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

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lee, Bo Yon, E-mail: boyonlee@gmail.com; Shim, Sang Woo; Kim, Young Sun

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 andmore » 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.« less

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

PubMed

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).

Asterless Licenses Daughter Centrioles to Duplicate for the First Time in Drosophila Embryos

PubMed Central

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

2014-01-01

Summary 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. PMID:24835456

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

PubMed

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.

Atypical centrioles are present in Tribolium sperm.

PubMed

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.

Ultrastructural diversity between centrioles of eukaryotes.

PubMed

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.

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

PubMed

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.

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

PubMed Central

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

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

PubMed

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.

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

PubMed

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.

Centrobin–tubulin interaction is required for centriole elongation and stability

PubMed Central

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

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

PubMed

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.

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

PubMed

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.

Asterless is required for centriole length control and sperm development

PubMed Central

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

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

PubMed

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.

Centriole triplet microtubules are required for stable centriole formation and inheritance in human cells

PubMed Central

Wang, Jennifer T; Kong, Dong; Hoerner, Christian R; Loncarek, Jadranka

2017-01-01

Centrioles are composed of long-lived microtubules arranged in nine triplets. However, the contribution of triplet microtubules to mammalian centriole formation and stability is unknown. Little is known of the mechanism of triplet microtubule formation, but experiments in unicellular eukaryotes indicate that delta-tubulin and epsilon-tubulin, two less-studied tubulin family members, are required. Here, we report that centrioles in delta-tubulin and epsilon-tubulin null mutant human cells lack triplet microtubules and fail to undergo centriole maturation. These aberrant centrioles are formed de novo each cell cycle, but are unstable and do not persist to the next cell cycle, leading to a futile cycle of centriole formation and disintegration. Disintegration can be suppressed by paclitaxel treatment. Delta-tubulin and epsilon-tubulin physically interact, indicating that these tubulins act together to maintain triplet microtubules and that these are necessary for inheritance of centrioles from one cell cycle to the next. PMID:28906251

What is the function of centrioles?

PubMed

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.

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

PubMed Central

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

2015-01-01

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. DOI: http://dx.doi.org/10.7554/eLife.10586.001 PMID:26609813

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

PubMed Central

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

2016-01-01

ABSTRACT 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. PMID:27764551

Centriole triplet microtubules are required for stable centriole formation and inheritance in human cells.

PubMed

Wang, Jennifer T; Kong, Dong; Hoerner, Christian R; Loncarek, Jadranka; Stearns, Tim

2017-09-14

Centrioles are composed of long-lived microtubules arranged in nine triplets. However, the contribution of triplet microtubules to mammalian centriole formation and stability is unknown. Little is known of the mechanism of triplet microtubule formation, but experiments in unicellular eukaryotes indicate that delta-tubulin and epsilon-tubulin, two less-studied tubulin family members, are required. Here, we report that centrioles in delta-tubulin and epsilon-tubulin null mutant human cells lack triplet microtubules and fail to undergo centriole maturation. These aberrant centrioles are formed de novo each cell cycle, but are unstable and do not persist to the next cell cycle, leading to a futile cycle of centriole formation and disintegration. Disintegration can be suppressed by paclitaxel treatment. Delta-tubulin and epsilon-tubulin physically interact, indicating that these tubulins act together to maintain triplet microtubules and that these are necessary for inheritance of centrioles from one cell cycle to the next.

Centriole amplification by mother and daughter centrioles differs in multiciliated cells.

PubMed

Al Jord, Adel; Lemaître, Anne-Iris; Delgehyr, Nathalie; Faucourt, Marion; Spassky, Nathalie; Meunier, Alice

2014-12-04

The semi-conservative centrosome duplication in cycling cells gives rise to a centrosome composed of a mother and a newly formed daughter centriole. Both centrioles are regarded as equivalent in their ability to form new centrioles and their symmetric duplication is crucial for cell division homeostasis. Multiciliated cells do not use the archetypal duplication program and instead form more than a hundred centrioles that are required for the growth of motile cilia and the efficient propelling of physiological fluids. The majority of these new centrioles are thought to appear de novo, that is, independently from the centrosome, around electron-dense structures called deuterosomes. Their origin remains unknown. Using live imaging combined with correlative super-resolution light and electron microscopy, we show that all new centrioles derive from the pre-existing progenitor cell centrosome through multiple rounds of procentriole seeding. Moreover, we establish that only the daughter centrosomal centriole contributes to deuterosome formation, and thus to over ninety per cent of the final centriole population. This unexpected centriolar asymmetry grants new perspectives when studying cilia-related diseases and pathological centriole amplification observed in cycling cells and associated with microcephaly and cancer.

Drosophila Ana1 is required for centrosome assembly and centriole elongation

PubMed Central

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

2016-01-01

ABSTRACT 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. PMID:27206860

Centriole Remodeling during Spermiogenesis in Drosophila.

PubMed

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.

Conserved molecular interactions in centriole-to-centrosome conversion.

PubMed

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.

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.

PubMed

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.

Cep152 interacts with Plk4 and is required for centriole duplication

PubMed Central

Hatch, Emily M.; Kulukian, Anita; Holland, Andrew J.; Cleveland, Don W.

2010-01-01

Centrioles are microtubule-based structures that organize the centrosome and nucleate cilia. Centrioles duplicate once per cell cycle, and duplication requires Plk4, a member of the Polo-like kinase family; however, the mechanism linking Plk4 activity and centriole formation is unknown. In this study, we show in human and frog cells that Plk4 interacts with the centrosome protein Cep152, the orthologue of Drosophila melanogaster Asterless. The interaction requires the N-terminal 217 residues of Cep152 and the crypto Polo-box of Plk4. Cep152 and Plk4 colocalize at the centriole throughout the cell cycle. Overexpression of Cep152 (1–217) mislocalizes Plk4, but both Cep152 and Plk4 are able to localize to the centriole independently of the other. Depletion of Cep152 prevents both normal centriole duplication and Plk4-induced centriole amplification and results in a failure to localize Sas6 to the centriole, an early step in duplication. Cep152 can be phosphorylated by Plk4 in vitro, suggesting that Cep152 acts with Plk4 to initiate centriole formation. PMID:21059850

Drosophila Ana1 is required for centrosome assembly and centriole elongation.

PubMed

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.

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

PubMed Central

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

2014-01-01

SUMMARY 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 re-duplication. These results lead to a proposed model that centrioles may duplicate via a template-based process to preserve their geometry and copy number. PMID:25017693

Isolation and proteomic analysis of Chlamydomonas centrioles.

PubMed

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.

p53 protects against genome instability following centriole duplication failure

PubMed Central

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

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

PubMed Central

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

2016-01-01

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. PMID:27002173

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

PubMed

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.

Tripeptidyl Peptidase II Is Required for c-MYC-Induced Centriole Overduplication and a Novel Therapeutic Target in c-MYC-Associated Neoplasms.

PubMed

Duensing, Stefan; Darr, Sebastian; Cuevas, Rolando; Melquiot, Nadja; Brickner, Anthony G; Duensing, Anette; Münger, Karl

2010-09-01

Centrosome aberrations are frequently detected in c-MYC-associated human malignancies. Here, we show that c-MYC-induced centrosome and centriole overduplication critically depend on the protease tripeptidyl peptidase II (TPPII). We found that TPPII localizes to centrosomes and that overexpression of TPPII, similar to c-MYC, can disrupt centriole duplication control and cause centriole multiplication, a process during which maternal centrioles nucleate the formation of more than a single daughter centriole. We report that inactivation of TPPII using chemical inhibitors or siRNA-mediated protein knockdown effectively reduced c-MYC-induced centriole overduplication. Remarkably, the potent and selective TPPII inhibitor butabindide not only potently suppressed centriole aberrations but also caused significant cell death and growth suppression in aggressive human Burkitt lymphoma cells with c-MYC overexpression. Taken together, these results highlight the role of TPPII in c-MYC-induced centriole overduplication and encourage further studies to explore TPPII as a novel antineoplastic drug target.

Tripeptidyl Peptidase II Is Required for c-MYC–Induced Centriole Overduplication and a Novel Therapeutic Target in c-MYC–Associated Neoplasms

PubMed Central

Duensing, Stefan; Darr, Sebastian; Cuevas, Rolando; Melquiot, Nadja; Brickner, Anthony G.; Duensing, Anette; Münger, Karl

2010-01-01

Centrosome aberrations are frequently detected in c-MYC–associated human malignancies. Here, we show that c-MYC–induced centrosome and centriole overduplication critically depend on the protease tripeptidyl peptidase II (TPPII). We found that TPPII localizes to centrosomes and that overexpression of TPPII, similar to c-MYC, can disrupt centriole duplication control and cause centriole multiplication, a process during which maternal centrioles nucleate the formation of more than a single daughter centriole. We report that inactivation of TPPII using chemical inhibitors or siRNA-mediated protein knockdown effectively reduced c-MYC–induced centriole overduplication. Remarkably, the potent and selective TPPII inhibitor butabindide not only potently suppressed centriole aberrations but also caused significant cell death and growth suppression in aggressive human Burkitt lymphoma cells with c-MYC overexpression. Taken together, these results highlight the role of TPPII in c-MYC–induced centriole overduplication and encourage further studies to explore TPPII as a novel antineoplastic drug target. PMID:21647238

Mother centrioles do a cartwheel to produce just one daughter.

PubMed

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.

Electron tomography study of isolated human centrioles.

PubMed

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 maturation requires regulated Plk1 activity during two consecutive cell cycles.

PubMed

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.

Centriole maturation requires regulated Plk1 activity during two consecutive cell cycles

PubMed Central

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

2014-01-01

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. PMID:25246616

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

PubMed

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.

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

PubMed

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.

The centriole duplication cycle

PubMed Central

Fırat-Karalar, Elif Nur; Stearns, Tim

2014-01-01

Centrosomes are the main microtubule-organizing centre of animal cells and are important for many critical cellular and developmental processes from cell polarization to cell division. At the core of the centrosome are centrioles, which recruit pericentriolar material to form the centrosome and act as basal bodies to nucleate formation of cilia and flagella. Defects in centriole structure, function and number are associated with a variety of human diseases, including cancer, brain diseases and ciliopathies. In this review, we discuss recent advances in our understanding of how new centrioles are assembled and how centriole number is controlled. We propose a general model for centriole duplication control in which cooperative binding of duplication factors defines a centriole ‘origin of duplication’ that initiates duplication, and passage through mitosis effects changes that license the centriole for a new round of duplication in the next cell cycle. We also focus on variations on the general theme in which many centrioles are created in a single cell cycle, including the specialized structures associated with these variations, the deuterosome in animal cells and the blepharoplast in lower plant cells. PMID:25047614

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

PubMed Central

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

Stable centrosomal roots disentangle to allow interphase centriole independence

PubMed Central

2018-01-01

The centrosome is a non–membrane-bound cellular compartment consisting of 2 centrioles surrounded by a protein coat termed the pericentriolar material (PCM). Centrioles generally remain physically associated together (a phenomenon called centrosome cohesion), yet how this occurs in the absence of a bounding lipid membrane is unclear. One model posits that pericentriolar fibres formed from rootletin protein directly link centrioles, yet little is known about the structure, biophysical properties, or assembly kinetics of such fibres. Here, I combine live-cell imaging of endogenously tagged rootletin with cell fusion and find previously unrecognised plasticity in centrosome cohesion. Rootletin forms large, diffusionally stable bifurcating fibres, which amass slowly on mature centrioles over many hours from anaphase. Nascent centrioles (procentrioles), in contrast, do not form roots and must be licensed to do so through polo-like kinase 1 (PLK1) activity. Transient separation of roots accompanies centriolar repositioning during the interphase, suggesting that centrioles organize as independent units, each containing discrete roots. Indeed, forced induction of duplicate centriole pairs allows independent reshuffling of individual centrioles between the pairs. Therefore collectively, these findings suggest that progressively nucleated polymers mediate the dynamic association of centrioles as either 1 or 2 interphase centrosomes, with implications for the understanding of how non–membrane-bound organelles self-organise. PMID:29649211

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

PubMed

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.

A homeostatic clock sets daughter centriole size in flies

PubMed Central

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

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

PubMed

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.

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

PubMed

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.

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

PubMed

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.

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

PubMed

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.

Two Polo-like kinase 4 binding domains in Asterless perform distinct roles in regulating kinase stability

PubMed Central

Klebba, Joseph E.; Galletta, Brian J.; Nye, Jonathan; Plevock, Karen M.; Buster, Daniel W.; Hollingsworth, Natalie A.; Slep, Kevin C.

2015-01-01

Plk4 (Polo-like kinase 4) and its binding partner Asterless (Asl) are essential, conserved centriole assembly factors that induce centriole amplification when overexpressed. Previous studies found that Asl acts as a scaffolding protein; its N terminus binds Plk4’s tandem Polo box cassette (PB1-PB2) and targets Plk4 to centrioles to initiate centriole duplication. However, how Asl overexpression drives centriole amplification is unknown. In this paper, we investigated the Asl–Plk4 interaction in Drosophila melanogaster cells. Surprisingly, the N-terminal region of Asl is not required for centriole duplication, but a previously unidentified Plk4-binding domain in the C terminus is required. Mechanistic analyses of the different Asl regions revealed that they act uniquely during the cell cycle: the Asl N terminus promotes Plk4 homodimerization and autophosphorylation during interphase, whereas the Asl C terminus stabilizes Plk4 during mitosis. Therefore, Asl affects Plk4 in multiple ways to regulate centriole duplication. Asl not only targets Plk4 to centrioles but also modulates Plk4 stability and activity, explaining the ability of overexpressed Asl to drive centriole amplification. PMID:25688134

Mps1 phosphorylation sites regulate the function of centrin 2 in centriole assembly.

PubMed

Yang, Ching-Hui; Kasbek, Christopher; Majumder, Shubhra; Yusof, Adlina Mohd; Fisk, Harold A

2010-12-01

The nondegradable Mps1(Δ12/13) protein drives centriole overproduction, suggesting that Mps1 phosphorylates a subset of centrosomal proteins to drive the assembly of new centrioles. Here we identify three Mps1 phosphorylation sites within the centriolar protein Centrin 2 (Cetn2). Although centrioles can be assembled in the absence of Cetn2, centriole assembly is attenuated in the absence of Cetn2. While wild-type Cetn2 can compensate for this attenuation, a nonphosphorylatable version cannot. In addition, overexpressing Cetn2 causes Mps1-dependent centriole overproduction that requires each of the three Mps1 phosphorylation sites within Cetn2 and is greatly exacerbated by mimicking phosphorylation at any of these sites. Wild-type Cetn2 generates excess foci that are competent as mitotic spindle poles in HsSas-6-depleted cells, suggesting that Cetn2 can organize a subset of centriolar proteins independently of cartwheels. However, centriole overproduction caused by a phosphomimetic Cetn2 mutant requires HsSas-6, suggesting that Cetn2 phosphorylation stimulates the canonical centriole assembly pathway. Moreover, in the absence of Cetn2, Mps1(Δ12/13) cannot drive the production of mature centrioles capable of recruiting γ-Tubulin, and a nonphosphorylatable Cetn2 mutant cannot compensate for this defect and exacerbates Cetn2 depletion. Together, our data suggest that Mps1-dependent phosphorylation of Cetn2 stimulates the canonical centriole assembly pathway.

Double life of centrioles: CP110 in the spotlight.

PubMed

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.

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

PubMed Central

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

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

PubMed

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.

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

PubMed

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.

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

PubMed

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.

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

PubMed

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.

Physics models of centriole replication.

PubMed

Cheng, Kang; Zou, Changhua

2006-01-01

Our previous pre-clinic experimental results have showed that the epithelialization can be enhanced by the externally applied rectangular pulsed electrical current stimulation (RPECS). The results are clinically significant for patients, especially for those difficult patients whose skin wounds need long periods to heal. However, the results also raise questions: How does the RPECS accelerate the epithelium cell proliferation? To answer these questions, we have previously developed several models for animal cells, in a view of physics, to explain mechanisms of mitosis and cytokinesis at a cellular level, and separation of nucleotide sequences and the unwinding of a double helix during DNA replication at a bio-molecular level. In this paper, we further model the mechanism of centriole replication during a natural and normal mitosis and cytokinesis to explore the mechanism of epithelialization enhanced with the externally applied RPECS at a bio-molecular level. Our models suggest: (1) Centriole replication is an information flowing. The direction of the information flowing is from centrioles to centrioles based on a cylindrical template of 9 x 3 protein microtubules (MTs) pattern. (2) A spontaneous and strong electromagnetic field (EMF) force is a pushing force that separates a mother and a daughter centrioles in centrosomes or in cells, while a pulling force of interacting fibers and pericentriolar materials delivers new babies. The newly born babies inherit the pattern information from their mother(s) and grow using microtubule fragments that come through the centrosome pores. A daughter centriole is always born and grows along stronger EMF. The EMF mostly determines centrioles positions and plays key role in centriole replication. We also hypothesize that the normal centriole replication could not been disturbed in centrosome in the epithelium cells by our RPECS, because the centrioles have two non-conducting envelope (cell and centrosome membranes), that protect the normal duplication. The induced electric field by externally applied RPECS could be mild compared with the spontaneous and natural electric field of the centrioles. Therefore, the centriole replication during the epithelium cellular proliferation may be directly, as well as indirectly (e.g., somatic reflex) accelerated by the RPECS.

Centriole distribution during tripolar mitosis in Chinese hamster ovary cells

PubMed Central

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

Centrioles: duplicating precariously.

PubMed

Pelletier, Laurence

2007-09-04

To assemble a mitotic spindle and accurately segregate chromosomes to progeny, a cell needs to precisely regulate its centrosome number, a feat largely accomplished through the tight control of centriole duplication. Recent work showing that the overexpression of centriolar proteins can lead to the formation of multiple centrioles in the absence of pre-existing centrioles challenges the idea that it is a self-replicating organelle.

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

PubMed

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.

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

PubMed Central

Roessler, Max

2017-01-01

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. PMID:28411189

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

PubMed Central

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

2016-01-01

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. PMID:27021213

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

PubMed

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.

VDAC3 regulates centriole assembly by targeting Mps1 to centrosomes.

PubMed

Majumder, Shubhra; Slabodnick, Mark; Pike, Amanda; Marquardt, Joseph; Fisk, Harold A

2012-10-01

Centrioles are duplicated during S-phase to generate the two centrosomes that serve as mitotic spindle poles during mitosis. The centrosomal pool of the Mps1 kinase is important for centriole assembly, but how Mps1 is delivered to centrosomes is unknown. Here we have identified a centrosome localization domain within Mps1 and identified the mitochondrial porin VDAC3 as a protein that binds to this region of Mps1. Moreover, we show that VDAC3 is present at the mother centriole and modulates centriole assembly by recruiting Mps1 to centrosomes.

VDAC3 regulates centriole assembly by targeting Mps1 to centrosomes

PubMed Central

Majumder, Shubhra; Slabodnick, Mark; Pike, Amanda; Marquardt, Joseph; Fisk, Harold A.

2012-01-01

Centrioles are duplicated during S-phase to generate the two centrosomes that serve as mitotic spindle poles during mitosis. The centrosomal pool of the Mps1 kinase is important for centriole assembly, but how Mps1 is delivered to centrosomes is unknown. Here we have identified a centrosome localization domain within Mps1 and identified the mitochondrial porin VDAC3 as a protein that binds to this region of Mps1. Moreover, we show that VDAC3 is present at the mother centriole and modulates centriole assembly by recruiting Mps1 to centrosomes. PMID:22935710

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

PubMed Central

2011-01-01

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. PMID:21609466

CEP295 interacts with microtubules and is required for centriole elongation.

PubMed

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.

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

PubMed

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.

CEP295 interacts with microtubules and is required for centriole elongation

PubMed Central

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

2016-01-01

ABSTRACT 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. PMID:27185865

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

NASA Astrophysics Data System (ADS)

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.

Re-examining the role of Drosophila Sas-4 in centrosome assembly using two-colour-3D-SIM FRAP

PubMed Central

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

2015-01-01

Centrosomes have many important functions and comprise a ‘mother’ and ‘daughter’ centriole surrounded by pericentriolar material (PCM). The mother centriole recruits and organises the PCM and templates the formation of the daughter centriole. It has been reported that several important Drosophila PCM-organising proteins are recruited to centrioles from the cytosol as part of large cytoplasmic ‘S-CAP’ complexes that contain the centriole protein Sas-4. In a previous paper (Conduit et al., 2014b) we showed that one of these proteins, Cnn, and another key PCM-organising protein, Spd-2, are recruited around the mother centriole before spreading outwards to form a scaffold that supports mitotic PCM assembly; the recruitment of Cnn and Spd-2 is dependent on another S-CAP protein, Asl. We show here, however, that Cnn, Spd-2 and Asl are not recruited to the mother centriole as part of a complex with Sas-4. Thus, PCM recruitment in fly embryos does not appear to require cytosolic S-CAP complexes. DOI: http://dx.doi.org/10.7554/eLife.08483.001 PMID:26530814

Basal body assembly in ciliates: the power of numbers

PubMed Central

Pearson, Chad G.; Winey, Mark

2009-01-01

Centrioles perform the dual functions of organizing both centrosomes and cilia. The biogenesis of nascent centrioles is an essential cellular event that is tightly coupled to the cell cycle so that each cell contains only two or four centrioles at any given point in the cell cycle. The assembly of centrioles and their analogs, basal bodies, is well characterized at the ultrastructural level whereby structural modules are built into a functional organelle. Genetic studies in model organisms combined with proteomic, bioinformatic, and identifying ciliary disease gene orthologs have revealed a wealth of molecules requiring further analysis to determine their roles in centriole duplication, assembly, and function. Nonetheless, at this stage our understanding of how molecular components interact to build new centrioles and basal bodies is limited. The ciliates, Tetrahymena and Paramecium, historically have been the subject of cytological and genetic study of basal bodies. Recent advances in the ciliate genetic and molecular toolkit have placed these model organisms in a favorable position to study the molecular mechanisms of centriole and basal body assembly. PMID:19192246

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

PubMed Central

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

Flies without centrioles.

PubMed

Basto, Renata; Lau, Joyce; Vinogradova, Tatiana; Gardiol, Alejandra; Woods, C Geoffrey; Khodjakov, Alexey; Raff, Jordan W

2006-06-30

Centrioles and centrosomes have an important role in animal cell organization, but it is uncertain to what extent they are essential for animal development. The Drosophila protein DSas-4 is related to the human microcephaly protein CenpJ and the C. elegans centriolar protein Sas-4. We show that DSas-4 is essential for centriole replication in flies. DSas-4 mutants start to lose centrioles during embryonic development, and, by third-instar larval stages, no centrioles or centrosomes are detectable. Mitotic spindle assembly is slow in mutant cells, and approximately 30% of the asymmetric divisions of larval neuroblasts are abnormal. Nevertheless, mutant flies develop with near normal timing into morphologically normal adults. These flies, however, have no cilia or flagella and die shortly after birth because their sensory neurons lack cilia. Thus, centrioles are essential for the formation of centrosomes, cilia, and flagella, but, remarkably, they are not essential for most aspects of Drosophila development.

Basal body stability and ciliogenesis requires the conserved component Poc1

PubMed Central

Osborn, Daniel P.S.; Giddings, Thomas H.; Beales, Philip L.; Winey, Mark

2009-01-01

Centrioles are the foundation for centrosome and cilia formation. The biogenesis of centrioles is initiated by an assembly mechanism that first synthesizes the ninefold symmetrical cartwheel and subsequently leads to a stable cylindrical microtubule scaffold that is capable of withstanding microtubule-based forces generated by centrosomes and cilia. We report that the conserved WD40 repeat domain–containing cartwheel protein Poc1 is required for the structural maintenance of centrioles in Tetrahymena thermophila. Furthermore, human Poc1B is required for primary ciliogenesis, and in zebrafish, DrPoc1B knockdown causes ciliary defects and morphological phenotypes consistent with human ciliopathies. T. thermophila Poc1 exhibits a protein incorporation profile commonly associated with structural centriole components in which the majority of Poc1 is stably incorporated during new centriole assembly. A second dynamic population assembles throughout the cell cycle. Our experiments identify novel roles for Poc1 in centriole stability and ciliogenesis. PMID:20008567

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

PubMed Central

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

Pix proteins and the evolution of centrioles.

