for clarifications that will require multidisciplinary efforts.

Continuation of mitosis after selective laser microbeam destruction of the centriolar region

Energy Technology Data Exchange (ETDEWEB)

Berns, N.W.; Richardson, S.M.

1977-12-01

The centriole regions of prophase PTK2 cells were irradiated with a laser microbeam. Cells continued through mitosis normally. Ultrastructural analysis revealed either an absence of centrioles or severely damaged centrioles at the irradiated poles. Microtubules appeared to focus into pericentriolar cloud material.

Continuation of mitosis after selective laser microbeam destruction of the centriolar region

International Nuclear Information System (INIS)

Berns, N.W.; Richardson, S.M.

1977-01-01

The centriole regions of prophase PTK2 cells were irradiated with a laser microbeam. Cells continued through mitosis normally. Ultrastructural analysis revealed either an absence of centrioles or severely damaged centrioles at the irradiated poles. Microtubules appeared to focus into pericentriolar cloud material

De novo formation of centrosomes in vertebrate cells arrested during S phase

NARCIS (Netherlands)

Khodjakov, A; Rieder, CL; Sluder, G; Cassels, G; Sibon, O; Wang, CL

2002-01-01

The centrosome usually replicates in a semiconservative fashion, i.e., new centrioles form in association with preexisting "maternal" centrioles. De novo formation of centrioles has been reported for a few highly specialized cell types but it has not been seen in vertebrate somatic cells. We find

An ultrastructural study of spermatogenesis and the mature ...

African Journals Online (AJOL)

1992-10-22

Oct 22, 1992 ... A pair of centrioles, the proximal and distal centrioles, are arranged orthogonally to one another. (Figure 7). The flagellum differentiates precociously from the dislal centriole and thus marks the longitudinal axis of the spennatocyte. Intercellular bridges (Figures 5 & 6) between the spenna- tocyles persist ...

Microtubules are organized independently of the centrosome in Drosophila neurons

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Nguyen Michelle M

2011-12-01

Full Text Available Abstract Background The best-studied arrangement of microtubules is that organized by the centrosome, a cloud of microtubule nucleating and anchoring proteins is clustered around centrioles. However, noncentrosomal microtubule arrays are common in many differentiated cells, including neurons. Although microtubules are not anchored at neuronal centrosomes, it remains unclear whether the centrosome plays a role in organizing neuronal microtubules. We use Drosophila as a model system to determine whether centrosomal microtubule nucleation is important in mature neurons. Results In developing and mature neurons, centrioles were not surrounded by the core nucleation protein γ-tubulin. This suggests that the centrioles do not organize functional centrosomes in Drosophila neurons in vivo. Consistent with this idea, centriole position was not correlated with a specific region of the cell body in neurons, and growing microtubules did not cluster around the centriole, even after axon severing when the number of growing plus ends is dramatically increased. To determine whether the centrosome was required for microtubule organization in mature neurons, we used two approaches. First, we used DSas-4 centriole duplication mutants. In these mutants, centrioles were present in many larval sensory neurons, but they were not fully functional. Despite reduced centriole function, microtubule orientation was normal in axons and dendrites. Second, we used laser ablation to eliminate the centriole, and again found that microtubule polarity in axons and dendrites was normal, even 3 days after treatment. Conclusion We conclude that the centrosome is not a major site of microtubule nucleation in Drosophila neurons, and is not required for maintenance of neuronal microtubule organization in these cells.

Centrosome isolation and analysis by mass spectrometry-based proteomics

DEFF Research Database (Denmark)

Jakobsen, Lis; Schrøder, Jacob Morville; Larsen, Katja M

2013-01-01

Centrioles are microtubule-based scaffolds that are essential for the formation of centrosomes, cilia, and flagella with important functions throughout the cell cycle, in physiology and during development. The ability to purify centriole-containing organelles on a large scale, combined with advan...... to isolate centrosomes from human cells and strategies to selectively identify and study the properties of the associated proteins using quantitative mass spectrometry-based proteomics.......Centrioles are microtubule-based scaffolds that are essential for the formation of centrosomes, cilia, and flagella with important functions throughout the cell cycle, in physiology and during development. The ability to purify centriole-containing organelles on a large scale, combined...... with advances in protein identification using mass spectrometry-based proteomics, have revealed multiple centriole-associated proteins that are conserved during evolution in eukaryotes. Despite these advances, the molecular basis for the plethora of processes coordinated by cilia and centrosomes is not fully...

A molecular mechanism of mitotic centrosome assembly in Drosophila

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Conduit, Paul T; Richens, Jennifer H; Wainman, Alan; Holder, James; Vicente, Catarina C; Pratt, Metta B; Dix, Carly I; Novak, Zsofia A; Dobbie, Ian M; Schermelleh, Lothar; Raff, Jordan W

2014-01-01

Centrosomes comprise a pair of centrioles surrounded by pericentriolar material (PCM). The PCM expands dramatically as cells enter mitosis, but it is unclear how this occurs. In this study, we show that the centriole protein Asl initiates the recruitment of DSpd-2 and Cnn to mother centrioles; both proteins then assemble into co-dependent scaffold-like structures that spread outwards from the mother centriole and recruit most, if not all, other PCM components. In the absence of either DSpd-2 or Cnn, mitotic PCM assembly is diminished; in the absence of both proteins, it appears to be abolished. We show that DSpd-2 helps incorporate Cnn into the PCM and that Cnn then helps maintain DSpd-2 within the PCM, creating a positive feedback loop that promotes robust PCM expansion around the mother centriole during mitosis. These observations suggest a surprisingly simple mechanism of mitotic PCM assembly in flies. DOI: http://dx.doi.org/10.7554/eLife.03399.001 PMID:25149451

[Ultrastructure of spermatogenesis and spermatozoa of Emallagma cheliferum Selys, 1875 (Coenagrionidae: Odonata)].

Science.gov (United States)

Gama, V; Zaha, A; Landim, C D; Ferreira, A

1976-06-01

The spermiogenesis in Enallagma cheliferum follows the usual patterns of differentiation in insects. Thus, the Golgi originates the acrosome; the "nebenkern", the mitochondrial structures that form a long tail with the axonema. The axonema has the configuration 9 + 9 + 2 and around the centriole a centriole adjunt is visible in the immature spermatide. The centriole adjunt differentiates into dense bodies as a "demi-lune" shape in the mature sperm.

Breaking the ties that bind: new advances in centrosome biology.

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Mardin, Balca R; Schiebel, Elmar

2012-04-02

The centrosome, which consists of two centrioles and the surrounding pericentriolar material, is the primary microtubule-organizing center (MTOC) in animal cells. Like chromosomes, centrosomes duplicate once per cell cycle and defects that lead to abnormalities in the number of centrosomes result in genomic instability, a hallmark of most cancer cells. Increasing evidence suggests that the separation of the two centrioles (disengagement) is required for centrosome duplication. After centriole disengagement, a proteinaceous linker is established that still connects the two centrioles. In G2, this linker is resolved (centrosome separation), thereby allowing the centrosomes to separate and form the poles of the bipolar spindle. Recent work has identified new players that regulate these two processes and revealed unexpected mechanisms controlling the centrosome cycle.

Novel asymmetrically localizing components of human centrosomes identified by complementary proteomics methods

DEFF Research Database (Denmark)

Jakobsen, Lis; Vanselow, Katja; Skogs, Marie

2011-01-01

by identifying a novel set of five proteins preferentially associated with mother or daughter centrioles, comprising genes implicated in cell polarity. Pulsed labelling demonstrates a remarkable variation in the stability of centrosomal protein complexes. These spatiotemporal proteomics data provide leads......Centrosomes in animal cells are dynamic organelles with a proteinaceous matrix of pericentriolar material assembled around a pair of centrioles. They organize the microtubule cytoskeleton and the mitotic spindle apparatus. Mature centrioles are essential for biogenesis of primary cilia that mediate...

Basal-body-associated macromolecules: a continuing debate.

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Pierre Mignot, J; Brugerolle, G; Didier, P; Bornens, M

1993-07-01

Controversy over the possibility that centrioles/basal bodies contain nucleic acids has overshadowed results demonstrating other macromolecules in the lumen of these organelles. Glycogen particles, which are known to be present within the lumen of the centriole/basal body of sperm cells, have now been found in basal bodies of protists belonging to three different groups. Here, we extend the debate on a role for RNA in basal body/centriole function and speculate on the origin and the function of centriolar glycogen.

Centrosome/Cell cycle uncoupling and elimination in the endoreduplicating intestinal cells of C. elegans.

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Yu Lu

Full Text Available The centrosome cycle is most often coordinated with mitotic cell division through the activity of various essential cell cycle regulators, consequently ensuring that the centriole is duplicated once, and only once, per cell cycle. However, this coupling can be altered in specific developmental contexts; for example, multi-ciliated cells generate hundreds of centrioles without any S-phase requirement for their biogenesis, while Drosophila follicle cells eliminate their centrosomes as they begin to endoreduplicate. In order to better understand how the centrosome cycle and the cell cycle are coordinated in a developmental context we use the endoreduplicating intestinal cell lineage of C. elegans to address how novel variations of the cell cycle impact this important process. In C. elegans, the larval intestinal cells undergo one nuclear division without subsequent cytokinesis, followed by four endocycles that are characterized by successive rounds of S-phase. We monitored the levels of centriolar/centrosomal markers and found that centrosomes lose their pericentriolar material following the nuclear division that occurs during the L1 stage and is thereafter never re-gained. The centrioles then become refractory to S phase regulators that would normally promote duplication during the first endocycle, after which they are eliminated during the L2 stage. Furthermore, we show that SPD-2 plays a central role in the numeral regulation of centrioles as a potential target of CDK activity. On the other hand, the phosphorylation on SPD-2 by Polo-like kinase, the transcriptional regulation of genes that affect centriole biogenesis, and the ubiquitin/proteasome degradation pathway, contribute collectively to the final elimination of the centrioles during the L2 stage.

Ultrastructure of spermiogenesis of Dipylidium caninum (Cestoda, Cyclophyllidea, Dipylidiidae), an intestinal parasite of Canis familiaris.

Science.gov (United States)

Miquel, J; Bâ, C T; Marchand, B

1998-09-01

We describe for the first time the ultrastructure of spermiogenesis of a representative of the family Dipylidiidae, Dipylidium caninum. Spermiogenesis begins with the formation of a differentiation zone. This conical area presents two centrioles with associated striated roots. One of the centrioles develops a flagellum and posteriorly a proximodistal fusion is produced between the axoneme and a median cytoplasmic process. In a final stage of spermiogenesis a condensation of electron-dense material between cortical microtubules is observed in the anterior part of the differentiation zone. This is the origin of the crest-like body that appears at the end of spermiogenesis. The presence of striated roots associated with centrioles constitutes the first report of these structures in the spermiogenesis of a cyclophyllidean cestode.

Using sea urchin gametes and zygotes to investigate centrosome duplication.

Science.gov (United States)

Sluder, Greenfield

2016-01-01

Centriole structure and function in the sea urchin zygote parallel those in mammalian somatic cells. Here, I briefly introduce the properties and attributes of the sea urchin system that make it an attractive platform for the study of centrosome and centriole duplication. These attributes apply to all echinoderms readily available from commercial suppliers: sea urchins, sand dollars, and starfish. I list some of the practical aspects of the system that make it a cost- and time-effective system for experimental work and then list properties that are a "tool kit" that can be used to conduct studies that would not be practical, or in some cases not possible, with mammalian somatic cells. Since centrioles organize and localize the pericentriolar material that nucleates the astral arrays of microtubules (Bobinnec et al. in J Cell Biol 143(6):1575-1589, 1998), the pattern of aster duplication over several cell cycles can be used as a reliable measure for centriole duplication (Sluder and Rieder in J Cell Biol 100(3):887-896, 1985). Descriptions of the methods my laboratory has used to handle and image echinoderm zygotes are reviewed in Sluder et al. (Methods Cell Biol 61:439-472, 1999). Also included is a bibliography of papers that describe additional methods.

LRRC45 Is a Centrosome Linker Component Required for Centrosome Cohesion

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Runsheng He

2013-09-01

Full Text Available During interphase, centrosomes are connected by a proteinaceous linker between the proximal ends of the centrioles, which is important for the centrosomes to function as a single microtubule-organizing center. However, the composition and regulation of centrosomal linker remain largely unknown. Here, we show that LRRC45 is a centrosome linker that localizes at the proximal ends of the centrioles and forms fiber-like structures between them. Depletion of LRRC45 results in centrosome splitting during interphase. Moreover, LRRC45 interacts with both C-Nap1 and rootletin and is phosphorylated by Nek2A at S661 during mitosis. After phosphorylation, both LRRC45 centrosomal localization and fiber-like structures are significantly reduced, which subsequently leads to centrosome separation. Thus, LRRC45 is a critical component of the proteinaceous linker between two centrioles and is required for centrosome cohesion.

Centriole polarisation to the immunological synapse directs secretion from cytolytic cells of both the innate and adaptive immune systems

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Arico Maurizo

2011-06-01

Full Text Available Abstract Background Cytolytic cells of the immune system destroy pathogen-infected cells by polarised exocytosis of secretory lysosomes containing the pore-forming protein perforin. Precise delivery of this lethal hit is essential to ensuring that only the target cell is destroyed. In cytotoxic T lymphocytes (CTLs, this is accomplished by an unusual movement of the centrosome to contact the plasma membrane at the centre of the immunological synapse formed between killer and target cells. Secretory lysosomes are directed towards the centrosome along microtubules and delivered precisely to the point of target cell recognition within the immunological synapse, identified by the centrosome. We asked whether this mechanism of directing secretory lysosome release is unique to CTL or whether natural killer (NK and invariant NKT (iNKT cytolytic cells of the innate immune system use a similar mechanism to focus perforin-bearing lysosome release. Results NK cells were conjugated with B-cell targets lacking major histocompatibility complex class I 721.221 cells, and iNKT cells were conjugated with glycolipid-pulsed CD1-bearing targets, then prepared for thin-section electron microscopy. High-resolution electron micrographs of the immunological synapse formed between NK and iNKT cytolytic cells with their targets revealed that in both NK and iNKT cells, the centrioles could be found associated (or 'docked' with the plasma membrane within the immunological synapse. Secretory clefts were visible within the synapses formed by both NK and iNKT cells, and secretory lysosomes were polarised along microtubules leading towards the docked centrosome. The Golgi apparatus and recycling endosomes were also polarised towards the centrosome at the plasma membrane within the synapse. Conclusions These results reveal that, like CTLs of the adaptive immune system, the centrosomes of NK and iNKT cells (cytolytic cells of the innate immune system direct secretory lysosomes to

Centriole polarisation to the immunological synapse directs secretion from cytolytic cells of both the innate and adaptive immune systems.

Science.gov (United States)

Stinchcombe, Jane C; Salio, Mariolina; Cerundolo, Vincenzo; Pende, Daniela; Arico, Maurizo; Griffiths, Gillian M

2011-06-28

Cytolytic cells of the immune system destroy pathogen-infected cells by polarised exocytosis of secretory lysosomes containing the pore-forming protein perforin. Precise delivery of this lethal hit is essential to ensuring that only the target cell is destroyed. In cytotoxic T lymphocytes (CTLs), this is accomplished by an unusual movement of the centrosome to contact the plasma membrane at the centre of the immunological synapse formed between killer and target cells. Secretory lysosomes are directed towards the centrosome along microtubules and delivered precisely to the point of target cell recognition within the immunological synapse, identified by the centrosome. We asked whether this mechanism of directing secretory lysosome release is unique to CTL or whether natural killer (NK) and invariant NKT (iNKT) cytolytic cells of the innate immune system use a similar mechanism to focus perforin-bearing lysosome release. NK cells were conjugated with B-cell targets lacking major histocompatibility complex class I 721.221 cells, and iNKT cells were conjugated with glycolipid-pulsed CD1-bearing targets, then prepared for thin-section electron microscopy. High-resolution electron micrographs of the immunological synapse formed between NK and iNKT cytolytic cells with their targets revealed that in both NK and iNKT cells, the centrioles could be found associated (or 'docked') with the plasma membrane within the immunological synapse. Secretory clefts were visible within the synapses formed by both NK and iNKT cells, and secretory lysosomes were polarised along microtubules leading towards the docked centrosome. The Golgi apparatus and recycling endosomes were also polarised towards the centrosome at the plasma membrane within the synapse. These results reveal that, like CTLs of the adaptive immune system, the centrosomes of NK and iNKT cells (cytolytic cells of the innate immune system) direct secretory lysosomes to the immunological synapse. Morphologically, the

Radiation damage to bull sperm motility. III. Further x-ray studies

International Nuclear Information System (INIS)

Rikmenspoel, R.

1975-01-01

The results of previous radiation experiments, which indicated that the centriole serves as a control center for bull sperm motility, appear to be in conflict with experiments showing that the bull sperm flagellum is an autonomous oscillator. To resolve this conflict experiments were conducted to calibrate absolutely the dose-response curves for the radiation damage, and to measure the force production and the mechanochemical energy conversion after irradiation in bull sperm. The results indicate that the centriole acts as a mechanical anchor for the contractile fibers

A novel family of katanin-like 2 protein isoforms (KATNAL2), interacting with nucleotide-binding proteins Nubp1 and Nubp2, are key regulators of different MT-based processes in mammalian cells.

Science.gov (United States)

Ververis, Antonis; Christodoulou, Andri; Christoforou, Maria; Kamilari, Christina; Lederer, Carsten W; Santama, Niovi

2016-01-01

Katanins are microtubule (MT)-severing AAA proteins with high phylogenetic conservation throughout the eukaryotes. They have been functionally implicated in processes requiring MT remodeling, such as spindle assembly in mitosis and meiosis, assembly/disassembly of flagella and cilia and neuronal morphogenesis. Here, we uncover a novel family of katanin-like 2 proteins (KATNAL2) in mouse, consisting of five alternatively spliced isoforms encoded by the Katnal2 genomic locus. We further demonstrate that in vivo these isoforms are able to interact with themselves, with each other and moreover directly and independently with MRP/MinD-type P-loop NTPases Nubp1 and Nubp2, which are integral components of centrioles, negative regulators of ciliogenesis and implicated in centriole duplication in mammalian cells. We find KATNAL2 localized on interphase MTs, centrioles, mitotic spindle, midbody and the axoneme and basal body of sensory cilia in cultured murine cells. shRNAi of Katnal2 results in inefficient cytokinesis and severe phenotypes of enlarged cells and nuclei, increased numbers of centrioles and the manifestation of aberrant multipolar mitotic spindles, mitotic defects, chromosome bridges, multinuclearity, increased MT acetylation and an altered cell cycle pattern. Silencing or stable overexpression of KATNAL2 isoforms drastically reduces ciliogenesis. In conclusion, KATNAL2s are multitasking enzymes involved in the same cell type in critically important processes affecting cytokinesis, MT dynamics, and ciliogenesis and are also implicated in cell cycle progression.

Estrutura fina do espermatozóide de Mellita quinquiesperforata Leske (Echinodermata do litoral norte do Brasil Fine structure of the spermatozoon of the Mellita quinquiesperforata (Echinodermata of the Northern littoral of Brazil

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Edilson Matos

2000-09-01

Full Text Available The fine structure of the spermatozoon in the Mellita quinquiesperforata Leske, 1778 was studied. Collected from the Amazon region, were described by light and transmission electron microscopy. The spermatozoon consisted of a acrosome complex, nucleus, four to six mitochondria, two centrioles and flagellum. The acrosome was composed of a acrosome complex, the acrosomal vesicle and the subacrosomal space. The nucleus are composed by dense material. The middle piece contains 4-6 mitochondria which are arranged around the centrioles, the 9p+0, whose axoneme has the classic 9p+2 microtubular construction.

TBCD links centriologenesis, spindle microtubule dynamics, and midbody abscission in human cells.

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Mónica López Fanarraga

2010-01-01

Full Text Available Microtubule-organizing centers recruit alpha- and beta-tubulin polypeptides for microtubule nucleation. Tubulin synthesis is complex, requiring five specific cofactors, designated tubulin cofactors (TBCs A-E, which contribute to various aspects of microtubule dynamics in vivo. Here, we show that tubulin cofactor D (TBCD is concentrated at the centrosome and midbody, where it participates in centriologenesis, spindle organization, and cell abscission. TBCD exhibits a cell-cycle-specific pattern, localizing on the daughter centriole at G1 and on procentrioles by S, and disappearing from older centrioles at telophase as the protein is recruited to the midbody. Our data show that TBCD overexpression results in microtubule release from the centrosome and G1 arrest, whereas its depletion produces mitotic aberrations and incomplete microtubule retraction at the midbody during cytokinesis. TBCD is recruited to the centriole replication site at the onset of the centrosome duplication cycle. A role in centriologenesis is further supported in differentiating ciliated cells, where TBCD is organized into "centriolar rosettes". These data suggest that TBCD participates in both canonical and de novo centriolar assembly pathways.

Unique properties of Drosophila spermatocyte primary cilia

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Maria Giovanna Riparbelli

2013-09-01

The primary cilium is an essential organelle required for animal development and adult homeostasis that is found on most animal cells. The primary cilium contains a microtubule-based axoneme cytoskeleton that typically grows from the mother centriole in G0/G1 phase of the cell cycle as a membrane-bound compartment that protrudes from the cell surface. A unique system of bidirectional transport, intraflagellar transport (IFT, maintains the structure and function of cilia. While the axoneme is dynamic, growing and shrinking at its tip, at the same time it is very stable to the effects of microtubule-targeting drugs. The primary cilia found on Drosophila spermatocytes diverge from the general rules of primary cilium biology in several respects. Among these unique attributes, spermatocyte cilia assemble from all four centrioles in an IFT-independent manner in G2 phase, and persist continuously through two cell divisions. Here, we show that Drosophila spermatocyte primary cilia are extremely sensitive to microtubule-targeting drugs, unlike their mammalian counterparts. Spermatocyte cilia and their axonemes fail to assemble or be maintained upon nocodazole treatment, while centriole replication appears unperturbed. On the other hand, paclitaxel (Taxol, a microtubule-stabilizing drug, disrupted transition zone assembly and anchoring to the plasma membrane while causing spermatocyte primary cilia to grow extensively long during the assembly/elongation phase, but did not overtly affect the centrioles. However, once assembled to their mature length, spermatocyte cilia appeared unaffected by Taxol. The effects of these drugs on axoneme dynamics further demonstrate that spermatocyte primary cilia are endowed with unique assembly properties.

A highly conserved Poc1 protein characterized in embryos of the hydrozoan Clytia hemisphaerica: localization and functional studies.

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Cécile Fourrage

Full Text Available Poc1 (Protein of Centriole 1 proteins are highly conserved WD40 domain-containing centriole components, well characterized in the alga Chlamydomonas, the ciliated protazoan Tetrahymena, the insect Drosophila and in vertebrate cells including Xenopus and zebrafish embryos. Functions and localizations related to the centriole and ciliary axoneme have been demonstrated for Poc1 in a range of species. The vertebrate Poc1 protein has also been reported to show an additional association with mitochondria, including enrichment in the specialized "germ plasm" region of Xenopus oocytes. We have identified and characterized a highly conserved Poc1 protein in the cnidarian Clytia hemisphaerica. Clytia Poc1 mRNA was found to be strongly expressed in eggs and early embryos, showing a punctate perinuclear localization in young oocytes. Fluorescence-tagged Poc1 proteins expressed in developing embryos showed strong localization to centrioles, including basal bodies. Anti-human Poc1 antibodies decorated mitochondria in Clytia, as reported in human cells, but failed to recognise endogenous or fluorescent-tagged Clytia Poc1. Injection of specific morpholino oligonucleotides into Clytia eggs prior to fertilization to repress Poc1 mRNA translation interfered with cell division from the blastula stage, likely corresponding to when neosynthesis normally takes over from maternally supplied protein. Cell cycle lengthening and arrest were observed, phenotypes consistent with an impaired centriolar biogenesis or function. The specificity of the defects could be demonstrated by injection of synthetic Poc1 mRNA, which restored normal development. We conclude that in Clytia embryos, Poc1 has an essentially centriolar localization and function.

Ndel1 suppresses ciliogenesis in proliferating cells by regulating the trichoplein-Aurora A pathway.

Science.gov (United States)

Inaba, Hironori; Goto, Hidemasa; Kasahara, Kousuke; Kumamoto, Kanako; Yonemura, Shigenobu; Inoko, Akihito; Yamano, Shotaro; Wanibuchi, Hideki; He, Dongwei; Goshima, Naoki; Kiyono, Tohru; Hirotsune, Shinji; Inagaki, Masaki

2016-02-15

Primary cilia protrude from the surface of quiescent cells and disassemble at cell cycle reentry. We previously showed that ciliary reassembly is suppressed by trichoplein-mediated Aurora A activation pathway in growing cells. Here, we report that Ndel1, a well-known modulator of dynein activity, localizes at the subdistal appendage of the mother centriole, which nucleates a primary cilium. In the presence of serum, Ndel1 depletion reduces trichoplein at the mother centriole and induces unscheduled primary cilia formation, which is reverted by forced trichoplein expression or coknockdown of KCTD17 (an E3 ligase component protein for trichoplein). Serum starvation induced transient Ndel1 degradation, subsequent to the disappearance of trichoplein at the mother centriole. Forced expression of Ndel1 suppressed trichoplein degradation and axonemal microtubule extension during ciliogenesis, similar to trichoplein induction or KCTD17 knockdown. Most importantly, the proportion of ciliated and quiescent cells was increased in the kidney tubular epithelia of newborn Ndel1-hypomorphic mice. Thus, Ndel1 acts as a novel upstream regulator of the trichoplein-Aurora A pathway to inhibit primary cilia assembly. © 2016 Inaba et al.

Electron Microscopy Structural Insights into CPAP Oligomeric Behavior

DEFF Research Database (Denmark)

Alvarez-Cabrera, Ana L; Delgado, Sandra; Gil-Carton, David

2017-01-01

Centrosomal P4.1-associated protein (CPAP) is a cell cycle regulated protein fundamental for centrosome assembly and centriole elongation. In humans, the region between residues 897-1338 of CPAP mediates interactions with other proteins and includes a homodimerization domain. CPAP mutations cause...... into clearly different regular 3D maps (putatively corresponding to dimers and tetramers) and direct observation of individual images representing other complexes of HsCPAP(897-1338) (i.e., putative flexible monomers and higher-order multimers), we report a dynamic oligomeric behavior of this protein, where...... of different oligomers of CPAP, suggesting further insights to understand how this protein works, contributing to the elucidation of control mechanisms for centriole biogenesis....

Sas-4 proteins are required during basal body duplication in Paramecium

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Gogendeau, Delphine; Hurbain, Ilse; Raposo, Graca; Cohen, Jean; Koll, France; Basto, Renata

2011-01-01

Centrioles and basal bodies are structurally related organelles composed of nine microtubule (MT) triplets. Studies performed in Caenorhabditis elegans embryos have shown that centriole duplication takes place in sequential way, in which different proteins are recruited in a specific order to assemble a procentriole. ZYG-1 initiates centriole duplication by triggering the recruitment of a complex of SAS-5 and SAS-6, which then recruits the final player, SAS-4, to allow the incorporation of MT singlets. It is thought that a similar mechanism (that also involves additional proteins) is present in other animal cells, but it remains to be investigated whether the same players and their ascribed functions are conserved during basal body duplication in cells that exclusively contain basal bodies. To investigate this question, we have used the multiciliated protist Paramecium tetraurelia. Here we show that in the absence of PtSas4, two types of defects in basal body duplication can be identified. In the majority of cases, the germinative disk and cartwheel, the first structures assembled during duplication, are not detected. In addition, if daughter basal bodies were formed, they invariably had defects in MT recruitment. Our results suggest that PtSas4 has a broader function than its animal orthologues. PMID:21289083

Non-Overlapping Distributions and Functions of the VDAC Family in Ciliogenesis

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Shubhra Majumder

2015-07-01

Full Text Available Centrosomes are major microtubule-organizing centers of animal cells that consist of two centrioles. In mitotic cells, centrosomes are duplicated to serve as the poles of the mitotic spindle, while in quiescent cells, centrosomes move to the apical membrane where the oldest centriole is transformed into a basal body to assemble a primary cilium. We recently showed that mitochondrial outer membrane porin VDAC3 localizes to centrosomes where it negatively regulates ciliogenesis. We show here that the other two family members, VDAC1 and VDAC2, best known for their function in mitochondrial bioenergetics, are also found at centrosomes. Like VDAC3, centrosomal VDAC1 is predominantly localized to the mother centriole, while VDAC2 localizes to centriolar satellites in a microtubule-dependent manner. Down-regulation of VDAC1 leads to inappropriate ciliogenesis, while its overexpression suppresses cilia formation, suggesting that VDAC1 and VDAC3 both negatively regulate ciliogenesis. However, this negative effect on ciliogenesis is not shared by VDAC2, which instead appears to promote maturation of primary cilia. Moreover, because overexpression of VDAC3 cannot compensate for depletion of VDAC1, our data suggest that while the entire VDAC family localizes to centrosomes, they have non-redundant functions in cilogenesis.