PubMed

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.

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

PubMed

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.

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

PubMed

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.

Post-Testicular Sperm Maturation: Centriole Pairs, Found in Upper Epididymis, are Destroyed Prior to Sperm’s Release at Ejaculation

PubMed Central

Simerly, C.; Castro, C.; Hartnett, C.; Lin, C. C.; Sukhwani, M.; Orwig, K.; Schatten, G.

2016-01-01

The fertilizing sperm’s lengthiest unchartered voyage is through the longest, least-investigated organ in a man’s body – the Epididymis. Over six meters long in men, ~80 meters in stallions and over one-hundred times a mouse’s body length, there are few functions known aside from sperm storage and nutrition. While spermatogenesis is completed in the testes, here we demonstrate sperm centriole reduction occurs within the epididymis. Investigations of GFP-CENTR mice and controls demonstrate both the presence of centriole pairs in the upper caput region of the epididymis and, the destruction, first, of the distal and, then, of the proximal centriole as the sperm transits to the cauda and vas deferens in preparation for its climactic release. These centrioles can neither recruit γ-tubulin nor nucleate microtubules when eggs are inseminated or microinjected, yet numerous maternally-nucleated cytasters are found. These sperm centrioles appear as vestigial basal bodies, destroyed in the mid-to-lower corpus. Post-testicular sperm maturation, in which sperm centrioles found in the caput are destroyed prior to ejaculation, is a newly discovered function for the epididymis. PMID:27534805

Structural basis of the 9-fold symmetry of centrioles.

PubMed

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.

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

PubMed

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.

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

PubMed Central

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

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

PubMed

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.

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

PubMed

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.

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

PubMed

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.

Centrioles want to move out and make cilia.

PubMed

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.

Drosophila Ana2 is a conserved centriole duplication factor

PubMed Central

Stevens, Naomi R.; Dobbelaere, Jeroen; Brunk, Kathrin; Franz, Anna

2010-01-01

In Caenorhabditis elegans, five proteins are required for centriole duplication: SPD-2, ZYG-1, SAS-5, SAS-6, and SAS-4. Functional orthologues of all but SAS-5 have been found in other species. In Drosophila melanogaster and humans, Sak/Plk4, DSas-6/hSas-6, and DSas-4/CPAP—orthologues of ZYG-1, SAS-6, and SAS-4, respectively—are required for centriole duplication. Strikingly, all three fly proteins can induce the de novo formation of centriole-like structures when overexpressed in unfertilized eggs. Here, we find that of eight candidate duplication factors identified in cultured fly cells, only two, Ana2 and Asterless (Asl), share this ability. Asl is now known to be essential for centriole duplication in flies, but no equivalent protein has been found in worms. We show that Ana2 is the likely functional orthologue of SAS-5 and that it is also related to the vertebrate STIL/SIL protein family that has been linked to microcephaly in humans. We propose that members of the SAS-5/Ana2/STIL family of proteins are key conserved components of the centriole duplication machinery. PMID:20123993

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

PubMed

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.

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

PubMed

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.

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

PubMed

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.

Life without centrioles: cilia in the spotlight.

PubMed

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.

Conserved TCP domain of Sas-4/CPAP is essential for pericentriolar material tethering during centrosome biogenesis

PubMed Central

Zheng, Xiangdong; Gooi, Li Ming; Wason, Arpit; Gabriel, Elke; Mehrjardi, Narges Zare; Yang, Qian; Zhang, Xingrun; Debec, Alain; Basiri, Marcus L.; Avidor-Reiss, Tomer; Pozniakovsky, Andrei; Poser, Ina; Šarić, Tomo; Hyman, Anthony A.; Li, Haitao; Gopalakrishnan, Jay

2014-01-01

Pericentriolar material (PCM) recruitment to centrioles forms a key step in centrosome biogenesis. Deregulation of this process leads to centrosome aberrations causing disorders, one of which is autosomal recessive primary microcephaly (MCPH), a neurodevelopmental disorder where brain size is reduced. During PCM recruitment, the conserved centrosomal protein Sas-4/CPAP/MCPH6, known to play a role in centriole formation, acts as a scaffold for cytoplasmic PCM complexes to bind and then tethers them to centrioles to form functional centrosomes. To understand Sas-4’s tethering role, we determined the crystal structure of its T complex protein 10 (TCP) domain displaying a solvent-exposed single-layer of β-sheets fold. This unique feature of the TCP domain suggests that it could provide an “extended surface-like” platform to tether the Sas-4–PCM scaffold to a centriole. Functional studies in Drosophila, human cells, and human induced pluripotent stem cell-derived neural progenitor cells were used to test this hypothesis, where point mutations within the 9–10th β-strands (β9–10 mutants including a MCPH-associated mutation) perturbed PCM tethering while allowing Sas-4/CPAP to scaffold cytoplasmic PCM complexes. Specifically, the Sas-4 β9–10 mutants displayed perturbed interactions with Ana2, a centrosome duplication factor, and Bld-10, a centriole microtubule-binding protein, suggesting a role for the β9–10 surface in mediating protein–protein interactions for efficient Sas-4–PCM scaffold centriole tethering. Hence, we provide possible insights into how centrosomal protein defects result in human MCPH and how Sas-4 proteins act as a vehicle to tether PCM complexes to centrioles independent of its well-known role in centriole duplication. PMID:24385583

Centrioles: active players or passengers during mitosis?

PubMed

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.

Cortical neurogenesis in the absence of centrioles.

PubMed

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.

Centriole duplication: A lesson in self-control.

PubMed

Holland, Andrew J; Lan, Weijie; Cleveland, Don W

2010-07-15

In interphase and mitosis, centrosomes play a major role in the spatial organization of the microtubule network. Alterations in centrosome number and structure are associated with genomic instability and occur in many cancers. Centrosome duplication is controlled by centriole replication. In most dividing animal cells, centrioles duplicate only once per cell cycle at a site adjacent to existing centrioles. The conserved protein kinase Polo-like kinase 4 (Plk4) has a key role in controlling centriole biogenesis. Overexpression of Plk4 drives centrosome amplification and is associated with tumorigenesis in flies. By contrast, haploinsufficiency of Plk4 promotes cytokinesis failure, leading to an increased incidence of tumors in mice. Recent studies have shown that Plk4 is a low abundance protein whose stability is linked to the activity of the enzyme. We discuss how this autoregulatory feedback loop acts to limit the damaging effects caused by too much or too little Plk4.

Structures of SAS-6 suggest its organization in centrioles.

PubMed

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.

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

PubMed Central

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

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

PubMed

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.

Loss of centrioles causes chromosomal instability in vertebrate somatic cells.

PubMed

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.

A molecular mechanism of mitotic centrosome assembly in Drosophila

PubMed Central

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

Cytoplasmic E2f4 forms organizing centres for initiation of centriole amplification during multiciliogenesis

PubMed Central

Mori, Munemasa; Hazan, Renin; Danielian, Paul S.; Mahoney, John E.; Li, Huijun; Lu, Jining; Miller, Emily S.; Zhu, Xueliang; Lees, Jacqueline A.; Cardoso, Wellington V.

2017-01-01

Abnormal development of multiciliated cells is a hallmark of a variety of human conditions associated with chronic airway diseases, hydrocephalus and infertility. Multiciliogenesis requires both activation of a specialized transcriptional program and assembly of cytoplasmic structures for large-scale centriole amplification that generates basal bodies. It remains unclear, however, what mechanism initiates formation of these multiprotein complexes in epithelial progenitors. Here we show that this is triggered by nucleocytoplasmic translocation of the transcription factor E2f4. After inducing a transcriptional program of centriole biogenesis, E2f4 forms apical cytoplasmic organizing centres for assembly and nucleation of deuterosomes. Using genetically altered mice and E2F4 mutant proteins we demonstrate that centriole amplification is crucially dependent on these organizing centres and that, without cytoplasmic E2f4, deuterosomes are not assembled, halting multiciliogenesis. Thus, E2f4 integrates nuclear and previously unsuspected cytoplasmic events of centriole amplification, providing new perspectives for the understanding of normal ciliogenesis, ciliopathies and cancer. PMID:28675157

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

PubMed

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.

PubMed

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.

Auditory Hair Cell Centrioles Undergo Confined Brownian Motion Throughout the Developmental Migration of the Kinocilium

PubMed Central

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

2013-01-01

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. PMID:23823223

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

PubMed

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.

Centriole, differentiation, and senescence.

PubMed

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.

The homo-oligomerisation of both Sas-6 and Ana2 is required for efficient centriole assembly in flies

PubMed Central

Cottee, Matthew A; Muschalik, Nadine; Johnson, Steven; Leveson, Joanna; Raff, Jordan W; Lea, Susan M

2015-01-01

Sas-6 and Ana2/STIL proteins are required for centriole duplication and the homo-oligomerisation properties of Sas-6 help establish the ninefold symmetry of the central cartwheel that initiates centriole assembly. Ana2/STIL proteins are poorly conserved, but they all contain a predicted Central Coiled-Coil Domain (CCCD). Here we show that the Drosophila Ana2 CCCD forms a tetramer, and we solve its structure to 0.8 Å, revealing that it adopts an unusual parallel-coil topology. We also solve the structure of the Drosophila Sas-6 N-terminal domain to 2.9 Å revealing that it forms higher-order oligomers through canonical interactions. Point mutations that perturb Sas-6 or Ana2 homo-oligomerisation in vitro strongly perturb centriole assembly in vivo. Thus, efficient centriole duplication in flies requires the homo-oligomerisation of both Sas-6 and Ana2, and the Ana2 CCCD tetramer structure provides important information on how these proteins might cooperate to form a cartwheel structure. DOI: http://dx.doi.org/10.7554/eLife.07236.001 PMID:26002084

Basal-body-associated macromolecules: a continuing debate.

PubMed

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.

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

PubMed

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.

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

PubMed

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.

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

PubMed

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.

Centriolar satellites assemble centrosomal microcephaly proteins to recruit CDK2 and promote centriole duplication

PubMed Central

Kodani, Andrew; Yu, Timothy W; Johnson, Jeffrey R; Jayaraman, Divya; Johnson, Tasha L; Al-Gazali, Lihadh; Sztriha, Lāszló; Partlow, Jennifer N; Kim, Hanjun; Krup, Alexis L; Dammermann, Alexander; Krogan, Nevan J; Walsh, Christopher A; Reiter, Jeremy F

2015-01-01

Primary microcephaly (MCPH) associated proteins CDK5RAP2, CEP152, WDR62 and CEP63 colocalize at the centrosome. We found that they interact to promote centriole duplication and form a hierarchy in which each is required to localize another to the centrosome, with CDK5RAP2 at the apex, and CEP152, WDR62 and CEP63 at sequentially lower positions. MCPH proteins interact with distinct centriolar satellite proteins; CDK5RAP2 interacts with SPAG5 and CEP72, CEP152 with CEP131, WDR62 with MOONRAKER, and CEP63 with CEP90 and CCDC14. These satellite proteins localize their cognate MCPH interactors to centrosomes and also promote centriole duplication. Consistent with a role for satellites in microcephaly, homozygous mutations in one satellite gene, CEP90, may cause MCPH. The satellite proteins, with the exception of CCDC14, and MCPH proteins promote centriole duplication by recruiting CDK2 to the centrosome. Thus, centriolar satellites build a MCPH complex critical for human neurodevelopment that promotes CDK2 centrosomal localization and centriole duplication. DOI: http://dx.doi.org/10.7554/eLife.07519.001 PMID:26297806

Centrioles, centrosomes, and cilia in health and disease.

PubMed

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.

Male gametogenesis without centrioles.

PubMed

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.

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

PubMed

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.

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

PubMed Central

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

2017-01-01

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. PMID:28476861

Choosing sides--asymmetric centriole and basal body assembly.

PubMed

Pearson, Chad G

2014-07-01

Centrioles and basal bodies (CBBs) are microtubule-rich cylindrical structures that nucleate and organize centrosomes and cilia, respectively. Despite their apparent ninefold rotational symmetry, the nine sets of triplet microtubules in CBBs possess asymmetries in their morphology and in the structures that associate with them. These asymmetries define the position of nascent CBB assembly, the orientation of ciliary beating, the orientation of spindle poles and the maintenance of cellular geometry. For some of these functions, the orientation of CBBs is first established during new CBB biogenesis when the daughter structure is positioned adjacent to the mother. The mother CBB organizes the surrounding environment that nascent CBBs are born into, thereby providing a nest for the new CBB to develop. Protists, including ciliates and algae, highlight the importance of this environment with the formation of asymmetrically placed scaffolds onto which new basal bodies assemble and are positioned. Recent studies illuminate the positioning of nascent centrioles relative to a modular pericentriolar material (PCM) environment and suggest that, like ciliates, centrosomes organize an immediate environment surrounding centrioles for their biogenesis and positioning. In this Commentary, I will explore the positioning of nascent CBB assembly as the first event in building cellular asymmetries and describe how the environment surrounding both basal bodies and centrioles may define asymmetric assembly. © 2014. Published by The Company of Biologists Ltd.

Choosing sides – asymmetric centriole and basal body assembly

PubMed Central

Pearson, Chad G.

2014-01-01

ABSTRACT Centrioles and basal bodies (CBBs) are microtubule-rich cylindrical structures that nucleate and organize centrosomes and cilia, respectively. Despite their apparent ninefold rotational symmetry, the nine sets of triplet microtubules in CBBs possess asymmetries in their morphology and in the structures that associate with them. These asymmetries define the position of nascent CBB assembly, the orientation of ciliary beating, the orientation of spindle poles and the maintenance of cellular geometry. For some of these functions, the orientation of CBBs is first established during new CBB biogenesis when the daughter structure is positioned adjacent to the mother. The mother CBB organizes the surrounding environment that nascent CBBs are born into, thereby providing a nest for the new CBB to develop. Protists, including ciliates and algae, highlight the importance of this environment with the formation of asymmetrically placed scaffolds onto which new basal bodies assemble and are positioned. Recent studies illuminate the positioning of nascent centrioles relative to a modular pericentriolar material (PCM) environment and suggest that, like ciliates, centrosomes organize an immediate environment surrounding centrioles for their biogenesis and positioning. In this Commentary, I will explore the positioning of nascent CBB assembly as the first event in building cellular asymmetries and describe how the environment surrounding both basal bodies and centrioles may define asymmetric assembly. PMID:24895399

Acentriolar mitosis activates a p53-dependent apoptosis pathway in the mouse embryo

PubMed Central

Bazzi, Hisham; Anderson, Kathryn V.

2014-01-01

Centrosomes are the microtubule-organizing centers of animal cells that organize interphase microtubules and mitotic spindles. Centrioles are the microtubule-based structures that organize centrosomes, and a defined set of proteins, including spindle assembly defective-4 (SAS4) (CPAP/CENPJ), is required for centriole biogenesis. The biological functions of centrioles and centrosomes vary among animals, and the functions of mammalian centrosomes have not been genetically defined. Here we use a null mutation in mouse Sas4 to define the cellular and developmental functions of mammalian centrioles in vivo. Sas4-null embryos lack centrosomes but survive until midgestation. As expected, Sas4−/− mutants lack primary cilia and therefore cannot respond to Hedgehog signals, but other developmental signaling pathways are normal in the mutants. Unlike mutants that lack cilia, Sas4−/− embryos show widespread apoptosis associated with global elevated expression of p53. Cell death is rescued in Sas4−/− p53−/− double-mutant embryos, demonstrating that mammalian centrioles prevent activation of a p53-dependent apoptotic pathway. Expression of p53 is not activated by abnormalities in bipolar spindle organization, chromosome segregation, cell-cycle profile, or DNA damage response, which are normal in Sas4−/− mutants. Instead, live imaging shows that the duration of prometaphase is prolonged in the mutants while two acentriolar spindle poles are assembled. Independent experiments show that prolonging spindle assembly is sufficient to trigger p53-dependent apoptosis. We conclude that a short delay in the prometaphase caused by the absence of centrioles activates a previously undescribed p53-dependent cell death pathway in the rapidly dividing cells of the mouse embryo. PMID:24706806

The Centrosome-Specific Phosphorylation of Cnn by Polo/Plk1 Drives Cnn Scaffold Assembly and Centrosome Maturation

PubMed Central

Conduit, Paul T.; Feng, Zhe; Richens, Jennifer H.; Baumbach, Janina; Wainman, Alan; Bakshi, Suruchi D.; Dobbelaere, Jeroen; Johnson, Steven; Lea, Susan M.; Raff, Jordan W.

2014-01-01

Summary Centrosomes are important cell organizers. They consist of a pair of centrioles surrounded by pericentriolar material (PCM) that expands dramatically during mitosis—a process termed centrosome maturation. How centrosomes mature remains mysterious. Here, we identify a domain in Drosophila Cnn that appears to be phosphorylated by Polo/Plk1 specifically at centrosomes during mitosis. The phosphorylation promotes the assembly of a Cnn scaffold around the centrioles that is in constant flux, with Cnn molecules recruited continuously around the centrioles as the scaffold spreads slowly outward. Mutations that block Cnn phosphorylation strongly inhibit scaffold assembly and centrosome maturation, whereas phosphomimicking mutations allow Cnn to multimerize in vitro and to spontaneously form cytoplasmic scaffolds in vivo that organize microtubules independently of centrosomes. We conclude that Polo/Plk1 initiates the phosphorylation-dependent assembly of a Cnn scaffold around centrioles that is essential for efficient centrosome maturation in flies. PMID:24656740

TSKS concentrates in spermatid centrioles during flagellogenesis.

PubMed

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.

The centrosome-specific phosphorylation of Cnn by Polo/Plk1 drives Cnn scaffold assembly and centrosome maturation.

PubMed

Conduit, Paul T; Feng, Zhe; Richens, Jennifer H; Baumbach, Janina; Wainman, Alan; Bakshi, Suruchi D; Dobbelaere, Jeroen; Johnson, Steven; Lea, Susan M; Raff, Jordan W

2014-03-31

Centrosomes are important cell organizers. They consist of a pair of centrioles surrounded by pericentriolar material (PCM) that expands dramatically during mitosis-a process termed centrosome maturation. How centrosomes mature remains mysterious. Here, we identify a domain in Drosophila Cnn that appears to be phosphorylated by Polo/Plk1 specifically at centrosomes during mitosis. The phosphorylation promotes the assembly of a Cnn scaffold around the centrioles that is in constant flux, with Cnn molecules recruited continuously around the centrioles as the scaffold spreads slowly outward. Mutations that block Cnn phosphorylation strongly inhibit scaffold assembly and centrosome maturation, whereas phosphomimicking mutations allow Cnn to multimerize in vitro and to spontaneously form cytoplasmic scaffolds in vivo that organize microtubules independently of centrosomes. We conclude that Polo/Plk1 initiates the phosphorylation-dependent assembly of a Cnn scaffold around centrioles that is essential for efficient centrosome maturation in flies. Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.

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

PubMed

Roque, Helio; Saurya, Saroj; Pratt, Metta B; Johnson, Errin; Raff, Jordan W

2018-02-01

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.

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

PubMed

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.

Centriolar satellite– and hMsd1/SSX2IP-dependent microtubule anchoring is critical for centriole assembly

PubMed Central

Hori, Akiko; Peddie, Christopher J.; Collinson, Lucy M.; Toda, Takashi

2015-01-01

Centriolar satellites are numerous electron-dense granules dispersed around the centrosome. Mutations in their components are linked to various human diseases, but their molecular roles remain elusive. In particular, the significance of spatial communication between centriolar satellites and the centrosome is unknown. hMsd1/SSX2IP localizes to both the centrosome and centriolar satellites and is required for tethering microtubules to the centrosome. Here we show that hMsd1/SSX2IP-mediated microtubule anchoring is essential for proper centriole assembly and duplication. On hMsd1/SSX2IP knockdown, the centriolar satellites become stuck at the microtubule minus end near the centrosome. Intriguingly, these satellites contain many proteins that normally localize to the centrosome. Of importance, microtubule structures, albeit not being anchored properly, are still required for the emergence of abnormal satellites, as complete microtubule depolymerization results in the disappearance of these aggregates from the vicinity of the centrosome. We highlighted, using superresolution and electron microscopy, that under these conditions, centriole structures are faulty. Remarkably, these cells are insensitive to Plk4 overproduction–induced ectopic centriole formation, yet they accelerate centrosome reduplication upon hydroxyurea arrest. Finally, the appearance of satellite aggregates is cancer cell specific. Together our findings provide novel insights into the mechanism of centriole assembly and microtubule anchoring. PMID:25833712

Taking Centrioles to the Elimination Round.

PubMed

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.

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

PubMed

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.

Do centrioles generate a polar ejection force?

PubMed

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.

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

PubMed Central

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

Centrioles in flies: the exception to the rule?

PubMed

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.

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

PubMed

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.

Centrin 3 is an inhibitor of centrosomal Mps1 and antagonizes centrin 2 function

PubMed Central

Sawant, Dwitiya B.; Majumder, Shubhra; Perkins, Jennifer L.; Yang, Ching-Hui; Eyers, Patrick A.; Fisk, Harold A.

2015-01-01

Centrins are a family of small, calcium-binding proteins with diverse cellular functions that play an important role in centrosome biology. We previously identified centrin 2 and centrin 3 (Cetn2 and Cetn3) as substrates of the protein kinase Mps1. However, although Mps1 phosphorylation sites control the function of Cetn2 in centriole assembly and promote centriole overproduction, Cetn2 and Cetn3 are not functionally interchangeable, and we show here that Cetn3 is both a biochemical inhibitor of Mps1 catalytic activity and a biological inhibitor of centrosome duplication. In vitro, Cetn3 inhibits Mps1 autophosphorylation at Thr-676, a known site of T-loop autoactivation, and interferes with Mps1-dependent phosphorylation of Cetn2. The cellular overexpression of Cetn3 attenuates the incorporation of Cetn2 into centrioles and centrosome reduplication, whereas depletion of Cetn3 generates extra centrioles. Finally, overexpression of Cetn3 reduces Mps1 Thr-676 phosphorylation at centrosomes, and mimicking Mps1-dependent phosphorylation of Cetn2 bypasses the inhibitory effect of Cetn3, suggesting that the biological effects of Cetn3 are due to the inhibition of Mps1 function at centrosomes. PMID:26354417

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

PubMed

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.

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

PubMed

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.

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

PubMed

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.

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

PubMed

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.

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

PubMed

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.

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

PubMed

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.

Using sea urchin gametes and zygotes to investigate centrosome duplication.

PubMed

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.

VDAC3 and Mps1 negatively regulate ciliogenesis.

PubMed

Majumder, Shubhra; Fisk, Harold A

2013-03-01

Centrosomes serve to organize new centrioles in cycling cells, whereas in quiescent cells they assemble primary cilia. We have recently shown that the mitochondrial porin VDAC3 is also a centrosomal protein that is predominantly associated with the mother centriole and modulates centriole assembly by recruiting Mps1 to centrosomes. Here, we show that depletion of VDAC3 causes inappropriate ciliogenesis in cycling cells, while expression of GFP-VDAC3 suppresses ciliogenesis in quiescent cells. Mps1 also negatively regulates ciliogenesis, and the inappropriate ciliogenesis caused by VDAC3 depletion can be bypassed by targeting Mps1 to centrosomes independently of VDAC3. Thus, our data show that a VDAC3-Mps1 module at the centrosome promotes ciliary disassembly during cell cycle entry and suppresses cilia assembly in proliferating cells. Our data also suggests that VDAC3 might be a link between mitochondrial dysfunction and ciliopathies in mammalian cells.

Hierarchical assembly of centriole subdistal appendages via centrosome binding proteins CCDC120 and CCDC68.