Spermatic characteristics and sperm evolution on the subfamily Stevardiinae (Ostariophysi: Characiformes: Characidae

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Clarianna Martins Baicere-Silva

Full Text Available The monophyly and phylogenetic relationships among the members of Clade A characids (sensu Malabarba & Weitzman, later redefined and named as the Stevardiinae (sensu Mirande, have been primarily supported by traditional morphological and molecular data. Herein were examined, described and compared spermiogenesis and sperm ultrastructure of 12 species of the genera Boehlkea, Bryconacidnus, Bryconamericus, Creagrutus, Cyanocharax, Hemibrycon, Knodus, Odontostoechus, Piabina, and Rhinobrycon in order to evaluate possible phylogenetic signals and their potential use in recovering relationships of the Stevardiinae. All examined species demonstrated a nuclear rotation equal or less than 95º resulting in a lateral position of the double nuclear fossa and flagellum. In all species, sperm nuclei are slightly elongate toward the flagellum, the proximal centriole is partially inside the nuclear fossa and lies anterior and oblique to the distal centriole, and the midpiece is short and strongly asymmetric. All species analyzed herein and other species previously examined for these systems in the Stevardiinae share homologous sperm characteristics as evidenced by spermiogenesis, further supporting the monophyly of this clade. Spermatozoa of the Stevardiinae further show three morphotypes (M1, M2, M3 of arrangement of centrioles, flagellum, nucleus and midpiece, hypothesized as successively derived in a series of transformation from the most basal morphotype (M1.

Bug22p, a Conserved Centrosomal/Ciliary Protein Also Present in Higher Plants, Is Required for an Effective Ciliary Stroke in Paramecium ▿ †

Science.gov (United States)

Laligné, C.; Klotz, C.; Garreau de Loubresse, N.; Lemullois, M.; Hori, M.; Laurent, F. X.; Papon, J. F.; Louis, B.; Cohen, J.; Koll, F.

2010-01-01

Centrioles, cilia, and flagella are ancestral conserved organelles of eukaryotic cells. Among the proteins identified in the proteomics of ciliary proteins in Paramecium, we focus here on a protein, Bug22p, previously detected by cilia and basal-body high-throughput studies but never analyzed per se. Remarkably, this protein is also present in plants, which lack centrioles and cilia. Bug22p sequence alignments revealed consensus positions that distinguish species with centrioles/cilia from plants. In Paramecium, antibody and green fluorescent protein (GFP) fusion labeling localized Bug22p in basal bodies and cilia, and electron microscopy immunolabeling refined the localization to the terminal plate of the basal bodies, the transition zone, and spots along the axoneme, preferentially between the membrane and the microtubules. RNA interference (RNAi) depletion of Bug22p provoked a strong decrease in swimming speed, followed by cell death after a few days. High-speed video microscopy and morphological analysis of Bug22p-depleted cells showed that the protein plays an important role in the efficiency of ciliary movement by participating in the stroke shape and rigidity of cilia. The defects in cell swimming and growth provoked by RNAi can be complemented by expression of human Bug22p. This is the first reported case of complementation by a human gene in a ciliate. PMID:20118210

Bug22p, a conserved centrosomal/ciliary protein also present in higher plants, is required for an effective ciliary stroke in Paramecium.

Science.gov (United States)

Laligné, C; Klotz, C; de Loubresse, N Garreau; Lemullois, M; Hori, M; Laurent, F X; Papon, J F; Louis, B; Cohen, J; Koll, F

2010-04-01

Centrioles, cilia, and flagella are ancestral conserved organelles of eukaryotic cells. Among the proteins identified in the proteomics of ciliary proteins in Paramecium, we focus here on a protein, Bug22p, previously detected by cilia and basal-body high-throughput studies but never analyzed per se. Remarkably, this protein is also present in plants, which lack centrioles and cilia. Bug22p sequence alignments revealed consensus positions that distinguish species with centrioles/cilia from plants. In Paramecium, antibody and green fluorescent protein (GFP) fusion labeling localized Bug22p in basal bodies and cilia, and electron microscopy immunolabeling refined the localization to the terminal plate of the basal bodies, the transition zone, and spots along the axoneme, preferentially between the membrane and the microtubules. RNA interference (RNAi) depletion of Bug22p provoked a strong decrease in swimming speed, followed by cell death after a few days. High-speed video microscopy and morphological analysis of Bug22p-depleted cells showed that the protein plays an important role in the efficiency of ciliary movement by participating in the stroke shape and rigidity of cilia. The defects in cell swimming and growth provoked by RNAi can be complemented by expression of human Bug22p. This is the first reported case of complementation by a human gene in a ciliate.

Cellular Mechanisms of Ciliary Length Control

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Jacob Keeling

2016-01-01

Full Text Available Cilia and flagella are evolutionarily conserved, membrane-bound, microtubule-based organelles on the surface of most eukaryotic cells. They play important roles in coordinating a variety of signaling pathways during growth, development, cell mobility, and tissue homeostasis. Defects in ciliary structure or function are associated with multiple human disorders called ciliopathies. These diseases affect diverse tissues, including, but not limited to the eyes, kidneys, brain, and lungs. Many processes must be coordinated simultaneously in order to initiate ciliogenesis. These include cell cycle, vesicular trafficking, and axonemal extension. Centrioles play a central role in both cell cycle progression and ciliogenesis, making the transition between basal bodies and mitotic spindle organizers integral to both processes. The maturation of centrioles involves a functional shift from cell division toward cilium nucleation which takes place concurrently with its migration and fusion to the plasma membrane. Several proteinaceous structures of the distal appendages in mother centrioles are required for this docking process. Ciliary assembly and maintenance requires a precise balance between two indispensable processes; so called assembly and disassembly. The interplay between them determines the length of the resulting cilia. These processes require a highly conserved transport system to provide the necessary substances at the tips of the cilia and to recycle ciliary turnover products to the base using a based microtubule intraflagellar transport (IFT system. In this review; we discuss the stages of ciliogenesis as well as mechanisms controlling the lengths of assembled cilia.

A PLK4 mutation causing azoospermia in a man with Sertoli cell-only syndrome.

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Miyamoto, T; Bando, Y; Koh, E; Tsujimura, A; Miyagawa, Y; Iijima, M; Namiki, M; Shiina, M; Ogata, K; Matsumoto, N; Sengoku, K

2016-01-01

About 15% of couples wishing to have children are infertile; approximately half these cases involve a male factor. Polo-like kinase 4 (PLK-4) is a member of the polo protein family and a key regulator of centriole duplication. Male mice with a point mutation in the Plk4 gene show azoospermia associated with germ cell loss. Mutational analysis of 81 patients with azoospermia and Sertoli cell-only syndrome (SCOS) identified one man with a heterozygous 13-bp deletion in the Ser/Thr kinase domain of PLK4. Division of centrioles occurred in wild-type PLK4-transfected cells, but was hampered in PLK-4-mutant transfectants, which also showed abnormal nuclei. Thus, this PLK4 mutation might be a cause of human SCOS and nonobstructive azoospermia. © 2015 American Society of Andrology and European Academy of Andrology.

Centriolar CPAP/SAS-4 Imparts Slow Processive Microtubule Growth

NARCIS (Netherlands)

Sharma, Ashwani; Aher, Amol; Dynes, Nicola J; Frey, Daniel; Katrukha, Eugene A; Jaussi, Rolf; Grigoriev, Ilya; Croisier, Marie; Kammerer, Richard A; Akhmanova, Anna; Gönczy, Pierre; Steinmetz, Michel O

2016-01-01

Centrioles are fundamental and evolutionarily conserved microtubule-based organelles whose assembly is characterized by microtubule growth rates that are orders of magnitude slower than those of cytoplasmic microtubules. Several centriolar proteins can interact with tubulin or microtubules, but how

The fine structure of the sperm and spermatid differentiation in the ...

African Journals Online (AJOL)

process of spermatogenesis for any of the South African ... So-Arc. Tydskr. ... acrosome: ar, axial rod; av, acrosomaJ vesicle; c, collir; dc, distal centriole; H. head; M, mid-piece: m, .... posite end of the cell to the acrosomal vesicle so forming the.

Centrosome Clustering in the Development of Bovine Binucleate Trophoblast Giant Cells.

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Klisch, Karl; Schraner, Elisabeth M; Boos, Alois

2017-01-01

Binucleate trophoblast giant cells (BNC) are the characteristic feature of the ruminant placenta. During their development, BNC pass through 2 acytokinetic mitoses and become binucleate with 2 tetraploid nuclei. In this study, we investigate the number and location of centrosomes in bovine BNC. Centrosomes typically consist of 2 centrioles surrounded by electron-dense pericentriolar material. Duplication of centrosomes is tightly linked to the cell cycle, which ensures that the number of centrosomes remains constant in proliferating diploid cells. Alterations of the cell cycle, which affect the number of chromosome sets, also affect the number of centrosomes. In this study, we use placentomal tissue from pregnant cows (gestational days 80-230) for immunohistochemical staining of γ-tubulin (n = 3) and transmission electron microscopy (n = 3). We show that mature BNC have 4 centrosomes with 8 centrioles, clustered in the angle between the 2 cell nuclei. During the second acytokinetic mitosis, the centrosomes must be clustered to form the poles of a bipolar spindle. In rare cases, centrosome clustering fails and tripolar mitosis leads to the formation of trinucleate "BNC". Generally, centrosome clustering occurs in polyploid tumor cells, which have an increased number of centrioles, but it is absent in proliferating diploid cells. Thus, inhibition of centrosome clustering in tumor cells is a novel promising strategy for cancer treatment. BNC are a cell population in which centrosome clustering occurs as part of the normal life history. Thus, they might be a good model for the study of the molecular mechanisms of centrosome clustering. © 2016 S. Karger AG, Basel.

Dishevelled is a NEK2 kinase substrate controlling dynamics of centrosomal linker proteins

Czech Academy of Sciences Publication Activity Database

Červenka, I.; Valnohová, J.; Bernatík, Ondřej; Harnoš, J.; Radsetoulal, M.; Šedová, K.; Hanakova, K.; Potěšil, D.; Sedláčková, M.; Salašová, A.; Steinhart, Z.; Angers, S.; Schulte, G.; Hampl, A.; Zdráhal, Z.; Bryja, Vítězslav

2016-01-01

Roč. 113, č. 33 (2016), s. 9304-9309 ISSN 0027-8424 R&D Projects: GA ČR GP13-31488P Institutional support: RVO:68081707 Keywords : cell-cycle * centriole duplication * beta-catenin * cohesion Subject RIV: BO - Biophysics Impact factor: 9.661, year: 2016

Transmission electron microscopy of apical cells of Sphacelaria spp. (Sphacelariales, Phaeophyceae)

NARCIS (Netherlands)

Prud’homme van Reine, W.F.; Star, W.

1981-01-01

The ultrastructure of apical cells of six species of Sphacelaria (S. arctica, S. cirrosa, S. nana, S. racemosa, S. radicans, and S. rigidula) is studied here. In most details such as ultrastructure of chloroplasts, mitochondria, microbodies, nuclei and centrioles all Sphacelaria species studied are

OFIP/KIAA0753 forms a complex with OFD1 and FOR20 at pericentriolar satellites and centrosomes and is mutated in one individual with oral-facial-digital syndrome

NARCIS (Netherlands)

Chevrier, V.; Bruel, A.L.; Dam, T.J. van; Franco, B.; Scalzo, M. Lo; Lembo, F.; Audebert, S.; Baudelet, E.; Isnardon, D.; Bole, A.; Borg, J.P.; Kuentz, P.; Thevenon, J.; Burglen, L.; Faivre, L.; Riviere, J.B.; Huynen, M.A.; Birnbaum, D.; Rosnet, O.; Thauvin-Robinet, C.

2016-01-01

Oral-facial-digital (OFD) syndromes are rare heterogeneous disorders characterized by the association of abnormalities of the face, the oral cavity and the extremities, some due to mutations in proteins of the transition zone of the primary cilia or the closely associated distal end of centrioles.

Phosphatase PP2A and microtubule-mediated pulling forces disassemble centrosomes during mitotic exit

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Stephen J. Enos

2018-01-01

Full Text Available Centrosomes are microtubule-nucleating organelles that facilitate chromosome segregation and cell division in metazoans. Centrosomes comprise centrioles that organize a micron-scale mass of protein called pericentriolar material (PCM from which microtubules nucleate. During each cell cycle, PCM accumulates around centrioles through phosphorylation-mediated assembly of PCM scaffold proteins. During mitotic exit, PCM swiftly disassembles by an unknown mechanism. Here, we used Caenorhabditis elegans embryos to determine the mechanism and importance of PCM disassembly in dividing cells. We found that the phosphatase PP2A and its regulatory subunit SUR-6 (PP2ASUR-6, together with cortically directed microtubule pulling forces, actively disassemble PCM. In embryos depleted of these activities, ∼25% of PCM persisted from one cell cycle into the next. Purified PP2ASUR-6 could dephosphorylate the major PCM scaffold protein SPD-5 in vitro. Our data suggest that PCM disassembly occurs through a combination of dephosphorylation of PCM components and force-driven fragmentation of the PCM scaffold.

Primordial dwarfism: an update.

Science.gov (United States)

Alkuraya, Fowzan S

2015-02-01

To review the recent advances in the clinical and molecular characterization of primordial dwarfism, an extreme growth deficiency disorder that has its onset during embryonic development and persists throughout life. The last decade has witnessed an unprecedented acceleration in the discovery of genes mutated in primordial dwarfism, from one gene to more than a dozen genes. These genetic discoveries have confirmed the notion that primordial dwarfism is caused by defects in basic cellular processes, most notably centriolar biology and DNA damage response. Fortunately, the increasing number of reported clinical primordial dwarfism subtypes has been accompanied by more accurate molecular classification. Qualitative defects of centrioles with resulting abnormal mitosis dynamics, reduced proliferation, and increased apoptosis represent the predominant molecular pathogenic mechanism in primordial dwarfism. Impaired DNA damage response is another important mechanism, which we now know is not mutually exclusive to abnormal centrioles. Molecular characterization of primordial dwarfism is helping families by enabling more reproductive choices and may pave the way for the future development of therapeutics.

Ultrastructure of the spermatozoon of Potamonautes perlatus ...

African Journals Online (AJOL)

... thickened ring, the latter trend being taken to completion in grapsid Thoracotremata. Unknown in other Brachyura are (iv) a rim-like electron pale expansion around the operculum and (v) the elongate condition of at least one of the two centrioles. These five unusual features are regarded as apomorphies of Potamonautes.

Spermatogenesis and sperm ultrastructure in the polychaete genus Ophryotrocha (Dorvilleidae)

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Pfannenstiel, Hans-Dieter; Grünig, Charlotte

1990-06-01

The details of spermatogenesis and spermiogenesis are described for Ophryotrocha puerilis. The ultrastructure of mature sperm is shown for O. puerilis, O. hartmanni, O. gracilis, O. diadema, O. labronica, and O. notoglandulata. Clusters of sixteen cells each are proliferated by two stem cells in each setigerous segment of O. puerilis representing the very early stages of both oogenesis and spermatogenesis. In each spermatocyte-I cluster, the cells are interconnected by cytoplasmic bridges. Early, clusters are enveloped by peritoneal sheath cells. These transient gonad walls break down prior to meiosis. The meiotic processes may start in the clusters with the cells still interconnected, or during breakdown of the original cluster, giving rise to smaller subclusters of both spermatocytes I and spermatocytes II with various numbers of cells. Finally, spermatid tetrads are present. As spermiogenesis progresses, the tetrads disintegrate. Golgi vesicles in both spermatocytes and spermatids contain electron-dense material, presumably preacrosomal. The acrosome is formed by such vesicles. In the six species studied here, the acrosomes appear to be of a similar overall structure but are of different shape. Centrioles are usually located beneath the acrosome. The distal centriole forms the basal body of a flagellum-like cytoplasmic process. The microtubules of these flagellar equivalents do not show a normal ciliar arrangement. The flagellar equivalent appears to be non-motile. In O. hartmanni and in O. notoglandulata, a flagellar equivalent is missing. Microtubules originating from the proximal end of the distal centriole stretch to the nuclear envelope. This feature appears to be especially conspicuous in O. puerilis and in O. labronica. In O. labronica and in O. notoglandulata, bundles of microtubules paralleling the cell perimeter appear to stabilise the sperm. Various numbers of mitochondria are either randomly distributed around the nucleus or accumulate on one side

Proximity Interactions among Basal Body Components in Trypanosoma brucei Identify Novel Regulators of Basal Body Biogenesis and Inheritance

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Hung Quang Dang

2017-01-01

Full Text Available The basal body shares similar architecture with centrioles in animals and is involved in nucleating flagellar axonemal microtubules in flagellated eukaryotes. The early-branching Trypanosoma brucei possesses a motile flagellum nucleated from the basal body that consists of a mature basal body and an adjacent pro-basal body. Little is known about the basal body proteome and its roles in basal body biogenesis and flagellar axoneme assembly in T. brucei. Here, we report the identification of 14 conserved centriole/basal body protein homologs and 25 trypanosome-specific basal body proteins. These proteins localize to distinct subdomains of the basal body, and several of them form a ring-like structure surrounding the basal body barrel. Functional characterization of representative basal body proteins revealed distinct roles in basal body duplication/separation and flagellar axoneme assembly. Overall, this work identified novel proteins required for basal body duplication and separation and uncovered new functions of conserved basal body proteins in basal body duplication and separation, highlighting an unusual mechanism of basal body biogenesis and inheritance in this early divergent eukaryote.

Casein kinase II is required for proper cell division and acts as a negative regulator of centrosome duplication in Caenorhabditis elegans embryos

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Jeffrey C. Medley

2017-01-01

Full Text Available Centrosomes are the primary microtubule-organizing centers that orchestrate microtubule dynamics during the cell cycle. The correct number of centrosomes is pivotal for establishing bipolar mitotic spindles that ensure accurate segregation of chromosomes. Thus, centrioles must duplicate once per cell cycle, one daughter per mother centriole, the process of which requires highly coordinated actions among core factors and modulators. Protein phosphorylation is shown to regulate the stability, localization and activity of centrosome proteins. Here, we report the function of Casein kinase II (CK2 in early Caenorhabditis elegans embryos. The catalytic subunit (KIN-3/CK2α of CK2 localizes to nuclei, centrosomes and midbodies. Inactivating CK2 leads to cell division defects, including chromosome missegregation, cytokinesis failure and aberrant centrosome behavior. Furthermore, depletion or inhibiting kinase activity of CK2 results in elevated ZYG-1 levels at centrosomes, restoring centrosome duplication and embryonic viability to zyg-1 mutants. Our data suggest that CK2 functions in cell division and negatively regulates centrosome duplication in a kinase-dependent manner.

Ultrastructure of spermatozoa in cobia, Rachycentron canadum (Linnaeus, 1766).

Science.gov (United States)

Dhanasekar, Krishnamoorthy; Selvakumar, Narasimman; Munuswamy, Natesan

2018-02-01

Ultrastructure and development of spermatozoa in cobia, Rachycentron canadum are described. Sections through the testis show different developmental stages viz, Spermatocytes, spermatids and sperm. Spermatozoa of R. canadum exhibit the configuration of uniflagellated, anacrosomal Type I aquasperm, typical for externally fertilizing fish. Mature spermatozoon is seen with a prominent head and long cylindrical flagellum. Ultrastructure of sperm shows invaginated 'U' shaped nucleus and other organelles. The mitochondrial matrix is electron-dense with irregular arrangement of the cristae. The nucleus reveals a deep invagination (nuclear fossa) in which the centriolar complex is located. The centriolar complex lies inside the nuclear fossa and is composed of a proximal and a distal centriole. The two centrioles are placed perpendicular to each other. The flagellum has a typical eukaryotic organization (microtubule doublets 9 + 2 pattern) and measures around 36.21 ± 0.42 μm in length. This study for the first time provides a comprehensive detail on the ultrastructure and developmental process of sperm in cobia, R. canadum. Copyright © 2017 Elsevier B.V. All rights reserved.

Interplay between microtubule bundling and sorting factors ensures acentriolar spindle stability during C. elegans oocyte meiosis.

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Timothy J Mullen

2017-09-01

Full Text Available In many species, oocyte meiosis is carried out in the absence of centrioles. As a result, microtubule organization, spindle assembly, and chromosome segregation proceed by unique mechanisms. Here, we report insights into the principles underlying this specialized form of cell division, through studies of C. elegans KLP-15 and KLP-16, two highly homologous members of the kinesin-14 family of minus-end-directed kinesins. These proteins localize to the acentriolar oocyte spindle and promote microtubule bundling during spindle assembly; following KLP-15/16 depletion, microtubule bundles form but then collapse into a disorganized array. Surprisingly, despite this defect we found that during anaphase, microtubules are able to reorganize into a bundled array that facilitates chromosome segregation. This phenotype therefore enabled us to identify factors promoting microtubule organization during anaphase, whose contributions are normally undetectable in wild-type worms; we found that SPD-1 (PRC1 bundles microtubules and KLP-18 (kinesin-12 likely sorts those bundles into a functional orientation capable of mediating chromosome segregation. Therefore, our studies have revealed an interplay between distinct mechanisms that together promote spindle formation and chromosome segregation in the absence of structural cues such as centrioles.

Spermatozoan ultrastructure in the trigonioid bivalve Neotrigonia margaritacea Lamarck (Mollusca): Comparison with other bivalves, especially Trigonioida and Unionoida

Science.gov (United States)

Healy, J. M.

1996-06-01

Spermatozoa of the trigonioid bivalve Neotrigonia margaritacea (Lamarck) (Trigoniidae, Trigonioida) are examined ultrastructurally. A cluster of discoidal, proacrosomal vesicles (between 9 to 15 in number) constitutes the acrosomal complex at the nuclear apex. The nucleus is short (2.4 2.6 μm long, maximum diameter 2.2 μm), blunt-conical in shape, and exhibits irregular lacunae within its contents. Five or sometimes four round mitochondria are impressed into shallow depressions in the base of the nucleus as is a discrete centriolar fossa. The mitochondria surround two orthogonally arranged centrioles to form, collectively, the midpiece region. The distal centriole, anchored by nine satellite fibres to the plasma membrane, acts as a basal body to the sperm flagellum. The presence of numerous proacrosomal vesicles instead of a single, conical acrosomal vesicle sets Neotrigonia (and the Trigonioida) apart from other bivalves, with the exception of the Unionoida which are also known to exhibit this multivesicular condition. Spermatozoa of N. margaritacea are very similar to those of the related species Neotrigonia bednalli (Verco) with the exception that the proacrosomal vesicles of N. margaritacea are noticeably larger than those of N. bednalli.

Ultrastructure of spermiogenesis in Vampyroteuthis infernalis Chun — a relict cephalopod mollusc

Science.gov (United States)

Healy, John M.

1990-03-01

Spermiogenesis in the relict deep-sea cephalopod Vampyroteuthis infernalis Chun is examined using transmission electron microscopy (TEM), and the results compared with available data on other cephalopods. Early spermatids of Vampyroteuthis exhibit an ovoid nucleus (with dense irregular patches), numerous mitochondria and a pair of triplet substructure centrioles (arranged parallel to each other). Subsequently, the following morphological changes take place: (1) nuclear contents condense into a fibrous reticulum, then into thick fibres; (2) the acrosomal vesicle (presumably Golgi-derived) positions itself in a shallow depression at the nuclear apex; (3) the flagellum forms from one of the two centrioles; (4) mitochondria cluster around the flagellum at the base of the nucleus; (5) a dense, fibrous plug forms within the basal invagination of the nucleus. Microtubules surround the acrosome and condensing nucleus of spermatids. The dense plug is of special systematic importance since it also occurs in spermatids and spermatozoa of Octopus spp., but not in any investigated species of the Sepiida, Sepiolida or Teuthida. Late spermatids and mature spermatozoa of Vampyroteuthis strongly resemble developing spermatids of Octopus, suggesting a close phylogenetic relationship between Vampyroteuthis (and the Vampyromorpha) and octopods.

CENP-W plays a role in maintaining bipolar spindle structure.

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Agnieszka Kaczmarczyk

Full Text Available The CENP-W/T complex was previously reported to be required for mitosis. HeLa cells depleted of CENP-W displayed profound mitotic defects, with mitotic timing delay, disorganized prometaphases and multipolar spindles as major phenotypic consequences. In this study, we examined the process of multipolar spindle formation induced by CENP-W depletion. Depletion of CENP-W in HeLa cells labeled with histone H2B and tubulin fluorescent proteins induced rapid fragmentation of originally bipolar spindles in a high proportion of cells. CENP-W depletion was associated with depletion of Hec1 at kinetochores. The possibility of promiscuous centrosomal duplication was ruled out by immunofluorescent examination of centrioles. However, centrioles were frequently observed to be abnormally split. In addition, a large proportion of the supernumerary poles lacked centrioles, but were positively stained with different centrosomal markers. These observations suggested that perturbation in spindle force distribution caused by defective kinetochores could contribute to a mechanical mechanism for spindle pole disruption. 'Spindle free' nocodazole arrested cells did not exhibit pole fragmentation after CENP-W depletion, showing that pole fragmentation is microtubule dependent. Inhibition of centrosome separation by monastrol reduced the incidence of spindle pole fragmentation, indicating that Eg5 plays a role in spindle pole disruption. Surprisingly, CENP-W depletion rescued the monopolar spindle phenotype of monastrol treatment, with an increased frequency of bipolar spindles observed after CENP-W RNAi. We overexpressed the microtubule cross-linking protein TPX2 to create spindle poles stabilized by the microtubule cross-linking activity of TPX2. Spindle pole fragmentation was suppressed in a TPX2-dependent fashion. We propose that CENP-W, by influencing proper kinetochore assembly, particularly microtubule docking sites, can confer spindle pole resistance to traction

CENP-W Plays a Role in Maintaining Bipolar Spindle Structure

Science.gov (United States)

Kaczmarczyk, Agnieszka; Sullivan, Kevin F.

2014-01-01

The CENP-W/T complex was previously reported to be required for mitosis. HeLa cells depleted of CENP-W displayed profound mitotic defects, with mitotic timing delay, disorganized prometaphases and multipolar spindles as major phenotypic consequences. In this study, we examined the process of multipolar spindle formation induced by CENP-W depletion. Depletion of CENP-W in HeLa cells labeled with histone H2B and tubulin fluorescent proteins induced rapid fragmentation of originally bipolar spindles in a high proportion of cells. CENP-W depletion was associated with depletion of Hec1 at kinetochores. The possibility of promiscuous centrosomal duplication was ruled out by immunofluorescent examination of centrioles. However, centrioles were frequently observed to be abnormally split. In addition, a large proportion of the supernumerary poles lacked centrioles, but were positively stained with different centrosomal markers. These observations suggested that perturbation in spindle force distribution caused by defective kinetochores could contribute to a mechanical mechanism for spindle pole disruption. ‘Spindle free’ nocodazole arrested cells did not exhibit pole fragmentation after CENP-W depletion, showing that pole fragmentation is microtubule dependent. Inhibition of centrosome separation by monastrol reduced the incidence of spindle pole fragmentation, indicating that Eg5 plays a role in spindle pole disruption. Surprisingly, CENP-W depletion rescued the monopolar spindle phenotype of monastrol treatment, with an increased frequency of bipolar spindles observed after CENP-W RNAi. We overexpressed the microtubule cross-linking protein TPX2 to create spindle poles stabilized by the microtubule cross-linking activity of TPX2. Spindle pole fragmentation was suppressed in a TPX2-dependent fashion. We propose that CENP-W, by influencing proper kinetochore assembly, particularly microtubule docking sites, can confer spindle pole resistance to traction forces exerted

EB1 is required for primary cilia assembly in fibroblasts

DEFF Research Database (Denmark)

Schrøder, Jacob M; Schneider, Linda; Christensen, Søren T

2007-01-01

EB1 is a small microtubule (MT)-binding protein that associates preferentially with MT plus ends and plays a role in regulating MT dynamics. EB1 also targets other MT-associated proteins to the plus end and thereby regulates interactions of MTs with the cell cortex, mitotic kinetochores, and diff...... that localization of EB1 at the centriole/basal body is required for primary cilia assembly in fibroblasts....

RNG1 is a Late Marker of the Apical Polar Ring in Toxoplasma gondii

Science.gov (United States)

Tran, Johnson Q.; de Leon, Jessica C.; Li, Catherine; Huynh, My-Hang; Beatty, Wandy; Morrissette, Naomi S.

2010-01-01

The asexually proliferating stages of apicomplexan parasites cause acute symptoms of diseases such as malaria, cryptosporidiosis and toxoplasmosis. These stages are characterized by the presence of two independent microtubule organizing centers (MTOCs). Centrioles are found at the poles of the intranuclear spindle. The apical polar ring (APR), a MTOC unique to apicomplexans, organizes subpellicular microtubules which impose cell shape and apical polarity on these protozoa. Here we describe the characteristics of a novel protein that localizes to the APR of Toxoplasma gondii which we have named ring-1 (RNG1). There are related RNG1 proteins in Neospora caninum and Sarcocystis neurona but no obvious homologs in Plasmodium spp., Cryptosporidium spp. or Babesia spp. RNG1 is a small, low-complexity, detergent-insoluble protein that assembles at the APR very late in the process of daughter parasite replication. We were unable to knock-out the RNG1 gene, suggesting that its gene product is essential. Tagged RNG1 lines have also allowed us to visualize the APR during growth of Toxoplasma in the microtubule-disrupting drug oryzalin. Oryzalin inhibits nuclear division and cytokinesis although Toxoplasma growth continues, and similar to earlier observations of unchecked centriole duplication in oryzalin-treated parasites, the APR continues to duplicate during aberrant parasite growth. PMID:20658557

Transcriptional program of ciliated epithelial cells reveals new cilium and centrosome components and links to human disease.

Directory of Open Access Journals (Sweden)

Ramona A Hoh

Full Text Available Defects in the centrosome and cilium are associated with a set of human diseases having diverse phenotypes. To further characterize the components that define the function of these organelles we determined the transcriptional profile of multiciliated tracheal epithelial cells. Cultures of mouse tracheal epithelial cells undergoing differentiation in vitro were derived from mice expressing GFP from the ciliated-cell specific FOXJ1 promoter (FOXJ1:GFP. The transcriptional profile of ciliating GFP+ cells from these cultures was defined at an early and a late time point during differentiation and was refined by subtraction of the profile of the non-ciliated GFP- cells. We identified 649 genes upregulated early, when most cells were forming basal bodies, and 73 genes genes upregulated late, when most cells were fully ciliated. Most, but not all, of known centrosome proteins are transcriptionally upregulated early, particularly Plk4, a master regulator of centriole formation. We found that three genes associated with human disease states, Mdm1, Mlf1, and Dyx1c1, are upregulated during ciliogenesis and localize to centrioles and cilia. This transcriptome for mammalian multiciliated epithelial cells identifies new candidate centrosome and cilia proteins, highlights similarities between components of motile and primary cilia, and identifies new links between cilia proteins and human disease.

Association of TCTP with Centrosome and Microtubules

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Mariusz K. Jaglarz

2012-01-01

Full Text Available Translationally Controlled Tumour Protein (TCTP associates with microtubules (MT, however, the details of this association are unknown. Here we analyze the relationship of TCTP with MTs and centrosomes in Xenopus laevis and mammalian cells using immunofluorescence, tagged TCTP expression and immunoelectron microscopy. We show that TCTP associates both with MTs and centrosomes at spindle poles when detected by species-specific antibodies and by Myc-XlTCTP expression in Xenopus and mammalian cells. However, when the antibodies against XlTCTP were used in mammalian cells, TCTP was detected exclusively in the centrosomes. These results suggest that a distinct pool of TCTP may be specific for, and associate with, the centrosomes. Double labelling for TCTP and γ-tubulin with immuno-gold electron microscopy in Xenopus laevis oogonia shows localization of TCTP at the periphery of the γ-tubulin-containing pericentriolar material (PCM enveloping the centriole. TCTP localizes in the close vicinity of, but not directly on the MTs in Xenopus ovary suggesting that this association requires unidentified linker proteins. Thus, we show for the first time: (1 the association of TCTP with centrosomes, (2 peripheral localization of TCTP in relation to the centriole and the γ-tubulin-containing PCM within the centrosome, and (3 the indirect association of TCTP with MTs.

Markiana nigripinnis (Perugia, 1891 as a putative member of the subfamily Stevardiinae (Characiformes: Characidae: spermatic evidence

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Clarianna Martins Baicere-Silva

Full Text Available The genus Markiana was until recently recognized as incertae sedis in the family Characidae, even though alternative placements for this genus have been advanced since its original description. More recently, it was hypothesized that Markiana nigripinnis is part of a clade informally named the Astyanax clade, indicating the putative close relationship of Markiana with the genus Astyanax. Examination of sperm ultrastructure of representatives of Astyanax and M. nigripinnis shows no evidence for this hypothesized close relationship. Rather, the spermatozoa of M. nigripinnis share characters found in spermatozoa of the non-inseminating members of the subfamily Stevardiinae, such as an angle of nuclear rotation equal to 85º resulting in a lateral position of the double nuclear fossa and flagellum. As with the non-inseminating Stevardiinae, sperm nuclei are also slightly elongate toward the flagellum, the proximal centriole is partially inside the nuclear fossa and anterior and oblique to the distal centriole, and the midpiece is short and strongly asymmetric. Additionally, M. nigripinnis shares with the other members of the Stevardiinae the presence of only four teeth in the inner row of the premaxillary and a short triangular ectopterygoid, which is never more than twice the length of the palatine.

Homozygous STIL mutation causes holoprosencephaly and microcephaly in two siblings.

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Charlotte Mouden

Full Text Available Holoprosencephaly (HPE is a frequent congenital malformation of the brain characterized by impaired forebrain cleavage and midline facial anomalies. Heterozygous mutations in 14 genes have been identified in HPE patients that account for only 30% of HPE cases, suggesting the existence of other HPE genes. Data from homozygosity mapping and whole-exome sequencing in a consanguineous Turkish family were combined to identify a homozygous missense mutation (c.2150G>A; p.Gly717Glu in STIL, common to the two affected children. STIL has a role in centriole formation and has previously been described in rare cases of microcephaly. Rescue experiments in U2OS cells showed that the STIL p.Gly717Glu mutation was not able to fully restore the centriole duplication failure following depletion of endogenous STIL protein indicating the deleterious role of the mutation. In situ hybridization experiments using chick embryos demonstrated that expression of Stil was in accordance with a function during early patterning of the forebrain. It is only the second time that a STIL homozygous mutation causing a recessive form of HPE was reported. This result also supports the genetic heterogeneity of HPE and increases the panel of genes to be tested for HPE diagnosis.

Ploidy Manipulation of Zebrafish Embryos with Heat Shock 2 Treatment

OpenAIRE

Baars, Destiny L.; Takle, Kendra A.; Heier, Jonathon; Pelegri, Francisco

2016-01-01

Manipulation of ploidy allows for useful transformations, such as diploids to tetraploids, or haploids to diploids. In the zebrafish Danio rerio, specifically the generation of homozygous gynogenetic diploids is useful in genetic analysis because it allows the direct production of homozygotes from a single heterozygous mother. This article describes a modified protocol for ploidy duplication based on a heat pulse during the first cell cycle, Heat Shock 2 (HS2). Through inhibition of centriole...

Disease: H01880 [KEGG MEDICUS

Lifescience Database Archive (English)

Full Text Available H01880 Autosomal recessive microcephaly and chorioretinopathy Autosomal-recessive ...ent in MCCRP patients are variable, but the chorioretinopathy is a constant feature and includes typical pun...ark DB ... TITLE ... Chorioretinopathy with hereditary microcephaly. ... JOURNAL ... Arch Ophthalmol 75:597-600 (1... autosomal recessive microcephaly and chorioretinopathy. ... JOURNAL ... Eur J Hum Genet 24:1702-1706 (2016) DOI... ... Mutations in PLK4, encoding a master regulator of centriole biogenesis, cause microcephaly, growth failure and retinopathy

Phosphorylation of CPAP by Aurora-A Maintains Spindle Pole Integrity during Mitosis

OpenAIRE

En-Ju Chou; Liang-Yi Hung; Chieh-Ju C. Tang; Wen-Bin Hsu; Hsin-Yi Wu; Pao-Chi Liao; Tang K. Tang

2016-01-01

CPAP is required for centriole elongation during S/G2 phase, but the role of CPAP in mitosis is incompletely understood. Here, we show that CPAP maintains spindle pole integrity through its phosphorylation by Aurora-A during mitosis. Depletion of CPAP induced a prolonged delay in mitosis, pericentriolar material (PCM) dispersion, and multiple mitotic abnormalities. Further studies demonstrated that CPAP directly interacts with and is phosphorylated by Aurora-A at serine 467 during mitosis. In...

Dissecting the function and assembly of acentriolar microtubule organizing centers in Drosophila cells in vivo.

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Janina Baumbach

2015-05-01

Full Text Available Acentriolar microtubule organizing centers (aMTOCs are formed during meiosis and mitosis in several cell types, but their function and assembly mechanism is unclear. Importantly, aMTOCs can be overactive in cancer cells, enhancing multipolar spindle formation, merotelic kinetochore attachment and aneuploidy. Here we show that aMTOCs can form in acentriolar Drosophila somatic cells in vivo via an assembly pathway that depends on Asl, Cnn and, to a lesser extent, Spd-2--the same proteins that appear to drive mitotic centrosome assembly in flies. This finding enabled us to ablate aMTOC formation in acentriolar cells, and so perform a detailed genetic analysis of the contribution of aMTOCs to acentriolar mitotic spindle formation. Here we show that although aMTOCs can nucleate microtubules, they do not detectably increase the efficiency of acentriolar spindle assembly in somatic fly cells. We find that they are required, however, for robust microtubule array assembly in cells without centrioles that also lack microtubule nucleation from around the chromatin. Importantly, aMTOCs are also essential for dynein-dependent acentriolar spindle pole focusing and for robust cell proliferation in the absence of centrioles and HSET/Ncd (a kinesin essential for acentriolar spindle pole focusing in many systems. We propose an updated model for acentriolar spindle pole coalescence by the molecular motors Ncd/HSET and dynein in conjunction with aMTOCs.

Cyclin G2 is a centrosome-associated nucleocytoplasmic shuttling protein that influences microtubule stability and induces a p53-dependent cell cycle arrest

International Nuclear Information System (INIS)

Arachchige Don, Aruni S.; Dallapiazza, Robert F.; Bennin, David A.; Brake, Tiffany; Cowan, Colleen E.; Horne, Mary C.

2006-01-01

Cyclin G2 is an atypical cyclin that associates with active protein phosphatase 2A. Cyclin G2 gene expression correlates with cell cycle inhibition; it is significantly upregulated in response to DNA damage and diverse growth inhibitory stimuli, but repressed by mitogenic signals. Ectopic expression of cyclin G2 promotes cell cycle arrest, cyclin dependent kinase 2 inhibition and the formation of aberrant nuclei [Bennin, D. A., Don, A. S., Brake, T., McKenzie, J. L., Rosenbaum, H., Ortiz, L., DePaoli-Roach, A. A., and Horne, M. C. (2002). Cyclin G2 associates with protein phosphatase 2A catalytic and regulatory B' subunits in active complexes and induces nuclear aberrations and a G 1 /S-phase cell cycle arrest. J Biol Chem 277, 27449-67]. Here we report that endogenous cyclin G2 copurifies with centrosomes and microtubules (MT) and that ectopic G2 expression alters microtubule stability. We find exogenous and endogenous cyclin G2 present at microtubule organizing centers (MTOCs) where it colocalizes with centrosomal markers in a variety of cell lines. We previously reported that cyclin G2 forms complexes with active protein phosphatase 2A (PP2A) and colocalizes with PP2A in a detergent-resistant compartment. We now show that cyclin G2 and PP2A colocalize at MTOCs in transfected cells and that the endogenous proteins copurify with isolated centrosomes. Displacement of the endogenous centrosomal scaffolding protein AKAP450 that anchors PP2A at the centrosome resulted in the depletion of centrosomal cyclin G2. We find that ectopic expression of cyclin G2 induces microtubule bundling and resistance to depolymerization, inhibition of polymer regrowth from MTOCs and a p53-dependent cell cycle arrest. Furthermore, we determined that a 100 amino acid carboxy-terminal region of cyclin G2 is sufficient to both direct GFP localization to centrosomes and induce cell cycle inhibition. Colocalization of endogenous cyclin G2 with only one of two GFP-centrin-tagged centrioles, the

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LENUS (Irish Health Repository)

2010-11-15

DNA damage can induce centrosome overduplication in a manner that requires G2-to-M checkpoint function, suggesting that genotoxic stress can decouple the centrosome and chromosome cycles. How this happens is unclear. Using live-cell imaging of cells that express fluorescently tagged NEDD1\\/GCP-WD and proliferating cell nuclear antigen, we found that ionizing radiation (IR)-induced centrosome amplification can occur outside S phase. Analysis of synchronized populations showed that significantly more centrosome amplification occurred after irradiation of G2-enriched populations compared with G1-enriched or asynchronous cells, consistent with G2 phase centrosome amplification. Irradiated and control populations of G2 cells were then fused to test whether centrosome overduplication is allowed through a diffusible stimulatory signal, or the loss of a duplication-inhibiting signal. Irradiated G2\\/irradiated G2 cell fusions showed significantly higher centrosome amplification levels than irradiated G2\\/unirradiated G2 fusions. Chicken-human cell fusions demonstrated that centrosome amplification was limited to the irradiated partner. Our finding that only the irradiated centrosome can duplicate supports a model where a centrosome-autonomous inhibitory signal is lost upon irradiation of G2 cells. We observed centriole disengagement after irradiation. Although overexpression of dominant-negative securin did not affect IR-induced centrosome amplification, Plk1 inhibition reduced radiation-induced amplification. Together, our data support centriole disengagement as a licensing signal for DNA damage-induced centrosome amplification.

The primary cilium as a multiple cellular signaling scaffold in development and disease

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Hyuk Wan Ko*

2012-08-01

Full Text Available Primary cilia, single hair-like appendage on the surface of themost mammalian cells, were once considered to be vestigialcellular organelles for a past century because of their tinystructure and unknown function. Although they lack ancestralmotility function of cilia or flagella, they share common groundwith multiciliated motile cilia and flagella on internal structuresuch as microtubule based nine outer doublets nucleated from thebase of mother centrioles called basal body. Making cilia,ciliogenesis, in cells depends on the cell cycle stage due to reuseof centrioles for cell division forming mitotic spindle pole (Mphase and assembling cilia from basal body (starting G1 phaseand maintaining most of interphase. Ciliary assembly requiredtwo conflicting processes such as assembly and disassembly andbalance between these two processes determines the length ofcilia. Both process required highly conserved transport system tosupply needed substance to grow tip of cilia and bring ciliaryturnover product back to the base of cilia using motor protein,kinesin and dynein, and transport protein complex, IFT particles.Disruption of ciliary structure or function causes multiple humandisorder called ciliopathies affecting disease of diverse ciliatedtissues ranging from eye, kidney, respiratory tract and brain.Recent explosion of research on the primary cilia and theirinvolvement on animal development and disease attracts scientificinterest on how extensively the function of cilia related to specificcell physiology and signaling pathway. In this review, I introducegeneral features of primary cilia and recent progress inunderstanding of the ciliary length control and signaling pathwaystransduced through primary cilia in vertebrates.

The fine structure of the sperm of the round goby (Neogobius melanostomus)

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Allen, Jeffrey D.; Walker, Glenn K.; Nichols, Susan J.; Sorenson, Dorothy

2004-01-01

The fine structural details of the spermatozoon of the round goby are presented for the first time in this study. Scanning and transmission electron microscopic examination of testis reveals an anacrosomal spermatozoon with a slightly elongate head and uniformly compacted chromatin. The midpiece contains a single, spherical mitochondrion. Two perpendicularly oriented centrioles lie in a deep, eccentric nuclear fossa with no regularly observed connection to the nucleus. The flagellum develops bilateral fins soon after emerging from the fossa; each extends approximately 1 A?m from the axoneme and persists nearly the length of the flagellum.

The Paramyxea Levine 1979: An original example of evolution towards multicellularity

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Desportes, Isabelle

1984-03-01

The Paramyxea are parasitic in marine invertebrates. Their development is a sporulation involving the differentiation within a stem cell of several sporonts which produce spores made of cells enclosed inside each other. Three genera are recognized according to the number of spores and sporal cells, and the taxonomic position of the host (Polychaeta, Mollusca, Crustacea). The Paramyxea exhibit both protistan and metazoan characters. Their nine singlets centrioles are observed in different Protoctists whereas the fact that their sporal cells acquire distinctive cytological features may be interpreted as an evolution towards multicellularity.

Role of delta-tubulin and the C-tubule in assembly of Paramecium basal bodies

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Beisson Janine

2001-03-01

Full Text Available Abstract Background A breakthrough in the understanding of centriole assembly was provided by the characterization of the UNI3 gene in Chlamydomonas. Deletion of this gene, found to encode a novel member of the tubulin superfamily, delta-tubulin, results in the loss of the C-tubule, in the nine microtubule triplets which are the hallmark of centrioles and basal bodies. Delta-tubulin homologs have been identified in the genomes of mammals and protozoa, but their phylogenetic relationships are unclear and their function is not yet known. Results Using the method of gene-specific silencing, we have inactivated the Paramecium delta-tubulin gene, which was recently identified. This inactivation leads to loss of the C-tubule in all basal bodies, without any effect on ciliogenesis. This deficiency does not directly affect basal body duplication, but perturbs the cortical cytoskeleton, progressively leading to mislocalization and loss of basal bodies and to altered cell size and shape. Furthermore, additional loss of B- and even A-tubules at one or more triplet sites are observed: around these incomplete cylinders, the remaining doublets are nevertheless positioned according to the native ninefold symmetry. Conclusions The fact that in two distinct phyla, delta-tubulin plays a similar role provides a new basis for interpreting phylogenetic relationships among delta-tubulins. The role of delta-tubulin in C-tubule assembly reveals that tubulins contribute subtle specificities at microtubule nucleation sites. Our observations also demonstrate the existence of a prepattern for the ninefold symmetry of the organelle which is maintained even if less than 9 triplets develop.

A new organellar complex in rat sympathetic neurons.

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Matt S Ramer

Full Text Available Membranous compartments of neurons such as axons, dendrites and modified primary cilia are defining features of neuronal phenotype. This is unlike organelles deep to the plasma membrane, which are for the most part generic and not related directly to morphological, neurochemical or functional specializations. However, here we use multi-label immunohistochemistry combined with confocal and electron microscopy to identify a very large (approximately 6 microns in diameter, entirely intracellular neuronal organelle which occurs singly in a ubiquitous but neurochemically distinct and morphologically simple subset of sympathetic ganglion neurons. Although usually toroidal, it also occurs as twists or rods depending on its intracellular position: tori are most often perinuclear whereas rods are often found in axons. These 'loukoumasomes' (doughnut-like bodies bind a monoclonal antibody raised against beta-III-tubulin (SDL.3D10, although their inability to bind other beta-III-tubulin monoclonal antibodies indicate that the responsible antigen is not known. Position-morphology relationships within neurons and their expression of non-muscle heavy chain myosin suggest a dynamic structure. They associate with nematosomes, enigmatic nucleolus-like organelles present in many neural and non-neural tissues, which we now show to be composed of filamentous actin. Loukoumasomes also separately interact with mother centrioles forming the basal body of primary cilia. They express gamma tubulin, a microtubule nucleator which localizes to non-neuronal centrosomes, and cenexin, a mother centriole-associated protein required for ciliogenesis. These data reveal a hitherto undescribed organelle, and depict it as an intracellular transport machine, shuttling material between the primary cilium, the nematosome, and the axon.

Revisiting the theoretical basis of the endosymbiotic origin of plastids in the original context of Lynn Margulis on the origin of mitosing, eukaryotic cells.

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Sato, Naoki

2017-12-07

Fifty years ago, Lynn Margulis proposed a comprehensive hypothesis on the origin of eukaryotic cells with an emphasis on the origin of mitosis. This hypothesis postulated that the eukaryotic cell is a composite of different parts as a result of the symbiosis of various different bacteria. In this hypothesis, she integrated previously proposed ideas that mitochondria and chloroplasts were descendants of endosymbionts that originated from aerobic bacteria and blue-green algae (now cyanobacteria), respectively. However, the major part of her hypothesis, which she believed to be original, was the origin of mitosis. The core of her postulate involved a chromosome partition mechanism dependent on DNA-microtubule binding, which originated from a hypothetical centriole-DNA complex, with an ability to replicate. Surprisingly, her complete lack of real experimental works in the cytoskeleton, cell motility, or paleontology did not prevent this 29-year-old junior scientist from assembling archival knowledge and constructing a narrative on the evolution of all organisms. Whether the centriole-DNA complex originated from a spirochete or not was a minor anecdote in this initial postulate. Unfortunately, this hypothesis on the origin of mitosis, which she believed to be a holistic unity, testable by experiments, was entirely refuted. Despite falsification of her original narrative as a whole, her success as a founder of endosymbiotic theory on the origin of mitochondria and chloroplasts is undoubted. We will discuss the reasons for her success in terms of the historical situation in the latter half of the 20th century. Copyright © 2017 Elsevier Ltd. All rights reserved.

Simulated hypogravity and synaptogenesis in culture

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Gruener, R.

1985-01-01

A study on the effects of simulated microgravity on spinal neurons and myocytes cultured from X. laevis, is performed. Horizontal clinorotation at 1-10 rpm lasted from 16-36 hours, a sufficient time for cells to proceed through ontogenetic maturation. Late appearance of striations, retarded consumpton of yolk platelets and fewer and thinner neurites indicate subnormal expression of cell functions. Furthermore, these cells do not respond normally to environmental cues like trophic substances or surface contact. The observed delay in cell maturation is consistent with a hypothesis that cellular graviperception may effect the centriole and cytoskeleton.

15 years of research on Oral-Facial-Digital syndromes: from 1 to 16 causal genes

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Bruel, Ange-Line; Franco, Brunella; Duffourd, Yannis; Thevenon, Julien; Jego, Laurence; Lopez, Estelle; Deleuze, Jean-François; Doummar, Diane; Giles, Rachel H.; Johnson, Colin A.; Huynen, Martijn A.; Chevrier, Véronique; Burglen, Lydie; Morleo, Manuela; Desguerres, Isabelle; Pierquin, Geneviève; Doray, Bérénice; Gilbert-Dussardier, Brigitte; Reversade, Bruno; Steichen-Gersdorf, Elisabeth; Baumann, Clarisse; Panigrahi, Inusha; Fargeot-Espaliat, Anne; Dieux, Anne; David, Albert; Goldenberg, Alice; Bongers, Ernie; Gaillard, Dominique; Argente, Jesús; Aral, Bernard; Gigot, Nadège; St-Onge, Judith; Birnbaum, Daniel; Phadke, Shubha R.; Cormier-Daire, Valérie; Eguether, Thibaut; Pazour, Gregory J.; Herranz-Pérez, Vicente; Lee, Jaclyn S.; Pasquier, Laurent; Loget, Philippe; Saunier, Sophie; Mégarbané, André; Rosnet, Olivier; Leroux, Michel R.; Wallingford, John B.; Blacque, Oliver E.; Nachury, Maxence V.; Attie-Bitach, Tania; Rivière, Jean-Baptiste; Faivre, Laurence; Thauvin-Robinet, Christel

2017-01-01

Oral-facial-digital syndromes (OFDS) gather rare genetic disorders characterized by facial, oral and digital abnormalities associated with a wide range of additional features (polycystic kidney disease, cerebral malformations and several others) to delineate a growing list of OFD subtypes. The most frequent, OFD type I, is caused by a heterozygous mutation in the OFD1 gene encoding a centrosomal protein. The wide clinical heterogeneity of OFDS suggests the involvement of other ciliary genes. For 15 years, we have aimed to identify the molecular bases of OFDS. This effort has been greatly helped by the recent development of whole exome sequencing (WES). Here, we present all our published and unpublished results for WES in 24 OFDS cases. We identified causal variants in five new genes (C2CD3, TMEM107, INTU, KIAA0753, IFT57) and related the clinical spectrum of four genes in other ciliopathies (C5orf42, TMEM138, TMEM231, WDPCP) to OFDS. Mutations were also detected in two genes previously implicated in OFDS. Functional studies revealed the involvement of centriole elongation, transition zone and intraflagellar transport defects in OFDS, thus characterizing three ciliary protein modules: the complex KIAA0753-FOPNL-OFD1, a regulator of centriole elongation; the MKS module, a major component of the transition zone; and the CPLANE complex necessary for IFT-A assembly. OFDS now appear to be a distinct subgroup of ciliopathies with wide heterogeneity, which makes the initial classification obsolete. A clinical classification restricted to the three frequent/well-delineated subtypes could be proposed, and for patients who do not fit one of these 3 main subtypes, a further classification could be based on the genotype. PMID:28289185

Actin and Arp2/3 localize at the centrosome of interphase cells

Energy Technology Data Exchange (ETDEWEB)

Hubert, Thomas; Vandekerckhove, Joel; Gettemans, Jan, E-mail: jan.gettemans@vib-ugent.be

2011-01-07

Research highlights: {yields} Actin was detected at the centrosome with the anti-actin antibody 1C7 that recognizes antiparallel ('lower dimer') actin dimers. {yields} Centrosomal actin was found in interphase but not mitotic MDA-MB-231 cells. {yields} Neither the anti-actin antibody C4 that binds to globular, monomer actin, nor the anti-actin antibody 2G2 that recognizes the nuclear conformation of actin detect actin at the centrosome. {yields} The Arp2/3 complex transiently localizes at the pericentriolar matrix but not at the centrioles of interphase HEK 293T cells. -- Abstract: Although many actin binding proteins such as cortactin and the Arp2/3 activator WASH localize at the centrosome, the presence and conformation of actin at the centrosome has remained elusive. Here, we report the localization of actin at the centrosome in interphase but not in mitotic MDA-MB-231 cells. Centrosomal actin was detected with the anti-actin antibody 1C7 that recognizes antiparallel ('lower dimer') actin dimers. In addition, we report the transient presence of the Arp2/3 complex at the pericentriolar matrix but not at the centrioles of interphase HEK 293T cells. Overexpression of an Arp2/3 component resulted in expansion of the pericentriolar matrix and selective accumulation of the Arp2/3 component in the pericentriolar matrix. Altogether, we hypothesize that the centrosome transiently recruits Arp2/3 to perform processes such as centrosome separation prior to mitotic entry, whereas the observed constitutive centrosomal actin staining in interphase cells reinforces the current model of actin-based centrosome reorientation toward the leading edge in migrating cells.

Actin and Arp2/3 localize at the centrosome of interphase cells

International Nuclear Information System (INIS)

Hubert, Thomas; Vandekerckhove, Joel; Gettemans, Jan

2011-01-01

Research highlights: → Actin was detected at the centrosome with the anti-actin antibody 1C7 that recognizes antiparallel ('lower dimer') actin dimers. → Centrosomal actin was found in interphase but not mitotic MDA-MB-231 cells. → Neither the anti-actin antibody C4 that binds to globular, monomer actin, nor the anti-actin antibody 2G2 that recognizes the nuclear conformation of actin detect actin at the centrosome. → The Arp2/3 complex transiently localizes at the pericentriolar matrix but not at the centrioles of interphase HEK 293T cells. -- Abstract: Although many actin binding proteins such as cortactin and the Arp2/3 activator WASH localize at the centrosome, the presence and conformation of actin at the centrosome has remained elusive. Here, we report the localization of actin at the centrosome in interphase but not in mitotic MDA-MB-231 cells. Centrosomal actin was detected with the anti-actin antibody 1C7 that recognizes antiparallel ('lower dimer') actin dimers. In addition, we report the transient presence of the Arp2/3 complex at the pericentriolar matrix but not at the centrioles of interphase HEK 293T cells. Overexpression of an Arp2/3 component resulted in expansion of the pericentriolar matrix and selective accumulation of the Arp2/3 component in the pericentriolar matrix. Altogether, we hypothesize that the centrosome transiently recruits Arp2/3 to perform processes such as centrosome separation prior to mitotic entry, whereas the observed constitutive centrosomal actin staining in interphase cells reinforces the current model of actin-based centrosome reorientation toward the leading edge in migrating cells.

The conserved Wdr8-hMsd1/SSX2IP complex localises to the centrosome and ensures proper spindle length and orientation

International Nuclear Information System (INIS)

Hori, Akiko; Morand, Agathe; Ikebe, Chiho; Frith, David; Snijders, Ambrosius P.; Toda, Takashi

2015-01-01

The centrosome plays a pivotal role in a wide range of cellular processes and its dysfunction is causally linked to many human diseases including cancer and developmental and neurological disorders. This organelle contains more than one hundred components, and yet many of them remain uncharacterised. Here we identified a novel centrosome protein Wdr8, based upon the structural conservation of the fission yeast counterpart. We showed that Wdr8 constitutively localises to the centrosome and super resolution microscopy uncovered that this protein is enriched at the proximal end of the mother centriole. Furthermore, we identified hMsd1/SSX2IP, a conserved spindle anchoring protein, as one of Wdr8 interactors by mass spectrometry. Wdr8 formed a complex and partially colocalised with hMsd1/SSX2IP. Intriguingly, knockdown of Wdr8 or hMsd1/SSX2IP displayed very similar mitotic defects, in which spindle microtubules became shortened and misoriented. Indeed, Wdr8 depletion resulted in the reduced recruitment of hMsd1/SSX2IP to the mitotic centrosome, though the converse is not true. Together, we propose that the conserved Wdr8-hMsd1/SSX2IP complex plays a critical role in controlling proper spindle length and orientation. - Highlights: • Human Wdr8 is a centrosomal protein enriched in the proximal end of the centriole. • Wdr8 and hMsd1/SSX2IP form a complex conserved in fungi. • Depletion of Wdr8 results in shorter, tilted spindle microtubules. • Depletion phenotypes of Wdr8 are very similar to those of hMsd1/SSX2IP knockdown.

Multiple requirements of PLK1 during mouse oocyte maturation.

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Petr Solc

Full Text Available Polo-like kinase 1 (PLK1 orchestrates multiple events of cell division. Although PLK1 function has been intensively studied in centriole-containing and rapidly cycling somatic cells, much less is known about its function in the meiotic divisions of mammalian oocytes, which arrest for a long period of time in prophase before meiotic resumption and lack centrioles for spindle assembly. Here, using specific small molecule inhibition combined with live mouse oocyte imaging, we comprehensively characterize meiotic PLK1's functions. We show that PLK1 becomes activated at meiotic resumption on microtubule organizing centers (MTOCs and later at kinetochores. PLK1 is required for efficient meiotic resumption by promoting nuclear envelope breakdown. PLK1 is also needed to recruit centrosomal proteins to acentriolar MTOCs to promote normal spindle formation, as well as for stable kinetochore-microtubule attachment. Consequently, PLK1 inhibition leads to metaphase I arrest with misaligned chromosomes activating the spindle assembly checkpoint (SAC. Unlike in mitosis, the metaphase I arrest is not bypassed by the inactivation of the SAC. We show that PLK1 is required for the full activation of the anaphase promoting complex/cyclosome (APC/C by promoting the degradation of the APC/C inhibitor EMI1 and is therefore essential for entry into anaphase I. Moreover, our data suggest that PLK1 is required for proper chromosome segregation and the maintenance of chromosome condensation during the meiosis I-II transition, independently of the APC/C. Thus, our results define the meiotic roles of PLK1 in oocytes and reveal interesting differential requirements of PLK1 between mitosis and oocyte meiosis in mammals.