PubMed

Huang, Ning; Xia, Yuqing; Zhang, Donghui; Wang, Song; Bao, Yitian; He, Runsheng; Teng, Junlin; Chen, Jianguo

2017-04-19

In animal cells, the centrosome is the main microtubule-organizing centre where microtubules are nucleated and anchored. The centriole subdistal appendages (SDAs) are the key structures that anchor microtubules in interphase cells, but the composition and assembly mechanisms of SDAs are not well understood. Here, we reveal that centrosome-binding proteins, coiled-coil domain containing (CCDC) 120 and CCDC68 are two novel SDA components required for hierarchical SDA assembly in human cells. CCDC120 is anchored to SDAs by ODF2 and recruits CEP170 and Ninein to the centrosome through different coiled-coil domains at its N terminus. CCDC68 is a CEP170-interacting protein that competes with CCDC120 in recruiting CEP170 to SDAs. Furthermore, CCDC120 and CCDC68 are required for centrosome microtubule anchoring. Our findings elucidate the molecular basis for centriole SDA hierarchical assembly and microtubule anchoring in human interphase cells.

Centrosomes are autocatalytic droplets of pericentriolar material organized by centrioles

NASA Astrophysics Data System (ADS)

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.

Centrin 3 is an inhibitor of centrosomal Mps1 and antagonizes centrin 2 function.

PubMed

Sawant, Dwitiya B; Majumder, Shubhra; Perkins, Jennifer L; Yang, Ching-Hui; Eyers, Patrick A; Fisk, Harold A

2015-11-01

Centrins are a family of small, calcium-binding proteins with diverse cellular functions that play an important role in centrosome biology. We previously identified centrin 2 and centrin 3 (Cetn2 and Cetn3) as substrates of the protein kinase Mps1. However, although Mps1 phosphorylation sites control the function of Cetn2 in centriole assembly and promote centriole overproduction, Cetn2 and Cetn3 are not functionally interchangeable, and we show here that Cetn3 is both a biochemical inhibitor of Mps1 catalytic activity and a biological inhibitor of centrosome duplication. In vitro, Cetn3 inhibits Mps1 autophosphorylation at Thr-676, a known site of T-loop autoactivation, and interferes with Mps1-dependent phosphorylation of Cetn2. The cellular overexpression of Cetn3 attenuates the incorporation of Cetn2 into centrioles and centrosome reduplication, whereas depletion of Cetn3 generates extra centrioles. Finally, overexpression of Cetn3 reduces Mps1 Thr-676 phosphorylation at centrosomes, and mimicking Mps1-dependent phosphorylation of Cetn2 bypasses the inhibitory effect of Cetn3, suggesting that the biological effects of Cetn3 are due to the inhibition of Mps1 function at centrosomes. © 2015 Sawant et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

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.

PubMed

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.

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

PubMed

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.

PubMed

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.

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

PubMed

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.

Centrosomes are autocatalytic droplets of pericentriolar material organized by centrioles.

PubMed

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

2014-07-01

Centrosomes are highly dynamic, spherical organelles without a membrane. Their physical nature and their assembly are not understood. Using the concept of phase separation, we propose a theoretical description of centrosomes as liquid droplets. In our model, centrosome material occurs in a form soluble in the cytosol and a form that tends to undergo phase separation from the cytosol. We show 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 nonequilibrium feature also allows for multiple stable centrosomes, a situation that 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 Caenorhabditis elegans embryo, and it also accounts for data acquired in experiments with aberrant numbers of centrosomes and altered cell volumes. Furthermore, the model can describe unequal centrosome sizes observed in cells with perturbed centrioles. We also propose an interpretation of the molecular details of the involved proteins in the case of C. elegans. Our example suggests a general picture of the organization of membraneless organelles.

From stem cell to embryo without centrioles.

PubMed

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.

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

PubMed Central

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

2013-01-01

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. PMID:24189274

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

PubMed

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.

Centriole assembly and the role of Mps1: defensible or dispensable?

PubMed

Pike, Amanda N; Fisk, Harold A

2011-04-14

The Mps1 protein kinase is an intriguing and controversial player in centriole assembly. Originally shown to control duplication of the budding yeast spindle pole body, Mps1 is present in eukaryotes from yeast to humans, the nematode C. elegans being a notable exception, and has also been shown to regulate the spindle checkpoint and an increasing number of cellular functions relating to genomic stability. While its function in the spindle checkpoint appears to be both universally conserved and essential in most organisms, conservation of its originally described function in spindle pole duplication has proven controversial, and it is less clear whether Mps1 is essential for centrosome duplication outside of budding yeast. Recent studies of Mps1 have identified at least two distinct functions for Mps1 in centriole assembly, while simultaneously supporting the notion that Mps1 is dispensable for the process. However, the fact that at least one centrosomal substrate of Mps1 is conserved from yeast to humans down to the phosphorylation site, combined with evidence demonstrating the exquisite control exerted over centrosomal Mps1 levels suggest that the notion of being essential may not be the most important of distinctions.

Centrosomes are autocatalytic droplets of pericentriolar material organized by centrioles

PubMed Central

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

2014-01-01

Centrosomes are highly dynamic, spherical organelles without a membrane. Their physical nature and their assembly are not understood. Using the concept of phase separation, we propose a theoretical description of centrosomes as liquid droplets. In our model, centrosome material occurs in a form soluble in the cytosol and a form that tends to undergo phase separation from the cytosol. We show 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 nonequilibrium feature also allows for multiple stable centrosomes, a situation that 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 Caenorhabditis elegans embryo, and it also accounts for data acquired in experiments with aberrant numbers of centrosomes and altered cell volumes. Furthermore, the model can describe unequal centrosome sizes observed in cells with perturbed centrioles. We also propose an interpretation of the molecular details of the involved proteins in the case of C. elegans. Our example suggests a general picture of the organization of membraneless organelles. PMID:24979791

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

PubMed Central

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

Basal body structure and composition in the apicomplexans Toxoplasma and Plasmodium.

PubMed

Francia, Maria E; Dubremetz, Jean-Francois; Morrissette, Naomi S

2015-01-01

The phylum Apicomplexa encompasses numerous important human and animal disease-causing parasites, including the Plasmodium species, and Toxoplasma gondii, causative agents of malaria and toxoplasmosis, respectively. Apicomplexans proliferate by asexual replication and can also undergo sexual recombination. Most life cycle stages of the parasite lack flagella; these structures only appear on male gametes. Although male gametes (microgametes) assemble a typical 9+2 axoneme, the structure of the templating basal body is poorly defined. Moreover, the relationship between asexual stage centrioles and microgamete basal bodies remains unclear. While asexual stages of Plasmodium lack defined centriole structures, the asexual stages of Toxoplasma and closely related coccidian apicomplexans contain centrioles that consist of nine singlet microtubules and a central tubule. There are relatively few ultra-structural images of Toxoplasma microgametes, which only develop in cat intestinal epithelium. Only a subset of these include sections through the basal body: to date, none have unambiguously captured organization of the basal body structure. Moreover, it is unclear whether this basal body is derived from pre-existing asexual stage centrioles or is synthesized de novo. Basal bodies in Plasmodium microgametes are thought to be synthesized de novo, and their assembly remains ill-defined. Apicomplexan genomes harbor genes encoding δ- and ε-tubulin homologs, potentially enabling these parasites to assemble a typical triplet basal body structure. Moreover, the UNIMOD components (SAS6, SAS4/CPAP, and BLD10/CEP135) are conserved in these organisms. However, other widely conserved basal body and flagellar biogenesis elements are missing from apicomplexan genomes. These differences may indicate variations in flagellar biogenesis pathways and in basal body arrangement within the phylum. As apicomplexan basal bodies are distinct from their metazoan counterparts, it may be possible to selectively target parasite structures in order to inhibit microgamete motility which drives generation of genetic diversity in Toxoplasma and transmission for Plasmodium.

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

PubMed Central

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.

PubMed

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.

Ultrastructural differentiation of sperm tail region in Diplometopon zarudnyi (an amphisbaenian reptile)

PubMed Central

Al-Dokhi, O.; Mukhtar, Ahmed; Al-Dosary, A.; Al-Sadoon, M.K.

2015-01-01

Diplometopon zarudnyi, a worm lizard belongs to amphisbaenia under trogonophidae family. This species exists in limited areas of the Arabian Peninsula and is an oscillating digger found in sub-surface soils. The present study aimed to investigate the sperm tail differentiation in D. zarudnyi. Ten male adults of D. zarudnyi were collected from Riyadh during April–May 2011. To study the sperm tail at the ultrastructural level the testes were fixed in 3% glutaraldehyde, than post fixed in 1% osmium tetaroxide followed by dehydration in ethanol grades; samples were cleared in propylene oxide and embedded in resin. Tail formation begins by the moving of centrioles and mitochondria towards the posterior pole of sperm head. Simultaneously many microtubules of the midpiece axoneme were enclosed by a thick layer of granular material. Mitochondria of midpiece lie alongside the proximal centriole which forms a very short neck region and possess tubular cristae internally and concentric layers of cristae superficially. During this course a fibrous sheath surrounds the axoneme of mid and principal piece. At the end dissolution of longitudinal manchette takes place. The mitochondria then rearrange themselves around the proximal and distal centrioles to form a neck region. Later, the fibrous sheath surrounds the proximal portion of the flagella. This part along with sperm head of D. zarudnyi provides a classical model that could be used in future for evolutionary and phylogenetic purposes of class reptilia. PMID:26155090

STED nanoscopy of the centrosome linker reveals a CEP68-organized, periodic rootletin network anchored to a C-Nap1 ring at centrioles.

PubMed

Vlijm, Rifka; Li, Xue; Panic, Marko; Rüthnick, Diana; Hata, Shoji; Herrmannsdörfer, Frank; Kuner, Thomas; Heilemann, Mike; Engelhardt, Johann; Hell, Stefan W; Schiebel, Elmar

2018-03-06

The centrosome linker proteins C-Nap1, rootletin, and CEP68 connect the two centrosomes of a cell during interphase into one microtubule-organizing center. This coupling is important for cell migration, cilia formation, and timing of mitotic spindle formation. Very little is known about the structure of the centrosome linker. Here, we used stimulated emission depletion (STED) microscopy to show that each C-Nap1 ring at the proximal end of the two centrioles organizes a rootletin ring and, in addition, multiple rootletin/CEP68 fibers. Rootletin/CEP68 fibers originating from the two centrosomes form a web-like, interdigitating network, explaining the flexible nature of the centrosome linker. The rootletin/CEP68 filaments are repetitive and highly ordered. Staggered rootletin molecules (N-to-N and C-to-C) within the filaments are 75 nm apart. Rootletin binds CEP68 via its C-terminal spectrin repeat-containing region in 75-nm intervals. The N-to-C distance of two rootletin molecules is ∼35 to 40 nm, leading to an estimated minimal rootletin length of ∼110 nm. CEP68 is important in forming rootletin filaments that branch off centrioles and to modulate the thickness of rootletin fibers. Thus, the centrosome linker consists of a vast network of repeating rootletin units with C-Nap1 as ring organizer and CEP68 as filament modulator. Copyright © 2018 the Author(s). Published by PNAS.

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

PubMed

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.

Is cohesin required for spindle-pole-body/centrosome cohesion?

PubMed Central

Jin, Hui; Avey, Martin

2012-01-01

Centrosomes are microtubule-organizing centers that nucleate spindle microtubules during cell division. In budding yeast, the centrosome, often referred to as the spindle pole body, shares structural components with the centriole, the central core of the animal centrosome. The parental centrosome is duplicated when DNA replication takes place. Like sister chromatids tethered together by cohesin, duplicated centrosomes are linked and then separate to form the bipolar spindle necessary for chromosome segregation. Recent studies have shown that cohesin is also localized to the animal centrosome and is perhaps directly involved in engaging paired centrioles. Here we discuss the potential role of cohesin in mediating spindle-pole-body cohesion in the context of yeast meiosis. We propose that the coordination of chromosome segregation with centrosome cohesion and duplication is mediated by the antagonistic interaction between the Aurora kinase and the Polo kinase and that the role of cohesin in centrosome regulation appears to be indirect in budding yeast. PMID:22482005

Calibrated mitotic oscillator drives motile ciliogenesis.

PubMed

Al Jord, Adel; Shihavuddin, Asm; Servignat d'Aout, Raphaël; Faucourt, Marion; Genovesio, Auguste; Karaiskou, Anthi; Sobczak-Thépot, Joëlle; Spassky, Nathalie; Meunier, Alice

2017-11-10

Cell division and differentiation depend on massive and rapid organelle remodeling. The mitotic oscillator, centered on the cyclin-dependent kinase 1-anaphase-promoting complex/cyclosome (CDK1-APC/C) axis, spatiotemporally coordinates this reorganization in dividing cells. Here we discovered that nondividing cells could also implement this mitotic clocklike regulatory circuit to orchestrate subcellular reorganization associated with differentiation. We probed centriole amplification in differentiating mouse-brain multiciliated cells. These postmitotic progenitors fine-tuned mitotic oscillator activity to drive the orderly progression of centriole production, maturation, and motile ciliation while avoiding the mitosis commitment threshold. Insufficient CDK1 activity hindered differentiation, whereas excessive activity accelerated differentiation yet drove postmitotic progenitors into mitosis. Thus, postmitotic cells can redeploy and calibrate the mitotic oscillator to uncouple cytoplasmic from nuclear dynamics for organelle remodeling associated with differentiation. Copyright © 2017 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.

PARP-3 localizes preferentially to the daughter centriole and interferes with the G1/S cell cycle progression.

PubMed

Augustin, Angélique; Spenlehauer, Catherine; Dumond, Hélène; Ménissier-De Murcia, Josiane; Piel, Matthieu; Schmit, Anne-Catherine; Apiou, Françoise; Vonesch, Jean-Luc; Kock, Michael; Bornens, Michel; De Murcia, Gilbert

2003-04-15

A novel member of the poly(ADP-ribose) polymerase (PARP) family, hPARP-3, is identified here as a core component of the centrosome. hPARP-3 is preferentially localized to the daughter centriole throughout the cell cycle. The N-terminal domain (54 amino acids) of hPARP-3 is responsible for its centrosomal localization. Full-length hPAPR-3 (540 amino acids, with an apparent mass of 67 kDa) synthesizes ADP-ribose polymers during its automodification. Overexpression of hPARP-3 or its N-terminal domain does not influence centrosomal duplication or amplification but interferes with the G1/S cell cycle progression. PARP-1 also resides for part of the cell cycle in the centrosome and interacts with hPARP-3. The presence of both PARP-1 and PARP-3 at the centrosome may link the DNA damage surveillance network to the mitotic fidelity checkpoint.

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

PubMed

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.

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

PubMed

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.

Ultrastructure of spermatozoa of tench Tinca tinca observed by means of scanning and transmission electron microscopy.

PubMed

Psenicka, M; Rodina, M; Nebesarova, J; Linhart, O

2006-09-15

Structure of tench (Tinca tinca L.) spermatozoa was investigated by means of scanning electron microscopy (SEM) and transmission electron microscopy (TEM). Spermatozoa of 26.1+/-3.8 microm total length possessed typical primitive simple structure, called "aqua sperm", without acrosomal head structures. It was probably the smallest spermatozoon described among cyprinid fishes. Heads were mostly composed of dense and slightly granular material, which appeared to be fairly homogeneous except for the occasional appearance of vacuoles. The midpiece remained separated from the flagellum by the cytoplasmic channel; it was cylindric/cone-shaped, 0.86+/-0.27 microm in length and 1.17+/-0.24 microm in width at proximal part. The proximal centriole was located in the "implantation fossa". The distal centriole appeared almost tangential to the nucleus and it functioned as a basal body for the flagellum. It had an orientation of 140 degrees with respect to the distal centriole. The sperm flagellum with 25.45+/-2.47 microm of total length had no any fin. The diameter of the flagellum perpendicular to the plane of the doublet of central microtubules was 173.67+/-20.45 nm and horizontal plane of the central microtubules was 200.71+/-20.45 nm. Peripheral doublets and the central doublet of microtubules measured 23.39+/-3.18 and 35.88+/-4.44 nm in width, respectively. The diameter of a microtubule was only 9.14+/-2.97 nm. A vesicle was attached to the most basal region of the flagellum and located just under plasma membrane of the flagellum.

Sperm-cell ultrastructure of North American sturgeons. IV. The pallid sturgeon (Scaphirhynchus albus Forbes and Richardson, 1905)

USGS Publications Warehouse

DiLauro, M.N.; Walsh, R.A.; Peiffer, M.; Bennett, R.M.

2001-01-01

Sperm-cell morphology and ultrastructure in the pallid sturgeon (Scaphirhynchus albus) were examined using transmission and scanning electron microscopy. Metrics and structure were compared with similar metrics obtained from other published descriptions of sturgeon sperm cells. General morphology was found to be similar to that of sperm cells of the white (Acipenser transmontanus), lake (A. fulvescens), stellate (A. stellatus), Chinese (A. sinensis), Russian (A. gueldenstaedti colchicus), and shortnose (A. brevirostrum) sturgeons, which all shared a gradual tapering of the nuclear diameter from posterior to anterior, unlike that of the Atlantic sturgeon (A. oxyrhynchus). The sperm cell of the pallid sturgeon was similar in size to that of the Atlantic sturgeon, being only slightly larger. The sperm cell of the pallid sturgeon differed from those of other sturgeons chiefly in the acrosomal region, where the posterolateral projections (PLP) have the shape of an acute triangle and are arranged in a spiral about the longitudinal axis of the cell. The PLP were longer than those of other sturgeons, being twice the length of those of the Atlantic sturgeon and 58% longer than those of the lake sturgeon. Also, in cross section the acrosome had the shape of a hollow cone rather than the cap of an oak tree acorn, as was found in ultrastructural studies of other sturgeons. In addition, we were able to confirm that the structural arrangement of the distal centriole of the midpiece is identical with that of the proximal centriole: nine sets of microtubular triplets around the periphery of the centriole. This information is of potential use to fishery biologists, forensic biologists, zoologists, reproductive physiologists, taxonomists, evolutionary biologists, and aquaculturists.

A comparative overview of the sperm centriolar complex in mammals and birds: Variations on a theme.

PubMed

Soley, John T

2016-06-01

This paper presents an overview of the structure, function and anomalies of the sperm centriolar complex (CC) on a comparative basis between mammals and birds. The information is based on selected references from the literature supplemented by original observations on spermiogenesis and sperm structure in disparate mammalian (cheetah and cane rat) and avian (ostrich, rhea and emu) species. Whereas the basic structure of the CC (a diplosome surrounded by pericentriolar material) is similar in Aves and Mammalia, certain differences are apparent. Centriole reduction does not generally occur in birds, but when present as in oscines, involves the loss of the proximal centriole. In ratites, the distal centriole forms the core of the entire midpiece and incorporates the outer dense fibres in addition to initiating axoneme formation. The elements of the connecting piece are not segmented in birds and less complex in basic design than in mammals. The functions of the various components of the CC appear to be similar in birds and mammals. Despite obvious differences in sperm head shape, the centrosomal anomalies afflicting both vertebrate groups demonstrate structural uniformity across species and display a similar range of defects. Most abnormalities result from defective migration and alignment of the CC relative to the nucleus. The most severe manifestation is that of acephalic sperm, while angled tail attachment, abaxial and multiflagellate sperm reflect additional defective forms. The stump-tail defect is not observed in birds. A comparison of defective sperm formation and centrosomal dysfunction at the molecular level is currently difficult owing to the paucity of relevant information on avian sperm. Copyright © 2016 Elsevier B.V. All rights reserved.

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

NASA Astrophysics Data System (ADS)

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, often directly under the acrosome.

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

PubMed

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.

Small organelle, big responsibility: the role of centrosomes in development and disease

PubMed Central

Chavali, Pavithra L.; Pütz, Monika; Gergely, Fanni

2014-01-01

The centrosome, a key microtubule organizing centre, is composed of centrioles, embedded in a protein-rich matrix. Centrosomes control the internal spatial organization of somatic cells, and as such contribute to cell division, cell polarity and migration. Upon exiting the cell cycle, most cell types in the human body convert their centrioles into basal bodies, which drive the assembly of primary cilia, involved in sensing and signal transduction at the cell surface. Centrosomal genes are targeted by mutations in numerous human developmental disorders, ranging from diseases exclusively affecting brain development, through global growth failure syndromes to diverse pathologies associated with ciliary malfunction. Despite our much-improved understanding of centrosome function in cellular processes, we know remarkably little of its role in the organismal context, especially in mammals. In this review, we examine how centrosome dysfunction impacts on complex physiological processes and speculate on the challenges we face when applying knowledge generated from in vitro and in vivo model systems to human development. PMID:25047622

Thrombocytogenesis by megakaryocyte; Interpretation by protoplatelet hypothesis

PubMed Central

KOSAKI, Goro; KAMBAYASHI, Junichi

2011-01-01

Serial transmission electron microscopy of human megakaryocytes (MKs) revealed their polyploidization and gradual maturation through consecutive transition in characteristics of various organelles and others. At the beginning of differentiation, MK with ploidy 32N, e.g., has 16 centrosomes in the cell center surrounded by 32N nucleus. Each bundle of microtubules (MTs) emanated from the respective centrosome supports and organizes 16 equally volumed cytoplasmic compartments which together compose one single 32N MK. During the differentiation, single centriole separated from the centriole pair, i.e., centrosome, migrates to the most periphery of the cell through MT bundle, corresponding to a half of the interphase array originated from one centrosome, supporting one “putative cytoplasmic compartment” (PCC). Platelet demarcation membrane (DM) is constructed on the boundary surface between neighbouring PCCs. Matured PCC, composing of a tandem array of platelet territories covered by a sheet of DM is designated as protoplatelet. Eventually, the rupture of MK results in release of platelets from protoplatelets. PMID:21558761

Loss and Rebirth of the Animal Microtubule Organizing Center: How Maternal Expression of Centrosomal Proteins Cooperates with the Sperm Centriole in Zygotic Centrosome Reformation.

PubMed

Inoue, Daigo; Wittbrodt, Joachim; Gruss, Oliver J

2018-04-01

Centrosomes are the main microtubule organizing centers in animal cells. In particular during embryogenesis, they ensure faithful spindle formation and proper cell divisions. As metazoan centrosomes are eliminated during oogenesis, they have to be reassembled upon fertilization. Most metazoans use the sperm centrioles as templates for new centrosome biogenesis while the egg's cytoplasm re-prepares all components for on-going centrosome duplication in rapidly dividing embryonic cells. We discuss our knowledge and the experimental challenges to analyze zygotic centrosome reformation, which requires genetic experiments to enable scrutinizing respective male and female contributions. Male and female knockout animals and mRNA injection to mimic maternal expression of centrosomal proteins could point a way to the systematic molecular dissection of the process. The most recent data suggest that timely expression of centrosome components in oocytes is the key to zygotic centrosome reformation that uses male sperm as coordinators for de novo centrosome production. © 2018 WILEY Periodicals, Inc.

Primordial dwarfism: an update.

PubMed

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

PubMed

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.

Small organelle, big responsibility: the role of centrosomes in development and disease.

PubMed

Chavali, Pavithra L; Pütz, Monika; Gergely, Fanni

2014-09-05

The centrosome, a key microtubule organizing centre, is composed of centrioles, embedded in a protein-rich matrix. Centrosomes control the internal spatial organization of somatic cells, and as such contribute to cell division, cell polarity and migration. Upon exiting the cell cycle, most cell types in the human body convert their centrioles into basal bodies, which drive the assembly of primary cilia, involved in sensing and signal transduction at the cell surface. Centrosomal genes are targeted by mutations in numerous human developmental disorders, ranging from diseases exclusively affecting brain development, through global growth failure syndromes to diverse pathologies associated with ciliary malfunction. Despite our much-improved understanding of centrosome function in cellular processes, we know remarkably little of its role in the organismal context, especially in mammals. In this review, we examine how centrosome dysfunction impacts on complex physiological processes and speculate on the challenges we face when applying knowledge generated from in vitro and in vivo model systems to human development. © 2014 The Author(s) Published by the Royal Society. All rights reserved.

Reconstruction From Multiple Particles for 3D Isotropic Resolution in Fluorescence Microscopy.