The conserved Wdr8-hMsd1/SSX2IP complex localises to the centrosome and ensures proper spindle length and orientation

Energy Technology Data Exchange (ETDEWEB)

Hori, Akiko; Morand, Agathe; Ikebe, Chiho; Frith, David; Snijders, Ambrosius P.; Toda, Takashi, E-mail: takashi-toda@hiroshima-u.ac.jp

2015-12-04

The centrosome plays a pivotal role in a wide range of cellular processes and its dysfunction is causally linked to many human diseases including cancer and developmental and neurological disorders. This organelle contains more than one hundred components, and yet many of them remain uncharacterised. Here we identified a novel centrosome protein Wdr8, based upon the structural conservation of the fission yeast counterpart. We showed that Wdr8 constitutively localises to the centrosome and super resolution microscopy uncovered that this protein is enriched at the proximal end of the mother centriole. Furthermore, we identified hMsd1/SSX2IP, a conserved spindle anchoring protein, as one of Wdr8 interactors by mass spectrometry. Wdr8 formed a complex and partially colocalised with hMsd1/SSX2IP. Intriguingly, knockdown of Wdr8 or hMsd1/SSX2IP displayed very similar mitotic defects, in which spindle microtubules became shortened and misoriented. Indeed, Wdr8 depletion resulted in the reduced recruitment of hMsd1/SSX2IP to the mitotic centrosome, though the converse is not true. Together, we propose that the conserved Wdr8-hMsd1/SSX2IP complex plays a critical role in controlling proper spindle length and orientation. - Highlights: • Human Wdr8 is a centrosomal protein enriched in the proximal end of the centriole. • Wdr8 and hMsd1/SSX2IP form a complex conserved in fungi. • Depletion of Wdr8 results in shorter, tilted spindle microtubules. • Depletion phenotypes of Wdr8 are very similar to those of hMsd1/SSX2IP knockdown.

Pericentriolar Targeting of the Mouse Mammary Tumor Virus GAG Protein.

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Guangzhi Zhang

Full Text Available The Gag protein of the mouse mammary tumor virus (MMTV is the chief determinant of subcellular targeting. Electron microscopy studies show that MMTV Gag forms capsids within the cytoplasm and assembles as immature particles with MMTV RNA and the Y box binding protein-1, required for centrosome maturation. Other betaretroviruses, such as Mason-Pfizer monkey retrovirus (M-PMV, assemble adjacent to the pericentriolar region because of a cytoplasmic targeting and retention signal in the Matrix protein. Previous studies suggest that the MMTV Matrix protein may also harbor a similar cytoplasmic targeting and retention signal. Herein, we show that a substantial fraction of MMTV Gag localizes to the pericentriolar region. This was observed in HEK293T, HeLa human cell lines and the mouse derived NMuMG mammary gland cells. Moreover, MMTV capsids were observed adjacent to centrioles when expressed from plasmids encoding either MMTV Gag alone, Gag-Pro-Pol or full-length virus. We found that the cytoplasmic targeting and retention signal in the MMTV Matrix protein was sufficient for pericentriolar targeting, whereas mutation of the glutamine to alanine at position 56 (D56/A resulted in plasma membrane localization, similar to previous observations from mutational studies of M-PMV Gag. Furthermore, transmission electron microscopy studies showed that MMTV capsids accumulate around centrioles suggesting that, similar to M-PMV, the pericentriolar region may be a site for MMTV assembly. Together, the data imply that MMTV Gag targets the pericentriolar region as a result of the MMTV cytoplasmic targeting and retention signal, possibly aided by the Y box protein-1 required for the assembly of centrosomal microtubules.

The C-terminal region of A-kinase anchor protein 350 (AKAP350A) enables formation of microtubule-nucleation centers and interacts with pericentriolar proteins.

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Kolobova, Elena; Roland, Joseph T; Lapierre, Lynne A; Williams, Janice A; Mason, Twila A; Goldenring, James R

2017-12-15

Microtubules in animal cells assemble (nucleate) from both the centrosome and the cis-Golgi cisternae. A-kinase anchor protein 350 kDa (AKAP350A, also called AKAP450/CG-NAP/AKAP9) is a large scaffolding protein located at both the centrosome and Golgi apparatus. Previous findings have suggested that AKAP350 is important for microtubule dynamics at both locations, but how this scaffolding protein assembles microtubule nucleation machinery is unclear. Here, we found that overexpression of the C-terminal third of AKAP350A, enhanced GFP-AKAP350A(2691-3907), induces the formation of multiple microtubule-nucleation centers (MTNCs). Nevertheless, these induced MTNCs lacked "true" centriole proteins, such as Cep135. Mapping analysis with AKAP350A truncations demonstrated that AKAP350A contains discrete regions responsible for promoting or inhibiting the formation of multiple MTNCs. Moreover, GFP-AKAP350A(2691-3907) recruited several pericentriolar proteins to MTNCs, including γ-tubulin, pericentrin, Cep68, Cep170, and Cdk5RAP2. Proteomic analysis indicated that Cdk5RAP2 and Cep170 both interact with the microtubule nucleation-promoting region of AKAP350A, whereas Cep68 interacts with the distal C-terminal AKAP350A region. Yeast two-hybrid assays established a direct interaction of Cep170 with AKAP350A. Super-resolution and deconvolution microscopy analyses were performed to define the association of AKAP350A with centrosomes, and these studies disclosed that AKAP350A spans the bridge between centrioles, co-localizing with rootletin and Cep68 in the linker region. siRNA-mediated depletion of AKAP350A caused displacement of both Cep68 and Cep170 from the centrosome. These results suggest that AKAP350A acts as a scaffold for factors involved in microtubule nucleation at the centrosome and coordinates the assembly of protein complexes associating with the intercentriolar bridge.

The centrosomal linker and microtubules provide dual levels of spatial coordination of centrosomes.

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Marko Panic

2015-05-01

Full Text Available The centrosome is the principal microtubule organizing center in most animal cells. It consists of a pair of centrioles surrounded by pericentriolar material. The centrosome, like DNA, duplicates exactly once per cell cycle. During interphase duplicated centrosomes remain closely linked by a proteinaceous linker. This centrosomal linker is composed of rootletin filaments that are anchored to the centrioles via the protein C-Nap1. At the onset of mitosis the linker is dissolved by Nek2A kinase to support the formation of the bipolar mitotic spindle. The importance of the centrosomal linker for cell function during interphase awaits characterization. Here we assessed the phenotype of human RPE1 C-Nap1 knockout (KO cells. The absence of the linker led to a modest increase in the average centrosome separation from 1 to 2.5 μm. This small impact on the degree of separation is indicative of a second level of spatial organization of centrosomes. Microtubule depolymerisation or stabilization in C-Nap1 KO cells dramatically increased the inter-centrosomal separation (> 8 μm. Thus, microtubules position centrosomes relatively close to one another in the absence of linker function. C-Nap1 KO cells had a Golgi organization defect with a two-fold expansion of the area occupied by the Golgi. When the centrosomes of C-Nap1 KO cells showed considerable separation, two spatially distinct Golgi stacks could be observed. Furthermore, migration of C-Nap1 KO cells was slower than their wild type RPE1 counterparts. These data show that the spatial organization of centrosomes is modulated by a combination of centrosomal cohesion and microtubule forces. Furthermore a modest increase in centrosome separation has major impact on Golgi organization and cell migration.

Katanin localization requires triplet microtubules in Chlamydomonas reinhardtii.

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Jessica M Esparza

Full Text Available Centrioles and basal bodies are essential for a variety of cellular processes that include the recruitment of proteins to these structures for both centrosomal and ciliary function. This recruitment is compromised when centriole/basal body assembly is defective. Mutations that cause basal body assembly defects confer supersensitivity to Taxol. These include bld2, bld10, bld12, uni3, vfl1, vfl2, and vfl3. Flagellar motility mutants do not confer sensitivity with the exception of mutations in the p60 (pf19 and p80 (pf15 subunits of the microtubule severing protein katanin. We have identified additional pf15 and bld2 (ε-tubulin alleles in screens for Taxol sensitivity. Null pf15 and bld2 alleles are viable and are not essential genes in Chlamydomonas. Analysis of double mutant strains with the pf15-3 and bld2-6 null alleles suggests that basal bodies in Chlamydomonas may recruit additional proteins beyond katanin that affect spindle microtubule stability. The bld2-5 allele is a hypomorphic allele and its phenotype is modulated by nutritional cues. Basal bodies in bld2-5 cells are missing proximal ends. The basal body mutants show aberrant localization of an epitope-tagged p80 subunit of katanin. Unlike IFT proteins, katanin p80 does not localize to the transition fibers of the basal bodies based on an analysis of the uni1 mutant as well as the lack of colocalization of katanin p80 with IFT74. We suggest that the triplet microtubules are likely to play a key role in katanin p80 recruitment to the basal body of Chlamydomonas rather than the transition fibers that are needed for IFT localization.

Dissecting the sequential assembly and localization of intraflagellar transport particle complex B in Chlamydomonas.

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Elizabeth A Richey

Full Text Available Intraflagellar transport (IFT, the key mechanism for ciliogenesis, involves large protein particles moving bi-directionally along the entire ciliary length. IFT particles contain two large protein complexes, A and B, which are constructed with proteins in a core and several peripheral proteins. Prior studies have shown that in Chlamydomonas reinhardtii, IFT46, IFT52, and IFT88 directly interact with each other and are in a subcomplex of the IFT B core. However, ift46, bld1, and ift88 mutants differ in phenotype as ift46 mutants are able to form short flagella, while the other two lack flagella completely. In this study, we investigated the functional differences of these individual IFT proteins contributing to complex B assembly, stability, and basal body localization. We found that complex B is completely disrupted in bld1 mutant, indicating an essential role of IFT52 for complex B core assembly. Ift46 mutant cells are capable of assembling a relatively intact complex B, but such complex is highly unstable and prone to degradation. In contrast, in ift88 mutant cells the complex B core still assembles and remains stable, but the peripheral proteins no longer attach to the B core. Moreover, in ift88 mutant cells, while complex A and the anterograde IFT motor FLA10 are localized normally to the transition fibers, complex B proteins instead are accumulated at the proximal ends of the basal bodies. In addition, in bld2 mutant, the IFT complex B proteins still localize to the proximal ends of defective centrioles which completely lack transition fibers. Taken together, these results revealed a step-wise assembly process for complex B, and showed that the complex first localizes to the proximal end of the centrioles and then translocates onto the transition fibers via an IFT88-dependent mechanism.

Basal body and striated rootlet changes in primate macular retinal pigmented epithelium after low level diffuse argon laser radiation. Final report 1981-1982

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Schuschereba, S.T.; Zwick, H.; Stuck, B.E.; Beatrice, E.S.

1982-09-01

Basal bodies or centrioles (BB - microtubule organizing centers) and striated rootlets (SR - bundles of 60 A action-like filaments) have a close association in primate retinal pigmented epithelial (RPE) cells. The frequency of occurrence of these structures was evaluated in the macular RPE after repeated exposure to low level diffuse argon laser radiation (DALR). The awake chaired animal's head was restrained and positioned near the center of the 0.75 m hemisphere which was diffusely irradiated with 514.5 nm laser radiation. The right eye of each subject was occluded during the two-hour exposure session. The first subject received 24 cumulative hours of exposure, the second, 40 hours and the third, 42 hours.

Spermatic characteristics and sperm evolution on the subfamily Stevardiinae (Ostariophysi: Characiformes: Characidae

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Clarianna Martins Baicere-Silva

2011-06-01

Full Text Available The monophyly and phylogenetic relationships among the members of Clade A characids (sensu Malabarba & Weitzman, later redefined and named as the Stevardiinae (sensu Mirande, have been primarily supported by traditional morphological and molecular data. Herein were examined, described and compared spermiogenesis and sperm ultrastructure of 12 species of the genera Boehlkea, Bryconacidnus, Bryconamericus, Creagrutus, Cyanocharax, Hemibrycon, Knodus, Odontostoechus, Piabina, and Rhinobrycon in order to evaluate possible phylogenetic signals and their potential use in recovering relationships of the Stevardiinae. All examined species demonstrated a nuclear rotation equal or less than 95º resulting in a lateral position of the double nuclear fossa and flagellum. In all species, sperm nuclei are slightly elongate toward the flagellum, the proximal centriole is partially inside the nuclear fossa and lies anterior and oblique to the distal centriole, and the midpiece is short and strongly asymmetric. All species analyzed herein and other species previously examined for these systems in the Stevardiinae share homologous sperm characteristics as evidenced by spermiogenesis, further supporting the monophyly of this clade. Spermatozoa of the Stevardiinae further show three morphotypes (M1, M2, M3 of arrangement of centrioles, flagellum, nucleus and midpiece, hypothesized as successively derived in a series of transformation from the most basal morphotype (M1.A monofilia e filogenia dos membros do Clado A (sensu Malabarba & Weitzman, mais tarde redefinido e nomeado Stevardiinae (sensu Mirande, é suportada por dados morfológicos e moleculares. Aqui são examinadas, descritas e comparadas a espermiogênese e ultraestrutura do espermatozoide de 12 espécies dos gêneros Boehlkea, Bryconacidnus, Bryconamericus, Creagrutus, Cyanocharax, Hemibrycon, Knodus, Odontostoechus, Piabina e Rhinobrycon, a fim de avaliar possíveis sinais filogenéticos e seu uso

Ultrastrukturelle Untersuchungen zur Morphologie und Genese der Spermien von Archaeogastropoda

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Kohnert, R.; Storch, V.

1983-03-01

The sperm cells of Patella coerulea (Patellacea), Monodonta turbinata, and Gibbula tumida (Trochacea) were investigated by means of transmission electron microscopy. They belong to the primitive type (sensu Franzén) and have more features in common with primitive Bivalvia sperms than with Neritacea. Their head contains an apical acrosome and a roundish nucleus followed by 4 or 5 mitochondria and a centriolar apparatus which consists of two centrioles, one of which bears a flagellum. The sperm cells of Monodonta and Gibbula are very similar to each other and differ mainly in size; Patella exhibits more differences (very small acrosome, subacrosomal space, variable number of spherical mitochondria (origin of spermic dimorphism ?). The development of the sperm cells shows no peculiarities.

The effects of 60Co γ-ray irradiation on cytoplasmic microtubules of mouse macrophages and lymphocytes

International Nuclear Information System (INIS)

Li Qianqian; Mao Zijun; Yin Zhiwei; Hu Yumin

1989-05-01

The effects of 60 Co γ-ray irradiation on cytoplasmic microtubules of mouse macrophages and lymphocytes were investigated by immunofluorescence microscopy and scanning electron microscope. The results indicated. (1) microtubule organization of the irradiated cells remarkably differed from that of the control since the doses over 4 Gy; (2) 144 hours after irradiation the alterations of microtubules have been shown to be basically r epaired ; (3) the cytoplasmic microtubules and centrioles disappeared under transmission electron microscope, the membranes irradiated and microvilli showed changes under scanning electron microscope too. From these observations and those of other workers who studied the radiation effect on extracted microtubule proteins in vitro, the authors support that 60 Co γ-ray irradiation can inhabits cytoplasmic microtubule assembling

Recent advances in pericentriolar material organization: ordered layers and scaffolding gels.

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Fry, Andrew M; Sampson, Josephina; Shak, Caroline; Shackleton, Sue

2017-01-01

The centrosome is an unusual organelle that lacks a surrounding membrane, raising the question of what limits its size and shape. Moreover, while electron microscopy (EM) has provided a detailed view of centriole architecture, there has been limited understanding of how the second major component of centrosomes, the pericentriolar material (PCM), is organized. Here, we summarize exciting recent findings from super-resolution fluorescence imaging, structural biology, and biochemical reconstitution that together reveal the presence of ordered layers and complex gel-like scaffolds in the PCM. Moreover, we discuss how this is leading to a better understanding of the process of microtubule nucleation, how alterations in PCM size are regulated in cycling and differentiated cells, and why mutations in PCM components lead to specific human pathologies.

Disruption of mouse Cenpj, a regulator of centriole biogenesis, phenocopies Seckel syndrome

OpenAIRE

McIntyre, Rebecca E; Lakshminarasimhan Chavali, Pavithra; Ismail, Ozama; Carragher, Damian M; Sanchez-Andrade, Gabriela; Forment, Josep V; Fu, Beiyuan; Del Castillo Velasco-Herrera, Martin; Edwards, Andrew; van der Weyden, Louise; Yang, Fengtang; Ramirez-Solis, Ramiro; Estabel, Jeanne; Gallagher, Ferdia A; Logan, Darren W

2012-01-01

Disruption of the centromere protein J gene, CENPJ (CPAP, MCPH6, SCKL4), which is a highly conserved and ubiquitiously expressed centrosomal protein, has been associated with primary microcephaly and the microcephalic primordial dwarfism disorder Seckel syndrome. The mechanism by which disruption of CENPJ causes the proportionate, primordial growth failure that is characteristic of Seckel syndrome is unknown. By generating a hypomorphic allele of Cenpj, we have developed a mouse (Cenpj(tm/tm)...

Exposure of spermatozoa to dibutyl phthalate induces abnormal embryonic development in a marine invertebrate Galeolaria caespitosa (Polychaeta: Serpulidae).

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Lu, Yonggang; Lin, Minjie; Aitken, Robert John

2017-10-01

In this study, we have investigated the impact of dibutyl phthalate (DBP) on early embryogenesis in a sessile marine invertebrate, Galeolaria caespitosa. DBP was found to induce sperm dysfunction as well as impaired and defective embryogenesis characterised by a particular pattern of abnormality. Thus, after the first cleavage, one blastomere in these abnormal embryos was able to carry out further mitoses, while the other arrested. Analysis of microtubules, chromosomes and actin filaments demonstrated that the mitotic spindles in the abnormal embryos were irregularly bent, shortened and unable to anchor to the cortex, resulting in the defective segregation of chromosomes. Within the non-dividing blastomeres, karyokinesis was found to continue at a slow pace as indicated by the presence of multiple sets of abnormal mitotic spindles. However, cytokinesis had been disrupted in these arrested cells due to a failure to assemble the contractile actin ring, as a result of which one pole of the embryos remained as one large, undivided cell. DBP was found to suppress the activity of superoxide dismutase in spermatozoa and, in association with this change, DBP-treated cells experienced oxidative stress as indicated by the presence of lipid aldehydes, such as 4-hydroxynonenal (4-HNE) in the sperm acrosome and neck. Adduction of lipid aldehydes at the level of the acrosome would be expected to impede the acrosome reaction and account for the significant decrease in fertilisation rates. 4-HNE generated as a consequence of lipid peroxidation in the sperm neck resulted in alkylation of the sperm centrioles. Such paternally damaged centrioles were inherited by the embryos and disrupted cytoskeletal protein organisation during early cleavage, generating the observed abnormalities in embryonic development. This research emphasises the vulnerability of spermatozoa to oxidative damage and highlights novel potential mechanisms for reproductive toxicity involving the alkylation of

A novel component of the mitochondrial genome segregation machinery in trypanosomes

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Anneliese Hoffmann

2016-07-01

Full Text Available We recently described a new component (TAC102 of the mitochondrial genome segregation machinery (mtGSM in the protozoan parasite Trypanosoma brucei. T. brucei belongs to a group of organisms that contain a single mitochondrial organelle with a single mitochondrial genome (mt-genome per cell. The mt-genome consists of 5000 minicircles (1 kb and 25 maxicircles (23 kb that are catenated into a large network. After replication of the network its segregation is driven by the separating basal bodies, which are homologous structures to the centrioles organizing the spindle apparatus in many eukaryotes. The structure connecting the basal body to the mt-genome was named the Tripartite Attachment Complex (TAC owing its name to the distribution across three areas in the cell including the two mitochondrial membranes.

Symbiosis and the origin of eukaryotic motility

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Margulis, L.; Hinkle, G.

1991-01-01

Ongoing work to test the hypothesis of the origin of eukaryotic cell organelles by microbial symbioses is discussed. Because of the widespread acceptance of the serial endosymbiotic theory (SET) of the origin of plastids and mitochondria, the idea of the symbiotic origin of the centrioles and axonemes for spirochete bacteria motility symbiosis was tested. Intracellular microtubular systems are purported to derive from symbiotic associations between ancestral eukaryotic cells and motile bacteria. Four lines of approach to this problem are being pursued: (1) cloning the gene of a tubulin-like protein discovered in Spirocheata bajacaliforniesis; (2) seeking axoneme proteins in spirochets by antibody cross-reaction; (3) attempting to cultivate larger, free-living spirochetes; and (4) studying in detail spirochetes (e.g., Cristispira) symbiotic with marine animals. Other aspects of the investigation are presented.

Holographic View of the Brain Memory Mechanism Based on Evanescent Superluminal Photons

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Takaaki Musha

2012-08-01

Full Text Available D. Pollen and M. Trachtenberg proposed the holographic brain theory to help explain the existence of photographic memories in some people. They suggested that such individuals had more vivid memories because they somehow could access a very large region of their memory holograms. Hameroff suggested in his paper that cylindrical neuronal microtubule cavities, or centrioles, function as waveguides for the evanescent photons for quantum signal processing. The supposition is that microtubular structures of the brain function as a coherent fiber bundle set used to store holographic images, as would a fiber-optic holographic system. In this paper, the author proposes that superluminal photons propagating inside the microtubules via evanescent waves could provide the access needed to record or retrieve a quantum coherent entangled holographic memory.

Recent advances in pericentriolar material organization: ordered layers and scaffolding gels [version 1; referees: 3 approved

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Andrew M. Fry

2017-08-01

Full Text Available The centrosome is an unusual organelle that lacks a surrounding membrane, raising the question of what limits its size and shape. Moreover, while electron microscopy (EM has provided a detailed view of centriole architecture, there has been limited understanding of how the second major component of centrosomes, the pericentriolar material (PCM, is organized. Here, we summarize exciting recent findings from super-resolution fluorescence imaging, structural biology, and biochemical reconstitution that together reveal the presence of ordered layers and complex gel-like scaffolds in the PCM. Moreover, we discuss how this is leading to a better understanding of the process of microtubule nucleation, how alterations in PCM size are regulated in cycling and differentiated cells, and why mutations in PCM components lead to specific human pathologies.

Characterization of membrane protein trafficking and cellular signaling at the primary cilium

DEFF Research Database (Denmark)

Mogensen, Johanne Bay

Primary cilia are microtubule-based, non-motile, sensory organelles emerging in a single copy from the surface of most quiescent cells in vertebrates. They emanate from the centrosomal mother centriole and are assembled and maintained by a bidirectional transport process termed intraflagellar...... transport. Specific receptors, ion channels and downstream signaling components are localized along the cilium-centrosome axis, enabling the cilium to function as a hot spot for the balanced coordination of multiple signaling pathways to control cell cycle entry, differentiation and migration during...... differentiation of mouse stem cells into cardiomyocytes. These results support the conclusion that Tab2 functions at the primary cilium to coordinate specified signaling events, which when defective may lead to congenital heart disease Collectively, the results presented in this PhD thesis provide new insights...

Ultrastructure of Spermatogenesis of the Paradise Fish, Macropodus opercularis

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Tsung-Han Lee

2006-09-01

Full Text Available The intricate process of spermatogenesis in the paradise fish, Macropodus opercularis, was studied. In this species, the unrestricted or lobular type testes lining the caudal side of the body cavity are translucent and slender. Spermatogonia occur along the length of the tubules and the development of sperm takes place within cysts formed by Sertoli cells. Spermiogenesis involves preparatory morphological events followed by conspicuous modifications such as the movement of the centrioles, completion of the nuclear condensation, reduction of the cytoplasm, and the final differentiation of the flagellar complex. Mature spermatozoon has an oval nucleus, condensed chromatin, and typical 9 + 2 flagellar axoneme but lack acrosome. The role of the material in the nucleus and the cytoplasm as it reaches the Sertoli cell in the control of spermatogenesis is discussed.

Biflagellate spermatozoon structure of the hermaphrodite fish Satanoperca jurupari (Heckel, 1840 (Teleostei, Cichlidae from the Amazon River

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E. MATOS

Full Text Available The ultrastructural features of the sperm were studied in the hermaphroditic teleost Satanoperca jurupari HECKEL, 1840 from Amazon River. Spermatocytes, spermatids and sperm develop in the testicular cysts among the different oocyte stages. Different stages of early spermatocyte development, mainly the ones with synaptonemal complexes were often observed. The mature spermatozoa belong to the introsperm type, with a short head (~ 3 mm long and 1.3 mum wide without acrosome, short midpiece (~ 1.2 mum long and 1.8 mum wide containing several mitochondria surrounding two centrioles and forming a mitochondrial collar. They have two flagella (each ~15 mum long each of which has a common 9 + 2 microtubular pattern. Each flagellum has two opposite lateral cytoplasmic extensions that begin about 3 mum the midpiece still close to the end piece of flagellum.

Biflagellate spermatozoon structure of the hermaphrodite fish Satanoperca jurupari (Heckel, 1840 (Teleostei, Cichlidae from the Amazon River

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MATOS E.

2002-01-01

Full Text Available The ultrastructural features of the sperm were studied in the hermaphroditic teleost Satanoperca jurupari HECKEL, 1840 from Amazon River. Spermatocytes, spermatids and sperm develop in the testicular cysts among the different oocyte stages. Different stages of early spermatocyte development, mainly the ones with synaptonemal complexes were often observed. The mature spermatozoa belong to the introsperm type, with a short head (~ 3 mm long and 1.3 mum wide without acrosome, short midpiece (~ 1.2 mum long and 1.8 mum wide containing several mitochondria surrounding two centrioles and forming a mitochondrial collar. They have two flagella (each ~15 mum long each of which has a common 9 + 2 microtubular pattern. Each flagellum has two opposite lateral cytoplasmic extensions that begin about 3 mum the midpiece still close to the end piece of flagellum.

Primary Cilia, Signaling Networks and Cell Migration

DEFF Research Database (Denmark)

Veland, Iben Rønn

Primary cilia are microtubule-based, sensory organelles that emerge from the centrosomal mother centriole to project from the surface of most quiescent cells in the human body. Ciliary entry is a tightly controlled process, involving diffusion barriers and gating complexes that maintain a unique...... this controls directional cell migration as a physiological response. The ciliary pocket is a membrane invagination with elevated activity of clathrin-dependent endocytosis (CDE). In paper I, we show that the primary cilium regulates TGF-β signaling and the ciliary pocket is a compartment for CDE...... on formation of the primary cilium and CDE at the pocket region. The ciliary protein Inversin functions as a molecular switch between canonical and non-canonical Wnt signaling. In paper II, we show that Inversin and the primary cilium control Wnt signaling and are required for polarization and cell migration...

Ultrastructural characterization of primary cilia in pathologically characterized human glioblastoma multiforme (GBM) tumors.

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Moser, Joanna J; Fritzler, Marvin J; Rattner, Jerome B

2014-01-01

Primary cilia are non-motile sensory cytoplasmic organelles that are involved in cell cycle progression. Ultrastructurally, the primary cilium region is complex, with normal ciliogenesis progressing through five distinct morphological stages in human astrocytes. Defects in early stages of ciliogenesis are key features of astrocytoma/glioblastoma cell lines and provided the impetus for the current study which describes the morphology of primary cilia in molecularly characterized human glioblastoma multiforme (GBM) tumors. Seven surgically resected human GBM tissue samples were molecularly characterized according to IDH1/2 mutation status, EGFR amplification status and MGMT promoter methylation status and were examined for primary cilia expression and structure using indirect immunofluorescence and electron microscopy. We report for the first time that primary cilia are disrupted in the early stages of ciliogenesis in human GBM tumors. We confirm that immature primary cilia and basal bodies/centrioles have aberrant ciliogenesis characteristics including absent paired vesicles, misshaped/swollen vesicular hats, abnormal configuration of distal appendages, and discontinuity of centriole microtubular blades. Additionally, the transition zone plate is able to form in the absence of paired vesicles on the distal end of the basal body and when a cilium progresses beyond the early stages of ciliogenesis, it has electron dense material clumped along the transition zone and a darkening of the microtubules at the proximal end of the cilium. Primary cilia play a role in a variety of human cancers. Previously primary cilia structure was perturbed in cultured cell lines derived from astrocytomas/glioblastomas; however there was always some question as to whether these findings were a cell culture phenomena. In this study we confirm that disruptions in ciliogenesis at early stages do occur in GBM tumors and that these ultrastructural findings bear resemblance to those previously

Phylogeny of the Acanthocephala based on morphological characters.