PubMed

Fortun, Denis; Guichard, Paul; Hamel, Virginie; Sorzano, Carlos Oscar S; Banterle, Niccolo; Gonczy, Pierre; Unser, Michael

2018-05-01

The imaging of proteins within macromolecular complexes has been limited by the low axial resolution of optical microscopes. To overcome this problem, we propose a novel computational reconstruction method that yields isotropic resolution in fluorescence imaging. The guiding principle is to reconstruct a single volume from the observations of multiple rotated particles. Our new operational framework detects particles, estimates their orientation, and reconstructs the final volume. The main challenge comes from the absence of initial template and a priori knowledge about the orientations. We formulate the estimation as a blind inverse problem, and propose a block-coordinate stochastic approach to solve the associated non-convex optimization problem. The reconstruction is performed jointly in multiple channels. We demonstrate that our method is able to reconstruct volumes with 3D isotropic resolution on simulated data. We also perform isotropic reconstructions from real experimental data of doubly labeled purified human centrioles. Our approach revealed the precise localization of the centriolar protein Cep63 around the centriole microtubule barrel. Overall, our method offers new perspectives for applications in biology that require the isotropic mapping of proteins within macromolecular assemblies.

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

PubMed

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.

Disease-Associated Mutations in CEP120 Destabilize the Protein and Impair Ciliogenesis.

PubMed

Joseph, Nimesh; Al-Jassar, Caezar; Johnson, Christopher M; Andreeva, Antonina; Barnabas, Deepak D; Freund, Stefan M V; Gergely, Fanni; van Breugel, Mark

2018-05-29

Ciliopathies are a group of genetic disorders caused by a failure to form functional cilia. Due to a lack of structural information, it is currently poorly understood how ciliopathic mutations affect protein functionality to give rise to the underlying disease. Using X-ray crystallography, we show that the ciliopathy-associated centriolar protein CEP120 contains three C2 domains. The point mutations V194A and A199P, which cause Joubert syndrome (JS) and Jeune asphyxiating thoracic dystrophy (JATD), respectively, both reduce the thermostability of the second C2 domain by targeting residues that point toward its hydrophobic core. Genome-engineered cells homozygous for these mutations have largely normal centriole numbers but show reduced CEP120 levels, compromised recruitment of distal centriole markers, and deficient cilia formation. Our results provide insight into the disease mechanism of two ciliopathic mutations in CEP120, identify putative binding partners of CEP120 C2B, and suggest a complex genotype-phenotype relation of the CEP120 ciliopathy alleles. Copyright © 2018 MRC Laboratory of Molecular Biology. Published by Elsevier Inc. All rights reserved.

CENP-W Plays a Role in Maintaining Bipolar Spindle Structure

PubMed Central

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 by motor proteins during chromosome congression. Taken together, our findings are consistent with a model in which centrosome integrity is controlled by the pathways regulating kinetochore-microtubule attachment stability. PMID:25329824

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

PubMed

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.

Electron Microscopy Structural Insights into CPAP Oligomeric Behavior: A Plausible Assembly Process of a Supramolecular Scaffold of the Centrosome

PubMed Central

Alvarez-Cabrera, Ana L.; Delgado, Sandra; Gil-Carton, David; Mortuza, Gulnahar B.; Montoya, Guillermo; Sorzano, Carlos O. S.; Tang, Tang K.; Carazo, Jose M.

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 primary autosomal recessive microcephaly and Seckel syndrome. Despite of the biological/clinical relevance of CPAP, its mechanistic behavior remains unclear and its C-terminus (the G-box/TCP domain) is the only part whose structure has been solved. This situation is perhaps due in part to the challenges that represent obtaining the protein in a soluble, homogeneous state for structural studies. Our work constitutes a systematic structural analysis on multiple oligomers of HsCPAP897−1338, using single-particle electron microscopy (EM) of negatively stained (NS) samples. Based on image classification into clearly different regular 3D maps (putatively corresponding to dimers and tetramers) and direct observation of individual images representing other complexes of HsCPAP897−1338 (i.e., putative flexible monomers and higher-order multimers), we report a dynamic oligomeric behavior of this protein, where different homo-oligomers coexist in variable proportions. We propose that dimerization of the putative homodimer forms a putative tetramer which could be the structural unit for the scaffold that either tethers the pericentriolar material to centrioles or promotes procentriole elongation. A coarse fitting of atomic models into the NS 3D maps at resolutions around 20 Å is performed only to complement our experimental data, allowing us to hypothesize on the oligomeric composition of the different complexes. In this way, the current EM work represents an initial step toward the structural characterization of different oligomers of CPAP, suggesting further insights to understand how this protein works, contributing to the elucidation of control mechanisms for centriole biogenesis. PMID:28396859

LINE-1 Mediated Insertion into Poc1a (Protein of Centriole 1 A) Causes Growth Insufficiency and Male Infertility in Mice

PubMed Central

Geister, Krista A.; Brinkmeier, Michelle L.; Cheung, Leonard Y.; Wendt, Jennifer; Oatley, Melissa J.; Burgess, Daniel L.; Kozloff, Kenneth M.; Cavalcoli, James D.; Oatley, Jon M.; Camper, Sally A.

2015-01-01

Skeletal dysplasias are a common, genetically heterogeneous cause of short stature that can result from disruptions in many cellular processes. We report the identification of the lesion responsible for skeletal dysplasia and male infertility in the spontaneous, recessive mouse mutant chagun. We determined that Poc1a, encoding protein of the centriole 1a, is disrupted by the insertion of a processed Cenpw cDNA, which is flanked by target site duplications, suggestive of a LINE-1 retrotransposon-mediated event. Mutant fibroblasts have impaired cilia formation and multipolar spindles. Male infertility is caused by defective spermatogenesis early in meiosis and progressive germ cell loss. Spermatogonial stem cell transplantation studies revealed that Poc1a is essential for normal function of both Sertoli cells and germ cells. The proliferative zone of the growth plate is small and disorganized because chondrocytes fail to re-align after cell division and undergo increased apoptosis. Poc1a and several other genes associated with centrosome function can affect the skeleton and lead to skeletal dysplasias and primordial dwarfisms. This mouse mutant reveals how centrosome dysfunction contributes to defects in skeletal growth and male infertility. PMID:26496357

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

NASA Astrophysics Data System (ADS)

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.

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

NASA Astrophysics Data System (ADS)

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.

Centrioles are freed from cilia by severing prior to mitosis.

PubMed

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.

LINE-1 Mediated Insertion into Poc1a (Protein of Centriole 1 A) Causes Growth Insufficiency and Male Infertility in Mice.

PubMed

Geister, Krista A; Brinkmeier, Michelle L; Cheung, Leonard Y; Wendt, Jennifer; Oatley, Melissa J; Burgess, Daniel L; Kozloff, Kenneth M; Cavalcoli, James D; Oatley, Jon M; Camper, Sally A

2015-10-01

Skeletal dysplasias are a common, genetically heterogeneous cause of short stature that can result from disruptions in many cellular processes. We report the identification of the lesion responsible for skeletal dysplasia and male infertility in the spontaneous, recessive mouse mutant chagun. We determined that Poc1a, encoding protein of the centriole 1a, is disrupted by the insertion of a processed Cenpw cDNA, which is flanked by target site duplications, suggestive of a LINE-1 retrotransposon-mediated event. Mutant fibroblasts have impaired cilia formation and multipolar spindles. Male infertility is caused by defective spermatogenesis early in meiosis and progressive germ cell loss. Spermatogonial stem cell transplantation studies revealed that Poc1a is essential for normal function of both Sertoli cells and germ cells. The proliferative zone of the growth plate is small and disorganized because chondrocytes fail to re-align after cell division and undergo increased apoptosis. Poc1a and several other genes associated with centrosome function can affect the skeleton and lead to skeletal dysplasias and primordial dwarfisms. This mouse mutant reveals how centrosome dysfunction contributes to defects in skeletal growth and male infertility.

Molecular basis for CPAP-tubulin interaction in controlling centriolar and ciliary length

PubMed Central

Zheng, Xiangdong; Ramani, Anand; Soni, Komal; Gottardo, Marco; Zheng, Shuangping; Ming Gooi, Li; Li, Wenjing; Feng, Shan; Mariappan, Aruljothi; Wason, Arpit; Widlund, Per; Pozniakovsky, Andrei; Poser, Ina; Deng, Haiteng; Ou, Guangshuo; Riparbelli, Maria; Giuliano, Callaini; Hyman, Anthony A.; Sattler, Michael; Gopalakrishnan, Jay; Li, Haitao

2016-01-01

Centrioles and cilia are microtubule-based structures, whose precise formation requires controlled cytoplasmic tubulin incorporation. How cytoplasmic tubulin is recognized for centriolar/ciliary-microtubule construction remains poorly understood. Centrosomal-P4.1-associated-protein (CPAP) binds tubulin via its PN2-3 domain. Here, we show that a C-terminal loop-helix in PN2-3 targets β-tubulin at the microtubule outer surface, while an N-terminal helical motif caps microtubule's α-β surface of β-tubulin. Through this, PN2-3 forms a high-affinity complex with GTP-tubulin, crucial for defining numbers and lengths of centriolar/ciliary-microtubules. Surprisingly, two distinct mutations in PN2-3 exhibit opposite effects on centriolar/ciliary-microtubule lengths. CPAPF375A, with strongly reduced tubulin interaction, causes shorter centrioles and cilia exhibiting doublet- instead of triplet-microtubules. CPAPEE343RR that unmasks the β-tubulin polymerization surface displays slightly reduced tubulin-binding affinity inducing over-elongation of newly forming centriolar/ciliary-microtubules by enhanced dynamic release of its bound tubulin. Thus CPAP regulates delivery of its bound-tubulin to define the size of microtubule-based cellular structures using a ‘clutch-like' mechanism. PMID:27306797

Homozygous STIL Mutation Causes Holoprosencephaly and Microcephaly in Two Siblings

PubMed Central

Mouden, Charlotte; de Tayrac, Marie; Dubourg, Christèle; Rose, Sophie; Carré, Wilfrid; Hamdi-Rozé, Houda; Babron, Marie-Claude; Akloul, Linda; Héron-Longe, Bénédicte; Odent, Sylvie; Dupé, Valérie; Giet, Régis; David, Véronique

2015-01-01

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. PMID:25658757

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

PubMed Central

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.

PubMed

Hoh, Ramona A; Stowe, Timothy R; Turk, Erin; Stearns, Tim

2012-01-01

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.

Transcriptional Program of Ciliated Epithelial Cells Reveals New Cilium and Centrosome Components and Links to Human Disease

PubMed Central

Hoh, Ramona A.; Stowe, Timothy R.; Turk, Erin; Stearns, Tim

2012-01-01

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. PMID:23300604

The NIMA-like kinase Nek2 is a key switch balancing cilia biogenesis and resorption in the development of left-right asymmetry.

PubMed

Endicott, S Joseph; Basu, Basudha; Khokha, Mustafa; Brueckner, Martina

2015-12-01

Vertebrate left-right (LR) asymmetry originates at a transient left-right organizer (LRO), a ciliated structure where cilia play a crucial role in breaking symmetry. However, much remains unknown about the choreography of cilia biogenesis and resorption at this organ. We recently identified a mutation affecting NEK2, a member of the NIMA-like serine-threonine kinase family, in a patient with congenital heart disease associated with abnormal LR development. Here, we report how Nek2 acts through cilia to influence LR patterning. Both overexpression and knockdown of nek2 in Xenopus result in abnormal LR development and reduction of LRO cilia count and motility, phenotypes that are modified by interaction with the Hippo signaling pathway. nek2 knockdown leads to a centriole defect at the LRO, consistent with the known role of Nek2 in centriole separation. Nek2 overexpression results in premature ciliary resorption in cultured cells dependent on function of the tubulin deacetylase Hdac6. Finally, we provide evidence that the known interaction between Nek2 and Nup98, a nucleoporin that localizes to the ciliary base, is important for regulating cilium resorption. Together, these data show that Nek2 is a switch balancing ciliogenesis and resorption in the development of LR asymmetry. © 2015. Published by The Company of Biologists Ltd.

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

PubMed

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.

Dissecting the Function and Assembly of Acentriolar Microtubule Organizing Centers in Drosophila Cells In Vivo

PubMed Central

Baumbach, Janina; Novak, Zsofia Anna; Raff, Jordan W.; Wainman, Alan

2015-01-01

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. PMID:26020779

CYTODIFFERENTIATION DURING SPERMIOGENESIS IN LUMBRICUS TERRESTRIS

PubMed Central

Anderson, W. A.; Weissman, A.; Ellis, R. A.

1967-01-01

The structural changes during spermiogenesis were studied on developing spermatids in seminal vesicles and receptacles of Lumbricus terrestris fixed in glutaraldehyde-osmium tetroxide and embedded in Epon-Araldite. The centriole plays a prominent role in the morphogenesis and organization of the microtubules of the manchette and flagellum. Microtubules arising from the centriole extend anteriorly to encase the developing middle piece, the nucleus, and the acrosome. The manchette not only provides a supporting framework for the cell during elongation, but also may provide the motive force for the elimination of both nucleoplasm and cytoplasm. The manchette participates in segregation and elimination of the nuclear vesicle that contains the nonchromatin nucleoplasm. Compartmentalization and conservation may also be a function of the manchette since those elements which remain within the framework of microtubules are retained, while all the cytoplasm outside the manchette is discarded. At maturation, the endoplasmic reticulum plays a key role in dismantling the manchette and reducing the cytoplasm external to it. During the early stages of middle-piece formation, six ovoid mitochondria aggregate at the posterior pole of the spermatid nucleus. Concurrent with manchette formation, the mitochondria are compressed laterally into elongate wedge-shaped components, and their outer limiting membranes fuse to form an hexagonal framework that surrounds the dense intramitochondrial matrices. Dense glycogen granules are arranged linearly between the peripheral flagellar tubules and the outer membrane of the mature sperm tail. PMID:10976199

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

PubMed Central

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

Hook2 is involved in the morphogenesis of the primary cilium

PubMed Central

Baron Gaillard, Carole L.; Pallesi-Pocachard, Emilie; Massey-Harroche, Dominique; Richard, Fabrice; Arsanto, Jean-Pierre; Chauvin, Jean-Paul; Lecine, Patrick; Krämer, Helmut; Borg, Jean-Paul; Le Bivic, André

2011-01-01

Primary cilia originate from the centrosome and play essential roles in several cellular, developmental, and pathological processes, but the underlying mechanisms of ciliogenesis are not fully understood. Given the involvement of the adaptor protein Hook2 in centrosomal homeostasis and protein transport to pericentrosomal aggresomes, we explored its role in ciliogenesis. We found that in human retinal epithelial cells, Hook2 localizes at the Golgi apparatus and centrosome/basal body, a strategic partitioning for ciliogenesis. Of importance, Hook2 depletion disrupts ciliogenesis at a stage before the formation of the ciliary vesicle at the distal tip of the mother centriole. Using two hybrid and immunoprecipitation assays and a small interfering RNA strategy, we found that Hook2 interacts with and stabilizes pericentriolar material protein 1 (PCM1), which was reported to be essential for the recruitment of Rab8a, a GTPase that is believed to be crucial for membrane transport to the primary cilium. Of interest, GFP::Rab8a coimmunoprecipitates with endogenous Hook2 and PCM1. Finally, GFP::Rab8a can overcome Hook2 depletion, demonstrating a functional interaction between Hook2 and these two important regulators of ciliogenesis. The data indicate that Hook2 interacts with PCM1 in a complex that also contains Rab8a and regulates a limiting step required for further initiation of ciliogenesis after centriole maturation. PMID:21998199

Sperm with fibrous sheath dysplasia and anomalies in head-neck junction: focus on centriole and centrin 1.

PubMed

Moretti, E; Pascarelli, N A; Belmonte, G; Renieri, T; Collodel, G

2017-09-01

Spermatozoa with a rare combination of two monomorphic sperm defects, dysplasia of the fibrous sheath (DFS) and alterations in head-mid-piece junction were analysed. The main focus was to explore the status of the centriole, a key organisation during fertilisation, using the centrin 1, a calcium-binding protein linked to this structure. The sperm quality was examined by light, scanning and transmission electron microscopy (SEM, TEM); immunocytochemistry was performed for tubulin, A-kinase anchor protein 4 (AKAP4) and centrin 1. Spermatozoa showed DFS defect associated with anomalies in head-tail attachment detected by SEM and TEM. Immunolocalisation of tubulin, AKAP4 and centrin 1 confirmed these alterations. Centrin 1 was visible in 67% of spermatozoa (in only 13% centrin localised in a normal position); in the majority of sperm centrin 1's location was altered, sometimes bent; often four spots, indicating the presence of two implantation fossae, were detected. At the centriolar level, immunoreactive fragments, frequently invading the entire short and thick tail, were observed. Centrin 1 is an essential component of the spermatozoa connecting piece and plays a role in centrosome dynamics during sperm morphogenesis and in zygotes and early embryos during spindle assembly. It is important to shed light on these rare conditions in order to better manage the patients during assisted reproductive technology. © 2016 Blackwell Verlag GmbH.

Coordinate regulation of the mother centriole component nlp by nek2 and plk1 protein kinases.

PubMed

Rapley, Joseph; Baxter, Joanne E; Blot, Joelle; Wattam, Samantha L; Casenghi, Martina; Meraldi, Patrick; Nigg, Erich A; Fry, Andrew M

2005-02-01

Mitotic entry requires a major reorganization of the microtubule cytoskeleton. Nlp, a centrosomal protein that binds gamma-tubulin, is a G(2)/M target of the Plk1 protein kinase. Here, we show that human Nlp and its Xenopus homologue, X-Nlp, are also phosphorylated by the cell cycle-regulated Nek2 kinase. X-Nlp is a 213-kDa mother centriole-specific protein, implicating it in microtubule anchoring. Although constant in abundance throughout the cell cycle, it is displaced from centrosomes upon mitotic entry. Overexpression of active Nek2 or Plk1 causes premature displacement of Nlp from interphase centrosomes. Active Nek2 is also capable of phosphorylating and displacing a mutant form of Nlp that lacks Plk1 phosphorylation sites. Importantly, kinase-inactive Nek2 interferes with Plk1-induced displacement of Nlp from interphase centrosomes and displacement of endogenous Nlp from mitotic spindle poles, while active Nek2 stimulates Plk1 phosphorylation of Nlp in vitro. Unlike Plk1, Nek2 does not prevent association of Nlp with gamma-tubulin. Together, these results provide the first example of a protein involved in microtubule organization that is coordinately regulated at the G(2)/M transition by two centrosomal kinases. We also propose that phosphorylation by Nek2 may prime Nlp for phosphorylation by Plk1.

Simulated hypogravity and synaptogenesis in culture

NASA Technical Reports Server (NTRS)

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.

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

NASA Astrophysics Data System (ADS)

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.

Interaction between ROCK II and nucleophosmin/B23 in the regulation of centrosome duplication.

PubMed

Ma, Zhiyong; Kanai, Masayuki; Kawamura, Kenji; Kaibuchi, Kozo; Ye, Keqiang; Fukasawa, Kenji

2006-12-01

Nucleophosmin (NPM)/B23 has been implicated in the regulation of centrosome duplication. NPM/B23 localizes between two centrioles in the unduplicated centrosome. Upon phosphorylation on Thr(199) by cyclin-dependent kinase 2 (CDK2)/cyclin E, the majority of centrosomal NPM/B23 dissociates from centrosomes, but some NPM/B23 phosphorylated on Thr(199) remains at centrosomes. It has been shown that Thr(199) phosphorylation of NPM/B23 is critical for the physical separation of the paired centrioles, an initial event of the centrosome duplication process. Here, we identified ROCK II kinase, an effector of Rho small GTPase, as a protein that localizes to centrosomes and physically interacts with NPM/B23. Expression of the constitutively active form of ROCK II promotes centrosome duplication, while down-regulation of ROCK II expression results in the suppression of centrosome duplication, especially delaying the initiation of centrosome duplication during the cell cycle. Moreover, ROCK II regulates centrosome duplication in its kinase and centrosome localization activity-dependent manner. We further found that ROCK II kinase activity is significantly enhanced by binding to NPM/B23 and that NPM/B23 acquires a higher binding affinity to ROCK II upon phosphorylation on Thr(199). Moreover, physical interaction between ROCK II and NPM/B23 in vivo occurs in association with CDK2/cyclin E activation and the emergence of Thr(199)-phosphorylated NPM/B23. All these findings point to ROCK II as the effector of the CDK2/cyclin E-NPM/B23 pathway in the regulation of centrosome duplication.

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

USGS Publications Warehouse

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.

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

PubMed

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.

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

PubMed Central

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

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hori, Akiko; Morand, Agathe; Ikebe, Chiho

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, asmore » 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.« less

Primary Cilia: Highly Sophisticated Biological Sensors

PubMed Central

Abou Alaiwi, Wissam A.; Lo, Shao T.; Nauli, Surya M.

2009-01-01

Primary cilia, thin hair-like structures protruding from the apical surface of most mammalian cells, have gained the attention of many researchers over the past decade. Primary cilia are microtubule-filled sensory organelles that are enclosed within the ciliary membrane. They originate at the cell surface from the mother centriole that becomes the mature basal body. In this review, we will discuss recent literatures on the roles of cilia as sophisticated sensory organelles. With particular emphasis on vascular endothelia and renal epithelia, the mechanosensory role of cilia in sensing fluid shear stress will be discussed. Also highlighted is the ciliary involvement in cell cycle regulation, development, cell signaling and cancer. Finally, primary cilia-related disorders will be briefly described. PMID:22423203

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

PubMed

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.

Parthenogenesis in non-rodent species: developmental competence and differentiation plasticity.

PubMed

Brevini, T A L; Pennarossa, G; Vanelli, A; Maffei, S; Gandolfi, F

2012-03-01

An oocyte can activate its developmental process without the intervention of the male counterpart. This form of reproduction, known as parthenogenesis, occurs spontaneously in a variety of lower organisms, but not in mammals. However, it must be noted that mammalian oocytes can be activated in vitro, mimicking the intracellular calcium wave induced by the spermatozoon at fertilization, which triggers cleavage divisions and embryonic development. The resultant parthenotes are not capable of developing to term and arrest their growth at different stages, depending on the species. It is believed that this arrest is due to genomic imprinting, which causes the repression of genes normally expressed by the paternal allele. Human parthenogenetic embryos have recently been proposed as an alternative, less controversial source of embryonic stem cell lines, based on their inherent inability to form a new individual. However many aspects related to the biology of parthenogenetic embryos and parthenogenetically derived cell lines still need to be elucidated. Limited information is available in particular on the consequences of the lack of centrioles and on the parthenote's ability to assemble a new embryonic centrosome in the absence of the sperm centriole. Indeed, in lower species, successful parthenogenesis largely depends upon the oocyte's ability to regenerate complete and functional centrosomes in the absence of the material supplied by a male gamete, while the control of this event appears to be less stringent in mammalian cells. In an attempt to better elucidate some of these aspects, parthenogenetic cell lines, recently derived in our laboratory, have been characterized for their pluripotency. In vitro and in vivo differentiation plasticity have been assessed, demonstrating the ability of these cells to differentiate into cell types derived from the three germ layers. These results confirmed common features between uni- and bi-parental embryonic stem cells. However data obtained with parthenogenetic cells indicate the presence of an intrinsic deregulation of the mechanisms controlling proliferation vs. differentiation and suggest their uni-parental origin as a possible cause. Copyright © 2012 Elsevier Inc. All rights reserved.

Electron microscopy observations on testis and spermatozoa of Leptodactylus chaquensis (Anura, Leptodactylidae).