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Monks, S

2001-02-01

Only four previous studies of relationships among acanthocephalans have included cladistic analyses, and knowledge of the phylogeny of the group has not kept pace with that of other taxa. The purpose of this study is to provide a more comprehensive analysis of the phylogenetic relationships among members of the phylum Acanthocephala using morphological characters. The most appropriate outgroups are those that share a common early cell-cleavage pattern (polar placement of centrioles), such as the Rotifera, rather than the Priapulida (meridional placement of centrioles) to provide character polarity based on common ancestry rather than a general similarity likely due to convergence of body shapes. The phylogeny of 22 species of the Acanthocephala was evaluated based on 138 binary and multistate characters derived from comparative morphological and ontogenetic studies. Three assumptions of cement gland structure were tested: (i) the plesiomorphic type of cement glands in the Rotifera, as the sister group, is undetermined; (ii) non-syncytial cement glands are plesiomorphic; and (iii) syncytial cement glands are plesiomorphic. The results were used to test an early move of Tegorhynchus pectinarius to Koronacantha and to evaluate the relationship between Tegorhynchus and Illiosentis. Analysis of the data-set for each of these assumptions of cement gland structure produced the same single most parsimonious tree topology. Using Assumptions i and ii for the cement glands, the trees were the same length (length = 404 steps, CI = 0.545, CIX = 0.517, HI = 0.455, HIX = 0.483, RI = 0.670, RC = 0.365). Using Assumption iii, the tree was three steps longer (length = 408 steps, CI = 0.539, CIX = 0.512, HI = 0.461, HIX = 0.488, RI = 0.665, RC = 0.359). The tree indicates that the Palaeacanthocephala and Eoacanthocephala both are monophyletic and are sister taxa. The members of the Archiacanthocephala are basal to the other two clades, but do not themselves form a clade. The results

The Role of Microtubule End Binding (EB) Proteins in Ciliogenesis

DEFF Research Database (Denmark)

Schrøder, Jacob Morville

cellular organelles (Lansbergen and Akhmanova, 2006). EB1 also localizes to centrosomes and is required for centrosomal MT anchoring and organization of the MT network (Askham et al., 2002). Further, EB1 has been shown to localize to the flagellar tip and proximal region of the basal bodies......, are required for assembly of primary cilia in cultured human cells. The EB3 - siRNA ciliary phenotype could be rescued by GFP-EB1 expression, and GFP-EB3 over expression resulted in elongated cilia. Transmission electron microscopy (TEM) revealed that EB3-depleted cells possess stumpy cilia, a disorganized...... centrosomal MT array and abnormally long centriole-associated rootlet filaments. Cells lacking EB1 also had stumpy cilia and a disorganized centrosomal MT array, but rootlet filaments appeared normal. Further, live imaging revealed increased release frequency of MTs from the centrosome upon EB1 or EB3...

Ploidy Manipulation of Zebrafish Embryos with Heat Shock 2 Treatment

Science.gov (United States)

Baars, Destiny L.; Pelegri, Francisco

2016-01-01

Manipulation of ploidy allows for useful transformations, such as diploids to tetraploids, or haploids to diploids. In the zebrafish Danio rerio, specifically the generation of homozygous gynogenetic diploids is useful in genetic analysis because it allows the direct production of homozygotes from a single heterozygous mother. This article describes a modified protocol for ploidy duplication based on a heat pulse during the first cell cycle, Heat Shock 2 (HS2). Through inhibition of centriole duplication, this method results in a precise cell division stall during the second cell cycle. The precise one-cycle division stall, coupled to unaffected DNA duplication, results in whole genome duplication. Protocols associated with this method include egg and sperm collection, UV treatment of sperm, in vitro fertilization and heat pulse to cause a one-cell cycle division delay and ploidy duplication. A modified version of this protocol could be applied to induce ploidy changes in other animal species. PMID:28060351

The ultrastructure of the mullet Mugil curema Valenciennes (Teleostei, Mugilidae spermatozoa

Directory of Open Access Journals (Sweden)

Daura Regina Eiras-Stofella

1993-01-01

Full Text Available The structure of the spermatozoon of Mugil curema Valenciennes, 1836 was studied using scanning and transmission electron microscopy. The spermatic head is rounded and formed by the nucleus containing granular chromatin, firmly packed resulting in a mass extremely electron dense. The acrossome is absent. The midpiece is characterized by the presence of two centrioles, a plasmatic canal, very few vesicles, and several mitochondria (9-10 with aproximately 0.50µm in diameter. The head and the midpiece are aproximately 1.56µm in diameter. The flagellum conforms to the 9 + 0 flagellar pattern near the transition region in its midpiece and is 9 + 2 from there on up to the distal region of the axoneme. The electron density in the A tubules 1, 2, 5 and 6 shows the asymetry of this spermatozoa. Its spermatic cell differs ultrastructuraly from those of other Mugilidae species mainly because it has the highest number of mitochondria.

Mutations in DZIP1L, which encodes a ciliary transition zone protein, cause autosomal recessive polycystic kidney disease

Science.gov (United States)

Lu, Hao; Galeano, Maria C. Rondón; Ott, Elisabeth; Kaeslin, Geraldine; Kausalya, P. Jaya; Kramer, Carina; Ortiz-Brüchle, Nadina; Hilger, Nadescha; Metzis, Vicki; Hiersche, Milan; Tay, Shang Yew; Tunningley, Robert; Vij, Shubha; Courtney, Andrew D.; Whittle, Belinda; Wühl, Elke; Vester, Udo; Hartleben, Björn; Neuber, Steffen; Frank, Valeska; Little, Melissa H.; Epting, Daniel; Papathanasiou, Peter; Perkins, Andrew C.; Wright, Graham D.; Hunziker, Walter; Gee, Heon Yung; Otto, Edgar A.; Zerres, Klaus; Hildebrandt, Friedhelm; Roy, Sudipto; Wicking, Carol; Bergmann, Carsten

2017-01-01

Autosomal recessive polycystic kidney disease (ARPKD), usually considered to be a genetically homogeneous disease caused by mutations in PKHD1, has been associated with ciliary dysfunction. Here, we describe mutations in the DAZ interacting protein 1-like (DZIP1L) gene in patients with ARPKD, findings we have further validated by loss-of-function studies in mice and zebrafish. DZIP1L localizes to centrioles and at the distal end of basal bodies, and interacts with septin2, a protein implicated in maintenance of the periciliary diffusion barrier at the ciliary transition zone. Consistent with a defect in the diffusion barrier, we found that the ciliary membrane translocation of the PKD proteins, polycystin-1 and −2, is compromised in DZIP1L mutant cells. Together, these data provide the first conclusive evidence that ARPKD is not a homogeneous disorder, and establishes DZIP1L as a second gene involved in its pathogenesis. PMID:28530676

Mutations in DZIP1L, which encodes a ciliary-transition-zone protein, cause autosomal recessive polycystic kidney disease.

Science.gov (United States)

Lu, Hao; Galeano, Maria C Rondón; Ott, Elisabeth; Kaeslin, Geraldine; Kausalya, P Jaya; Kramer, Carina; Ortiz-Brüchle, Nadina; Hilger, Nadescha; Metzis, Vicki; Hiersche, Milan; Tay, Shang Yew; Tunningley, Robert; Vij, Shubha; Courtney, Andrew D; Whittle, Belinda; Wühl, Elke; Vester, Udo; Hartleben, Björn; Neuber, Steffen; Frank, Valeska; Little, Melissa H; Epting, Daniel; Papathanasiou, Peter; Perkins, Andrew C; Wright, Graham D; Hunziker, Walter; Gee, Heon Yung; Otto, Edgar A; Zerres, Klaus; Hildebrandt, Friedhelm; Roy, Sudipto; Wicking, Carol; Bergmann, Carsten

2017-07-01

Autosomal recessive polycystic kidney disease (ARPKD), usually considered to be a genetically homogeneous disease caused by mutations in PKHD1, has been associated with ciliary dysfunction. Here, we describe mutations in DZIP1L, which encodes DAZ interacting protein 1-like, in patients with ARPKD. We further validated these findings through loss-of-function studies in mice and zebrafish. DZIP1L localizes to centrioles and to the distal ends of basal bodies, and interacts with septin2, a protein implicated in maintenance of the periciliary diffusion barrier at the ciliary transition zone. In agreement with a defect in the diffusion barrier, we found that the ciliary-membrane translocation of the PKD proteins polycystin-1 and polycystin-2 is compromised in DZIP1L-mutant cells. Together, these data provide what is, to our knowledge, the first conclusive evidence that ARPKD is not a homogeneous disorder and further establish DZIP1L as a second gene involved in ARPKD pathogenesis.

The Centrosome and Its Duplication Cycle

Science.gov (United States)

Fu, Jingyan; Hagan, Iain M.; Glover, David M.

2015-01-01

The centrosome was discovered in the late 19th century when mitosis was first described. Long recognized as a key organelle of the spindle pole, its core component, the centriole, was realized more than 50 or so years later also to comprise the basal body of the cilium. Here, we chart the more recent acquisition of a molecular understanding of centrosome structure and function. The strategies for gaining such knowledge were quickly developed in the yeasts to decipher the structure and function of their distinctive spindle pole bodies. Only within the past decade have studies with model eukaryotes and cultured cells brought a similar degree of sophistication to our understanding of the centrosome duplication cycle and the multiple roles of this organelle and its component parts in cell division and signaling. Now as we begin to understand these functions in the context of development, the way is being opened up for studies of the roles of centrosomes in human disease. PMID:25646378

Phosphorylation of CPAP by Aurora-A Maintains Spindle Pole Integrity during Mitosis

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En-Ju Chou

2016-03-01

Full Text Available CPAP is required for centriole elongation during S/G2 phase, but the role of CPAP in mitosis is incompletely understood. Here, we show that CPAP maintains spindle pole integrity through its phosphorylation by Aurora-A during mitosis. Depletion of CPAP induced a prolonged delay in mitosis, pericentriolar material (PCM dispersion, and multiple mitotic abnormalities. Further studies demonstrated that CPAP directly interacts with and is phosphorylated by Aurora-A at serine 467 during mitosis. Interestingly, the dispersal of the PCM was effectively rescued by ectopic expression of wild-type CPAP or a phospho-mimic CPAP-S467D mutant, but not a non-phosphorylated CPAP-S467A mutant. Finally, we found that CPAP-S467D has a low affinity for microtubule binding but a high affinity for PCM proteins. Together, our results support a model wherein CPAP is required for proper mitotic progression, and phosphorylation of CPAP by Aurora-A is essential for maintaining spindle pole integrity.

GAS2L1 Is a Centriole-Associated Protein Required for Centrosome Dynamics and Disjunction.

NARCIS (Netherlands)

Au, F.K.; Jia, Y.; Jiang, K.; Grigoriev, I.S.; Hau, B.K.; Shen, Y.; Du, S.; Akhmanova, A.S.; Qi, R.Z.

2017-01-01

Mitotic spindle formation and chromosome segregation require timely separation of the two duplicated centrosomes, and this process is initiated in late G2 by centrosome disjunction. Here we report that GAS2L1, a microtubule- and actin-binding protein, associates with the proximal end of mature

Studying protein assembly with reversible Brownian dynamics of patchy particles

International Nuclear Information System (INIS)

Klein, Heinrich C. R.; Schwarz, Ulrich S.

2014-01-01

Assembly of protein complexes like virus shells, the centriole, the nuclear pore complex, or the actin cytoskeleton is strongly determined by their spatial structure. Moreover, it is becoming increasingly clear that the reversible nature of protein assembly is also an essential element for their biological function. Here we introduce a computational approach for the Brownian dynamics of patchy particles with anisotropic assemblies and fully reversible reactions. Different particles stochastically associate and dissociate with microscopic reaction rates depending on their relative spatial positions. The translational and rotational diffusive properties of all protein complexes are evaluated on-the-fly. Because we focus on reversible assembly, we introduce a scheme which ensures detailed balance for patchy particles. We then show how the macroscopic rates follow from the microscopic ones. As an instructive example, we study the assembly of a pentameric ring structure, for which we find excellent agreement between simulation results and a macroscopic kinetic description without any adjustable parameters. This demonstrates that our approach correctly accounts for both the diffusive and reactive processes involved in protein assembly

Emerging connection between centrosome and DNA repair machinery

International Nuclear Information System (INIS)

Shimada, Mikio; Komatsu, Kenshi

2009-01-01

Centrosomes function in proper cell division in animal cells. The centrosome consists of a pair of centrioles and the surrounding pericentriolar matrix (PCM). After cytokinesis, daughter cells each acquire one centrosome, which subsequently duplicates at the G1/S phase in a manner that is dependent upon CDK2/cyclin-E activity. Defects in the regulation of centrosome duplication lead to tumorigenesis through abnormal cell division and resulting inappropriate chromosome segregation. Therefore, maintenance of accurate centrosome number is important for cell fate. Excess number of centrosomes can be induced by several factors including ionizing radiation (IR). Recent studies have shown that several DNA repair proteins localize to the centrosome and are involved in the regulation of centrosome number possibly through cell cycle checkpoints or direct modification of centrosome proteins. Furthermore, it has been reported that the development of microcephaly is likely caused by defective expression of centrosome proteins, such as ASPM, which are also involved in the response to IR. The present review highlights centrosome duplication in association with genotoxic stresses and the regulatory mechanism mediated by DNA repair proteins. (author)

Primary cilium - antenna-like structure on the surface of most mammalian cell types

International Nuclear Information System (INIS)

Dvorak, J; Kasaova, L; Filip, S; Petera, J; Sitorova, V; Nikolov, D Hadzi; Ryska, A; Mokry, J; Richter, I

2011-01-01

The primary cilium is a sensory solitary non-motile microtubule-based organelle protruding in the quiescent phase of the cell cycle from the surface of the majority of human cells, including embryonic cells, stem cells and stromal cells of malignant tumors. The presence of a primary cilium on the surface of a cell is transient, limited to the quiescent G 1 (G 0 ) phase and the beginning of the S phase of the cell cycle. The primary cilium is formed from the mother centriole. Primary cilia are key coordinators of signaling pathways during development and tissue homeostasis and, when deffective, they are a major cause of human diseases and developmental disorders, now commonly referred to as ciliopathies. Most cancer cells do not possess a primary cilium. The loss of the primary cilium is a regular feature of neoplastic transformation in the majority of solid tumors. The primary cilium could serve as a tumor suppressor organelle. The aim of this paper was to provide a review of the current knowledge of the primary cilium.

Chemically induced aneuploidy in mammalian cells: mechanisms and biological significance in cancer

Energy Technology Data Exchange (ETDEWEB)

Oshimura, M.; Barrett, J.C.

1986-01-01

A literature review with over 200 references examines the growing body of evidence from human and animal cancer cytogenetics that aneuploidy is an important chromosome change in carcinogenesis. Evidence from in vitro cell transformation studies supports the idea that aneuploidy has a direct effect on the conversion of a normal cell to a preneoplastic or malignant cell. Induction of an aneuploid state in a preneoplastic or neoplastic cell could have any of the following four biological effects: a change in gene dosage, a change in gene balance, expression of a recessive mutation, or a change in genetic instability (which could secondarily lead to neoplasia). There are a number of possible mechanisms by which chemicals might induce aneuploidy, including effects on microtubules, damage to essential elements for chromosome function reduction in chromosome condensation or pairing, induction of chromosome interchanges, unresolved recombination structures, increased chromosome stickiness, damage to centrioles, impairment of chromosome alignment ionic alterations during mitosis, damage to the nuclear membrane, and a physical disruption of chromosome segregation. Therefore, a number of different targets exist for chemically induced aneuploidy.

Ultrastructural study of spermatogenesis in Phoronopsis harmeri (Lophophorata, Phoronida)

Science.gov (United States)

Reunov, Arkadiy; Klepal, Waltraud

2004-02-01

The process of sperm development in Phoronopsis harmeri was studied by electron microscopy. Developing spermatogenical cells are aggregated around the capillaries of the haemal plexus. The spermatogonia, which are situated around the capillary walls of the caeca, are remarkable for the presence of germ-line vesicles and contain their centrioles near the cell membrane. The spermatocytes and spermatids are flagellated cells arranged in clusters. During spermiogenesis the basal body/flagellum complex migrates to the apical pole of the spermatid. The acrosome-like structure arises from material produced by the Golgi complex. It lacks a surrounding membrane and has a fibrillar content. The nucleus elongates and the condensation of chromatin is caused by an activation of 'initiation centres'. The late spermatid and the spermatozoon appear as two-armed 'V'-shaped cells in which one arm contains the nucleus and posteriorly located mitochondria, and the other one is the axoneme. Spermatogenesis of P. harmeri is an interesting example of gamete differentiation where advanced sperm structure is combined with a plesiomorphic pattern of sperm development characterized as 'flagellate spermatogenesis'.

Downregulation of Protein 4.1R impairs centrosome function,bipolar spindle organization and anaphase

Energy Technology Data Exchange (ETDEWEB)

Spence, Jeffrey R.; Go, Minjoung M.; Bahmanyar, S.; Barth,A.I.M.; Krauss, Sharon Wald

2006-03-17

Centrosomes nucleate and organize interphase MTs and areinstrumental in the assembly of the mitotic bipolar spindle. Here wereport that two members of the multifunctional protein 4.1 family havedistinct distributions at centrosomes. Protein 4.1R localizes to maturecentrioles whereas 4.1G is a component of the pericentriolar matrixsurrounding centrioles. To selectively probe 4.1R function, we used RNAinterference-mediated depletion of 4.1R without decreasing 4.1Gexpression. 4.1R downregulation reduces MT anchoring and organization atinterphase and impairs centrosome separation during prometaphase.Metaphase chromosomes fail to properly condense/align and spindleorganization is aberrant. Notably 4.1R depletion causes mislocalizationof its binding partner NuMA (Nuclear Mitotic Apparatus Protein),essential for spindle pole focusing, and disrupts ninein. Duringanaphase/telophase, 4.1R-depleted cells have lagging chromosomes andaberrant MT bridges. Our data provide functional evidence that 4.1R makescrucial contributions to centrosome integrity and to mitotic spindlestructure enabling mitosis and anaphase to proceed with the coordinatedprecision required to avoid pathological events.

POC1A truncation mutation causes a ciliopathy in humans characterized by primordial dwarfism.

Science.gov (United States)

Shaheen, Ranad; Faqeih, Eissa; Shamseldin, Hanan E; Noche, Ramil R; Sunker, Asma; Alshammari, Muneera J; Al-Sheddi, Tarfa; Adly, Nouran; Al-Dosari, Mohammed S; Megason, Sean G; Al-Husain, Muneera; Al-Mohanna, Futwan; Alkuraya, Fowzan S

2012-08-10

Primordial dwarfism (PD) is a phenotype characterized by profound growth retardation that is prenatal in onset. Significant strides have been made in the last few years toward improved understanding of the molecular underpinning of the limited growth that characterizes the embryonic and postnatal development of PD individuals. These include impaired mitotic mechanics, abnormal IGF2 expression, perturbed DNA-damage response, defective spliceosomal machinery, and abnormal replication licensing. In three families affected by a distinct form of PD, we identified a founder truncating mutation in POC1A. This gene is one of two vertebrate paralogs of POC1, which encodes one of the most abundant proteins in the Chlamydomonas centriole proteome. Cells derived from the index individual have abnormal mitotic mechanics with multipolar spindles, in addition to clearly impaired ciliogenesis. siRNA knockdown of POC1A in fibroblast cells recapitulates this ciliogenesis defect. Our findings highlight a human ciliopathy syndrome caused by deficiency of a major centriolar protein. Copyright © 2012 The American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.

Tripolar mitosis in human cells and embryos: occurrence, pathophysiology and medical implications.

Science.gov (United States)

Kalatova, Beata; Jesenska, Renata; Hlinka, Daniel; Dudas, Marek

2015-01-01

Tripolar mitosis is a specific case of cell division driven by typical molecular mechanisms of mitosis, but resulting in three daughter cells instead of the usual count of two. Other variants of multipolar mitosis show even more mitotic poles and are relatively rare. In nature, this phenomenon was frequently observed or suspected in multiple common cancers, infected cells, the placenta, and in early human embryos with impaired pregnancy-yielding potential. Artificial causes include radiation and various toxins. Here we combine several pieces of the most recent evidence for the existence of different types of multipolar mitosis in preimplantation embryos together with a detailed review of the literature. The related molecular and cellular mechanisms are discussed, including the regulation of centriole duplication, mitotic spindle biology, centromere functions, cell cycle checkpoints, mitotic autocorrection mechanisms, and the related complicating factors in healthy and affected cells, including post-mitotic cell-cell fusion often associated with multipolar cell division. Clinical relevance for oncology and embryo selection in assisted reproduction is also briefly discussed in this context. Copyright © 2014 Elsevier GmbH. All rights reserved.

Phosphorylation of CPAP by Aurora-A Maintains Spindle Pole Integrity during Mitosis.

Science.gov (United States)

Chou, En-Ju; Hung, Liang-Yi; Tang, Chieh-Ju C; Hsu, Wen-Bin; Wu, Hsin-Yi; Liao, Pao-Chi; Tang, Tang K

2016-03-29

CPAP is required for centriole elongation during S/G2 phase, but the role of CPAP in mitosis is incompletely understood. Here, we show that CPAP maintains spindle pole integrity through its phosphorylation by Aurora-A during mitosis. Depletion of CPAP induced a prolonged delay in mitosis, pericentriolar material (PCM) dispersion, and multiple mitotic abnormalities. Further studies demonstrated that CPAP directly interacts with and is phosphorylated by Aurora-A at serine 467 during mitosis. Interestingly, the dispersal of the PCM was effectively rescued by ectopic expression of wild-type CPAP or a phospho-mimic CPAP-S467D mutant, but not a non-phosphorylated CPAP-S467A mutant. Finally, we found that CPAP-S467D has a low affinity for microtubule binding but a high affinity for PCM proteins. Together, our results support a model wherein CPAP is required for proper mitotic progression, and phosphorylation of CPAP by Aurora-A is essential for maintaining spindle pole integrity. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

Primary cilium - antenna-like structure on the surface of most mammalian cell types

Science.gov (United States)

Dvorak, J.; Sitorova, V.; Hadzi Nikolov, D.; Mokry, J.; Richter, I.; Kasaova, L.; Filip, S.; Ryska, A.; Petera, J.

2011-12-01

The primary cilium is a sensory solitary non-motile microtubule-based organelle protruding in the quiescent phase of the cell cycle from the surface of the majority of human cells, including embryonic cells, stem cells and stromal cells of malignant tumors. The presence of a primary cilium on the surface of a cell is transient, limited to the quiescent G1(G0) phase and the beginning of the S phase of the cell cycle. The primary cilium is formed from the mother centriole. Primary cilia are key coordinators of signaling pathways during development and tissue homeostasis and, when deffective, they are a major cause of human diseases and developmental disorders, now commonly referred to as ciliopathies. Most cancer cells do not possess a primary cilium. The loss of the primary cilium is a regular feature of neoplastic transformation in the majority of solid tumors. The primary cilium could serve as a tumor suppressor organelle. The aim of this paper was to provide a review of the current knowledge of the primary cilium.

Chirality of the cytoskeleton in the origins of cellular asymmetry

Science.gov (United States)

2016-01-01

Self-assembly of two important components of the cytoskeleton of eukaryotic cells, actin microfilaments and microtubules (MTs) results in polar filaments of one chirality. As is true for bacterial flagella, in actin microfilaments, screw direction is important for assembly processes and motility. For MTs, polar orientation within the cell is paramount. The alignment of these elements in the cell cytoplasm gives rise to emergent properties, including the potential for cell differentiation and specialization. Complex MTs with a characteristic chirality are found in basal bodies and centrioles; this chirality is preserved in cilia. In motile cilia, it is reflected in the direction of the effective stroke. The positioning of the basal body or cilia on the cell surface depends on polarity proteins. In evolution, survival depends on global polarity information relayed to the cell in part by orientation of the MT and actin filament cytoskeletons and the chirality of the basal body to determine left and right coordinates within a defined anterior–posterior cell and tissue axis. This article is part of the themed issue ‘Provocative questions in left–right asymmetry’. PMID:27821520

Studying protein assembly with reversible Brownian dynamics of patchy particles

Energy Technology Data Exchange (ETDEWEB)

Klein, Heinrich C. R. [Institute for Theoretical Physics, Heidelberg University, 69120 Heidelberg (Germany); Schwarz, Ulrich S., E-mail: ulrich.schwarz@bioquant.uni-heidelberg.de [Institute for Theoretical Physics, Heidelberg University, 69120 Heidelberg (Germany); BioQuant, Heidelberg University, 69120 Heidelberg (Germany)

2014-05-14

Assembly of protein complexes like virus shells, the centriole, the nuclear pore complex, or the actin cytoskeleton is strongly determined by their spatial structure. Moreover, it is becoming increasingly clear that the reversible nature of protein assembly is also an essential element for their biological function. Here we introduce a computational approach for the Brownian dynamics of patchy particles with anisotropic assemblies and fully reversible reactions. Different particles stochastically associate and dissociate with microscopic reaction rates depending on their relative spatial positions. The translational and rotational diffusive properties of all protein complexes are evaluated on-the-fly. Because we focus on reversible assembly, we introduce a scheme which ensures detailed balance for patchy particles. We then show how the macroscopic rates follow from the microscopic ones. As an instructive example, we study the assembly of a pentameric ring structure, for which we find excellent agreement between simulation results and a macroscopic kinetic description without any adjustable parameters. This demonstrates that our approach correctly accounts for both the diffusive and reactive processes involved in protein assembly.

The origin of the eukaryotic cell

Science.gov (United States)

Hartman, H.

1984-01-01

The endosymbiotic hypothesis for the origin of the eukaryotic cell has been applied to the origin of the mitochondria and chloroplasts. However as has been pointed out by Mereschowsky in 1905, it should also be applied to the nucleus as well. If the nucleus, mitochondria and chloroplasts are endosymbionts, then it is likely that the organism that did the engulfing was not a DNA-based organism. In fact, it is useful to postulate that this organism was a primitive RNA-based organism. This hypothesis would explain the preponderance of RNA viruses found in eukaryotic cells. The centriole and basal body do not have a double membrane or DNA. Like all MTOCs (microtubule organising centres), they have a structural or morphic RNA implicated in their formation. This would argue for their origin in the early RNA-based organism rather than in an endosymbiotic event involving bacteria. Finally, the eukaryotic cell uses RNA in ways quite unlike bacteria, thus pointing to a greater emphasis of RNA in both control and structure in the cell. The origin of the eukaryotic cell may tell us why it rather than its prokaryotic relative evolved into the metazoans who are reading this paper.

Effects of spaceflight conditions on fertilization and embryogenesis in the sea urchin Lytechinus pictus

Science.gov (United States)

Schatten, H.; Chakrabarti, A.; Taylor, M.; Sommer, L.; Levine, H.; Anderson, K.; Runco, M.; Kemp, R.

1999-01-01

Calcium loss and muscle atrophy are two of the main metabolic changes experienced by astronauts and crew members during exposure to microgravity in space. Calcium and cytoskeletal events were investigated within sea urchin embryos which were cultured in space under both microgravity and 1 g conditions. Embryos were fixed at time-points ranging from 3 h to 8 days after fertilization. Investigative emphasis was placed upon: (1) sperm-induced calcium-dependent exocytosis and cortical granule secretion, (2) membrane fusion of cortical granule and plasma membranes; (3) microfilament polymerization and microvilli elongation; and (5) embryonic development into morula, blastula, gastrula, and pluteus stages. For embryos cultured under microgravity conditions, the processes of cortical granule discharge, fusion of cortical granule membranes with the plasma membrane, elongation of microvilli and elevation of the fertilization coat were reduced in comparison with embryos cultured at 1 g in space and under normal conditions on Earth. Also, 4% of all cells undergoing division in microgravity showed abnormalities in the centrosome-centriole complex. These abnormalities were not observed within the 1 g flight and ground control specimens, indicating that significant alterations in sea urchin development processes occur under microgravity conditions. Copyright 1999 Academic Press.

The Drosophila Pericentrin-like-protein (PLP cooperates with Cnn to maintain the integrity of the outer PCM

Directory of Open Access Journals (Sweden)

Jennifer H. Richens

2015-08-01

Full Text Available Centrosomes comprise a pair of centrioles surrounded by a matrix of pericentriolar material (PCM. In vertebrate cells, Pericentrin plays an important part in mitotic PCM assembly, but the Drosophila Pericentrin-like protein (PLP appears to have a more minor role in mitotic fly cells. Here we investigate the function of PLP during the rapid mitotic cycles of the early Drosophila embryo. Unexpectedly, we find that PLP is specifically enriched in the outer-most regions of the PCM, where it largely co-localizes with the PCM scaffold protein Cnn. In the absence of PLP the outer PCM appears to be structurally weakened, and it rapidly disperses along the centrosomal microtubules (MTs. As a result, centrosomal MTs are subtly disorganized in embryos lacking PLP, although mitosis is largely unperturbed and these embryos develop and hatch at near-normal rates. Y2H analysis reveals that PLP can potentially form multiple interactions with itself and with the PCM recruiting proteins Asl, Spd-2 and Cnn. A deletion analysis suggests that PLP participates in a complex network of interactions that ultimately help to strengthen the PCM.

Live imaging of individual cell divisions in mouse neuroepithelium shows asymmetry in cilium formation and Sonic hedgehog response

Directory of Open Access Journals (Sweden)

Piotrowska-Nitsche Karolina

2012-05-01

Full Text Available Abstract Background Primary cilia are microtubule-based sensory organelles that play important roles in developmental signaling pathways. Recent work demonstrated that, in cell culture, the daughter cell that inherits the older mother centriole generates a primary cilium and responds to external stimuli prior to its sister cell. This asynchrony in timing of cilia formation could be especially critical during development as cell divisions are required for both differentiation and maintenance of progenitor cell niches. Methods Here we integrate several fluorescent markers and use ex vivo live imaging of a single cell division within the mouse E8.5 neuroepithelium to reveal both the formation of a primary cilium and the transcriptional response to Sonic hedgehog in the daughter cells. Results We show that, upon cell division, cilia formation and the Sonic hedgehog response are asynchronous between the daughter cells. Conclusions Our results demonstrate that we can directly observe single cell divisions within the developing neuroepithelium and concomitantly monitor cilium formation or Sonic hedgehog response. We expect this method to be especially powerful in examining whether cellular behavior can lead to both differentiation and maintenance of cells in a progenitor niche.