PubMed

Iruzubieta Villagra, Lucrecia; Ramos, Inés; Cisint, Susana; Crespo, Claudia A; Fernández, Silvia N

2018-02-01

Scanning and transmission electron microscopy were used to investigate the fine structure of the testis and spermatozoa of toad Leptodactylus chaquensis. Our observations indicate that germinal compartment contains Sertoli (SC) and germ cells. The tight junctions and desmosomes among SC indicate the existence of an hematotesticular barrier in charge of maintaining the differences in the composition of the germinal and interstitial compartments. During spermatogenesis, SC acts as a structural support for germ cells. Secondary spermatogonias, spermatocytes and spermatids are joined by cytoplasmic bridges that allow communication between cells in the same cyst. Spermatids at the subcellular level two well-defined morphological stages can be observed: primary spermatids are rounded cells with an acrosomal vesicle attached to the nucleus which has a diameter of about 4.39±0.36μm. Secondary spermatids are elongated with a nucleus of about 19.50±0.92μm in diameter and the acrosome and the axoneme are located in opposite poles of the cells. At the apical end of the spermatozoon we can observe a large arrowhead-shaped acrosome (6.26±0.28μm in length) that takes up about one third of the gamete head with 17.33±0.29μm in length. The proximal centriole is located in the nuclear fossa while the distal centriole gives rise to the flagellar axoneme. The flagellum exhibits a typical pattern of "9+2" and adjacent to it is the axial fiber, and an undulating membrane stretches between both structures. Transmission electron microscopy observations allowed us to produce a diagram of the structure of the spermatozoon of L. chaquensis. Leydig cells, located in the interstitial compartment, show scarce cytoplasm, mitochondria and large-sized lipid droplets which would provide the raw matter for the synthesis of steroid hormones. Copyright © 2017 Elsevier Ltd. All rights reserved.

Fifteen years of research on oral-facial-digital syndromes: from 1 to 16 causal genes.

PubMed

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; Goldstein, 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

2017-06-01

Oral-facial-digital syndromes (OFDS) gather rare genetic disorders characterised 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 OFDS 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 cases with OFDS. We identified causal variants in five new genes ( C2CD3 , TMEM107 , INTU , KIAA0753 and IFT57 ) and related the clinical spectrum of four genes in other ciliopathies ( C5orf42 , TMEM138 , TMEM231 and 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 characterising three ciliary protein modules: the complex KIAA0753-FOPNL-OFD1, a regulator of centriole elongation; the Meckel-Gruber syndrome 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 three main subtypes, a further classification could be based on the genotype. © Article author(s) (or their employer(s) unless otherwise stated in the text of the article) 2017. All rights reserved. No commercial use is permitted unless otherwise expressly granted.

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

PubMed

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 subcellular structures in spermatozoa by lipid aldehydes. Copyright © 2017 Elsevier B.V. All rights reserved.

Opposing effects of pericentrin and microcephalin on the pericentriolar material regulate CHK1 activation in the DNA damage response.

PubMed

Antonczak, A K; Mullee, L I; Wang, Y; Comartin, D; Inoue, T; Pelletier, L; Morrison, C G

2016-04-14

Genotoxic stresses lead to centrosome amplification, a frequently-observed feature in cancer that may contribute to genome instability and to tumour cell invasion. Here we have explored how the centrosome controls DNA damage responses. For most of the cell cycle, centrosomes consist of two centrioles embedded in the proteinaceous pericentriolar material (PCM). Recent data indicate that the PCM is not an amorphous assembly of proteins, but actually a highly organised scaffold around the centrioles. The large coiled-coil protein, pericentrin, participates in PCM assembly and has been implicated in the control of DNA damage responses (DDRs) through its interactions with checkpoint kinase 1 (CHK1) and microcephalin (MCPH1). CHK1 is required for DNA damage-induced centrosome amplification, whereas MCPH1 deficiency greatly increases the amplification seen after DNA damage. We found that the PCM showed a marked expansion in volume and a noticeable change in higher-order organisation after ionising radiation treatment. PCM expansion was dependent on CHK1 kinase activity and was potentiated by MCPH1 deficiency. Furthermore, pericentrin deficiency or mutation of a separase cleavage site blocked DNA damage-induced PCM expansion. The extent of nuclear CHK1 activation after DNA damage reflected the level of PCM expansion, with a reduction in pericentrin-deficient or separase cleavage site mutant-expressing cells, and an increase in MCPH1-deficient cells that was suppressed by the loss of pericentrin. Deletion of the nuclear export signal of CHK1 led to its hyperphosphorylation after irradiation and reduced centrosome amplification. Deletion of the nuclear localisation signal led to low CHK1 activation and low centrosome amplification. From these data, we propose a feedback loop from the PCM to the nuclear DDR in which CHK1 regulates pericentrin-dependent PCM expansion to control its own activation.

Ultrastructure of spermatozoa of Onthophagus taurus (Coleoptera, Scarabaeidae) exhibits heritable variation

NASA Astrophysics Data System (ADS)

Werner, Michael; Simmons, Leigh W.

2011-03-01

Sperm competition is thought to be an important selective pressure shaping sperm form and function. However, few studies have moved beyond gross examinations of sperm morphology. Sperm length is subject to sexual selection via sperm competition in the scarab beetle Onthophagus taurus. Here, the structure and ultrastructure of spermatozoa in this species were investigated using light and electron microscopy. Spermatozoa were found to be filiform, measuring about 1,200 mm in length. The sperm head consists of a three-layered acrosome and a nuclear region bearing the anterior extension of the centriole adjunct. Acrosome and nuclear regions are bilaterally symmetric, with their axes of symmetry being orthogonal to each other. Head and flagellar structures are connected by a well-developed centriole adjunct. The sperm heads are asymmetrically surrounded by accessory material and embedded into the cytoplasm of the spermatocyst cell. The accessory material is produced inside the spermatids and then transferred to the outside due to a new membrane formed around the sperm's organelles. The old spermatid membrane separates the accessory material from the cyst cell. The flagellum contains a 9+9+2 axoneme, two accessory bodies, and two mitochondrial derivatives of unequal size. The major mitochondrial derivative is significantly larger than the minor one. The axoneme is arranged in a sinusoidal manner parallel along the major mitochondrial derivative. The spermatozoa show no progressive motility when released in buffer solution which is likely to be the result of the flagellar arrangement and the structure of the major mitochondrial derivative. The cross-sectional area of the minor and the major mitochondrial derivatives show different patterns of genetic variation. The data provide the first estimates of genetic variation in sperm ultrastructure for any species, and give evidence for the persistence of genetic variation in ultrastructure required for the rapid and divergent evolution that characterizes spermatozoa generally.

Ultrastrukturelle Untersuchungen zur Morphologie und Genese der Spermien von Archaeogastropoda

NASA Astrophysics Data System (ADS)

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.

Symbiosis and the origin of eukaryotic motility

NASA Technical Reports Server (NTRS)

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.

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

PubMed

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.

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

PubMed Central

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. PMID:29026530

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

PubMed

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 observed in cell cultures. This is the first study to demonstrate that defects in cilia expression and function are a true hallmark of GBM tumors and correlate with their unrestrained growth. A review of the current ultrastructural profiles in the literature provides suggestions as to the best possible candidate protein that underlies defects in the early stages of ciliogenesis within GBM tumors.

Evolution of centrosomes and the nuclear lamina: Amoebozoan assets.

PubMed

Gräf, Ralph; Batsios, Petros; Meyer, Irene

2015-06-01

The current eukaryotic tree of life groups most eukaryotes into one of five supergroups, the Opisthokonta, Amoebozoa, Archaeplastida, Excavata and SAR (Stramenopile, Alveolata, Rhizaria). Molecular and comparative morphological analyses revealed that the last eukaryotic common ancestor (LECA) already contained a rather sophisticated equipment of organelles including a mitochondrion, an endomembrane system, a nucleus with a lamina, a microtubule-organizing center (MTOC), and a flagellar apparatus. Recent studies of MTOCs, basal bodies/centrioles, and nuclear envelope organization of organisms in different supergroups have clarified our picture of how the nucleus and MTOCs co-evolved from LECA to extant eukaryotes. In this review we summarize these findings with special emphasis on valuable contributions of research on a lamin-like protein, nuclear envelope proteins, and the MTOC in the amoebozoan model organism Dictyostelium discoideum. Copyright © 2015 Elsevier GmbH. All rights reserved.

Regulation of spindle integrity and mitotic fidelity by BCCIP

PubMed Central

Huhn, S C; Liu, J; Ye, C; Lu, H; Jiang, X; Feng, X; Ganesan, S; White, E; Shen, Z

2017-01-01

Centrosomes together with the mitotic spindle ensure the faithful distribution of chromosomes between daughter cells, and spindle orientation is a major determinant of cell fate during tissue regeneration. Spindle defects are not only an impetus of chromosome instability but are also a cause of developmental disorders involving defective asymmetric cell division. In this work, we demonstrate BCCIP, especially BCCIPα, as a previously unidentified component of the mitotic spindle pole and the centrosome. We demonstrate that BCCIP localizes proximal to the mother centriole and participates in microtubule organization and then redistributes to the spindle pole to ensure faithful spindle architecture. We find that BCCIP depletion leads to morphological defects, disoriented mitotic spindles, chromosome congression defects and delayed mitotic progression. Our study identifies BCCIP as a novel factor critical for microtubule regulation and explicates a mechanism utilized by BCCIP in tumor suppression. PMID:28394342

The Rho-associated protein kinase p160ROCK is required for centrosome positioning

PubMed Central

Chevrier, Véronique; Piel, Matthieu; Collomb, Nora; Saoudi, Yasmina; Frank, Ronald; Paintrand, Michel; Narumiya, Shuh; Bornens, Michel; Job, Didier

2002-01-01

The p160–Rho-associated coiled-coil–containing protein kinase (ROCK) is identified as a new centrosomal component. Using immunofluorescence with a variety of p160ROCK antibodies, immuno EM, and depletion with RNA interference, p160ROCK is principally bound to the mother centriole (MC) and an intercentriolar linker. Inhibition of p160ROCK provoked centrosome splitting in G1 with the MC, which is normally positioned at the cell center and shows little motion during G1, displaying wide excursions around the cell periphery, similar to its migration toward the midbody during cytokinesis. p160ROCK inhibition late after anaphase in mitosis triggered MC migration to the midbody followed by completion of cell division. Thus, p160ROCK is required for centrosome positioning and centrosome-dependent exit from mitosis. PMID:12034773

Spermiogenesis in birds

PubMed Central

Aire, Tom A

2014-01-01

Current knowledge on avian spermiogenesis, including strengths and weaknesses, has been reviewed. Information on avian spermiogenesis considerably lags behind that in mammals because of the paucity of reports in birds. Spermiogenesis in passerine birds has received even much less attention than in non-passerine birds. Mechanisms underlying morphogenesis of the acrosome and nucleus, and roles of microtubular assemblies are poorly understood. The proximal centriole found in non-passerine birds, but hitherto considered to be absent in passerine birds, has recently been described in spermatids and mature spermatozoa of 2 passeridan species, including the Masked weaver for which new and detailed spermiogenetic information is provided in this review. A great deal more studies on spermiogenesis, and spermatogenesis generally, in various avian species are required to considerably enhance knowledge of this phenomenon, contribute to comparative spermatology, provide a basis for appropriate applied studies, and contribute to understanding of phylogeny in this vast order of vertebrates. PMID:26413401

Cilia/Ift protein and motor -related bone diseases and mouse models.

PubMed

Yuan, Xue; Yang, Shuying

2015-01-01

Primary cilia are essential cellular organelles projecting from the cell surface to sense and transduce developmental signaling. They are tiny but have complicated structures containing microtubule (MT)-based internal structures (the axoneme) and mother centriole formed basal body. Intraflagellar transport (Ift) operated by Ift proteins and motors are indispensable for cilia formation and function. Mutations in Ift proteins or Ift motors cause various human diseases, some of which have severe bone defects. Over the last few decades, major advances have occurred in understanding the roles of these proteins and cilia in bone development and remodeling by examining cilia/Ift protein-related human diseases and establishing mouse transgenic models. In this review, we describe current advances in the understanding of the cilia/Ift structure and function. We further summarize cilia/Ift-related human diseases and current mouse models with an emphasis on bone-related phenotypes, cilia morphology, and signaling pathways.

[On the nervous system of a parasitic cnidarian Polypodium hydriforme].

PubMed

Raĭkova, E V

2013-01-01

Nerve cells in a parasitic cnidarian Polypodium hydriforme at the parasitic and free-living stages of the life cycle have been localized immunocytochemically using antibodies to FMRF-amide, and their ultrastructure has been described. Ganglion cells form a net under epidermis consisting of bi- and tripolar neurons which cross the mesoglea and usually contact muscle cells and cnidocytes. Fusiform sensory and neurosecretory cells, especially characteristic to sensory tentacles, are interspersed among epidermal cells. All three types of nerve cells have dense cored vesicles about 80-120 nm in diameter. The sensory cells demonstrate a sensory flagellum-like immobile structure. Neurosecretory and sensory cells form septate junctions with epidermal cells. Ganglion cells show gap junctions between them. A centriole encircled by a fragment of nuclear envelope which is a marker of ectodermal lineage cells in Polypodium has been described in the cytoplasm of a sensory cell, thus proving the ectodermal nature of the nervous system.

Ploidy Manipulation of Zebrafish Embryos with Heat Shock 2 Treatment

PubMed Central

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

Phylogeny of the Acanthocephala based on morphological characters.

PubMed

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 provide strong support for the Palaeacanthocephala and the Eoacanthocephala and the hypothesis that the Eoacanthocephala is the most primitive group is not supported. Little support for the Archiacanthocephala as a monophyletic group was provided by the analysis. Support is provided for the recognition of Tegorhynchus and Illiosentis as distinct taxa, as well as the transfer of T. pectinarius to Koronacantha.

The Centrosome and Its Duplication Cycle

PubMed Central

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

A comparative ultrastructural study of spermatozoa of the teiid lizards Cnemidophorus gularis gularis, Cnemidophorus ocellifer, and Kentropyx altamazonica (Reptilia, Squamata, Teiidae).

PubMed

Teixeira, R D; Scheltinga, D M; Trauth, S E; Colli, G R; Báo, S N

2002-06-01

The ultrastructure of the spermatozoa of Cnemidophorus gularis gularis, Cnemidophorus ocellifer, and Kentropyx altamazonica is described for the first time. Mature spermatozoa of Cnemidophorus spp. and K. altamazonica differ in the occurrence of a perforatorial base plate, the enlargement of axonemal fibers 3 and 8, and shape of mitochondria. The comparisons of the ultrastructure sperm of Cnemidophorus spp. and K. altamazonica with Ameiva ameiva [J. Morphol. (2002) in press] suggest that Ameiva and Cnemidophorus are more similar to each other than either is to Kentropyx. Statistical analyses reveal that sperm of all three species studied are significantly different in the following dimensions: head, acrosome, distal centriole length, and nuclear shoulders width. There was no variable statistically different between the Cnemidophorus spp. only. The length of the tail, midpiece, entire sperm, and nuclear rostrum are significantly different between K. altamazonica and Cnemidophorus spp. Our results indicate that sperm ultrastructure presents intra and intergeneric variability.

GAK, a regulator of clathrin-mediated membrane traffic, also controls centrosome integrity and chromosome congression.

PubMed

Shimizu, Hiroyuki; Nagamori, Ippei; Yabuta, Norikazu; Nojima, Hiroshi

2009-09-01

Cyclin G-associated kinase (GAK) is an association partner of clathrin heavy chain (CHC) and is essential for clathrin-mediated membrane trafficking. Here, we report two novel functions of GAK: maintenance of proper centrosome maturation and of mitotic chromosome congression. Indeed, GAK knockdown by siRNA caused cell-cycle arrest at metaphase, which indicates that GAK is required for proper mitotic progression. We found that this impaired mitotic progression was due to activation of the spindle-assembly checkpoint, which senses protruded, misaligned or abnormally condensed chromosomes in GAK-siRNA-treated cells. GAK knockdown also caused multi-aster formation, which was due to abnormal fragmentation of pericentriolar material, but not of the centrioles. Moreover, GAK and CHC cooperated in the same pathway and interacted in mitosis to regulate the formation of a functional spindle. Taken together, we conclude that GAK and clathrin function cooperatively not only in endocytosis, but also in mitotic progression.

Three-dimensional structure of basal body triplet revealed by electron cryo-tomography

PubMed Central

Li, Sam; Fernandez, Jose-Jesus; Marshall, Wallace F; Agard, David A

2012-01-01

Basal bodies and centrioles play central roles in microtubule (MT)-organizing centres within many eukaryotes. They share a barrel-shaped cylindrical structure composed of nine MT triplet blades. Here, we report the structure of the basal body triplet at 33 Å resolution obtained by electron cryo-tomography and 3D subtomogram averaging. By fitting the atomic structure of tubulin into the EM density, we built a pseudo-atomic model of the tubulin protofilaments at the core of the triplet. The 3D density map reveals additional densities that represent non-tubulin proteins attached to the triplet, including a large inner circular structure in the basal body lumen, which functions as a scaffold to stabilize the entire basal body barrel. We found clear longitudinal structural variations along the basal body, suggesting a sequential and coordinated assembly mechanism. We propose a model in which δ-tubulin and other components participate in the assembly of the basal body. PMID:22157822

Spermatozoon structure and motility in the anuran Lepidobatrachus laevis.

PubMed

Waggener, W L; Carroll, E J

1998-02-01

Synthetic human gonadotropin releasing hormone (GnRH) injections were used for induction of spermatozoon release followed by cloacal lavage or mechanical stimulation of sperm release in Lepidobatrachus laevis. Light microscopic observations of Lepidobatrachus laevis spermatozoa indicated an acrosomal segment with a length of 4.1 microm delineated by an indentation, a nuclear region of 12.6 microm in length and a midpiece of 0.87 microm in length. The tail was 54.9 microm long by 1.35 microm wide with two lateral axial fibers and a central undulating membrane. At the electron microscopic level, the unusual tail had two complete axonemes that emanated from the distal centriole. The tail also contained two axial fibers 77 nm in diameter medial to the axonemes and was connected by an undulating membrane. An unusual accessory cell adherent to the head of the spermatozoon was noted in freshly obtained suspensions of spermatozoa. Spermatozoa with the accessory cell were motile and a subsequent loss of motility was correlated with the shedding of the accessory cell.

Shugoshins function as a guardian for chromosomal stability in nuclear division.

PubMed

Yao, Yixin; Dai, Wei

2012-07-15

Accurate chromosome segregation during mitosis and meiosis is regulated and secured by several distinctly different yet intricately connected regulatory mechanisms. As chromosomal instability is a hallmark of a majority of tumors as well as a cause of infertility for germ cells, extensive research in the past has focused on the identification and characterization of molecular components that are crucial for faithful chromosome segregation during cell division. Shugoshins, including Sgo1 and Sgo2, are evolutionarily conserved proteins that function to protect sister chromatid cohesion, thus ensuring chromosomal stability during mitosis and meiosis in eukaryotes. Recent studies reveal that Shugoshins in higher animals play an essential role not only in protecting centromeric cohesion of sister chromatids and assisting bi-orientation attachment at the kinetochores, but also in safeguarding centriole cohesion/engagement during early mitosis. Many molecular components have been identified that play essential roles in modulating/mediating Sgo functions. This review primarily summarizes recent advances on the mechanisms of action of Shugoshins in suppressing chromosomal instability during nuclear division in eukaryotic organisms.

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

PubMed

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.

CEP63 deficiency promotes p53-dependent microcephaly and reveals a role for the centrosome in meiotic recombination.

PubMed

Marjanović, Marko; Sánchez-Huertas, Carlos; Terré, Berta; Gómez, Rocío; Scheel, Jan Frederik; Pacheco, Sarai; Knobel, Philip A; Martínez-Marchal, Ana; Aivio, Suvi; Palenzuela, Lluís; Wolfrum, Uwe; McKinnon, Peter J; Suja, José A; Roig, Ignasi; Costanzo, Vincenzo; Lüders, Jens; Stracker, Travis H

2015-07-09

CEP63 is a centrosomal protein that facilitates centriole duplication and is regulated by the DNA damage response. Mutations in CEP63 cause Seckel syndrome, a human disease characterized by microcephaly and dwarfism. Here we demonstrate that Cep63-deficient mice recapitulate Seckel syndrome pathology. The attrition of neural progenitor cells involves p53-dependent cell death, and brain size is rescued by the deletion of p53. Cell death is not the result of an aberrant DNA damage response but is triggered by centrosome-based mitotic errors. In addition, Cep63 loss severely impairs meiotic recombination, leading to profound male infertility. Cep63-deficient spermatocytes display numerical and structural centrosome aberrations, chromosome entanglements and defective telomere clustering, suggesting that a reduction in centrosome-mediated chromosome movements underlies recombination failure. Our results provide novel insight into the molecular pathology of microcephaly and establish a role for the centrosome in meiotic recombination.

CEP63 deficiency promotes p53-dependent microcephaly and reveals a role for the centrosome in meiotic recombination

PubMed Central

Marjanović, Marko; Sánchez-Huertas, Carlos; Terré, Berta; Gómez, Rocío; Scheel, Jan Frederik; Pacheco, Sarai; Knobel, Philip A.; Martínez-Marchal, Ana; Aivio, Suvi; Palenzuela, Lluís; Wolfrum, Uwe; McKinnon, Peter J.; Suja, José A.; Roig, Ignasi; Costanzo, Vincenzo; Lüders, Jens; Stracker, Travis H.

2015-01-01

CEP63 is a centrosomal protein that facilitates centriole duplication and is regulated by the DNA damage response. Mutations in CEP63 cause Seckel syndrome, a human disease characterized by microcephaly and dwarfism. Here we demonstrate that Cep63 deficient mice recapitulate Seckel syndrome pathology. The attrition of neural progenitor cells involves p53-dependent cell death and brain size is rescued by the deletion of p53. Cell death is not the result of an aberrant DNA damage response but is triggered by centrosome-based mitotic errors. In addition, Cep63 loss severely impairs meiotic recombination, leading to profound male infertility. Cep63 deficient spermatocytes display numerical and structural centrosome aberrations, chromosome entanglements and defective telomere clustering, suggesting that a reduction in centrosome-mediated chromosome movements underlies recombination failure. Our results provide novel insight into the molecular pathology of microcephaly and establish a role for the centrosome in meiotic recombination. PMID:26158450

POC1A Truncation Mutation Causes a Ciliopathy in Humans Characterized by Primordial Dwarfism

PubMed Central

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.

2012-01-01

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. PMID:22840364

Spermatological characters of the pseudophyllidean cestode Bothriocephalus scorpii (Müller, 1776).

PubMed

Levron, Céline; Brunanská, Magdaléna; Poddubnaya, Larisa G

2006-06-01

Spermiogenesis of Bothriocephalus scorpii (Cestoda, Pseudophyllidea) includes an orthogonal development of two flagella, followed by a flagellar rotation and a proximo-distal fusion with the median cytoplasmic process. The fusion occurs at the level of four attachment zones. The presence of dense material in the apical region of the differentiation zone in the early stage of spermiogenesis appears to be a characteristic feature for the Pseudophyllidea. The mature spermatozoon possesses two axonemes of 9+"1" pattern of the Trepaxonemata, nucleus, cortical microtubules, electron-dense granules and crested body. The anterior part of the gamete exhibits a centriole surrounded by electron-dense tubular structures arranged as incomplete spiral. When the crested body disappears, the electron-dense tubular structures are arranged into a ring encircling the axoneme. The electron-dense tubular structures and their arrangement appear to be a specific feature for the clade "Bothriocephalidea". The organization of the posterior extremity of the gamete with the nucleus is described for the first time in the Pseudophyllidea.

A Conserved Role for Girdin in Basal Body Positioning and Ciliogenesis.

PubMed

Nechipurenko, Inna V; Olivier-Mason, Anique; Kazatskaya, Anna; Kennedy, Julie; McLachlan, Ian G; Heiman, Maxwell G; Blacque, Oliver E; Sengupta, Piali

2016-09-12

Primary cilia are ubiquitous sensory organelles that mediate diverse signaling pathways. Cilia position on the cell surface is determined by the location of the basal body (BB) that templates the cilium. The mechanisms that regulate BB positioning in the context of ciliogenesis are largely unknown. Here we show that the conserved signaling and scaffolding protein Girdin localizes to the proximal regions of centrioles and regulates BB positioning and ciliogenesis in Caenorhabditis elegans sensory neurons and human RPE-1 cells. Girdin depletion alters localization of the intercentriolar linker and ciliary rootlet component rootletin, and rootletin knockdown in RPE-1 cells mimics Girdin-dependent phenotypes. C. elegans Girdin also regulates localization of the apical junction component AJM-1, suggesting that in nematodes Girdin may position BBs via rootletin- and AJM-1-dependent anchoring to the cytoskeleton and plasma membrane, respectively. Together, our results describe a conserved role for Girdin in BB positioning and ciliogenesis. Copyright © 2016 Elsevier Inc. All rights reserved.