Ultrastructure of photo-sensory cells and pigment epithelium in the retina of the Antarctic fish Notothenia neglecta Nybelin (Nototheniidae

Directory of Open Access Journals (Sweden)

Lucelia Donatti

2002-03-01

Full Text Available The Antarctic nototheniid Notothenia neglecta is the dominant fish in its habitat in Admiralty Bay, King George Island. They are predators, often ambush feeders, with accurate visual behaviour. For that reason, the ultrastructure of retinal photoreceptive cells and the pigment epithelium was analysed through electron microscopy. Their retina has a pigment epithelium, five different photoreceptors : rods, short single, long single, double, and triple cones, and neurones and support cells. The pigment epithelium is characterised by infoldings of the basal membrane, basal mitochondria, smooth reticule, large amount of microtubules, melanin granules, phagosomes and detached membranes of photoreceptors. Cones show bimembranous discs in the outer segment, an accessory outer segment, a connecting cilium, calycal processes, microtubules in the inferior ellipsoid and myoid, centrioles in the ellipsoid, interdigitating myoid fins and apical microvilli of Muller cells in the myoid and elliposid region. All these features allow all sorts of adaptations to the environmental photic variations, and situate N. neglecta among fish with a complex retina, with cells that are arranged in ten layers, allowing horizontal and vertical integration among them. This allows optimal visual behaviour and perception of food and environment in every Antarctic season.

Ultrastructural dynamics of human reproduction, from ovulation to fertilization and early embryo development.

Science.gov (United States)

Familiari, Giuseppe; Heyn, Rosemarie; Relucenti, Michela; Nottola, Stefania A; Sathananthan, A Henry

2006-01-01

This study describes the updated, fine structure of human gametes, the human fertilization process, and human embryos, mainly derived from assisted reproductive technology (ART). As clearly shown, the ultrastructure of human reproduction is a peculiar multistep process, which differs in part from that of other mammalian models, having some unique features. Particular attention has been devoted to the (1) sperm ultrastructure, likely "Tygerberg (Kruger) strict morphology criteria"; (2) mature oocyte, in which the MII spindle is barrel shaped, anastral, and lacking centrioles; (3) three-dimensional microarchitecture of the zona pellucida with its unique supramolecular filamentous organization; (4) sperm-egg interactions with the peculiarity of the sperm centrosome that activates the egg and organizes the sperm aster and mitotic spindles of the embryo; and (5) presence of viable cumulus cells whose metabolic activity is closely related to egg and embryo behavior in in vitro as well as in vivo conditions, in a sort of extraovarian "microfollicular unit." Even if the ultrastructural morphodynamic features of human fertilization are well understood, our knowledge about in vivo fertilization is still very limited and the complex sequence of in vivo biological steps involved in human reproduction is only partially reproduced in current ART procedures.

Microgravity effects during fertilization, cell division, development, and calcium metabolism in sea urchins

Science.gov (United States)

Schatten, Heide

1996-01-01

The overall objectives of this project are to explore the role of microgravity during fertilization, early development, cytoskeletal organization, and skeletal calcium deposition in a model development system: the sea urchin eggs and embryos. While pursuing these objectives, we have also helped to develop, test, and fly the Aquatic Research Facility (ARF) system. Cells were fixed at preselected time points to preserve the structures and organelles of interest with regards to cell biology events during development. The protocols used for the analysis of the results had been developed during the earlier part of this research and were applied for post-flight analysis using light and (immuno)fluorescence microscopy, scanning electron microscopy, and transmission electron microscopy. The structures of interest are: microtubules during fertilization, cell division, and cilia movement; microfilaments during cell surface restructuring and cell division; centrosomes and centrioles during cell division, cell differentiation, and cilia formation and movement; membranes, Golgi, endoplasmic reticulum, mitochondria, and chromosomes at all stages of development; and calcium deposits during spicule formation in late-stage embryos. In addition to further explore aspects important or living in space, several aspects of this research are also aimed at understanding diseases that affect humans on Earth which may be accelerated in space.

Ultrastructure of the spermatozoa of Corystes cassivelaunus (Corystidae), Platepistoma nanum (Cancridae) and Cancer pagurus (Cancridae) supports recognition of the Corystoidea (Crustacea, Brachyura, Heterotremata)

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Jamieson, B. G. M.; Guinot, D.; Tudge, C. C.; Richer de Forges, B.

1997-03-01

A combination of characters, not individually unique, possessed by the corystid, Corystes cassivelaunus, and the two cancrids, Platepistoma nanum and Cancer pagurus, defines a corystoid-type of spermatozoon: the basally bulbous, anteriorly narrowing perforatorium, the extent of this almost to the plasma membrane through a widely perforate operculum, and the simple inner acrosome zone, lacking an acrosome ray zone. The sperm of the two cancrids are closely similar, that of the corystid differing, for instance, in the less pointed, and less tapered, form of the perforatorium. This relative uniformity of spermatozoal ultrastructure in the cancrid+corystid assemblage so far investigated supports inclusion of the two families in the superfamily Corystoidea by Guinot (1978). The combination of perforation of the operculum and absence of an acrosome ray zone (at least in a clearly recognizable form) are features of the Potamidae which possibly indicate that the latter family, modified for a freshwater existence, is related to the cancrid+corystid assemblage. Some elongation of the centrioles, apparent at least in Corystes, may be a further link with potamids in which they are greatly elongated. The coenospermial spermatophores of cancridoids are a notable difference from the cleistospermia of potamids; but the latter is probably an apomorphic modification for fertilization biology.

Three-dimensional organization of micronuclei induced by colchicine in PtK sub 1 cells

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Geraud, G.; Laquerriere, F.; Masson, C.; Arnoult, J.; Labidi, B.; Hernandez-Verdun, D. (Univ. Pierre et Marie Curie, Paris (France))

1989-03-01

In PtK{sub 1} cells micronucleated by colchicine, the authors previously demonstrated that some micronuclei contain a single chromosome. Here, they investigated interphase chromosome organization in micronucleated PtK{sub 1} cells using conventional electron microscopy and three-dimensional computer reconstruction. The distribution of micronuclei was not always polarized, but in some cells they formed a ring. When this occurred, centrioles and Golgi apparatus were located inside the ring. On freeze-fracture replicas, they observed that nuclear pore distinction among the micronuclei was heterogeneous, and on thin sections some micronuclei displayed an incomplete nuclear envelope, with gaps in the double membrane and areas without lamina or condensed chromatin. By autoradiography, they showed that the fibrillar dots were not sites of active transcription. They applied three dimensional reconstruction to one micronucleated cell containing 22 micronuclei whose size indicated that each micronucleus probably contained one chromosome. In this cell they demonstrated that only the smallest micronuclei had an incomplete nuclear envelope. The presence in micronuclei of either nucleoli or fibrillar dots was found to be mutually exclusive. Taken together, these findings indicate that in the diploid nuclei of PtK{sub 1} cells, the three-dimensional organization of the nucleolar domain seems to be directly controlled by the X-chromosome.

Morphology of the Male Reproductive System and Spermiogenesis of Dendroctonus armandi Tsai and Li (Coleoptera: Curculionidae: Scolytinae).

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Wu, Yi-Fei; Wei, Lu-Sha; Anthony Torres, Mark; Zhang, Xu; Wu, Shao-Ping; Chen, Hui

2017-01-01

Studying the reproductive attributes of pests is central to understanding their life cycle history and in crafting management strategies to regulate, if not bring down, their population below threshold levels. In this article, the morphology of the male reproductive tract, topology of the spermatozoa, and salient features of spermiogenesis in the Chinese white pine beetle, Dendroctonus armandi Tsai and Li was studied to provide baseline information for further pest management studies. Results showed that male reproductive tract of this species differs from those documented in other Coleopterans by having 20 testicular tubules in each testis and the presence of two types of accessory glands. The spermatozoon is seen having peculiar characteristics such as an "h"-shaped acrosomal vesicle with a "puff"-like expansion, one centriole, one large spongy body, and two accessory bodies. Despite with some morphological differences of the male reproductive organ, spermatogenesis in this organism is similar to other Coleopterans. Overall, detailed studies regarding the components of the primary male reproductive organ of this beetle species would expand the knowledge on the less-understood biology of Coleopteran pests and would help in designing regulatory measures to conserve endemic and indigenous pine trees in China. © The Authors 2017. Published by Oxford University Press on behalf of Entomological Society of America.

Cep169, a Novel Microtubule Plus-End-Tracking Centrosomal Protein, Binds to CDK5RAP2 and Regulates Microtubule Stability.

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Yusuke Mori

Full Text Available The centrosomal protein, CDK5RAP2, is a microcephaly protein that regulates centrosomal maturation by recruitment of a γ-tubulin ring complex (γ-TuRC onto centrosomes. In this report, we identified a novel human centrosomal protein, Cep169, as a binding partner of CDK5RAP2, a member of microtubule plus-end-tracking proteins (+TIPs. Cep169 interacts directly with CDK5RAP2 through CM1, an evolutionarily conserved domain, and colocalizes at the pericentriolar matrix (PCM around centrioles with CDK5RAP2. In addition, Cep169 interacts with EB1 through SxIP-motif responsible for EB1 binding, and colocalizes with CDK5RAP2 at the microtubule plus-end. EB1-binding-deficient Cep169 abolishes EB1 interaction and microtubule plus-end attachment, indicating Cep169 as a novel member of +TIPs. We further show that ectopic expression of either Cep169 or CDK5RAP2 induces microtubule bundling and acetylation in U2OS cells, and depletion of Cep169 induces microtubule depolymerization in HeLa cells, although Cep169 is not required for assembly of γ-tubulin onto centrosome by CDK5RAP2. These results show that Cep169 targets microtubule tips and regulates stability of microtubules with CDK5RAP2.

TgICMAP1 is a novel microtubule binding protein in Toxoplasma gondii.

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Aoife T Heaslip

Full Text Available The microtubule cytoskeleton provides essential structural support for all eukaryotic cells and can be assembled into various higher order structures that perform drastically different functions. Understanding how microtubule-containing assemblies are built in a spatially and temporally controlled manner is therefore fundamental to understanding cell physiology. Toxoplasma gondii, a protozoan parasite, contains at least five distinct tubulin-containing structures, the spindle pole, centrioles, cortical microtubules, the conoid, and the intra-conoid microtubules. How these five structurally and functionally distinct sets of tubulin containing structures are constructed and maintained in the same cell is an intriguing problem. Previously, we performed a proteomic analysis of the T. gondii apical complex, a cytoskeletal complex located at the apical end of the parasite that is composed of the conoid, three ring-like structures, and the two short intra-conoid microtubules. Here we report the characterization of one of the proteins identified in that analysis, TgICMAP1. We show that TgICMAP1 is a novel microtubule binding protein that can directly bind to microtubules in vitro and stabilizes microtubules when ectopically expressed in mammalian cells. Interestingly, in T. gondii, TgICMAP1 preferentially binds to the intra-conoid microtubules, providing us the first molecular tool to investigate the intra-conoid microtubule assembly process during daughter construction.

Polo-like kinase phosphorylation determines Caenorhabditis elegans centrosome size and density by biasing SPD-5 toward an assembly-competent conformation

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Oliver Wueseke

2016-10-01

Full Text Available Centrosomes are major microtubule-organizing centers composed of centrioles surrounded by an extensive proteinacious layer called the pericentriolar material (PCM. In Caenorhabditis elegans embryos, the mitotic PCM expands by Polo-like kinase 1 (PLK-1 phosphorylation-accelerated assembly of SPD-5 molecules into supramolecular scaffolds. However, how PLK-1 phosphorylation regulates SPD-5 assembly is not known. We found that a mutant version of SPD-5 that is insensitive to PLK-1 phosphorylation (SPD-54A could localize to PCM but was unable to rescue the reduction in PCM size and density when wild-type SPD-5 levels were decreased. In vitro, purified SPD-54A self-assembled into functional supramolecular scaffolds over long time scales, suggesting that phosphorylation only controls the rate of SPD-5 scaffold assembly. Furthermore, the SPD-5 scaffold, once assembled, remained intact and supported microtubule nucleation in the absence of PLK-1 activity in vivo. We conclude that PLK-1 is required for rapid assembly of the PCM scaffold but not for scaffold maintenance or function. Based on this idea, we developed a theoretical model that adequately predicted PCM growth rates in different mutant conditions in vivo. We propose that PLK-1 phosphorylation-dependent conversion of SPD-5 into an assembly-competent form underlies PCM formation in vivo and that the rate of this conversion determines final PCM size and density.

contribution to carcinogenesis

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Aneta Białkowska

2014-01-01

Full Text Available The centrosomes are subcellular organelles composed of two centrioles surrounded by a pericentriolar material. In animal cells they are responsible for the organization of the interphase microtubule cytoskeleton including microtubule nucleation and elongation, their attachment and release. The centrosomes are also involved in the construction of the mitotic spindle and chromosome segregation. More than a century ago it was suggested that these structures might be involved in human diseases, including cancer. Cancer cells show a high frequency of centrosome aberrations, especially amplification. Centrosome defects may increase the incidence of multipolar mitoses that lead to chromosomal segregation abnormalities and aneuploidy, which is the predominant type of genomic instability found in human solid tumors. The number of these organelles in cells is strictly controlled and is dependent on the proper process of centrosome duplication. Multiple genes that are frequently found mutated in cancers encode proteins which participate in the regulation of centrosome duplication and the numeral integrity of centrosomes. In recent years there has been growing interest in the potential participation of centrosomes in the process of carcinogenesis, especially because centrosome abnormalities are observed in premalignant stages of cancer development. The common presence of abnormal centrosomes in cancer cells and the role these organelles play in the cells suggest that the factors controlling the number of centrosomes may be potential targets for cancer therapy.

SAS6-like protein in Plasmodium indicates that conoid-associated apical complex proteins persist in invasive stages within the mosquito vector.

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Wall, Richard J; Roques, Magali; Katris, Nicholas J; Koreny, Ludek; Stanway, Rebecca R; Brady, Declan; Waller, Ross F; Tewari, Rita

2016-06-24

The SAS6-like (SAS6L) protein, a truncated paralogue of the ubiquitous basal body/centriole protein SAS6, has been characterised recently as a flagellum protein in trypanosomatids, but associated with the conoid in apicomplexan Toxoplasma. The conoid has been suggested to derive from flagella parts, but is thought to have been lost from some apicomplexans including the malaria-causing genus Plasmodium. Presence of SAS6L in Plasmodium, therefore, suggested a possible role in flagella assembly in male gametes, the only flagellated stage. Here, we have studied the expression and role of SAS6L throughout the Plasmodium life cycle using the rodent malaria model P. berghei. Contrary to a hypothesised role in flagella, SAS6L was absent during gamete flagellum formation. Instead, SAS6L was restricted to the apical complex in ookinetes and sporozoites, the extracellular invasive stages that develop within the mosquito vector. In these stages SAS6L forms an apical ring, as we show is also the case in Toxoplasma tachyzoites. The SAS6L ring was not apparent in blood-stage invasive merozoites, indicating that the apical complex is differentiated between the different invasive forms. Overall this study indicates that a conoid-associated apical complex protein and ring structure is persistent in Plasmodium in a stage-specific manner.

Establishment and mitotic characterization of new Drosophila acentriolar cell lines from DSas-4 mutant

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Nicolas Lecland

2013-01-01

In animal cells the centrosome is commonly viewed as the main cellular structure driving microtubule (MT assembly into the mitotic spindle apparatus. However, additional pathways, such as those mediated by chromatin and augmin, are involved in the establishment of functional spindles. The molecular mechanisms involved in these pathways remain poorly understood, mostly due to limitations inherent to current experimental systems available. To overcome these limitations we have developed six new Drosophila cell lines derived from Drosophila homozygous mutants for DSas-4, a protein essential for centriole biogenesis. These cells lack detectable centrosomal structures, astral MT, with dispersed pericentriolar proteins D-PLP, Centrosomin and γ-tubulin. They show poorly focused spindle poles that reach the plasma membrane. Despite being compromised for functional centrosome, these cells could successfully undergo mitosis. Live-cell imaging analysis of acentriolar spindle assembly revealed that nascent MTs are nucleated from multiple points in the vicinity of chromosomes. These nascent MTs then grow away from kinetochores allowing the expansion of fibers that will be part of the future acentriolar spindle. MT repolymerization assays illustrate that acentriolar spindle assembly occurs “inside-out” from the chromosomes. Colchicine-mediated depolymerization of MTs further revealed the presence of a functional Spindle Assembly Checkpoint (SAC in the acentriolar cells. Finally, pilot RNAi experiments open the potential use of these cell lines for the molecular dissection of anastral pathways in spindle and centrosome assembly.

Centrosome Amplification Increases Single-Cell Branching in Post-mitotic Cells.

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Ricolo, Delia; Deligiannaki, Myrto; Casanova, Jordi; Araújo, Sofia J

2016-10-24

Centrosome amplification is a hallmark of cancer, although we are still far from understanding how this process affects tumorigenesis [1, 2]. Besides the contribution of supernumerary centrosomes to mitotic defects, their biological effects in the post-mitotic cell are not well known. Here, we exploit the effects of centrosome amplification in post-mitotic cells during single-cell branching. We show that Drosophila tracheal cells with extra centrosomes branch more than wild-type cells. We found that mutations in Rca1 and CycA affect subcellular branching, causing tracheal tip cells to form more than one subcellular lumen. We show that Rca1 and CycA post-mitotic cells have supernumerary centrosomes and that other mutant conditions that increase centrosome number also show excess of subcellular lumen branching. Furthermore, we show that de novo lumen formation is impaired in mutant embryos with fewer centrioles. The data presented here define a requirement for the centrosome as a microtubule-organizing center (MTOC) for the initiation of subcellular lumen formation. We propose that centrosomes are necessary to drive subcellular lumen formation. In addition, centrosome amplification increases single-cell branching, a process parallel to capillary sprouting in blood vessels [3]. These results shed new light on how centrosomes can contribute to pathology independently of mitotic defects. Copyright © 2016 Elsevier Ltd. All rights reserved.

Spastin-Interacting Protein NA14/SSNA1 Functions in Cytokinesis and Axon Development

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Chang, Jaerak; Blackstone, Craig

2014-01-01

Hereditary spastic paraplegias (HSPs) are a genetically diverse group of inherited neurological disorders (SPG1-72) with the cardinal feature of prominent lower-extremity spasticity due to a length-dependent axonopathy of corticospinal motor neurons. The most frequent form of autosomal dominant HSP results from mutations of the SPG4 gene product spastin. This is an ATPase associated with diverse cellular activities (AAA) protein that binds to and severs microtubules. While spastin participates in crucial cellular processes such as cytokinesis, endosomal tubulation, and axon development, its role in HSP pathogenesis remains unclear. Spastin interacts in cells with the NA14 protein, a major target for auto-antibodies in Sjögren's syndrome (nuclear autoantigen 1; SSNA1). Our analysis of endogenous spastin and NA14 proteins in HeLa cells and rat cortical neurons in primary culture revealed a clear distribution of both proteins to centrosomes, with NA14 localizing specifically to centrioles. Stable NA14 knockdown in cell lines dramatically affected cell division, in particular cytokinesis. Furthermore, overexpression of NA14 in neurons significantly increased axon outgrowth and branching, while also enhancing neuronal differentiation. We postulate that NA14 may act as an adaptor protein regulating spastin localization to centrosomes, temporally and spatially regulating the microtubule-severing activity of spastin that is particularly critical during the cell cycle and neuronal development. PMID:25390646

Spastin-interacting protein NA14/SSNA1 functions in cytokinesis and axon development.

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Uma Goyal

Full Text Available Hereditary spastic paraplegias (HSPs are a genetically diverse group of inherited neurological disorders (SPG1-72 with the cardinal feature of prominent lower-extremity spasticity due to a length-dependent axonopathy of corticospinal motor neurons. The most frequent form of autosomal dominant HSP results from mutations of the SPG4 gene product spastin. This is an ATPase associated with diverse cellular activities (AAA protein that binds to and severs microtubules. While spastin participates in crucial cellular processes such as cytokinesis, endosomal tubulation, and axon development, its role in HSP pathogenesis remains unclear. Spastin interacts in cells with the NA14 protein, a major target for auto-antibodies in Sjögren's syndrome (nuclear autoantigen 1; SSNA1. Our analysis of endogenous spastin and NA14 proteins in HeLa cells and rat cortical neurons in primary culture revealed a clear distribution of both proteins to centrosomes, with NA14 localizing specifically to centrioles. Stable NA14 knockdown in cell lines dramatically affected cell division, in particular cytokinesis. Furthermore, overexpression of NA14 in neurons significantly increased axon outgrowth and branching, while also enhancing neuronal differentiation. We postulate that NA14 may act as an adaptor protein regulating spastin localization to centrosomes, temporally and spatially regulating the microtubule-severing activity of spastin that is particularly critical during the cell cycle and neuronal development.

Bug22 influences cilium morphology and the post-translational modification of ciliary microtubules

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Teresa Mendes Maia

2014-01-01

Cilia and flagella are organelles essential for motility and sensing of environmental stimuli. Depending on the cell type, cilia acquire a defined set of functions and, accordingly, are built with an appropriate length and molecular composition. Several ciliary proteins display a high degree of conservation throughout evolution and mutations in ciliary genes are associated with various diseases such as ciliopathies and infertility. Here, we describe the role of the highly conserved ciliary protein, Bug22, in Drosophila. Previous studies in unicellular organisms have shown that Bug22 is required for proper cilia function, but its exact role in ciliogenesis has not been investigated yet. Null Bug22 mutant flies display cilia-associated phenotypes and nervous system defects. Furthermore, sperm differentiation is blocked at the individualization stage, due to impaired migration of the individualization machinery. Tubulin post-translational modifications (PTMs such as polyglycylation, polyglutamylation or acetylation, are determinants of microtubule (MT functions and stability in centrioles, cilia and neurons. We found defects in the timely incorporation of polyglycylation in sperm axonemal MTs of Bug22 mutants. In addition, we found that depletion of human Bug22 in RPE1 cells resulted in the appearance of longer cilia and reduced axonemal polyglutamylation. Our work identifies Bug22 as a protein that plays a conserved role in the regulation of PTMs of the ciliary axoneme.

Spermatogenesis-specific features of the meiotic program in Caenorhabditis elegans.

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Diane C Shakes

2009-08-01

Full Text Available In most sexually reproducing organisms, the fundamental process of meiosis is implemented concurrently with two differentiation programs that occur at different rates and generate distinct cell types, sperm and oocytes. However, little is known about how the meiotic program is influenced by such contrasting developmental programs. Here we present a detailed timeline of late meiotic prophase during spermatogenesis in Caenorhabditis elegans using cytological and molecular landmarks to interrelate changes in chromosome dynamics with germ cell cellularization, spindle formation, and cell cycle transitions. This analysis expands our understanding C. elegans spermatogenesis, as it identifies multiple spermatogenesis-specific features of the meiotic program and provides a framework for comparative studies. Post-pachytene chromatin of spermatocytes is distinct from that of oocytes in both composition and morphology. Strikingly, C. elegans spermatogenesis includes a previously undescribed karyosome stage, a common but poorly understood feature of meiosis in many organisms. We find that karyosome formation, in which chromosomes form a constricted mass within an intact nuclear envelope, follows desynapsis, involves a global down-regulation of transcription, and may support the sequential activation of multiple kinases that prepare spermatocytes for meiotic divisions. In spermatocytes, the presence of centrioles alters both the relative timing of meiotic spindle assembly and its ultimate structure. These microtubule differences are accompanied by differences in kinetochores, which connect microtubules to chromosomes. The sperm-specific features of meiosis revealed here illuminate how the underlying molecular machinery required for meiosis is differentially regulated in each sex.

Organizational changes of the daughter basal complex during the parasite replication of Toxoplasma gondii.

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Ke Hu

2008-01-01

Full Text Available The apicomplexans are a large group of parasitic protozoa, many of which are important human and animal pathogens, including Plasmodium falciparum and Toxoplasma gondii. These parasites cause disease only when they replicate, and their replication is critically dependent on the proper assembly of the parasite cytoskeletons during cell division. In addition to their importance in pathogenesis, the apicomplexan parasite cytoskeletons are spectacular structures. Therefore, understanding the cytoskeletal biogenesis of these parasites is important not only for parasitology but also of general interest to broader cell biology. Previously, we found that the basal end of T. gondii contains a novel cytoskeletal assembly, the basal complex, a cytoskeletal compartment constructed in concert with the daughter cortical cytoskeleton during cell division. This study focuses on key events during the biogenesis of the basal complex using high resolution light microscopy, and reveals that daughter basal complexes are established around the duplicated centrioles independently of the structural integrity of the daughter cortical cytoskeleton, and that they are dynamic "caps" at the growing ends of the daughters. Compartmentation and polarization of the basal complex is first revealed at a late stage of cell division upon the recruitment of an EF-hand containing calcium binding protein, TgCentrin2. This correlates with the constriction of the basal complex, a process that can be artificially induced by increasing cellular calcium concentration. The basal complex is therefore likely to be a new kind of centrin-based contractile apparatus.

Fine structure of acrosome biogenesis and of mature sperm in the bivalve molluscs Glycymeris sp. (Pteriomorphia) and Eurhomalea rufa (Heterodonta)

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Guerra, Rosa; Sousa, Mário; Torres, Artur; Oliveira, Elsa; Baldaia, Luis

2003-03-01

Proacrosomal vesicles form during the pachytene stage, being synthetized by the Golgi complex in Glycymeris sp., and by both the Golgi and the rough endoplasmic reticulum in Eurhomalea rufa. During early spermiogenesis, a single acrosomal vesicle forms and its apex becomes linked to the plasma membrane while it migrates. In Glycymeris sp., the acrosomal vesicle then turns cap-shaped (1.8 μm) and acquires a complex substructure. In E. rufa, proacrosomal vesicles differentiate their contents while still at the premeiotic stage; as the acrosomal vesicle matures and its contents further differentiate, it elongates and becomes longer than the nucleus (3.2 μm), while the subacrosomal space develops a perforatorium. Before condensation, chromatin turns fibrillar in Glycymeris sp., whereas it acquires a cordonal pattern in E. rufa. Accordingly, the sperm nucleus of Glycymeris sp. is conical and elongated (8.3 μm), and that of E. rufa is short and ovoid (1.1 μm). In the midpiece (Glycymeris sp.: 1.1 μm; E. rufa: 0.8 μm), both species have four mitochondria encircling two linked orthogonal (Glycymeris sp.) or orthogonal and tilted (30-40°; E. rufa) centrioles. In comparison with other Arcoida species, sperm of Glycymeris sp. appear distinct due to the presence of an elongated nucleus, a highly differentiated acrosome, and four instead of five mitochondria. The same occurs with E. rufa regarding other Veneracea species, with the acrosome of the mature sperm strongly resembling that of the recent Mytilinae.

The Aurora A-HP1γ pathway regulates gene expression and mitosis in cells from the sperm lineage.

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Leonard, Phoebe H; Grzenda, Adrienne; Mathison, Angela; Morbeck, Dean E; Fredrickson, Jolene R; de Assuncao, Thiago M; Christensen, Trace; Salisbury, Jeffrey; Calvo, Ezequiel; Iovanna, Juan; Coddington, Charles C; Urrutia, Raul; Lomberk, Gwen

2015-05-29

HP1γ, a well-known regulator of gene expression, has been recently identified to be a target of Aurora A, a mitotic kinase which is important for both gametogenesis and embryogenesis. The purpose of this study was to define whether the Aurora A-HP1γ pathway supports cell division of gametes and/or early embryos, using western blot, immunofluorescence, immunohistochemistry, electron microscopy, shRNA-based knockdown, site-directed mutagenesis, and Affymetrix-based genome-wide expression profiles. We find that the form of HP1γ phosphorylated by Aurora A, P-Ser83 HP1γ, is a passenger protein, which localizes to the spermatozoa centriole and axoneme. In addition, disruption in this pathway causes centrosomal abnormalities and aberrations in cell division. Expression profiling of male germ cell lines demonstrates that HP1γ phosphorylation is critical for the regulation of mitosis-associated gene expression networks. In female gametes, we observe that P-Ser83-HP1γ is not present in meiotic centrosomes of M2 oocytes, but after syngamy, it becomes detectable during cleavage divisions, coinciding with early embryonic genome activation. These results support the idea that phosphorylation of HP1γ by Aurora A plays a role in the regulation of gene expression and mitotic cell division in cells from the sperm lineage and in early embryos. Combined, this data is relevant to better understanding the function of HP1γ in reproductive biology.

Centrosome – a promising anti-cancer target

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Rivera-Rivera Y

2016-12-01

Full Text Available Yainyrette Rivera-Rivera, Harold I Saavedra Department of Pharmacology, Ponce Health Sciences University-School of Medicine, Ponce Research Institute, Ponce, Puerto Rico Abstract: The centrosome, an organelle discovered >100 years ago, is the main microtubule-organizing center in mammalian organisms. The centrosome is composed of a pair of centrioles surrounded by the pericentriolar material (PMC and plays a major role in the regulation of cell cycle transitions (G1-S, G2-M, and metaphase-anaphase, ensuring the normality of cell division. Hundreds of proteins found in the centrosome exert a variety of roles, including microtubule dynamics, nucleation, and kinetochore–microtubule attachments that allow correct chromosome alignment and segregation. Errors in these processes lead to structural (shape, size, number, position, and composition, functional (abnormal microtubule nucleation and disorganized spindles, and numerical (centrosome amplification [CA] centrosome aberrations causing aneuploidy and genomic instability. Compelling data demonstrate that centrosomes are implicated in cancer, because there are important oncogenic and tumor suppressor proteins that are localized in this organelle and drive centrosome aberrations. Centrosome defects have been found in pre-neoplasias and tumors from breast, ovaries, prostate, head and neck, lung, liver, and bladder among many others. Several drugs/compounds against centrosomal proteins have shown promising results. Other drugs have higher toxicity with modest or no benefits, and there are more recently developed agents being tested in clinical trials. All of this emerging evidence suggests that targeting centrosome aberrations may be a future avenue for therapeutic intervention in cancer research. Keywords: centrosomes, cell cycle, mitosis, CA, CIN, cancer therapy

Studies on the cytodifferentiation of the neuroblasts and visual cells in the chick embryo retina, using the electron-microscopic autoradiography of 3H-thymidine

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Mishima, H.; Fujita, H.

1978-01-01

Studies on the histogenetic analysis of cytodifferentiation of the neuroblast and visual cell in the chick embryo retina were made using the autoradiography of 3 H-thymidine. The posterior pole region of the eyeball was observed in all the animals used. The retina in a 4-day-old chick embryo consists exclusively of matrix cells forming the matrix layer. In a 5-day-old chick embryo retina, neuroblasts first differentiated from the matrix cells migrate into the outer part of the matrix layer, forming the mantle layer. The matrix cell is a homogeneous epithelial cell containing abundant free ribosomes and a poorly developed cytoplasmic membrane system in the cytoplasm. The characteristic sign of differentiation of the neuroblast is an appearance of elements of rough-surfaced endoplasmic reticulum and an indentation of the nucleus. The primitive visual cell having just lost its ability to synthesize DNA appears just beneath the pigment epithelium in a 7-day-old chick embryo, and all the cells lying beneath the pigment epithelium lose the ability to synthesize DNA at 10 days of incubation. The cytoplasmic process of the matrix cell is in contact with the adjacent one, making an apicolateral junction. When the matrix cell loses its ability to synthesize DNA, a big tentlike process extending over the level of the apicolateral junction appears. This phenomenon is considered to be a sign of differentiation from matrix cell to primitive visual cell, and this big tentlike process containing 2 centrioles is a primordium of the inner segment of the visual cell. (orig.) [de

In vivo and in vitro HeNe laser effects on phagocyte functions

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Ricevuti, G.; Mazzone, A.; Monaia, C.; Fratino, P.; Degiulio, R.; Dell' Acqua, R.; Leonardi, G.; Jucci, A.; Sacchi, S. (Univ. of Pavia (Italy))

1989-10-01

The goal of this work was to evaluate the effect of helium-neon (HeNe) laser irradiation on immunocompetent cells. We used the in vivo skin window method and in vitro granulocyte function tests. The study of cellular migration showed a marked decrease in vitro and in vivo in a dose-independent manner. Superoxide release was not modified by laser irradiation. The granulocyte's aggregation, when using PHA and PMA, presented a reduction that was statistically very significant, not as a subordinate dose. An increase of the release of ATP was demonstrated only at 4 joules and precedes granulocyte aggregation. When using Ca2+ ionophore A23187 as stimulus, laser irradiation at 1, 2 or 4J did not show any modification of granulocyte aggregation. The monoclonal antibody 60.1, which identifies a membrane antigen fundamental for aggregation and chemotaxis, is expressed in normal amounts on granulocyte membranes both before and after irradiation with a HeNe laser. In fact, laser irradiation preferentially attacks the area of the cellular centrosome that determines a modification of cellular morphology. The electron microscope and immunofluorescence study with a monoclonal antibody have pointed out a disorganization of the microtubules. The alteration of some of the granulocyte functions is correlated to the damage in the centrioles. The granulocyte mitochondrial system and surface membrane remain intact, and this explains the normal production and release of free radicals. Further experiments are necessary to evaluate the clinical application of lasers in various diseases with immunophagocytic pathogenesis.

Phylogenetic analysis of the Neks reveals early diversification of ciliary-cell cycle kinases.

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Jeremy D K Parker

2007-10-01

Full Text Available NIMA-related kinases (Neks have been studied in diverse eukaryotes, including the fungus Aspergillus and the ciliate Tetrahymena. In the former, a single Nek plays an essential role in cell cycle regulation; in the latter, which has more than 30 Neks in its genome, multiple Neks regulate ciliary length. Mammalian genomes encode an intermediate number of Neks, several of which are reported to play roles in cell cycle regulation and/or localize to centrosomes. Previously, we reported that organisms with cilia typically have more Neks than organisms without cilia, but were unable to establish the evolutionary history of the gene family.We have performed a large-scale analysis of the Nek family using Bayesian techniques, including tests of alternate topologies. We find that the Nek family had already expanded in the last common ancestor of eukaryotes, a ciliated cell which likely expressed at least five Neks. We suggest that Neks played an important role in the common ancestor in regulating cilia, centrioles, and centrosomes with respect to mitotic entry, and that this role continues today in organisms with cilia. Organisms that lack cilia generally show a reduction in the number of Nek clades represented, sometimes associated with lineage specific expansion of a single clade, as has occurred in the plants.This is the first rigorous phylogenetic analysis of a kinase family across a broad array of phyla. Our findings provide a coherent framework for the study of Neks and their roles in coordinating cilia and cell cycle progression.

Sperm morphology in five species of cicadettine cicadas (Hemiptera: Cicadomorpha: Cicadidae).

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Chawanji, Abraham S; Hodgson, Alan N; Villet, Martin H

2006-12-01

Mature spermatozoa from five species of cicadas of the subfamily Cicadettinae (Quintilia wealei, Melampsalta leucoptera, Stagira simplex, Xosopsaltria thunbergi and Monomatapa matoposa) were examined by light and electron microscopy. In each species sperm are elongate, aggregated into organized bundles with their heads embedded in a homogenous matrix to form spermatodesmata, and exhibit polymegaly. The head of the sperm consist of an anteriorly positioned conical acrosome that has a tubular substructure and a deep, posterior invagination that forms the subacrosomal space (eccentrically positioned anteriorly). The acrosome is flattened anteriorly; posteriorly it extends along either side of the nucleus as two tubular processes that gradually decrease in diameter. The filiform nucleus tapers anteriorly and intrudes into the subscrosomal space. Posteriorly the nucleus has a lateral invagination that houses material of the so-called centriolar adjunct. Posterior to the centriolar adjuct and the nucleus are two crystalline mitochondrial derivatives and a centriole, respectively, the latter giving rise to the axoneme, which has a 9 + 9 + 2 arrangement of microtubules. In these respects the sperm are similar to those of platypleurine cicadas. However, some features seem unique to cicadettines, including the structural organization of an enlarged centriolar adjunct and the dimensions of the tails. The enlarged centriolar adjunct has a lamella-like substructure and can be considered a synapomorphic character in the Cicadettinae. It is, therefore, potentially useful in the separation of this subfamily from the Cicadinae. In addition, the great length of the sperm nucleus of long-headed sperm in M. matoposa could be a synapomorphy of this genus and related taphurine and cicadettine species.

Oscillatory fluid flow influences primary cilia and microtubule mechanics.

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Espinha, Lina C; Hoey, David A; Fernandes, Paulo R; Rodrigues, Hélder C; Jacobs, Christopher R

2014-07-01

Many tissues are sensitive to mechanical stimuli; however, the mechanotransduction mechanism used by cells remains unknown in many cases. The primary cilium is a solitary, immotile microtubule-based extension present on nearly every mammalian cell which extends from the basal body. The cilium is a mechanosensitive organelle and has been shown to transduce fluid flow-induced shear stress in tissues, such as the kidney and bone. The majority of microtubules assemble from the mother centriole (basal body), contributing significantly to the anchoring of the primary cilium. Several studies have attempted to quantify the number of microtubules emanating from the basal body and the results vary depending on the cell type. It has also been shown that cellular response to shear stress depends on microtubular integrity. This study hypothesizes that changing the microtubule attachment of primary cilia in response to a mechanical stimulus could change primary cilia mechanics and, possibly, mechanosensitivity. Oscillatory fluid flow was applied to two different cell types and the microtubule attachment to the ciliary base was quantified. For the first time, an increase in microtubules around primary cilia both with time and shear rate in response to oscillatory fluid flow stimulation was demonstrated. Moreover, it is presented that the primary cilium is required for this loading-induced cellular response. This study has demonstrated a new role for the cilium in regulating alterations in the cytoplasmic microtubule network in response to mechanical stimulation, and therefore provides a new insight into how cilia may regulate its mechanics and thus the cells mechanosensitivity. Copyright © 2014 Wiley Periodicals, Inc.

Expression of XNOA 36 in the mitochondrial cloud of Xenopus laevis oocytes.

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Vaccaro, M C; Wilding, M; Dale, B; Campanella, C; Carotenuto, R

2012-08-01

In Xenopus laevis oocytes a mitochondrial cloud (MC) is found between the nucleus and the plasma membrane at stages I-II of oogenesis. The MC contains RNAs that are transported to the future vegetal pole at stage II of oogenesis. In particular, germinal plasm mRNAs are found in the Message Transport Organiser (METRO) region, the MC region opposite to the nucleus. At stages II-III, a second pathway transports Vg1 and VegT mRNAs to the area where the MC content merges with the vegetal cortex. Microtubules become polarized at the sites of migration of Vg1 and VegT mRNAs through an unknown signalling mechanism. In early meiotic stages, the centrioles are almost completely lost with their remnants being dispersed into the cytoplasm and the MC, which may contain a MTOC to be used in the later localization pathway of the mRNAs. In mammals, XNOA 36 encodes a member of a highly conserved protein family and localises to the nucleolus or in the centromeres. In the Xenopus late stage I oocyte, XNOA 36 mRNA is transiently segregated in one half of the oocyte, anchored by a cytoskeletal network that contains spectrin. Here we found that XNOA 36 transcript also localises to the nucleoli and in the METRO region. XNOA 36 protein immunolocalization, using an antibody employed for the library immunoscreening that depicted XNOA 36 expression colonies, labels the migrating MC, the cytoplasm of stage I oocytes and in particular the vegetal cortex facing the MC. The possible role of XNOA 36 in mRNA anchoring to the vegetal cortex or in participating in early microtubule reorganization is discussed.

Targeting of beta-arrestin2 to the centrosome and primary cilium: role in cell proliferation control.

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Anahi Molla-Herman

Full Text Available The primary cilium is a sensory organelle generated from the centrosome in quiescent cells and found at the surface of most cell types, from where it controls important physiological processes. Specific sets of membrane proteins involved in sensing the extracellular milieu are concentrated within cilia, including G protein coupled receptors (GPCRs. Most GPCRs are regulated by beta-arrestins, betaarr1 and betaarr2, which control both their signalling and endocytosis, suggesting that betaarrs may also function at primary cilium.In cycling cells, betaarr2 was observed at the centrosome, at the proximal region of the centrioles, in a microtubule independent manner. However, betaarr2 did not appear to be involved in classical centrosome-associated functions. In quiescent cells, both in vitro and in vivo, betaarr2 was found at the basal body and axoneme of primary cilia. Interestingly, betaarr2 was found to interact and colocalize with 14-3-3 proteins and Kif3A, two proteins known to be involved in ciliogenesis and intraciliary transport. In addition, as suggested for other centrosome or cilia-associated proteins, betaarrs appear to control cell cycle progression. Indeed, cells lacking betaarr2 were unable to properly respond to serum starvation and formed less primary cilia in these conditions.Our results show that betaarr2 is localized to the centrosome in cycling cells and to the primary cilium in quiescent cells, a feature shared with other proteins known to be involved in ciliogenesis or primary cilium function. Within cilia, betaarr2 may participate in the signaling of cilia-associated GPCRs and, therefore, in the sensory functions of this cell "antenna".

Differential expression of centrosomal proteins at different stages of human glioma

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Loh, Joon-Khim; Lieu, Ann-Shung; Chou, Chia-Hua; Lin, Fang-Yi; Wu, Chia-Hung; Howng, Sheng-Long; Chio, Chung-Ching; Hong, Yi-Ren

2010-01-01

High-grade gliomas have poor prognosis, requiring aggressive treatment. The aim of this study is to explore mitotic and centrosomal dysregulation in gliomas, which may provide novel targets for treatment. A case-control study was performed using 34 resected gliomas, which were separated into low- and high-grade groups. Normal human brain tissue was used as a control. Using immunohistochemical analysis, immunofluorescent microscopy, and RT-PCR, detection of centrins 1 and 2, γ-tubulin, hNinein, Aurora A, and Aurora B, expression was performed. Analysis of the GBM8401 glioma cell line was also undertaken to complement the in vivo studies. In high-grade gliomas, the cells had greater than two very brightly staining centrioles within large, atypical nuclei, and moderate-to-strong Aurora A staining. Comparing with normal human brain tissue, most of the mRNAs expression in gliomas for centrosomal structural proteins, including centrin 3, γ-tubulin, and hNinein isoforms 1, 2, 5 and 6, Aurora A and Aurora B were elevated. The significant different expression was observed between high- and low-grade glioma in both γ-tubulin and Aurora A mRNA s. In the high-grade glioma group, 78.6% of the samples had higher than normal expression of γ-tubulin mRNA, which was significantly higher than in the low-grade glioma group (18.2%, p < 0.05). Markers for mitotic dysregulation, such as supernumerary centrosomes and altered expression of centrosome-related mRNA and proteins were more frequently detected in higher grade gliomas. Therefore, these results are clinically useful for glioma staging as well as the development of novel treatments strategies

Differential expression of centrosomal proteins at different stages of human glioma

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Lin Fang-Yi

2010-06-01

Full Text Available Abstract Background High-grade gliomas have poor prognosis, requiring aggressive treatment. The aim of this study is to explore mitotic and centrosomal dysregulation in gliomas, which may provide novel targets for treatment. Methods A case-control study was performed using 34 resected gliomas, which were separated into low- and high-grade groups. Normal human brain tissue was used as a control. Using immunohistochemical analysis, immunofluorescent microscopy, and RT-PCR, detection of centrins 1 and 2, γ-tubulin, hNinein, Aurora A, and Aurora B, expression was performed. Analysis of the GBM8401 glioma cell line was also undertaken to complement the in vivo studies. Results In high-grade gliomas, the cells had greater than two very brightly staining centrioles within large, atypical nuclei, and moderate-to-strong Aurora A staining. Comparing with normal human brain tissue, most of the mRNAs expression in gliomas for centrosomal structural proteins, including centrin 3, γ-tubulin, and hNinein isoforms 1, 2, 5 and 6, Aurora A and Aurora B were elevated. The significant different expression was observed between high- and low-grade glioma in both γ-tubulin and Aurora A mRNA s. In the high-grade glioma group, 78.6% of the samples had higher than normal expression of γ-tubulin mRNA, which was significantly higher than in the low-grade glioma group (18.2%, p Conclusions Markers for mitotic dysregulation, such as supernumerary centrosomes and altered expression of centrosome-related mRNA and proteins were more frequently detected in higher grade gliomas. Therefore, these results are clinically useful for glioma staging as well as the development of novel treatments strategies.

PCM1 Depletion Inhibits Glioblastoma Cell Ciliogenesis and Increases Cell Death and Sensitivity to Temozolomide

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Lan B. Hoang-Minh

2016-10-01

Full Text Available A better understanding of the molecules implicated in the growth and survival of glioblastoma (GBM cells and their response to temozolomide (TMZ, the standard-of-care chemotherapeutic agent, is necessary for the development of new therapies that would improve the outcome of current GBM treatments. In this study, we characterize the role of pericentriolar material 1 (PCM1, a component of centriolar satellites surrounding centrosomes, in GBM cell proliferation and sensitivity to genotoxic agents such as TMZ. We show that PCM1 is expressed around centrioles and ciliary basal bodies in patient GBM biopsies and derived cell lines and that its localization is dynamic throughout the cell cycle. To test whether PCM1 mediates GBM cell proliferation and/or response to TMZ, we used CRISPR/Cas9 genome editing to generate primary GBM cell lines depleted of PCM1. These PCM1-depleted cells displayed reduced AZI1 satellite protein localization and significantly decreased proliferation, which was attributable to increased apoptotic cell death. Furthermore, PCM1-depleted lines were more sensitive to TMZ toxicity than control lines. The increase in TMZ sensitivity may be partly due to the reduced ability of PCM1-depleted cells to form primary cilia, as depletion of KIF3A also ablated GBM cells' ciliogenesis and increased their sensitivity to TMZ while preserving PCM1 localization. In addition, the co-depletion of KIF3A and PCM1 did not have any additive effect on TMZ sensitivity. Together, our data suggest that PCM1 plays multiple roles in GBM pathogenesis and that associated pathways could be targeted to augment current or future anti-GBM therapies.

Deficiency in origin licensing proteins impairs cilia formation: implications for the aetiology of Meier-Gorlin syndrome.

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Tom Stiff

Full Text Available Mutations in ORC1, ORC4, ORC6, CDT1, and CDC6, which encode proteins required for DNA replication origin licensing, cause Meier-Gorlin syndrome (MGS, a disorder conferring microcephaly, primordial dwarfism, underdeveloped ears, and skeletal abnormalities. Mutations in ATR, which also functions during replication, can cause Seckel syndrome, a clinically related disorder. These findings suggest that impaired DNA replication could underlie the developmental defects characteristic of these disorders. Here, we show that although origin licensing capacity is impaired in all patient cells with mutations in origin licensing component proteins, this does not correlate with the rate of progression through S phase. Thus, the replicative capacity in MGS patient cells does not correlate with clinical manifestation. However, ORC1-deficient cells from MGS patients and siRNA-mediated depletion of origin licensing proteins also have impaired centrosome and centriole copy number. As a novel and unexpected finding, we show that they also display a striking defect in the rate of formation of primary cilia. We demonstrate that this impacts sonic hedgehog signalling in ORC1-deficient primary fibroblasts. Additionally, reduced growth factor-dependent signaling via primary cilia affects the kinetics of cell cycle progression following cell cycle exit and re-entry, highlighting an unexpected mechanism whereby origin licensing components can influence cell cycle progression. Finally, using a cell-based model, we show that defects in cilia function impair chondroinduction. Our findings raise the possibility that a reduced efficiency in forming cilia could contribute to the clinical features of MGS, particularly the bone development abnormalities, and could provide a new dimension for considering developmental impacts of licensing deficiency.

Male Hypogonadism and Germ Cell Loss Caused by a Mutation in Polo-Like Kinase 4

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Harris, Rebecca M.; Weiss, Jeffrey

2011-01-01

The genetic etiologies of male infertility remain largely unknown. To identify genes potentially involved in spermatogenesis and male infertility, we performed genome-wide mutagenesis in mice with N-ethyl-N-nitrosourea and identified a line with dominant hypogonadism and patchy germ cell loss. Genomic mapping and DNA sequence analysis identified a novel heterozygous missense mutation in the kinase domain of Polo-like kinase 4 (Plk4), altering an isoleucine to asparagine at residue 242 (I242N). Genetic complementation studies using a gene trap line with disruption in the Plk4 locus confirmed that the putative Plk4 missense mutation was causative. Plk4 is known to be involved in centriole formation and cell cycle progression. However, a specific role in mammalian spermatogenesis has not been examined. PLK4 was highly expressed in the testes both pre- and postnatally. In the adult, PLK4 expression was first detected in stage VIII pachytene spermatocytes and was present through step 16 elongated spermatids. Because the homozygous Plk4I242N/I242N mutation was embryonic lethal, all analyses were performed using the heterozygous Plk4+/I242N mice. Testis size was reduced by 17%, and histology revealed discrete regions of germ cell loss, leaving only Sertoli cells in these defective tubules. Testis cord formation (embryonic day 13.5) was normal. Testis histology was also normal at postnatal day (P)1, but germ cell loss was detected at P10 and subsequent ages. We conclude that the I242N heterozygous mutation in PLK4 is causative for patchy germ cell loss beginning at P10, suggesting a role for PLK4 during the initiation of spermatogenesis. PMID:21791561

The ultrastructure of the spermatozoon of Paradynomene tuberculata Sakai, 1963 (Crustacea, Brachyura, Dynomenidae): Synapomorphies with dromiid sperm

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Jamieson, B. G. M.; Guinot, D.; de Forges, B. Richer

1993-10-01

The dynomenid spermatozoon, exemplified here by Paradynomene tuberculata, resembles the spermatozoa of the Dromiidae, Homolidae and lyreidine raninoids and differs markedly from those of other crabs (the heterotreme, thoracotremes, raninines and raninoidines) in the depressed, discoidal form of the acrosome and the capitate form of the perforatorium. Four or five apparent dynomenid—dromiid sperm synapomorphies are recognizable. (1) Dynomenids ( P. tuberculata) and dromiids differ from homolids and lyreidines in the greater depression of the acrosome (ratio of length to width=0.3); (2) the capitate head of the perforatorium is bilaterally prolonged in P. tuberculata as in dromiids though symmetrical in homolids; (3) dynomenid and dromiid sperm lack the—albeit variably developed—posterior median process of the nucleus seen in homolids, anomurans, raninoids and lower heterotremes; (4) P. tuberculata, like dromiids and less distinctly homolids, has an apical protuberance of subopercular material through the opercular perforation, unknown in other crabs, being distinct from the apical button of thoracotreme sperm; (5) a less certain synapomorphy is the anterolateral electron-pale peripheral zone of the acrosome. These synapomorphies endorse a sister-group relationship of dynomenids and dromiids, P. tuberculata sperm differs notably from the sperm of dromiids in the more complex zonation of the acrosome. The perforatorium lacks the radial rays (“spiked wheel”) of homolid sperm and does not show the “amoeboid” form seen in lyreidines. Absence of internal corrugations of the perforatorial chamber is a major difference from all examined raninids. Centrioles are only very tentatively identifiable. Nuclear arms are absent in glutaraldehyde fixed spermatozoa of P. tuberculata and have not been observed in the dromiid Petalomera lateralis but are present as three small radial vertices in the dromiid Dromidiopsis edwardsi and in homolids. P. tuberculata resembles

Genetically determined patozoospermia. Literature review and research results

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E. E. Bragina

2015-01-01

Full Text Available Genetic factors (chromosomal aberrations and point mutations are the cause of infertility in 10–15 % of men with impaired fertility. Homogeneous structural and functional defects in the sperm or the total terato-, asthenozoospermia – rare cases of genetically determined male infertility, are autosomal recessive diseases. Currently, described 4 types of «syndromic» spermopatology. 1. Primary ciliary dyskinesia (PCD in men with total asthenozoospermia. Affects axoneme structures (microtubules, dynein arms, radial spokes. It identified more than 20 chromosomal loci responsible for the development of the PCD. 2. Dysplasia of the fibrous sheath of sperm tail in men with asthenozoospermia. The shortened and thickened sperm tail observed with disorganization of vertical columns and cross ribs of the fibrous sheath. Candidate genes – genes family ACAP. 3. Globozoospermia in men with teratozoospermia characterized by the presence of sperm with round heads, primary lack of acrosome and disorganization middle part of the flagellum. Found mutations or deletions of genes SPATA16, PICK1 and DPY19L2. 4. Syndrome decapitated spermatozoa in men with teratozoospermia (microcephaly. Abnormalities in the spermiogenesis development of connecting part jf the tail and proximal (morphologically normal centrioles.In 2012–2014 years we have studied the ultrastructure of 2267 semen samples of men with impaired fertility. Globozoospermia revealed in 7 patients, dysplasia of the fibrous sheath – 13, decapitated sperm – in one. PCD was revealed in 4 patients (lack of axoneme dynein arms was found in 1 patient, absence of axoneme radial spokes – in 3 patients.The problem of genetically determined patozoospermya must be taken into account when the assisted reproductive technologies practises. There are few cases of successful assisted reproductive technologies with sperm of these patients. We don»t know the etiological factors of syndromic spermopatologe, so

Spermatozoal ultrastructure in four genera of Homolidae (crustacea, decapoda): Exemplified by Homologenus sp., Latreillopsis sp., Homolomannia sibogae and Paromolosis boasi

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Jamieson, B. G. M.; Guinot, D.; Forges, B. Richer De

1993-10-01

The spermatozoa of Homologenus sp., Latreillopsis sp., Homolomannia sibogae and Paromolopsis boasi confirm characteristics of a distinctive homolid spermatozoon previously established for Homola sp., Paromola sp. and Paromola petterdi. Homolid features are (1) moderate anteroposterior depression of the acrosome (ratio of length: width 0.4 0.6) as in lyreidine raninids (0.5), depression being greater in dromiids and dynomenids (both 0.3); (2) the capitate form of the perforatorium, shared with dromiids, dynomenids and lyreidine raninids; (3)the autapomorphic spiked-wheel form of the anterior expansion of the perforatorium; (4) horizontal zonation of the acrosome is possibly a unique synapomorphy of homolids with dromiids and dynomenids, and therefore an autapomorphy of the dromioid-homolid assemblage. In dromiids the posterior zone is proportionately the larger, while in homolids the anterior zone is the larger. The anterior zone is complexly subdivided in dynomenids; (5) the autapomorphic presence of numerous radial arranged extension of the acrosomal operculum into the perforatorium; (6) presence of nuclear arms, a symplesiomorphy of all investigated crabs, but small or questionably sometimes absent in Dromiidae; (7) absence of microtubules from the nuclear arms, as in dromiids, raninids, higher heterotremes and thoracotremes; (8) transient presence of a posterior median process of the nucleus. The process is not seen in dromiids but occurs in anomurans and lower heterotremes; (9) apical perforation of the operculum, also seen, apparently symplesiomorphically, in dromiids, raninids, and lower heterotreme families; (10) absence of an acrosome ray zone, probably homoplasic with absence in raninids; (11) location of most of the cytoplasm, including tortuous membranes and degenerating mitochondria, below the acrosome, also seen in Lyreidus; (12) presence, in at least some species, of centrioles, unknown in dromiids and raninids and variable in occurrence in

Cep70 and Cep131 contribute to ciliogenesis in zebrafish embryos

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Carl Matthias

2009-03-01

Full Text Available Abstract Background The centrosome is the cell's microtubule organising centre, an organelle with important roles in cell division, migration and polarity. However, cells can divide and flies can, for a large part of development, develop without them. Many centrosome proteins have been identified but the roles of most are still poorly understood. The centrioles of the centrosome are similar to the basal bodies of cilia, hair-like extensions of many cells that have important roles in cell signalling and development. In a number of human diseases, such Bardet-Biedl syndrome, centrosome/cilium proteins are mutated, leading to polycystic kidney disease, situs inversus, and neurological problems, amongst other symptoms. Results We describe zebrafish (Danio rerio embryos depleted for two uncharacterised, centrosome proteins, Cep70 and Cep131. The phenotype of these embryos resembles that of zebrafish mutants for intraflagellar transport proteins (IFTs, with kidney and ear development affected and left-right asymmetry randomised. These organs and processes are those affected in Bardet-Biedl syndrome and other similar diseases. Like these diseases, the root cause of the phenotype lies, in fact, in dysfunctional cilia, which are shortened but not eliminated in several tissues in the morphants. Centrosomes and basal bodies, on the other hand, are present. Both Cep70 and Cep131 possess a putative HDAC (histone deacetylase interacting domain. However, we could not detect in yeast two-hybrid assays any interaction with the deacetylase that controls cilium length, HDAC6, or any of the IFTs that we tested. Conclusion Cep70 and Cep131 contribute to ciliogenesis in many tissues in the zebrafish embryo: cilia are made in cep70 and cep131 morphant zebrafish embryos but are shortened. We propose that the role of these centrosomal/basal body proteins is in making the cilium and that they are involved in determination of the length of the axoneme.

Pharmacological inhibition of Polo Like Kinase 2 (PLK2) does not cause chromosomal damage or result in the formation of micronuclei

International Nuclear Information System (INIS)

Fitzgerald, Kent; Bergeron, Marcelle; Willits, Christopher; Bowers, Simeon; Aubele, Danielle L.; Goldbach, Erich; Tonn, George; Ness, Daniel; Olaharski, Andrew

2013-01-01

Polo Like Kinase 2 (PLK2) phosphorylates α-synuclein and is considered a putative therapeutic target for Parkinson's disease. Several lines of evidence indicate that PLK2 is involved with proper centriole duplication and cell cycle regulation, inhibition of which could impact chromosomal integrity during mitosis. The objectives of the series of experiments presented herein were to assess whether specific inhibition of PLK2 is genotoxic and determine if PLK2 could be considered a tractable pharmacological target for Parkinson's disease. Several selective PLK2 inhibitors, ELN 582175 and ELN 582646, and their inactive enantiomers, ELN 582176 and ELN 582647, did not significantly increase the number of micronuclei in the in vitro micronucleus assay. ELN 582646 was administered to male Sprague Dawley rats in an exploratory 14-day study where flow cytometric analysis of peripheral blood identified a dose-dependent increase in the number of micronucleated reticulocytes. A follow-up investigative study demonstrated that ELN 582646 administered to PLK2 deficient and wildtype mice significantly increased the number of peripheral micronucleated reticulocytes in both genotypes, suggesting that ELN 582646-induced genotoxicity is not through the inhibition of PLK2. Furthermore, significant reduction of retinal phosphorylated α-synuclein levels was observed at three non-genotoxic doses, additional data to suggest that pharmacological inhibition of PLK2 is not the cause of the observed genotoxicity. These data, in aggregate, indicate that PLK2 inhibition is a tractable CNS pharmacological target that does not cause genotoxicity at doses and exposures that engage the target in the sensory retina. - Highlights: • Active and inactive enantiomers test negative in the in vitro micronucleus test. • ELN 582646 significantly increased micronuclei at 100 and 300 mg/kg/day doses. • ELN 582646 significantly increased micronuclei in PLK2 knockout mice. • ELN 582646 decreased

A gene encoding the major beta tubulin of the mitotic spindle in Physarum polycephalum plasmodia

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Burland, T.G.; Paul, E.C.A.; Oetliker, M.; Dove, W.F.