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

NASA Astrophysics Data System (ADS)

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'.

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

NASA Astrophysics Data System (ADS)

Reunov, Arkadiy; Klepal, Waltraud

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'.

Mutations in the Katnb1 gene cause left-right asymmetry and heart defects.

PubMed

Furtado, Milena B; Merriner, D Jo; Berger, Silke; Rhodes, Danielle; Jamsai, Duangporn; O'Bryan, Moira K

2017-12-01

The microtubule-severing protein complex katanin is composed two subunits, the ATPase subunit, KATNA1, and the noncatalytic regulatory subunit, KATNB1. Recently, the Katnb1 gene has been linked to infertility, regulation of centriole and cilia formation in fish and mammals, as well as neocortical brain development. KATNB1 protein is expressed in germ cells in humans and mouse, mitotic/meiotic spindles and cilia, although the full expression pattern of the Katnb1 gene has not been described. Using a knockin-knockout mouse model of Katnb1 dysfunction we demonstrate that Katnb1 is ubiquitously expressed during embryonic development, although a stronger expression is seen in the crown cells of the gastrulation organizer, the murine node. Furthermore, null and hypomorphic Katnb1 gene mutations show a novel correlation between Katnb1 dysregulation and the development of impaired left-right signaling, including cardiac malformations. Katanin function is a critical regulator of heart development in mice. These findings are potentially relevant to human cardiac development. Developmental Dynamics 246:1027-1035, 2017. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

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

PubMed

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.

In silico prediction of a disease-associated STIL mutant and its affect on the recruitment of centromere protein J (CENPJ).

PubMed

Kumar, Ambuj; Rajendran, Vidya; Sethumadhavan, Rao; Purohit, Rituraj

2012-01-01

Human STIL (SCL/TAL1 interrupting locus) protein maintains centriole stability and spindle pole localisation. It helps in recruitment of CENPJ (Centromere protein J)/CPAP (centrosomal P4.1-associated protein) and other centrosomal proteins. Mutations in STIL protein are reported in several disorders, especially in deregulation of cell cycle cascades. In this work, we examined the non-synonymous single nucleotide polymorphisms (nsSNPs) reported in STIL protein for their disease association. Different SNP prediction tools were used to predict disease-associated nsSNPs. Our evaluation technique predicted rs147744459 (R242C) as a highly deleterious disease-associated nsSNP and its interaction behaviour with CENPJ protein. Molecular modelling, docking and molecular dynamics simulation were conducted to examine the structural consequences of the predicted disease-associated mutation. By molecular dynamic simulation we observed structural consequences of R242C mutation which affects interaction of STIL and CENPJ functional domains. The result obtained in this study will provide a biophysical insight into future investigations of pathological nsSNPs using a computational platform.

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

PubMed

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.

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

Spence, Jeffrey R.; Go, Minjoung M.; Bahmanyar, S.

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,more » 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.« less

Chirality of the cytoskeleton in the origins of cellular asymmetry

PubMed Central

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

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

NASA Astrophysics Data System (ADS)

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.

Functional genome-wide siRNA screen identifies KIAA0586 as mutated in Joubert syndrome

PubMed Central

Roosing, Susanne; Hofree, Matan; Kim, Sehyun; Scott, Eric; Copeland, Brett; Romani, Marta; Silhavy, Jennifer L; Rosti, Rasim O; Schroth, Jana; Mazza, Tommaso; Miccinilli, Elide; Zaki, Maha S; Swoboda, Kathryn J; Milisa-Drautz, Joanne; Dobyns, William B; Mikati, Mohamed A; İncecik, Faruk; Azam, Matloob; Borgatti, Renato; Romaniello, Romina; Boustany, Rose-Mary; Clericuzio, Carol L; D'Arrigo, Stefano; Strømme, Petter; Boltshauser, Eugen; Stanzial, Franco; Mirabelli-Badenier, Marisol; Moroni, Isabella; Bertini, Enrico; Emma, Francesco; Steinlin, Maja; Hildebrandt, Friedhelm; Johnson, Colin A; Freilinger, Michael; Vaux, Keith K; Gabriel, Stacey B; Aza-Blanc, Pedro; Heynen-Genel, Susanne; Ideker, Trey; Dynlacht, Brian D; Lee, Ji Eun; Valente, Enza Maria; Kim, Joon; Gleeson, Joseph G

2015-01-01

Defective primary ciliogenesis or cilium stability forms the basis of human ciliopathies, including Joubert syndrome (JS), with defective cerebellar vermis development. We performed a high-content genome-wide small interfering RNA (siRNA) screen to identify genes regulating ciliogenesis as candidates for JS. We analyzed results with a supervised-learning approach, using SYSCILIA gold standard, Cildb3.0, a centriole siRNA screen and the GTex project, identifying 591 likely candidates. Intersection of this data with whole exome results from 145 individuals with unexplained JS identified six families with predominantly compound heterozygous mutations in KIAA0586. A c.428del base deletion in 0.1% of the general population was found in trans with a second mutation in an additional set of 9 of 163 unexplained JS patients. KIAA0586 is an orthologue of chick Talpid3, required for ciliogenesis and Sonic hedgehog signaling. Our results uncover a relatively high frequency cause for JS and contribute a list of candidates for future gene discoveries in ciliopathies. DOI: http://dx.doi.org/10.7554/eLife.06602.001 PMID:26026149

Connections of intermediate filaments with the nuclear lamina and the cell periphery.

PubMed

Katsuma, Y; Swierenga, S H; Marceau, N; French, S W

1987-01-01

We investigated the relationship between intermediate filaments (IFs) and other detergent- and nuclease-resistant filamentous structures of cultured liver epithelial cells (T51B cell line) using whole mount unembedded preparations which were sequentially extracted with Triton X-100 and nucleases. Immunogold labelling and stereoscopic observation facilitated the examination of each filamentous structure and their three-dimensional relationships to each other. After solubilizing phospholipid, nucleic acid and soluble cellular protein, the resulting cytoskeleton preparation consisted of a network of cytokeratin and vimentin IFs linked by 3 nm filaments. The IFs were anchored to and determined the position of the nuclear lamina filaments (NLF) network and the centrioles. The NLF was composed of the nuclear lamina filaments measuring 3-6 nm in diameter which radiated from and anchored to the skeleton of the nuclear pores. The IFs located in the nuclear region appeared to be interwoven with the NLF. At the cell surface, the IFs seemed to be attached to the putative actin filament network. They formed a focally interrupted plexus-like structure at the cell periphery. Fragments of vimentin filaments were found among the filamentous network located at the cell surface, and some filaments terminated blindly there.

Preformed cell structure and cell heredity

PubMed Central

2008-01-01

This review will first recall the phenomena of “cortical inheritance” observed and genetically demonstrated in Paramecium 40 years ago, and later in other ciliates (Tetrahymena, Oxytricha, Paraurostyla), and will analyze the deduced concept of “cytotaxis” or “structural memory.” The significance of these phenomena, all related (but not strictly restricted) to the properties of ciliary basal bodies and their mode of duplication, will be interpreted in the light of present knowledge on the mechanism and control of basal body/centriole duplication. Then other phenomena described in a variety of organisms will be analyzed or mentioned which show the relevance of the concept of cytotaxis to other cellular processes, mainly (1) cytoskeleton assembly and organization with examples on ciliates, trypanosome, mammalian cells and plants, and (2) transmission of polarities with examples on yeast, trypanosome and metazoa. Finally, I will discuss some aspects of this particular type of non-DNA inheritance: (1) why so few documented examples if structural memory is a basic parameter in cell heredity, and (2) how are these phenomena (which all rely on protein/protein interactions, and imply a formatting role of preexisting proteinic complexes on neo-formed proteins and their assembly) related to prions? PMID:19164887

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

PubMed Central

Richens, Jennifer H.; Barros, Teresa P.; Lucas, Eliana P.; Peel, Nina; Pinto, David Miguel Susano; Wainman, Alan; Raff, Jordan W.

2015-01-01

ABSTRACT 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. PMID:26157019

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

PubMed

Richens, Jennifer H; Barros, Teresa P; Lucas, Eliana P; Peel, Nina; Pinto, David Miguel Susano; Wainman, Alan; Raff, Jordan W

2015-07-08

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. © 2015. Published by The Company of Biologists Ltd.

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

PubMed

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.

Caenorhabditis elegans glutamylating enzymes function redundantly in male mating.

PubMed

Chawla, Daniel G; Shah, Ruchi V; Barth, Zachary K; Lee, Jessica D; Badecker, Katherine E; Naik, Anar; Brewster, Megan M; Salmon, Timothy P; Peel, Nina

2016-09-15

Microtubule glutamylation is an important modulator of microtubule function and has been implicated in the regulation of centriole stability, neuronal outgrowth and cilia motility. Glutamylation of the microtubules is catalyzed by a family of tubulin tyrosine ligase-like (TTLL) enzymes. Analysis of individual TTLL enzymes has led to an understanding of their specific functions, but how activities of the TTLL enzymes are coordinated to spatially and temporally regulate glutamylation remains relatively unexplored. We have undertaken an analysis of the glutamylating TTLL enzymes in C. elegans We find that although all five TTLL enzymes are expressed in the embryo and adult worm, loss of individual enzymes does not perturb microtubule function in embryonic cell divisions. Moreover, normal dye-filling, osmotic avoidance and male mating behavior indicate the presence of functional amphid cilia and male-specific neurons. A ttll-4(tm3310); ttll-11(tm4059); ttll-5(tm3360) triple mutant, however, shows reduced male mating efficiency due to a defect in the response step, suggesting that these three enzymes function redundantly, and that glutamylation is required for proper function of the male-specific neurons. © 2016. Published by The Company of Biologists Ltd.

SPERMIOGENESIS IN THE CRAYFISH (PROCAMBARUS CLARKII)

PubMed Central

Moses, Montrose J.

1961-01-01

The sperm of the crayfish, Procambarus clarkii, is relatively simple among decapod sperm and was described in the first paper of this series (28). The present paper details the development of this sperm as followed with the light and electron microscopes. The process is divided into six stages for purposes of description. The main points of interest discussed are the absence of mitochondria or mitochondrial derivatives in the mature sperm, the development of a complex acrosome in the absence of highly organized characteristic Golgi apparatus but in the presence of small stacks of annulate lamellae, and the changes in the nucleus. Of the latter, the elaborate convoluted sheets of membrane that are extensions of the nuclear envelope are unique. The nucleus undergoes unusual changes in size and shape that are accompanied by several phases of organization of the chromatin. In the mature sperm the nucleus is empty-appearing and notably lacking in any apparent high degree of order. The entire development of the sperm is consonant with the idea that the fate of the mitochondria and centrioles, structures that figure prominently in the elaborate architecture of flagellate sperm, is associated with the lack of a flagellum. PMID:13773055

The origin of the eukaryotic cell

NASA Technical Reports Server (NTRS)

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.

Translocation of Tektin 3 to the equatorial segment of heads in bull spermatozoa exposed to dibutyryl cAMP and calyculin A.

PubMed

Tsukamoto, Mariko; Hiyama, Erina; Hirotani, Karen; Gotoh, Takafumi; Inai, Tetsuichiro; Iida, Hiroshi

2017-01-01

Tektins (TEKTs) are filamentous proteins associated with microtubules in cilia, flagella, basal bodies, and centrioles. Five TEKTs (TEKT1, -2, -3, -4, and -5) have been identified as components of mammalian sperm flagella. We previously reported that TKET1 and -3 are also present in the heads of rodent spermatozoa. The present study clearly demonstrates that TEKT2 is present at the acrosome cap whereas TEKT3 resides just beneath the plasma membrane of the post-acrosomal region of sperm heads in unactivated bull spermatozoa, and builds on the distributional differences of TEKT1, -2, and -3 on sperm heads. We also discovered that hyperactivation of bull spermatozoa by cell-permeable cAMP and calyculin A, a protein phosphatase inhibitor, promoted translocation of TEKT3 from the post-acrosomal region to the equatorial segment in sperm heads, and that TEKT3 accumulated at the equatorial segment is lost upon acrosome reaction. Thus, translocation of TEKT3 to the equatorial segment may be a capacitation- or hyperactivation-associated phenomenon in bull spermatozoa. Mol. Reprod. Dev. 84: 30-43, 2017. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

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

NASA Technical Reports Server (NTRS)

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.

An essential role of the basal body protein SAS-6 in Plasmodium male gamete development and malaria transmission

PubMed Central

Marques, Sara R; Ramakrishnan, Chandra; Carzaniga, Raffaella; Blagborough, Andrew M; Delves, Michael J; Talman, Arthur M; Sinden, Robert E

2015-01-01

Gametocytes are the sole Plasmodium parasite stages that infect mosquitoes; therefore development of functional gametes is required for malaria transmission. Flagellum assembly of the Plasmodium male gamete differs from that of most other eukaryotes in that it is intracytoplasmic but retains a key conserved feature: axonemes assemble from basal bodies. The centriole/basal body protein SAS-6 normally regulates assembly and duplication of these organelles and its depletion causes severe flagellar/ciliary abnormalities in a diverse array of eukaryotes. Since basal body and flagellum assembly are intimately coupled to male gamete development in Plasmodium, we hypothesized that SAS-6 disruption may cause gametogenesis defects and perturb transmission. We show that Plasmodium berghei sas6 knockouts display severely abnormal male gametogenesis presenting reduced basal body numbers, axonemal assembly defects and abnormal nuclear allocation. The defects in gametogenesis reduce fertilization and render Pbsas6 knockouts less infectious to mosquitoes. Additionally, we show that lack of Pbsas6 blocks transmission from mosquito to vertebrate host, revealing an additional yet undefined role in ookinete to sporulating oocysts transition. These findings underscore the vulnerability of the basal body/SAS-6 to malaria transmission blocking interventions. PMID:25154861

Blastocyst production after intracytoplasmic sperm injection with semen from a stallion with testicular degeneration.

PubMed

Roels, K; Smits, K; Ververs, C; Govaere, J; D'Herde, K; Van Soom, A

2018-06-01

In horse breeding, intracytoplasmic sperm injection (ICSI) has gained interest to obtain offspring from subfertile individuals. This paper presents a case report of a stallion with severe testicular degeneration. Semen analysis showed very low motility and 83.5% of detached heads. Histology of a testicular biopsy showed severely decreased spermatogenesis, while transmission electron microscopy of the sperm cells revealed no significant abnormalities. A total of 39 oocytes were fertilized by ICSI with frozen-thawed spermatozoa of this stallion: 25 oocytes with intact spermatozoa and 24 with detached heads. When using intact sperm cells, 8 out of the 25 oocytes cleaved, and 1 developed to the blastocyst stage 9 days after ICSI. None of the oocytes injected with a detached sperm head cleaved. Studies on the paternal influence on ICSI outcome are limited in the horse and further research is needed to define which stallion factors may influence ICSI results. Here, we report the possibility to produce a blastocyst by ICSI of a stallion suffering from testicular degeneration with a poor spermiogram, as long as an intact sperm cell containing a centriole is selected. © 2018 Blackwell Verlag GmbH.

Structural insights into SAM domain‐mediated tankyrase oligomerization

PubMed Central

DaRosa, Paul A.; Ovchinnikov, Sergey

2016-01-01

Abstract Tankyrase 1 (TNKS1; a.k.a. ARTD5) and tankyrase 2 (TNKS2; a.k.a ARTD6) are highly homologous poly(ADP‐ribose) polymerases (PARPs) that function in a wide variety of cellular processes including Wnt signaling, Src signaling, Akt signaling, Glut4 vesicle translocation, telomere length regulation, and centriole and spindle pole maturation. Tankyrase proteins include a sterile alpha motif (SAM) domain that undergoes oligomerization in vitro and in vivo. However, the SAM domains of TNKS1 and TNKS2 have not been structurally characterized and the mode of oligomerization is not yet defined. Here we model the SAM domain‐mediated oligomerization of tankyrase. The structural model, supported by mutagenesis and NMR analysis, demonstrates a helical, homotypic head‐to‐tail polymer that facilitates TNKS self‐association. Furthermore, we show that TNKS1 and TNKS2 can form (TNKS1 SAM‐TNKS2 SAM) hetero‐oligomeric structures mediated by their SAM domains. Though wild‐type tankyrase proteins have very low solubility, model‐based mutations of the SAM oligomerization interface residues allowed us to obtain soluble TNKS proteins. These structural insights will be invaluable for the functional and biophysical characterization of TNKS1/2, including the role of TNKS oligomerization in protein poly(ADP‐ribosyl)ation (PARylation) and PARylation‐dependent ubiquitylation. PMID:27328430

Structural insights into SAM domain-mediated tankyrase oligomerization.

PubMed

DaRosa, Paul A; Ovchinnikov, Sergey; Xu, Wenqing; Klevit, Rachel E

2016-09-01

Tankyrase 1 (TNKS1; a.k.a. ARTD5) and tankyrase 2 (TNKS2; a.k.a ARTD6) are highly homologous poly(ADP-ribose) polymerases (PARPs) that function in a wide variety of cellular processes including Wnt signaling, Src signaling, Akt signaling, Glut4 vesicle translocation, telomere length regulation, and centriole and spindle pole maturation. Tankyrase proteins include a sterile alpha motif (SAM) domain that undergoes oligomerization in vitro and in vivo. However, the SAM domains of TNKS1 and TNKS2 have not been structurally characterized and the mode of oligomerization is not yet defined. Here we model the SAM domain-mediated oligomerization of tankyrase. The structural model, supported by mutagenesis and NMR analysis, demonstrates a helical, homotypic head-to-tail polymer that facilitates TNKS self-association. Furthermore, we show that TNKS1 and TNKS2 can form (TNKS1 SAM-TNKS2 SAM) hetero-oligomeric structures mediated by their SAM domains. Though wild-type tankyrase proteins have very low solubility, model-based mutations of the SAM oligomerization interface residues allowed us to obtain soluble TNKS proteins. These structural insights will be invaluable for the functional and biophysical characterization of TNKS1/2, including the role of TNKS oligomerization in protein poly(ADP-ribosyl)ation (PARylation) and PARylation-dependent ubiquitylation. © 2016 The Protein Society.

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

NASA Astrophysics Data System (ADS)

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.

ELECTRON MICROSCOPY OF MITOSIS IN A RADIOSENSITIVE GIANT AMOEBA

DOE Office of Scientific and Technical Information (OSTI.GOV)

Daniels, E.W.; Roth, L.E.

1962-10-01

Pelomyxa illinoisensis amoebae, the large radiosensitive species, were fixed in OsO/sub 4/ and embedded in either Epon 812 or methacrylate. Ultrastructural morphology is demonstrated in subnuclear structures at interphase and during specific times in mitosis. Evidence of nuclear envelope breakdown and reconstruction is presented. Fragments of nuclear envelope membranes are traced throughout metaphase and anaphase to telophase. Annuli in the nuclear envelope and its fragments are demonstrated. P. illinoisensis is unique in mitochondrial arrangement during metaphase and anaphase-- mitochondria are aligned at the ends of fibrils distal to the chromosomes at the positions occupied by centrioles in other types ofmore » cells; there they remain until the end of anaphase. The radioresistant amoebae, Pelomyxa carolinensis and Amoeba proteus do not have polar mitochondria. P. illinoisensis also differs from the two radioresistant species in nucleolar morphology during interphase, and in the manner of nucleolar dissolution during prophase. On the other hand, helical coils are shown in the interphase nucleoplasm which appear similar to those in the radioresistant amoebae, P. carolinensis and A. groteus. A blister stage in the telophase of P. illinoisensis is described which is interpreted to be the result of a rapid nuclear expansion leading to interphase. This has not been observed in the radioresistant amoebae. (auth)« less

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

PubMed

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.

Antizyme restrains centrosome amplification by regulating the accumulation of Mps1 at centrosomes.

PubMed

Kasbek, Christopher; Yang, Ching-Hui; Fisk, Harold A

2010-11-15

Extra centrosomes are found in many tumors, and their appearance is an early event that can generate aberrant mitotic spindles and aneuploidy. Because the failure to appropriately degrade the Mps1 protein kinase correlates with centrosome overproduction in tumor-derived cells, defects in the factors that promote Mps1 degradation may contribute to extra centrosomes in tumors. However, while we have recently characterized an Mps1 degradation signal, the factors that regulate Mps1 centrosomal Mps1 are unknown. Antizyme (OAZ), a mediator of ubiquitin-independent degradation and a suspected tumor suppressor, was recently shown to localize to centrosomes and modulate centrosome overproduction, but the known OAZ substrates were not responsible for its effect on centrosomes. We have found that OAZ exerts its effect on centrosomes via Mps1. OAZ promotes the removal of Mps1 from centrosomes, and centrosome overproduction caused by reducing OAZ activity requires Mps1. OAZ binds to Mps1 via the Mps1 degradation signal and modulates the function of Mps1 in centrosome overproduction. Moreover, OAZ regulates the canonical centrosome duplication cycle, and reveals a function for Mps1 in procentriole assembly. Together, our data suggest that OAZ restrains the assembly of centrioles by controlling the levels of centrosomal Mps1 through the Cdk2-regulated Mps1 degradation signal.

Ultrastructural aspects of spermatogenesis, testes, and vas deferens in the parthenogenetic tapeworm Atractolytocestus huronensis Anthony, 1958 (Cestoda: Caryophyllidea), a carp parasite from Slovakia.

PubMed

Bruňanská, Magdaléna; Nebesářová, Jana; Oros, Mikuláš

2011-01-01

Spermatogenesis, testes, and vas deferens in the parthenogenetic monozoic tapeworm Atractolytocestus huronensis Anthony, 1958 (Cestoda: Caryophyllidea) from Slovakia, parasitizing the carp Cyprinus carpio L., have been investigated by means of transmission electron microscopy for the first time. The present results show that helminths with parthenogenetic and normal reproduction may share some common spermatology features, e.g., dense cytoplasm of the peripherally localized spermatogonia or a rosette type of spermatogenesis. In contrast to tapeworms with normal reproduction, the most prominent ultrastructural characteristic of the spermatocytes of A. huronensis is fragmentation of their nuclei. This clear feature of cell degeneration might be a consequence of the aberrant first meiotic division. Peripheral cortical microtubules and a single centriole, indicators of the ongoing spermiogenesis, were observed only very rarely in the early spermatids. Characteristics of normal spermiogenesis, i.e., apical dense material in the zone of differentiation in early stages of spermiogenesis, flagellar rotation, and proximo-distal fusion, were never found in the present study. The testes follicles are surrounded by a thin cytoplasmic sheath underlined by a basal lamina. Vas deferens is lined by flat epithelium with numerous surface lamellae and cilia. Mature, functional spermatozoa were not observed in the vas deferens of A. huronensis from Slovakia.

The Stil protein regulates centrosome integrity and mitosis through suppression of Chfr

PubMed Central

Castiel, Asher; Danieli, Michal Mark; David, Ahuvit; Moshkovitz, Sharon; Aplan, Peter D.; Kirsch, Ilan R.; Brandeis, Michael; Krämer, Alwin; Izraeli, Shai

2011-01-01

Stil (Sil, SCL/TAL1 interrupting locus) is a cytosolic and centrosomal protein expressed in proliferating cells that is required for mouse and zebrafish neural development and is mutated in familial microcephaly. Recently the Drosophila melanogaster ortholog of Stil was found to be important for centriole duplication. Consistent with this finding, we report here that mouse embryonic fibroblasts lacking Stil are characterized by slow growth, low mitotic index and absence of clear centrosomes. We hypothesized that Stil regulates mitosis through the tumor suppressor Chfr, an E3 ligase that blocks mitotic entry in response to mitotic stress. Mouse fibroblasts lacking Stil by genomic or RNA interference approaches, as well as E9.5 Stil−/− embryos, express high levels of the Chfr protein and reduced levels of the Chfr substrate Plk1. Exogenous expression of Stil, knockdown of Chfr or overexpression of Plk1 reverse the abnormal mitotic phenotypes of fibroblasts lacking Stil. We further demonstrate that Stil increases Chfr auto-ubiquitination and reduces its protein stability. Thus, Stil is required for centrosome organization, entry into mitosis and cell proliferation, and these functions are at least partially mediated by Chfr and its targets. This is the first identification of a negative regulator of the Chfr mitotic checkpoint. PMID:21245198

Mitosis in Barbulanympha. I. Spindle structure, formation, and kinetochore engagement

PubMed Central

1978-01-01

Successful culture of the obligatorily anaerobic symbionts residing in the hindgut of the wood-eating cockroach Cryptocercus punctulatus now permits continuous observation of mitosis in individual Barbulanympha cells. In Part I of this two-part paper, we report methods for culture of the protozoa, preparation of microscope slide cultures in which Barbulanympha survived and divided for up to 3 days, and an optical arrangement which permits observation and through-focus photographic recording of dividing cells, sequentially in differential interference contrast and rectified polarized light microscopy. We describe the following prophase events and structures: development of the astral rays and large extranuclear central spindle from the tips of the elongate-centrioles; the fine structure of spindle fibers and astral rays which were deduced in vivo from polarized light microscopy and seen as a particular array of microtubules in thin-section electron micrographs; formation of chromosomal spindle fibers by dynamic engagement of astral rays to the kinetochores embedded in the persistent nuclear envelope; and repetitive shortening of chromosomal spindle fibers which appear to hoist the nucleus to the spindle surface, cyclically jostle the kinetochores within the nuclear envelope, and churn the prophase chromosomes. The observations described here and in Part II have implications both for the evolution of mitosis and for understanding the mitotic process generally. PMID:681451

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

NASA Technical Reports Server (NTRS)

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.

Coiled-coil domain containing 42 (Ccdc42) is necessary for proper sperm development and male fertility in the mouse.

PubMed

Pasek, Raymond C; Malarkey, Erik; Berbari, Nicolas F; Sharma, Neeraj; Kesterson, Robert A; Tres, Laura L; Kierszenbaum, Abraham L; Yoder, Bradley K

2016-04-15

Spermiogenesis is the differentiation of spermatids into motile sperm consisting of a head and a tail. The head harbors a condensed elongated nucleus partially covered by the acrosome-acroplaxome complex. Defects in the acrosome-acroplaxome complex are associated with abnormalities in sperm head shaping. The head-tail coupling apparatus (HTCA), a complex structure consisting of two cylindrical microtubule-based centrioles and associated components, connects the tail or flagellum to the sperm head. Defects in the development of the HTCA cause sperm decapitation and disrupt sperm motility, two major contributors to male infertility. Here, we provide data indicating that mutations in the gene Coiled-coil domain containing 42 (Ccdc42) is associated with malformation of the mouse sperm flagella. In contrast to many other flagella and motile cilia genes, Ccdc42 expression is only observed in the brain and developing sperm. Male mice homozygous for a loss-of-function Ccdc42 allele (Ccdc42(KO)) display defects in the number and location of the HTCA, lack flagellated sperm, and are sterile. The testes enriched expression of Ccdc42 and lack of other phenotypes in mutant mice make it an ideal candidate for screening cases of azoospermia in humans. Copyright © 2016 Elsevier Inc. All rights reserved.

THE INFLUENCE OF X RAYS ON ORGANELLE INDUCTION AND DIFFERENTIATION IN GRASSHOPPER SPERMATOGENESIS

DOE Office of Scientific and Technical Information (OSTI.GOV)

Tahmisian, T.N.; Devine, R.L.

1961-01-01

The effects or x radiation on grasshopper spermatogenesis were studied by light and electron microscopy. The insects were irradiated at the second instar prior to the presence of maturation stages and observed at the last instar and imago stages. Dosages of 100 to 800 r retarded the differentiation of the nucleus and mitochondrial nebenkern in spermatids. Irradiation caused a curtailment and disorganization in the differentiation of the nebenkern from mitochondria. It also induced the formation of supernumerary centrioles, flagellar filaments, and acrosomes; nuclear disorganization as well as pycnosis and fragmentation also occurred. The nucleus appeared to be drawn toward eachmore » radioinduced supernumerary acrosome, with consequent multipolarity of the nucleus. Induction of a set of flagellar filaments was seen only where the centriolar structure was in contact with the nucleus. Details are given of an organelle that is composed of anastomosed and interwoven cytoplasmic strands. The results show that radioinduced changes in cell morphology are preceded by either alteration or curtailment of organelle differentiation. Furthermore, organelle differentiation during spermatogenesis was altered by doses that did not visibly affect the antecedent mitotic, maturation, or meiotic divisions; grasshoppers irradiated during the third instar showed the first effects of radiation at the last instar. (P.C.H.)« less

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

PubMed Central

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. PMID:26545777

Dry Preservation of Spermatozoa: Considerations for Different Species.

PubMed

Patrick, Jennifer; Comizzoli, Pierre; Elliott, Gloria

2017-04-01

The current gold standard for sperm preservation is storage at cryogenic temperatures. Dry preservation is an attractive alternative, eliminating the need for ultralow temperatures, reducing storage maintenance costs, and providing logistical flexibility for shipping. Many seeds and anhydrobiotic organisms are able to survive extended periods in a dry state through the accumulation of intracellular sugars and other osmolytes and are capable of returning to normal physiology postrehydration. Using techniques inspired by nature's adaptations, attempts have been made to dehydrate and dry preserve spermatozoa from a variety of species. Most of the anhydrous preservation research performed to date has focused on mouse spermatozoa, with only a small number of studies in nonrodent mammalian species. There is a significant difference between sperm function in rodent and nonrodent mammalian species with respect to centrosomal inheritance. Studies focused on reproductive technologies have demonstrated that in nonrodent species, the centrosome must be preserved to maintain sperm function as the spermatozoon centrosome contributes the dominant nucleating seed, consisting of the proximal centriole surrounded by pericentriolar components, onto which the oocyte's centrosomal material is assembled. Preservation techniques used for mouse sperm may therefore not necessarily be applicable to nonrodent spermatozoa. The range of technologies used to dehydrate sperm and the effect of processing and storage conditions on fertilization and embryogenesis using dried sperm are reviewed in the context of reproductive physiology and cellular morphology in different species.

Meier-Gorlin syndrome mutations disrupt an Orc1 CDK inhibitory domain and cause centrosome reduplication.

PubMed

Hossain, Manzar; Stillman, Bruce

2012-08-15

Like DNA replication, centrosomes are licensed to duplicate once per cell division cycle to ensure genetic stability. In addition to regulating DNA replication, the Orc1 subunit of the human origin recognition complex controls centriole and centrosome copy number. Here we report that Orc1 harbors a PACT centrosome-targeting domain and a separate domain that differentially inhibits the protein kinase activities of Cyclin E-CDK2 and Cyclin A-CDK2. A cyclin-binding motif (Cy motif) is required for Orc1 to bind Cyclin A and inhibit Cyclin A-CDK2 kinase activity but has no effect on Cyclin E-CDK2 kinase activity. In contrast, Orc1 inhibition of Cyclin E-CDK2 kinase activity occurs by a different mechanism that is affected by Orc1 mutations identified in Meier-Gorlin syndrome patients. The cyclin/CDK2 kinase inhibitory domain of Orc1, when tethered to the PACT domain, localizes to centrosomes and blocks centrosome reduplication. Meier-Gorlin syndrome mutations that disrupt Cyclin E-CDK2 kinase inhibition also allow centrosome reduplication. Thus, Orc1 contains distinct domains that control centrosome copy number and DNA replication. We suggest that the Orc1 mutations present in some Meier-Gorlin syndrome patients contribute to the pronounced microcephaly and dwarfism observed in these individuals by altering centrosome duplication in addition to DNA replication defects.

14-3-3γ Prevents Centrosome Amplification and Neoplastic Progression.

PubMed

Mukhopadhyay, Amitabha; Sehgal, Lalit; Bose, Arunabha; Gulvady, Anushree; Senapati, Parijat; Thorat, Rahul; Basu, Srikanta; Bhatt, Khyati; Hosing, Amol S; Balyan, Renu; Borde, Lalit; Kundu, Tapas K; Dalal, Sorab N

2016-06-02

More than 80% of malignant tumors show centrosome amplification and clustering. Centrosome amplification results from aberrations in the centrosome duplication cycle, which is strictly coordinated with DNA-replication-cycle. However, the relationship between cell-cycle regulators and centrosome duplicating factors is not well understood. This report demonstrates that 14-3-3γ localizes to the centrosome and 14-3-3γ loss leads to centrosome amplification. Loss of 14-3-3γ results in the phosphorylation of NPM1 at Thr-199, causing early centriole disjunction and centrosome hyper-duplication. The centrosome amplification led to aneuploidy and increased tumor formation in mice. Importantly, an increase in passage of the 14-3-3γ-knockdown cells led to an increase in the number of cells containing clustered centrosomes leading to the generation of pseudo-bipolar spindles. The increase in pseudo-bipolar spindles was reversed and an increase in the number of multi-polar spindles was observed upon expression of a constitutively active 14-3-3-binding-defective-mutant of cdc25C (S216A) in the 14-3-3γ knockdown cells. The increase in multi-polar spindle formation was associated with decreased cell viability and a decrease in tumor growth. Our findings uncover the molecular basis of regulation of centrosome duplication by 14-3-3γ and inhibition of tumor growth by premature activation of the mitotic program and the disruption of centrosome clustering.

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

PubMed Central

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

AN ELECTRON MICROSCOPE STUDY OF SPERMATID DIFFERENTIATION IN THE TOAD, BUFO ARENARUM HENSEL

PubMed Central

Burgos, Mario H.; Fawcett, Don W.

1956-01-01

The differentiation of the spermatids of Bufo arenarum has been described from a study of electron micrographs of thin sections of testis. The development of the acrosome from the Golgi complex takes place in much the same manner as in mammalian spermatogenesis but no acrosome granule is formed. A perforatorium is described for the first time in this species. It is formed by a convergence of dense filaments that arise between the nuclear membrane and the head cap. During maturation of the spermatid the chromatin undergoes striking physicochemical alterations. Fine chromatin granules uniformly dispersed in the karyoplasm are replaced by larger and larger aggregates and these ultimately coalesce to form a very dense sperm head. Two centrioles of cylindrical form are situated very near the base of the sperm head. The longitudinal fibrils of the tail flagellum take origin from one, and the dense fibrous substance of the undulating membrane is closely related to the other. Phase contrast cinematographic observations on the swimming movements of living toad sperm, when considered in relation to the fine structural components of the tail, suggest that there is a contractile component in the undulating membrane as well as in the axial fibrils. The differences in the structure of mammalian and amphibian sperm tails are discussed in relation to differences in the character of their movements. PMID:13331956

Amphiastral Mitotic Spindle Assembly in Vertebrate Cells Lacking Centrosomes

PubMed Central

Hornick, Jessica E.; Mader, Christopher C.; Tribble, Emily K.; Bagne, Cydney C.; Vaughan, Kevin T.; Shaw, Sidney L.; Hinchcliffe, Edward H.

2011-01-01

Summary The role of centrosomes/centrioles during mitotic spindle assembly in vertebrates remains controversial. In cell-free extracts and experimentally derived acentrosomal cells, randomly oriented microtubules (MTs) self-organize around mitotic chromosomes and assemble anastral spindles [1, 2, 3]. However, vertebrate somatic cells normally assemble a connected pair of polarized, astral MT arrays – termed an amphiaster (“a star on both sides” [4]) – that is formed by the splitting and separation of the microtubule-organizing center (MTOC) well before nuclear envelope breakdown (NEB) [5]. Whether amphiaster formation requires splitting of duplicated centrosomes is not known. We found that when centrosomes were removed from living vertebrate cells early in their cell cycle, an acentriolar MTOC re-assembled, and prior to NEB, a functional amphiastral spindle formed. Cytoplasmic dynein, dynactin, and pericentrin are all recruited to the interphase aMTOC, and the activity of kinesin-5 is needed for amphiaster formation. Mitosis proceeded on time and these karyoplasts divided in two. However, ~35% of aMTOCs failed to split/separate before NEB, and these entered mitosis with persistent monastral spindles. The chromatin-mediated RAN-GTP pathway could not restore bipolarity to monastral spindles, and these cells exited mitosis as single daughters. Our data reveal the novel finding that MTOC separation and amphiaster formation does not absolutely require the centrosome, but in its absence, the fidelity of bipolar spindle assembly is highly compromised. PMID:21439826

Centrosomal protein Dzip1l binds Cby, promotes ciliary bud formation, and acts redundantly with Bromi to regulate ciliogenesis in the mouse.

PubMed

Wang, Chengbing; Li, Jia; Takemaru, Ken-Ichi; Jiang, Xiaogang; Xu, Guoqiang; Wang, Baolin

2018-03-15

The primary cilium is a microtubule-based organelle required for Hedgehog (Hh) signaling and consists of a basal body, a ciliary axoneme and a compartment between the first two structures, called the transition zone (TZ). The TZ serves as a gatekeeper to control protein composition in cilia, but less is known about its role in ciliary bud formation. Here, we show that centrosomal protein Dzip1l is required for Hh signaling between Smoothened and Sufu. Dzip1l colocalizes with basal body appendage proteins and Rpgrip1l, a TZ protein. Loss of Dzip1l results in reduced ciliogenesis and dysmorphic cilia in vivo Dzip1l interacts with, and acts upstream of, Cby, an appendage protein, in ciliogenesis. Dzip1l also has overlapping functions with Bromi (Tbc1d32) in ciliogenesis, cilia morphogenesis and neural tube patterning. Loss of Dzip1l arrests ciliogenesis at the stage of ciliary bud formation from the TZ. Consistent with this, Dzip1l mutant cells fail to remove the capping protein Cp110 (Ccp110) from the distal end of mother centrioles and to recruit Rpgrip1l to the TZ. Therefore, Dzip1l promotes ciliary bud formation and is required for the integrity of the TZ. © 2018. Published by The Company of Biologists Ltd.

Never in mitosis gene A related kinase-6 attenuates pressure overload-induced activation of the protein kinase B pathway and cardiac hypertrophy.

PubMed

Bian, Zhouyan; Liao, Haihan; Zhang, Yan; Wu, Qingqing; Zhou, Heng; Yang, Zheng; Fu, Jinrong; Wang, Teng; Yan, Ling; Shen, Difei; Li, Hongliang; Tang, Qizhu

2014-01-01

Cardiac hypertrophy appears to be a specialized form of cellular growth that involves the proliferation control and cell cycle regulation. NIMA (never in mitosis, gene A)-related kinase-6 (Nek6) is a cell cycle regulatory gene that could induce centriole duplication, and control cell proliferation and survival. However, the exact effect of Nek6 on cardiac hypertrophy has not yet been reported. In the present study, the loss- and gain-of-function experiments were performed in Nek6 gene-deficient (Nek6-/-) mice and Nek6 overexpressing H9c2 cells to clarify whether Nek6 which promotes the cell cycle also mediates cardiac hypertrophy. Cardiac hypertrophy was induced by transthoracic aorta constriction (TAC) and then evaluated by echocardiography, pathological and molecular analyses in vivo. We got novel findings that the absence of Nek6 promoted cardiac hypertrophy, fibrosis and cardiac dysfunction, which were accompanied by a significant activation of the protein kinase B (Akt) signaling in an experimental model of TAC. Consistent with this, the overexpression of Nek6 prevented hypertrophy in H9c2 cells induced by angiotonin II and inhibited Akt signaling in vitro. In conclusion, our results demonstrate that the cell cycle regulatory gene Nek6 is also a critical signaling molecule that helps prevent cardiac hypertrophy and inhibits the Akt signaling pathway.

THE AXOSTYLE OF SACCINOBACULUS

PubMed Central

McIntosh, J. Richard; Ogata, Edward S.; Landis, Story C.

1973-01-01

The axostyle of the flagellate Saccinobaculus is a motile ribbon composed of microtubules, cross-bridged to form interconnected rows. We find a centriole-related row of dark-staining tubules near the nucleus at the anterior end of the axostyle. Other tubule rows bind parallel to this primary row, acquire ordered relationships, and become the tubules of the axostyle proper. The number of tubule rows is constant in Saccinobaculus lata from the region near the nucleus to within a few micrometers of the posterior tip of the cell. In Saccinobaculus ambloaxostylus a few tubule rows are added to the axostyle posterior to the nucleus, giving this axostyle a leaf spring construction. The tubules of S. lata are held in rows by links with a 140 Å periodicity along the tubule axis; bridges between rows of tubules are also seen but are not apparently periodic. Each tubule in S. ambloaxostylus shows an axial periodicity of 150 Å due to pairs of arms, one of which is always part of the intrarow link. Interrow bridges in this species run either from tubule to tubule or from tubule to the free arm, but as in S. lata they do not display an obvious axial periodicity. An average unit cell is presented for the axostyle of each species, and the relation of the intertubule links to the microtubule substructure is discussed. PMID:4682899

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

PubMed

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.

Dry Preservation of Spermatozoa: Considerations for Different Species

PubMed Central

Patrick, Jennifer; Comizzoli, Pierre

2017-01-01

The current gold standard for sperm preservation is storage at cryogenic temperatures. Dry preservation is an attractive alternative, eliminating the need for ultralow temperatures, reducing storage maintenance costs, and providing logistical flexibility for shipping. Many seeds and anhydrobiotic organisms are able to survive extended periods in a dry state through the accumulation of intracellular sugars and other osmolytes and are capable of returning to normal physiology postrehydration. Using techniques inspired by nature's adaptations, attempts have been made to dehydrate and dry preserve spermatozoa from a variety of species. Most of the anhydrous preservation research performed to date has focused on mouse spermatozoa, with only a small number of studies in nonrodent mammalian species. There is a significant difference between sperm function in rodent and nonrodent mammalian species with respect to centrosomal inheritance. Studies focused on reproductive technologies have demonstrated that in nonrodent species, the centrosome must be preserved to maintain sperm function as the spermatozoon centrosome contributes the dominant nucleating seed, consisting of the proximal centriole surrounded by pericentriolar components, onto which the oocyte's centrosomal material is assembled. Preservation techniques used for mouse sperm may therefore not necessarily be applicable to nonrodent spermatozoa. The range of technologies used to dehydrate sperm and the effect of processing and storage conditions on fertilization and embryogenesis using dried sperm are reviewed in the context of reproductive physiology and cellular morphology in different species. PMID:28398834

Structural protein 4.1 is located in mammalian centrosomes

PubMed Central

Krauss, Sharon Wald; Chasis, Joel Anne; Rogers, Catherine; Mohandas, Narla; Krockmalnic, Gabriela; Penman, Sheldon

1997-01-01

Structural protein 4.1 was first characterized as an important 80-kDa protein in the mature red cell membrane skeleton. It is now known to be a member of a family of protein isoforms detected at diverse intracellular sites in many nucleated mammalian cells. We recently reported that protein 4.1 isoforms are present at interphase in nuclear matrix and are rearranged during the cell cycle. Here we report that protein 4.1 epitopes are present in centrosomes of human and murine cells and are detected by using affinity-purified antibodies specific for 80-kDa red cell 4.1 and for 4.1 peptides. Immunofluorescence, by both conventional and confocal microscopy, showed that protein 4.1 epitopes localized in the pericentriolar region. Protein 4.1 epitopes remained in centrosomes after extraction of cells with detergent, salt, and DNase. Higher resolution electron microscopy of detergent-extracted cell whole mounts showed centrosomal protein 4.1 epitopes distributed along centriolar cylinders and on pericentriolar fibers, at least some of which constitute the filamentous network surrounding each centriole. Double-label electron microscopy showed that protein 4.1 epitopes were predominately localized in regions also occupied by epitopes for centrosome-specific autoimmune serum 5051 but were not found on microtubules. Our results suggest that protein 4.1 is an integral component of centrosome structure, in which it may play an important role in centrosome function during cell division and organization of cellular architecture. PMID:9207085

Discovery of 2-(1H-indol-5-ylamino)-6-(2,4-difluorophenylsulfonyl)-8-methylpyrido[2,3-d]pyrimidin-7(8H)-one (7ao) as a potent selective inhibitor of Polo like kinase 2 (PLK2)

PubMed Central

Reddy, M. V. Ramana; Akula, Balireddy; Jatiani, Shashidhar; Vasquez-Del Carpio, Rodrigo; Billa, Vinay K.; Mallireddigari, Muralidhar R.; Cosenza, Stephen C.; Subbaiah, D. R. C. Venkata; Bharathi, E. Vijaya; Pallela, Venkat R.; Ramkumar, Poornima; Jain, Rinku; Aggarwal, Aneel K.; Reddy, E. Premkumar

2018-01-01

Several families of protein kinases have been shown to play a critical role in the regulation of cell cycle progression, particularly progression through mitosis. These kinase families include the Aurora kinases, the Mps1 gene product and the Polo Like family of protein kinases (PLKs). The PLK family consists of five members and of these, the role of PLK1 in human cancer is well documented. PLK2 (SNK), which is highly homologous to PLK1, has been shown to play a critical role in centriole duplication and is also believed to play a regulatory role in the survival pathway by physically stabilizing the TSC1/2 complex in tumor cells under hypoxic conditions. As a part of our research program, we have developed a library of novel ATP mimetic chemotypes that are cytotoxic against a panel of cancer cell lines. We show that one of these chemotypes, the 6-arylsulfonyl pyridopyrimidinones, induces apoptosis of human tumor cell lines in nanomolar concentrations. The most potent of these compounds, 7ao, was found to be a highly specific inhibitor of PLK2 when profiled against a panel of 288 wild type, 55 mutant and 12 lipid kinases. Here, we describe the synthesis, structure activity relationship, in vitro kinase specificity and biological activity of the lead compound, 7ao. PMID:26762835

Spermatogenesis in Animals as Revealed by Electron Microscopy

PubMed Central

Yasuzumi, G.; Tanaka, Hiroaki

1958-01-01

This paper reports an electron microscope study of typical and atypical spermatogenesis in the pond snail, Cipangopaludina malteata. In the typical spermatid the nucleus undergoes profound changes as development proceeds, affecting both its form and internal fine structure. A large number of roughly parallel, dense filaments, arranged along the long axis of the nucleus, fuse with each other to form in the end the homogeneous helical body characteristic of the head of the adult spermatozoa. The nebenkern is apparently mitochondrial in nature and, in its early development, is similar to that of insects except that it appears as a double structure from the beginning. As differentiation proceeds, the mitochondria lose their membranes, and the residual, now denuded cristae, reorganize to give a parallel radial arrangement. In the last stages of development, the nebenkern derivations become applied to the sheath of the middle piece in a compact helical fashion. In the development of the atypical spermatozoa, the nucleus fails to differentiate and simply shrinks in volume until only a remnant, devoid of DNA, is left. The cytoplasm shows numerous vesicles containing small Feulgen-positive bodies, 80 to 130 mµ in diameter. These vesicles plus contents increase in number as spermatogenesis proceeds. The "head" structure of the atypical spermatozoa consists of a bundle (7 to 17) of tail flagella, each with a centriole at its anterior end. The end-piece of the atypical form appears brush-like and is made up of the free ends of the several flagella. PMID:13587559

Spermiogenesis and Taxonomical Values of Sperm Ultrastructures in Male Crassostrea ariakensis (Fujita & Wakiya, 1929) (Pteroirmorphia: Ostreidae) in the Estuary of the Seomjin River, Korea

PubMed Central

Son, Pal Won; Chung, Jae Seung; Kim, Jin Hee; Kim, Sung Han; Chung, Ee-Yung

2014-01-01

Characteristics of the developmental stages of spermatids during spermiogenesis and phylogenetic classicfication of the species using sperm ultrastructures in male Crassostrea ariakensis were investigated by transmission electron microscope observations. The morphology of the spermatozoon of this species has a primitive type and is similar to those of Ostreidae. Ultrastructures of mature sperms are composed of broad, modified cap-shaped acrosomal vesicle and an axial rod in subacrosomal materials on an oval nucleus, four spherical mitochondria in the sperm midpiece, and satellite fibres which appear near the distal centriole. The axoneme of the sperm tail shows a 9+2 structure. Accordingly, the ultrastructural characteristics of mature sperm of C. ariakensis resemble to those of other investigated ostreids in Ostreidae in the subclass Pteriomorphia. In this study, particularly, two transverse bands (stripes) appear at the anterior region of the acrosomal vesicle of this species, unlike two or three transverse bands (stripes) in C. gigas. It is assumed that differences in this acrosomal substructure are associated with the inability of fertilization between the genus Crassostrea and other genus species in Ostreidae. Therefore, we can use sperm ultrastructures and morphologies in the resolution of taxonomic relationships within the Ostreidae in the subclass Pteriomorphia. These spermatozoa, which contain several ultrastructures such as acrosomal vesicle, an axial rod in the sperm head part and four mitochondria and satellite fibres in the sperm midpiece, belong to the family Ostreidae in the subclass Pteriomorphia. PMID:25949188

Mutations in the Gene Encoding IFT Dynein Complex Component WDR34 Cause Jeune Asphyxiating Thoracic Dystrophy

PubMed Central

Schmidts, Miriam; Vodopiutz, Julia; Christou-Savina, Sonia; Cortés, Claudio R.; McInerney-Leo, Aideen M.; Emes, Richard D.; Arts, Heleen H.; Tüysüz, Beyhan; D’Silva, Jason; Leo, Paul J.; Giles, Tom C.; Oud, Machteld M.; Harris, Jessica A.; Koopmans, Marije; Marshall, Mhairi; Elçioglu, Nursel; Kuechler, Alma; Bockenhauer, Detlef; Moore, Anthony T.; Wilson, Louise C.; Janecke, Andreas R.; Hurles, Matthew E.; Emmet, Warren; Gardiner, Brooke; Streubel, Berthold; Dopita, Belinda; Zankl, Andreas; Kayserili, Hülya; Scambler, Peter J.; Brown, Matthew A.; Beales, Philip L.; Wicking, Carol; Duncan, Emma L.; Mitchison, Hannah M.