1988-03-01

The multinucleate plasmodium of Physarum polycephalum is unusual among eucaryotic cells in that it uses tubulins only in mitotic-spindle microtubules; cytoskeletal, flagellar, and centriolar microtubules are absent in this cell type. The authors identified a ..beta..-tubulin cDNA clone, ..beta..105, which is shown to correspond to the transcript of the betC ..beta..-tubulin locus and to encode ..beta..2 tubulin, the ..beta.. tubulin expressed specifically in the plasmodium and used exclusively in the mitotic spindle. Physarum amoebae utilize tubulins in the cytoskeleton, centrioles, and flagella, in addition to the mitotic spindle. Sequence analysis shows that ..beta..2 tubulin is only 83% identical to the two ..beta.. tubulins expressed in amoebae. This compares with 70 to 83% identity between Physarum ..beta..2 tubulin and the ..beta.. tubulins of yeasts, fungi, alga, trypanosome, fruit fly, chicken, and mouse. On the other hand, Physarum ..beta..2 tubulin is no more similar to, for example, Aspergillus ..beta.. tubulins than it is to those of Drosophila melanogaster or mammals. Several eucaryotes express at least one widely diverged ..beta.. tubulin as well as one or more ..beta.. tubulins that conform more closely to a consensus ..beta..-tubulin sequence. The authors suggest that ..beta..-tubulins diverge more when their expression pattern is restricted, especially when this restriction results in their use in fewer functions. This divergence among ..beta.. tubulins could have resulted through neutral drift. For example, exclusive use of Physarum ..beta..2 tubulin in the spindle may have allowed more amino acid substitutions than would be functionally tolerable in the ..beta.. tubulins that are utilized in multiple microtubular organelles. Alternatively, restricted use of ..beta.. tubulins may allow positive selection to operate more freely to refine ..beta..-tubulin function.

iLoc-Animal: a multi-label learning classifier for predicting subcellular localization of animal proteins.

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Lin, Wei-Zhong; Fang, Jian-An; Xiao, Xuan; Chou, Kuo-Chen

2013-04-05

Predicting protein subcellular localization is a challenging problem, particularly when query proteins have multi-label features meaning that they may simultaneously exist at, or move between, two or more different subcellular location sites. Most of the existing methods can only be used to deal with the single-label proteins. Actually, multi-label proteins should not be ignored because they usually bear some special function worthy of in-depth studies. By introducing the "multi-label learning" approach, a new predictor, called iLoc-Animal, has been developed that can be used to deal with the systems containing both single- and multi-label animal (metazoan except human) proteins. Meanwhile, to measure the prediction quality of a multi-label system in a rigorous way, five indices were introduced; they are "Absolute-True", "Absolute-False" (or Hamming-Loss"), "Accuracy", "Precision", and "Recall". As a demonstration, the jackknife cross-validation was performed with iLoc-Animal on a benchmark dataset of animal proteins classified into the following 20 location sites: (1) acrosome, (2) cell membrane, (3) centriole, (4) centrosome, (5) cell cortex, (6) cytoplasm, (7) cytoskeleton, (8) endoplasmic reticulum, (9) endosome, (10) extracellular, (11) Golgi apparatus, (12) lysosome, (13) mitochondrion, (14) melanosome, (15) microsome, (16) nucleus, (17) peroxisome, (18) plasma membrane, (19) spindle, and (20) synapse, where many proteins belong to two or more locations. For such a complicated system, the outcomes achieved by iLoc-Animal for all the aforementioned five indices were quite encouraging, indicating that the predictor may become a useful tool in this area. It has not escaped our notice that the multi-label approach and the rigorous measurement metrics can also be used to investigate many other multi-label problems in molecular biology. As a user-friendly web-server, iLoc-Animal is freely accessible to the public at the web-site .

[Ultrastructural features of the epidermis in turbellaria from the Lake Baikal Geocentrophora wagini (Lecithoepitheliata, Plathelminthes)].

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Drobysheva, I M; Timoshkin, O A

2006-01-01

The epidermis of Geocentrophora wagini was studied using transmission electron microscopy. The turbellarian body was entirely covered by cilia, whose density was higher on the ventral surface compared with the dorsal one. In all regions examined, the epidermis was made up of a one-layered insunk epithelium. The basal matrix, underlying the epidermis, was a well developed basement membrane (BM) with bilayered structure, overlying the muscle network of circular and longitudinal fibers. The double plasma membranes, extending from the apical surface of epidermis to BM, were linked by specialized cell junctions. This suggested that epidermis had a cellular rather than a cyncytial arrangement. Each insunk epidermal cell was made of two unequal parts: a comparatively thin surface plate attached to BM by hemiadherens junctions, and a massive nucleated portion located below the body wall musculature in the parenchyma. A thin cytoplasmic bridge connected the epidermal plate with the nucleated cell body. The epidermal plates were joined by belt-like junctions along their adjacent surfaces. Inconspicuous zonula adherens (ZA) had a most apical position, and prominent septate junction was arrayed proximally to this zonula. Except ZA, cell boundaries in epidermis were frequently flanked by rows of light tubules and vesicles. In the basal half of the epithelial sheet, they were occassionally accompanied by single cisternae of rough endoplasmic reticulum (RER). The ultrastructure of the insunk cell body and that of the surface plate showed a considerable similarity. The common features were distinctive profiles of RER and GA, the presence of epitheliosomes, light tubules and vesicles, centrioles and fibrous granules. Thus, ultrastructural features allow a rather reliable identification of epidermal cells in the parenchyma, despite the absence of any visible morphological association between cell body and its epidermal plate.

Pharmacological inhibition of Polo Like Kinase 2 (PLK2) does not cause chromosomal damage or result in the formation of micronuclei

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Fitzgerald, Kent, E-mail: Kent.fitzgerald@elan.com [Pharmacological Sciences, Elan Pharmaceuticals Inc., 180 Oyster Point Boulevard, South San Francisco, CA 94080 (United States); Bergeron, Marcelle, E-mail: Marcelle.bergeron@elan.com [Pharmacological Sciences, Elan Pharmaceuticals Inc., 180 Oyster Point Boulevard, South San Francisco, CA 94080 (United States); Willits, Christopher, E-mail: Chris.willits@elan.com [Pharmacological Sciences, Elan Pharmaceuticals Inc., 180 Oyster Point Boulevard, South San Francisco, CA 94080 (United States); Bowers, Simeon, E-mail: Simeon.bowers@elan.com [Chemistry, Elan Pharmaceuticals Inc., 180 Oyster Point Boulevard, South San Francisco, CA 94080 (United States); Aubele, Danielle L., E-mail: Danielle.aubele@elan.com [Chemistry, Elan Pharmaceuticals Inc., 180 Oyster Point Boulevard, South San Francisco, CA 94080 (United States); Goldbach, Erich, E-mail: Erich.goldbach@elan.com [Drug Metabolism and Pharmacokinetics, Elan Pharmaceuticals Inc., 180 Oyster Point Boulevard, South San Francisco, CA 94080 (United States); Tonn, George, E-mail: George.tonn@elan.com [Drug Metabolism and Pharmacokinetics, Elan Pharmaceuticals Inc., 180 Oyster Point Boulevard, South San Francisco, CA 94080 (United States); Ness, Daniel, E-mail: Dan.ness@elan.com [Nonclinical Safety Evaluation, Elan Pharmaceuticals Inc., 180 Oyster Point Boulevard, South San Francisco, CA 94080 (United States); Olaharski, Andrew, E-mail: andrew.olaharski@agios.com [Nonclinical Safety Evaluation, Elan Pharmaceuticals Inc., 180 Oyster Point Boulevard, South San Francisco, CA 94080 (United States)

2013-05-15

Polo Like Kinase 2 (PLK2) phosphorylates α-synuclein and is considered a putative therapeutic target for Parkinson's disease. Several lines of evidence indicate that PLK2 is involved with proper centriole duplication and cell cycle regulation, inhibition of which could impact chromosomal integrity during mitosis. The objectives of the series of experiments presented herein were to assess whether specific inhibition of PLK2 is genotoxic and determine if PLK2 could be considered a tractable pharmacological target for Parkinson's disease. Several selective PLK2 inhibitors, ELN 582175 and ELN 582646, and their inactive enantiomers, ELN 582176 and ELN 582647, did not significantly increase the number of micronuclei in the in vitro micronucleus assay. ELN 582646 was administered to male Sprague Dawley rats in an exploratory 14-day study where flow cytometric analysis of peripheral blood identified a dose-dependent increase in the number of micronucleated reticulocytes. A follow-up investigative study demonstrated that ELN 582646 administered to PLK2 deficient and wildtype mice significantly increased the number of peripheral micronucleated reticulocytes in both genotypes, suggesting that ELN 582646-induced genotoxicity is not through the inhibition of PLK2. Furthermore, significant reduction of retinal phosphorylated α-synuclein levels was observed at three non-genotoxic doses, additional data to suggest that pharmacological inhibition of PLK2 is not the cause of the observed genotoxicity. These data, in aggregate, indicate that PLK2 inhibition is a tractable CNS pharmacological target that does not cause genotoxicity at doses and exposures that engage the target in the sensory retina. - Highlights: • Active and inactive enantiomers test negative in the in vitro micronucleus test. • ELN 582646 significantly increased micronuclei at 100 and 300 mg/kg/day doses. • ELN 582646 significantly increased micronuclei in PLK2 knockout mice. • ELN 582646

The centrosomin CM2 domain is a multi-functional binding domain with distinct cell cycle roles.

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Citron, Y Rose; Fagerstrom, Carey J; Keszthelyi, Bettina; Huang, Bo; Rusan, Nasser M; Kelly, Mark J S; Agard, David A

2018-01-01

The centrosome serves as the main microtubule-organizing center in metazoan cells, yet despite its functional importance, little is known mechanistically about the structure and organizational principles that dictate protein organization in the centrosome. In particular, the protein-protein interactions that allow for the massive structural transition between the tightly organized interphase centrosome and the highly expanded matrix-like arrangement of the mitotic centrosome have been largely uncharacterized. Among the proteins that undergo a major transition is the Drosophila melanogaster protein centrosomin that contains a conserved carboxyl terminus motif, CM2. Recent crystal structures have shown this motif to be dimeric and capable of forming an intramolecular interaction with a central region of centrosomin. Here we use a combination of in-cell microscopy and in vitro oligomer assessment to show that dimerization is not necessary for CM2 recruitment to the centrosome and that CM2 alone undergoes significant cell cycle dependent rearrangement. We use NMR binding assays to confirm this intramolecular interaction and show that residues involved in solution are consistent with the published crystal structure and identify L1137 as critical for binding. Additionally, we show for the first time an in vitro interaction of CM2 with the Drosophila pericentrin-like-protein that exploits the same set of residues as the intramolecular interaction. Furthermore, NMR experiments reveal a calcium sensitive interaction between CM2 and calmodulin. Although unexpected because of sequence divergence, this suggests that centrosomin-mediated assemblies, like the mammalian pericentrin, may be calcium regulated. From these results, we suggest an expanded model where during interphase CM2 interacts with pericentrin-like-protein to form a layer of centrosomin around the centriole wall and that at the onset of mitosis this population acts as a nucleation site of intramolecular

Ultrastructural investigation and in vitro recapitulation of spermatid differentiation in a potential bio-indicator species - The marine invertebrate Galeolaria gemineoa (Polychaeta: Serpulidae.

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Yonggang Lu

Full Text Available Galeolaria gemineoa is a sessile broadcast-spawning marine invertebrate, whose spermatozoa have been regarded as a sensitive indicator for water quality monitoring. In this study, 10 steps of spermiogenesis have been identified at the ultrastructural level and this differentiation process has been recapitulated in vitro up to the point of spermiogenesis (step 7-9 spermatids. On completion of the second meiosis, newly formed spermatids were detached from the seminiferous epithelium and released to the lumen of each germinal chamber. These spermatids were present in pairs and interconnected by a cytoplasmic bridge throughout the entire spermiogenic process. On the basis of morphological events such as formation of the acrosome, elongation of the flagellum, and condensation of the nucleus, spermiogenesis has been temporally divided into Golgi phase, acrosomal phase and maturation phase. During the Golgi phase, proacrosomal vesicles appeared at the posterior pole of the spermatids and gradually fused into a proacrosomal vacuole. Simultaneously, the distal centriole docked onto the plasma membrane and gave rise to a formative flagellum. The acrosomal phase was characterised by differentiation of the acrosome, condensation of the chromatin and formation of a mitochondrial sheath surrounding the initial portion of the flagellum. During the maturation phase, the fully differentiated acrosome migrated to the anterior pole and excess cytoplasm was extruded from the spermatids in the form of residual bodies. In addition, we successfully induced step 1-3 spermatids to differentiate into the step 7-9 spermatids in both male germinal fluid and 10% foetal bovine serum in RPMI 1640 medium, but failed to replicate this process in female or boiled male germinal fluids. This finding supports our concept that spermatid differentiation in this species is dependent on intrinsic developmental programming and does not require input from accompanying nurse cells.

Dynamics of the association of heat shock protein HSPA6 (Hsp70B') and HSPA1A (Hsp70-1) with stress-sensitive cytoplasmic and nuclear structures in differentiated human neuronal cells.

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Shorbagi, Sadek; Brown, Ian R

2016-11-01

Heat shock proteins (Hsps) are cellular repair agents that counter the effects of protein misfolding that is a characteristic feature of neurodegenerative diseases. HSPA1A (Hsp70-1) is a widely studied member of the HSPA (Hsp70) family. The little-studied HSPA6 (Hsp70B') is present in the human genome and absent in mouse and rat; hence, it is missing in current animal models of neurodegenerative diseases. Differentiated human neuronal SH-SY5Y cells were employed to compare the dynamics of the association of YFP-tagged HSPA6 and HSPA1A with stress-sensitive cytoplasmic and nuclear structures. Following thermal stress, live-imaging confocal microscopy and Fluorescence Recovery After Photobleaching (FRAP) demonstrated that HSPA6 displayed a prolonged and more dynamic association, compared to HSPA1A, with centrioles that play critical roles in neuronal polarity and migration. HSPA6 and HSPA1A also targeted nuclear speckles, rich in RNA splicing factors, and the granular component of the nucleolus that is involved in rRNA processing and ribosomal subunit assembly. HSPA6 and HSPA1A displayed similar FRAP kinetics in their interaction with nuclear speckles and the nucleolus. Subsequently, during the recovery from neuronal stress, HSPA6, but not HSPA1A, localized with the periphery of nuclear speckles (perispeckles) that have been characterized as transcription sites. The stress-induced association of HSPA6 with perispeckles displayed the greatest dynamism compared to the interaction of HSPA6 or HSPA1A with other stress-sensitive cytoplasmic and nuclear structures. This suggests involvement of HSPA6 in transcriptional recovery of human neurons from cellular stress that is not apparent for HSPA1A.

Comparison of spermiogenesis in the externally fertilizing Hemigrammus erythrozonus and the inseminating Corynopoma riisei (Teleostei: Characiformes: Characidae

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Anna Pecio

Full Text Available Spermiogenesis and sperm ultrastructure were analyzed in two species of characids with different modes of fertilization: externally fertilizing Hemigrammus erythrozonus and inseminating Corynopoma riisei. Spermiogenesis in H. erythrozonus is characterized by lateral development of the flagellum, nuclear rotation, formation of a shallow nuclear fossa, condensation of the chromatin by elimination of the electron-lucent area from the peripheral region of the nucleus, and renewal of the nuclear membrane. Multilammelated membrane and multivesicular bodies were also observed during elimination of the excess cytoplasm. The spermatozoon exhibits characters typical of "aquasperm," i.e. a spherical head containing a spherical nucleus with highly condensed chromatin, several small mitochondria located at the base of the nucleus within a cytoplasmic collar that extends into a long cytoplasmic sleeve surrounding the anterior part of the single flagellum, which is contained within a cytoplasmic canal. The flagellum lacks fins. The proximal and distal centrioles are nearly parallel to one another, with the anterior tips of both located within shallow nuclear fossae. Spermiogenesis in C. riisei is characterized by nuclear elongation alongside the forming flagellum, formation of an elongate cytoplasmic canal, displacement and elongation of the mitochondria, and uniform condensation of chromatin throughout the nucleus through enlargement of the diameter of the chromatin granules. The spermatozoon has an elongate nucleus with two elongate mitochondria localized to one side. Mitochondria are also located posterior to the nucleus forming a mitochondrial region. The single flagellum, which lacks fins, is lateral to the nucleus and initially contained within the greatly elongate cytoplasmic canal before exiting the canal at its posterior terminus. The spermatozoon of C. riisei exhibits several characters typical of "introsperm," such as an elongate nucleus and

Knockdown of SCF(Skp2 function causes double-parked accumulation in the nucleus and DNA re-replication in Drosophila plasmatocytes.

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Paul T Kroeger

Full Text Available In Drosophila, circulating hemocytes are derived from the cephalic mesoderm during the embryonic wave of hematopoiesis. These cells are contributed to the larva and persist through metamorphosis into the adult. To analyze this population of hemocytes, we considered data from a previously published RNAi screen in the hematopoietic niche, which suggested several members of the SCF complex play a role in lymph gland development. eater-Gal4;UAS-GFP flies were crossed to UAS-RNAi lines to knockdown the function of all known SCF complex members in a plasmatocyte-specific fashion, in order to identify which members are novel regulators of plasmatocytes. This specific SCF complex contains five core members: Lin-19-like, SkpA, Skp2, Roc1a and complex activator Nedd8. The complex was identified by its very distinctive large cell phenotype. Furthermore, these large cells stained for anti-P1, a plasmatocyte-specific antibody. It was also noted that the DNA in these cells appeared to be over-replicated. Gamma-tubulin and DAPI staining suggest the cells are undergoing re-replication as they had multiple centrioles and excessive DNA content. Further experimentation determined enlarged cells were BrdU-positive indicating they have progressed through S-phase. To determine how these cells become enlarged and undergo re-replication, cell cycle proteins were analyzed by immunofluorescence. This analysis identified three proteins that had altered subcellular localization in these enlarged cells: Cyclin E, Geminin and Double-parked. Previous research has shown that Double-parked must be degraded to exit S-phase, otherwise the DNA will undergo re-replication. When Double-parked was titrated from the nucleus by an excess of its inhibitor, geminin, the enlarged cells and aberrant protein localization phenotypes were partially rescued. The data in this report suggests that the SCF(Skp2 complex is necessary to ubiquitinate Double-parked during plasmatocyte cell division

Function of donor cell centrosome in intraspecies and interspecies nuclear transfer embryos

International Nuclear Information System (INIS)

Zhong Zhisheng; Zhang Gang; Meng Xiaoqian; Zhang Yanling; Chen Dayuan; Schatten, Heide; Sun Qingyuan

2005-01-01

pericentriolar material surrounding a pair of centrioles, is degraded in the 1-cell reconstituted embryos after activation; (2) components of donor cell centrosomes contribute to the formation of the transient spindle and normal functional mitotic spindle, although the contribution of centrosomal material stored in the recipient ooplasm is not excluded; and (3) components of donor cell centrosomes involved in spindle assembly may not be species-specific

Regulation of mitotic spindle formation by the RhoA guanine nucleotide exchange factor ARHGEF10

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Satoh Takaya

2009-07-01

Full Text Available Abstract Background The Dbl family guanine nucleotide exchange factor ARHGEF10 was originally identified as the product of the gene associated with slowed nerve-conduction velocities of peripheral nerves. However, the function of ARHGEF10 in mammalian cells is totally unknown at a molecular level. ARHGEF10 contains no distinctive functional domains except for tandem Dbl homology-pleckstrin homology and putative transmembrane domains. Results Here we show that RhoA is a substrate for ARHGEF10. In both G1/S and M phases, ARHGEF10 was localized in the centrosome in adenocarcinoma HeLa cells. Furthermore, RNA interference-based knockdown of ARHGEF10 resulted in multipolar spindle formation in M phase. Each spindle pole seems to contain a centrosome consisting of two centrioles and the pericentriolar material. Downregulation of RhoA elicited similar phenotypes, and aberrant mitotic spindle formation following ARHGEF10 knockdown was rescued by ectopic expression of constitutively activated RhoA. Multinucleated cells were not increased upon ARHGEF10 knockdown in contrast to treatment with Y-27632, a specific pharmacological inhibitor for the RhoA effector kinase ROCK, which induced not only multipolar spindle formation, but also multinucleation. Therefore, unregulated centrosome duplication rather than aberration in cytokinesis may be responsible for ARHGEF10 knockdown-dependent multipolar spindle formation. We further isolated the kinesin-like motor protein KIF3B as a binding partner of ARHGEF10. Knockdown of KIF3B again caused multipolar spindle phenotypes. The supernumerary centrosome phenotype was also observed in S phase-arrested osteosarcoma U2OS cells when the expression of ARHGEF10, RhoA or KIF3B was abrogated by RNA interference. Conclusion Collectively, our results suggest that a novel RhoA-dependent signaling pathway under the control of ARHGEF10 has a pivotal role in the regulation of the cell division cycle. This pathway is not involved in

Cortical cytasters: a highly conserved developmental trait of Bilateria with similarities to Ctenophora

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Salinas-Saavedra Miguel

2011-12-01

Full Text Available Abstract Background Cytasters (cytoplasmic asters are centriole-based nucleation centers of microtubule polymerization that are observable in large numbers in the cortical cytoplasm of the egg and zygote of bilaterian organisms. In both protostome and deuterostome taxa, cytasters have been described to develop during oogenesis from vesicles of nuclear membrane that move to the cortical cytoplasm. They become associated with several cytoplasmic components, and participate in the reorganization of cortical cytoplasm after fertilization, patterning the antero-posterior and dorso-ventral body axes. Presentation of the hypothesis The specific resemblances in the development of cytasters in both protostome and deuterostome taxa suggest that an independent evolutionary origin is unlikely. An assessment of published data confirms that cytasters are present in several protostome and deuterostome phyla, but are absent in the non-bilaterian phyla Cnidaria and Ctenophora. We hypothesize that cytasters evolved in the lineage leading to Bilateria and were already present in the most recent common ancestor shared by protostomes and deuterostomes. Thus, cytasters would be an ancient and highly conserved trait that is homologous across the different bilaterian phyla. The alternative possibility is homoplasy, that is cytasters have evolved independently in different lineages of Bilateria. Testing the hypothesis So far, available published information shows that appropriate observations have been made in eight different bilaterian phyla. All of them present cytasters. This is consistent with the hypothesis of homology and conservation. However, there are several important groups for which there are no currently available data. The hypothesis of homology predicts that cytasters should be present in these groups. Increasing the taxonomic sample using modern techniques uniformly will test for evolutionary patterns supporting homology, homoplasy, or secondary loss of

Influence of maternal and own genotype at tanning dependence-related SNPs on sun exposure in childhood.

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Khouja, Jasmine; Lewis, Sarah J; Bonilla, Carolina

2018-04-12

Research suggests there may be a genetic influence on the likelihood of becoming tanning dependent (TD). The way in which mothers regulate their children's sun exposure may be affected by being TD. We investigated the associations between single nucleotide polymorphisms (SNPs) related to being TD and early sun exposure. Data from the Avon Longitudinal Study of Parents and Children (ALSPAC) were used. Associations between 17 TD related SNPs in children and their mothers and 10 sun exposure variables in children (assessed via questionnaire at age 8) were analyzed in logistic and ordinal logistic regressions. Analyses were adjusted for principal components of population structure and age (at time of questionnaire response). Models with additional adjustment for maternal or offspring genotypes were also tested. Secondary analyses included adjustment for sex and skin pigmentation. Among ALSPAC children, the rs29132 SNP in the Vesicle-associated membrane protein-associated protein A (VAPA) gene was associated with five sun exposure variables whilst the rs650662 SNP in the Opioid Receptor Mu 1 (OPRM1) gene was associated with three. The remaining SNPs did not show associations beyond what was expected by chance. After Bonferroni correction one SNP in the children was associated with an increased likelihood of using sun cream whilst in the sun at 8 years old (rs60050811 in the Spermatogenesis and Centriole Associated 1 (SPATC1) gene, OR per C allele = 1.34, 95% CI 1.11-1.62, p = .003). In the mothers, rs650662 in OPRM1 was associated with the use of a lower factor of sun cream in their children, (OR per A allele = 0.89, 95% CI 0.82-0.96, p = .002). Whilst rs2073478 in the Aldehyde Dehydrogenase 1 Family Member B1 (ALDH1B1) gene was associated with a reduced odds of their child using a sun block or cream with a 4 star rating (OR per T allele = 0.68, 95% CI 0.53-0.88, p = .003). Similar but weaker associations were observed for the main findings in

Tubulinlike protein from Spirochaeta bajacaliforniensis

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Bermudes, D.; Fracek, S. P. Jr; Laursen, R. A.; Margulis, L.; Obar, R.; Tzertzinis, G.

1987-01-01

Tubulin proteins are the fundamental subunits of all polymeric microtubule-based eukaryotic structures. Long, hollow structures each composed of 13 protofilaments as revealed by electron microscopy, microtubules (240 angstroms in diameter) are nearly ubiquitous in eukaryotes. These proteins have been the subject of intense biochemical and biophyiscal interest since the early 1970s and are of evolutionary interest as well. If tubulin-based structures (i.e., neurotubules, mitotic spindle tubules, centrioles, kinetosomes, axonemes, etc.) evolved from spirochetes by way of motility symbioses, tubulin homologies with spirochete proteins should be detectable. Tubulin proteins are widely thought to be limited to eukaryotes. Yet both azotobacters and spirochetes have shown immunological cross-reactivity with antitubulin antibodies. In neither of these studies was tubulin isolated nor any specific antigen identified as responsible for the immunoreactivity. Furthermore, although far less uniform in structure than eukaryotic microtubules, various cytoplasmic fibers and tubules (as seen by electron microscopy) have been reported in several types of prokaryotes (e.g., Spirochaeta; large termite spirochetes; treponemes; cyanobacteria; and Azotobacter. This work forms a part of our long-range study of the possible prokaryotic origin of tubulin and microtubules. Spirochetes are helically shaped gram-negative motile prokaryotes. They differ from all other bacterial in that the position of their flagella is periplasmic: their flagella lie between the inner and outer membranes of the gram-negative cell wall. Some of the largest spirochetes have longitudinally aligned 240 angstrom microtubules. Unfortunately, in spite of many attempts, all of the larger spirochetes (family Pillotaceae) with well-defined cytoplasmic tubules and antitubulin immunoreactivity are not cultivable. However, a newly described spirochete species (Spirochaeta bajacaliforniensis) possessing cytoplasmic fibers

Sobre a morfologia e ciclo evolutivo dos flagelados do genero Metasaccinobaculus n. gen. (Polymastigina, Oxymonadidae do termita Kalotermes (Neotermes Wagneri, Desneux, 1904, com a descrição de duas espécies novas

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Gilberto de Freitas

1945-10-01

fibrils. The axostyle is a ribbon-like structure, vigorously undulating when living, intensily stained by iron haematein and eosin, and fixed posteriorly to the body wall through a special tube-like stru¬cture. The system of chromophobic fibrils is contituted by rostellar fibrils which connect the undulating portion of the axostyle to the tip of the rostellum. The other system referred to as independent chromoprobic fibrils takes origin at the distal end of the rostellum perhaps in granules there found, runs back¬ward through this organelle and reaches the body, where it suffers sub-di¬visions into thinner bundles. The nucleus presents the chromatin arranged in large, thick and irregular structures: there is no karyosome. The young form is much smaller and its undulating axostyle is relatively very large in relation to the size of the body. It is attached to the body wall at posterior end. During growth, this point moves foreward. Near the point where the tube-like structure is fastened to the surface of the body, a sheath can be observed, constituted by a great number of very fine hyaline strands. The endoplasm is deeply chromophile, but free from brown spheres, which makes their appearence after the fixation of the organism. The nucleus, at the early stages, exhibit very thin granules of chromatin with a uniform dispersion. One or more structures surrounded by a clear halo and simulating a karyosome can be seen at this stage; nevertheless they soon disappear. 4 — Before mitosis which is quite similar to that of Oxymonas grandis the nucleus migrates to the posterior end of the body, due to the degenera¬tion of the neuromotor organellae. The nuclear membrane persists during the whole process. A central spindle develops within the nucleus as a straight rod, reaches the nuclear membrane assuming a convex barrel shape, and suffering a torsion in opposite sense on each pole. At sites where centrioles must lie, a clear circular space can be seen, the spindle fibrils