2013-01-01

Bidirectional (anterograde and retrograde) motor-based intraflagellar transport (IFT) governs cargo transport and delivery processes that are essential for primary cilia growth and maintenance and for hedgehog signaling functions. The IFT dynein-2 motor complex that regulates ciliary retrograde protein transport contains a heavy chain dynein ATPase/motor subunit, DYNC2H1, along with other less well functionally defined subunits. Deficiency of IFT proteins, including DYNC2H1, underlies a spectrum of skeletal ciliopathies. Here, by using exome sequencing and a targeted next-generation sequencing panel, we identified a total of 11 mutations in WDR34 in 9 families with the clinical diagnosis of Jeune syndrome (asphyxiating thoracic dystrophy). WDR34 encodes a WD40 repeat-containing protein orthologous to Chlamydomonas FAP133, a dynein intermediate chain associated with the retrograde intraflagellar transport motor. Three-dimensional protein modeling suggests that the identified mutations all affect residues critical for WDR34 protein-protein interactions. We find that WDR34 concentrates around the centrioles and basal bodies in mammalian cells, also showing axonemal staining. WDR34 coimmunoprecipitates with the dynein-1 light chain DYNLL1 in vitro, and mining of proteomics data suggests that WDR34 could represent a previously unrecognized link between the cytoplasmic dynein-1 and IFT dynein-2 motors. Together, these data show that WDR34 is critical for ciliary functions essential to normal development and survival, most probably as a previously unrecognized component of the mammalian dynein-IFT machinery. PMID:24183451

Discovery of 2-(1H-indol-5-ylamino)-6-(2,4-difluorophenylsulfonyl)-8-methylpyrido[2,3-d]pyrimidin-7(8H)-one (7ao) as a potent selective inhibitor of Polo like kinase 2 (PLK2).

PubMed

Reddy, M V Ramana; Akula, Balireddy; Jatiani, Shashidhar; Vasquez-Del Carpio, Rodrigo; Billa, Vinay K; Mallireddigari, Muralidhar R; Cosenza, Stephen C; Venkata Subbaiah, D R C; Bharathi, E Vijaya; Pallela, Venkat R; Ramkumar, Poornima; Jain, Rinku; Aggarwal, Aneel K; Reddy, E Premkumar

2016-02-15

Several families of protein kinases have been shown to play a critical role in the regulation of cell cycle progression, particularly progression through mitosis. These kinase families include the Aurora kinases, the Mps1 gene product and the Polo Like family of protein kinases (PLKs). The PLK family consists of five members and of these, the role of PLK1 in human cancer is well documented. PLK2 (SNK), which is highly homologous to PLK1, has been shown to play a critical role in centriole duplication and is also believed to play a regulatory role in the survival pathway by physically stabilizing the TSC1/2 complex in tumor cells under hypoxic conditions. As a part of our research program, we have developed a library of novel ATP mimetic chemotypes that are cytotoxic against a panel of cancer cell lines. We show that one of these chemotypes, the 6-arylsulfonyl pyridopyrimidinones, induces apoptosis of human tumor cell lines in nanomolar concentrations. The most potent of these compounds, 7ao, was found to be a highly specific inhibitor of PLK2 when profiled against a panel of 288 wild type, 55 mutant and 12 lipid kinases. Here, we describe the synthesis, structure activity relationship, in vitro kinase specificity and biological activity of the lead compound, 7ao. Copyright © 2015 Elsevier Ltd. All rights reserved.

Truncation of POC1A associated with short stature and extreme insulin resistance.

PubMed

Chen, Jian-Hua; Segni, Maria; Payne, Felicity; Huang-Doran, Isabel; Sleigh, Alison; Adams, Claire; Savage, David B; O'Rahilly, Stephen; Semple, Robert K; Barroso, Inês

2015-10-01

We describe a female proband with primordial dwarfism, skeletal dysplasia, facial dysmorphism, extreme dyslipidaemic insulin resistance and fatty liver associated with a novel homozygous frameshift mutation in POC1A, predicted to affect two of the three protein products of the gene. POC1A encodes a protein associated with centrioles throughout the cell cycle and implicated in both mitotic spindle and primary ciliary function. Three homozygous mutations affecting all isoforms of POC1A have recently been implicated in a similar syndrome of primordial dwarfism, although no detailed metabolic phenotypes were described. Primary cells from the proband we describe exhibited increased centrosome amplification and multipolar spindle formation during mitosis, but showed normal DNA content, arguing against mitotic skipping, cleavage failure or cell fusion. Despite evidence of increased DNA damage in cells with supernumerary centrosomes, no aneuploidy was detected. Extensive centrosome clustering both at mitotic spindles and in primary cilia mitigated the consequences of centrosome amplification, and primary ciliary formation was normal. Although further metabolic studies of patients with POC1A mutations are warranted, we suggest that POC1A may be added to ALMS1 and PCNT as examples of centrosomal or pericentriolar proteins whose dysfunction leads to extreme dyslipidaemic insulin resistance. Further investigation of links between these molecular defects and adipose tissue dysfunction is likely to yield insights into mechanisms of adipose tissue maintenance and regeneration that are critical to metabolic health. © 2015 Society for Endocrinology.

“Stop Ne(c)king around”: How interactomics contributes to functionally characterize Nek family kinases

PubMed Central

Meirelles, Gabriela Vaz; Perez, Arina Marina; de Souza, Edmárcia Elisa; Basei, Fernanda Luisa; Papa, Priscila Ferreira; Melo Hanchuk, Talita Diniz; Cardoso, Vanessa Bomfim; Kobarg, Jörg

2014-01-01

Aside from Polo and Aurora, a third but less studied kinase family involved in mitosis regulation is the never in mitosis-gene A (NIMA)-related kinases (Neks). The founding member of this family is the sole member NIMA of Aspergillus nidulans, which is crucial for the initiation of mitosis in that organism. All 11 human Neks have been functionally assigned to one of the three core functions established for this family in mammals: (1) centrioles/mitosis; (2) primary ciliary function/ciliopathies; and (3) DNA damage response (DDR). Recent findings, especially on Nek 1 and 8, showed however, that several Neks participate in parallel in at least two of these contexts: primary ciliary function and DDR. In the core section of this in-depth review, we report the current detailed functional knowledge on each of the 11 Neks. In the discussion, we return to the cross-connections among Neks and point out how our and other groups’ functional and interactomics studies revealed that most Neks interact with protein partners associated with two if not all three of the functional contexts. We then raise the hypothesis that Neks may be the connecting regulatory elements that allow the cell to fine tune and synchronize the cellular events associated with these three core functions. The new and exciting findings on the Nek family open new perspectives and should allow the Neks to finally claim the attention they deserve in the field of kinases and cell cycle biology. PMID:24921005

Oscillatory fluid flow influences primary cilia and microtubule mechanics.

PubMed

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.

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

NASA Astrophysics Data System (ADS)

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.

Biciliated ependymal cell proliferation contributes to spinal cord growth

PubMed Central

Alfaro-Cervello, Clara; Soriano-Navarro, Mario; Mirzadeh, Zaman; Alvarez-Buylla, Arturo; Garcia-Verdugo, Jose Manuel

2013-01-01

Two neurogenic regions have been described in the adult brain, the lateral ventricle subventricular zone and the dentate gyrus subgranular zone. It has been suggested that neural stem cells also line the central canal of the adult spinal cord. Using transmission and scanning electron microscopy and immunostaining, we describe here the organization and cell types of the central canal epithelium in adult mice. The identity of dividing cells was determined by three-dimensional ultrastructural reconstructions of [3H]thymidine-labeled cells and confocal analysis of bromodeoxyuridine labeling. The most common cell type lining the central canal had two long motile (9+2) cilia and was vimentin+, CD24+, FoxJ1+, Sox2+ and CD133+, but nestin- and glial fibrillary acidic protein (GFAP)-. These biciliated ependymal cells of the central canal (Ecc) resembled E2 cells of the lateral ventricles, but their basal bodies were different from that of E2 or E1 cells. Interestingly, we frequently found Ecc cells with two nuclei and four cilia, suggesting they are formed by incomplete cytokinesis or cell fusion. GFAP+ astrocytes with a single cilium and an orthogonally oriented centriole were also observed. The majority of dividing cells corresponded to biciliated Ecc cells. Central canal proliferation was most common during the active period of spinal cord growth. Pairs of labeled Ecc cells were observed within the central canal in adult mice 2.5 weeks post-labeling. Our work suggests that the vast majority of postnatal dividing cells in the central canal are Ecc cells and their proliferation is associated with the growth of the spinal cord. PMID:22434575

Morphology and ultrastructure of Brachymystax lenok tsinlingensis spermatozoa by scanning and transmission electron microscopy.

PubMed

Guo, Wei; Shao, Jian; Li, Ping; Wu, Jinming; Wei, Qiwei

2016-08-01

This study was conducted to investigate Brachymystax lenok tsinlingensis spermatozoa cell morphology and ultrastructure through scanning and transmission electron microscopy. Findings revealed that the spermatozoa can be differentiated into three major parts: a spherical head without an acrosome, a short mid-piece, and a long, cylindrical flagellum. The mean length of the spermatozoa was 36.11±2.84μm, with a spherical head length of 2.78±0.31μm. The mean anterior and posterior head widths were 2.20±0.42μm and 2.55±0.53μm, respectively. The nuclear fossa was positioned at the base of the nucleus that contained the anterior portion of flagellum and a centriolar complex (proximal and distal centrioles). The short mid-piece was located laterally to the nucleus and possessed just one spherical mitochondrion with a mean diameter of 0.65±0.14μm. The spermatozoa flagellum was long and cylindrical, and could be separated into two parts: a long main-piece and a short end-piece. The main piece of the flagellum had short irregular side-fins. The axoneme composed the typical '9+2' microtubular doublet structure and was enclosed by the cell membran e. This study confirmed that B. lenok tsinlingensis spermatozoa can be categorized as teleostean "Type I" spermatozoa; 'primitive' or 'ect-aquasperm type' spermatozoa. To the best of the authers knowledge, this was the first study conducted on the morphology and ultrastructure of B. lenok tsinlingensis spermatozoa. Copyright © 2016 Elsevier Ltd. All rights reserved.

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

PubMed

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.

Ultrastructure of spermatozoa of the lizard Ameiva ameiva, with considerations on polymorphism within the family Teiidae (Squamata).

PubMed

Giugliano, L G; Teixeira, R D; Colli, G R; Báo, S N

2002-09-01

A detailed description of sperm ultrastructure of the lizard Ameiva ameiva (Teiidae) is provided. Mature spermatozoa are characterized by: a depressed acrosome at the anterior portion; a unilateral ridge at the anterolateral portion; an acrosome vesicle divided into cortex and medulla; medulla divided into two regions with different electron-densities; paracrystalline subacrosomal material with radial organization in transverse section; a pointed prenuclear perforatorium; a stopper-like perforatorium base plate that appears embedded in the subacrosomal material; the presence of an epinuclear lucent zone surrounded by its own membrane; a large nuclear rostrum; round nuclear shoulders; a nuclear space at the nucleus tip; a bilateral stratified laminar structure; a central dense body within the proximal centriole; a short midpiece; an axonemal midpiece axial component; peripheral fibers 3 and 8 grossly enlarged at the anterior portion of axoneme; columnar mitochondria with linear cristae; solid dense bodies arranged as rings or spirals; a triangular-shaped annulus in transverse section; a fibrous sheath into the midpiece; a thin zone of cytoplasm at the anterior portion of the principal piece; and a slight decrease in diameter of the principal piece immediately after the annulus. Comparisons with Cnemidophorus sexlineatus and Micrablepharus maximiliani failed to identify unique sperm ultrastructure traits of Teiidae or Teiioidea (Teiidae + Gymnophthalmidae). High levels of polymorphism between Ameiva and Cnemidophorus, two closely related genera of the family Teiidae, were detected, suggesting that extensive sampling within squamate families is essential if sperm ultrastructure data are to be used in phylogenetic analyses at this taxonomic level. Copyright 2002 Wiley-Liss, Inc.

Mps1 as a link between centrosomes and genomic instability.

PubMed

Kasbek, Christopher; Yang, Ching-Hui; Fisk, Harold A

2009-10-01

Centrosomes are microtubule-organizing centers that must be precisely duplicated before mitosis. Centrosomes regulate mitotic spindle assembly, and the presence of excess centrosomes leads to the production of aberrant mitotic spindles which generate chromosome segregation errors. Many human tumors possess excess centrosomes that lead to the production of abnormal spindles in situ. In some tumors, these extra centrosomes appear before aneuploidy, suggesting that defects in centrosome duplication might promote genomic instability and tumorigenesis. The Mps1 protein kinase is required for centrosome duplication, and preventing the proteasome-dependent degradation of Mps1 at centrosomes increases its local concentration and causes the production of excess centrosomes during a prolonged S-phase. Here, we show that Mps1 degradation is misregulated in two tumor-derived cell lines, and that the failure to appropriately degrade Mps1 correlates with the ability of these cells to produce extra centrosomes during a prolonged S-phase. In the 21NT breast-tumor derived cell line, a mutant Mps1 protein that is normally constitutively degraded can accumulate at centrosomes and perturb centrosome duplication, suggesting that these cells have a defect in the mechanisms that target Mps1 to the proteasome. In contrast, the U2OS osteosarcoma cell line expresses a nondegradable form of Mps1, which we show causes the dose-dependent over duplication of centrioles even at very low levels of expression. Our data demonstrate that defects in Mps1 degradation can occur through multiple mechanisms, and suggest that Mps1 may provide a link between the control of centrosome duplication and genomic instability. (c) 2009 Wiley-Liss, Inc.

Mutations in ARL2BP, Encoding ADP-Ribosylation-Factor-Like 2 Binding Protein, Cause Autosomal-Recessive Retinitis Pigmentosa

PubMed Central

Davidson, Alice E.; Schwarz, Nele; Zelinger, Lina; Stern-Schneider, Gabriele; Shoemark, Amelia; Spitzbarth, Benjamin; Gross, Menachem; Laxer, Uri; Sosna, Jacob; Sergouniotis, Panagiotis I.; Waseem, Naushin H.; Wilson, Robert; Kahn, Richard A.; Plagnol, Vincent; Wolfrum, Uwe; Banin, Eyal; Hardcastle, Alison J.; Cheetham, Michael E.; Sharon, Dror; Webster, Andrew R.

2013-01-01

Retinitis pigmentosa (RP) is a genetically heterogeneous retinal degeneration characterized by photoreceptor death, which results in visual failure. Here, we used a combination of homozygosity mapping and exome sequencing to identify mutations in ARL2BP, which encodes an effector protein of the small GTPases ARL2 and ARL3, as causative for autosomal-recessive RP (RP66). In a family affected by RP and situs inversus, a homozygous, splice-acceptor mutation, c.101−1G>C, which alters pre-mRNA splicing of ARLBP2 in blood RNA, was identified. In another family, a homozygous c.134T>G (p.Met45Arg) mutation was identified. In the mouse retina, ARL2BP localized to the basal body and cilium-associated centriole of photoreceptors and the periciliary extension of the inner segment. Depletion of ARL2BP caused cilia shortening. Moreover, depletion of ARL2, but not ARL3, caused displacement of ARL2BP from the basal body, suggesting that ARL2 is vital for recruiting or anchoring ARL2BP at the base of the cilium. This hypothesis is supported by the finding that the p.Met45Arg amino acid substitution reduced binding to ARL2 and caused the loss of ARL2BP localization at the basal body in ciliated nasal epithelial cells. These data demonstrate a role for ARL2BP and ARL2 in primary cilia function and that this role is essential for normal photoreceptor maintenance and function. PMID:23849777

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

NASA Astrophysics Data System (ADS)

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 heterotremes.

Reorganization of microtubules in endosperm cells and cell fragments of the higher plant Haemanthus in vivo

PubMed Central

1986-01-01

The reorganization of the microtubular meshwork was studied in intact Haemanthus endosperm cells and cell fragments (cytoplasts). This higher plant tissue is devoid of a known microtubule organizating organelle. Observations on living cells were correlated with microtubule arrangements visualized with the immunogold method. In small fragments, reorganization did not proceed. In medium and large sized fragments, microtubular converging centers formed first. Then these converging centers reorganized into either closed bushy microtubular spiral or chromosome-free cytoplasmic spindles/phragmoplasts. Therefore, the final shape of organized microtubular structures, including spindle shaped, was determined by the initial size of the cell fragments and could be achieved without chromosomes or centrioles. Converging centers elongate due to the formation of additional structures resembling microtubular fir trees. These structures were observed at the pole of the microtubular converging center in anucleate fragments, accessory phragmoplasts in nucleated cells, and in the polar region of the mitotic spindle during anaphase. Therefore, during anaphase pronounced assembly of new microtubules occurs at the polar region of acentriolar spindles. Moreover, statistical analysis demonstrated that during the first two-thirds of anaphase, when chromosomes move with an approximately constant speed, kinetochore fibers shorten, while the length of the kinetochore fiber complex remains constant due to the simultaneous elongation of their integral parts (microtubular fir trees). The half-spindle shortens only during the last one-third of anaphase. These data contradict the presently prevailing view that chromosome-to-pole movements in acentriolar spindles of higher plants are concurrent with the shortening of the half-spindle, the self- reorganizing property of higher plant microtubules (tubulin) in vivo. It may be specific for cells without centrosomes and may be superimposed also on other microtubule-related processes. PMID:3941154

Fluorescence microscopy study on the cytoskeletal displacements during sperm differentiation in the bush-cricket Tylopsis liliifolia (Fabricius) (Orthoptera: Tettigoniidae).

PubMed

Viscuso, Renata; Federico, Concetta; Saccone, Salvatore; Bonaccorsi, Bianca; Vitale, Danilo G M

2016-02-01

A study by fluorescence microscopy has been carried out on male gametes from testicular follicles, seminal vesicles, spermatophores, and seminal receptacles of the bush-cricket Tylopsis liliifolia, focusing the attention on localization and movements of F-actin and α-tubulin during sperm differentiation, since data in this respect are lacking in the Orthoptera. F-actin and α-tubulin positivity was detected in the testicular follicles, in particular at the bridges connecting spermatids of a same clone and around their nucleus, during the first differentiation stages. During the following differentiation stages in the testes, F-actin was found at one of the spermatid poles and then, during nucleus elongation, at the whole acrosomal region. A peculiar F-actin-positivity was found at the flagellum, more markedly immediately posterior to the nucleus, at the basal body region of the gametes from the testicular follicles and from the other examined districts. Other interesting data from our investigations concerns the α-tubulin displacements during the differentiation stages of the spermatid and a constant absence of α-tubulin-positivity where the centrioles are located. No positivity was also found for both α-tubulin and nuclear markers at the anterior region of the gamete, where the acrosomal wings are localized. Our results, compared with what is so far known in literature for the insects, lead us to assert that microfilaments and microtubules undergo gradual displacements, markedly in the testicular follicles, during the morphogenesis of the male gamete of T. liliifolia aimed to its organization and motility and probably also to its interaction with the female gamete. © 2015 Wiley Periodicals, Inc.

LIM kinase function and renal growth: Potential role for LIM kinases in fetal programming of kidney development.

PubMed

Sparrow, Alexander J; Sweetman, Dylan; Welham, Simon J M

2017-10-01

Maternal dietary restriction during pregnancy impairs nephron development and results in offspring with fewer nephrons. Cell turnover in the early developing kidney is altered by exposure to maternal dietary restriction and may be regulated by the LIM-kinase family of enzymes. We set out to establish whether disturbance of LIM-kinase activity might play a role in the impairment of nephron formation. E12.5 metanephric kidneys and HK2 cells were grown in culture with the pharmacological LIM-kinase inhibitor BMS5. Organs were injected with DiI, imaged and cell numbers measured over 48h to assess growth. Cells undergoing mitosis were visualised by pH3 labelling. Growth of cultured kidneys reduced to 83% of controls after exposure to BMS5 and final cell number to 25% of control levels after 48h. Whilst control and BMS5 treated organs showed cells undergoing mitosis (100±11 cells/field vs 113±18 cells/field respectively) the proportion in anaphase was considerably diminished with BMS5 treatment (7.8±0.8% vs 0.8±0.6% respectively; P<0.01). This was consistent with effects on HK2 cells highlighting a severe impact of BMS5 on formation of the mitotic spindle and centriole positioning. DiI labelled cells migrated in 100% of control cultures vs 0% BMS5 treated organs. The number of nephrogenic precursor cells appeared depleted in whole organs and formation of new nephrons was blocked by exposure to BMS5. Pharmacological blockade of LIM-kinase function in the early developing kidney results in failure of renal development. This is likely due to prevention of dividing cells from completion of mitosis with their resultant loss. Copyright © 2017 Elsevier Inc. All rights reserved.

The Multiplicity of the Mitotic Centers and the Time-Course of Their Duplication and Separation

PubMed Central

Mazia, Daniel; Harris, Patricia J.; Bibring, Thomas

1960-01-01

In this study, the reproduction of the mitotic centers in the eggs of a sea urchin, Strongylocentrotus purpuratus and a sand dollar Dendraster excentricus has been studied by means of experimental designs that do not depend on the actual visualization of centrioles. The centers are defined in operational terms as potential poles. Blockage of mitosis by mercaptoethanol, it was found, inhibits the duplication of the centers, but does not inhibit the splitting and separation of centers that have already duplicated and thus potential poles could be realized as actual poles in multipolar divisions. At all times, the center is at least a duplex structure; that is, it contains two potential poles. The actual duplication process is the earliest event in a given mitotic cycle, taking place at very early interphase or in late telophase of the previous division. The splitting of the centers following duplication is a distinct process, dissociable from the duplication as such. Duplication and splitting normally occur at about the same time in the mitotic cycle, with a precession of the former. That is, as the two members of a pair of "old" centers split, each one gives rise to a new one, which remains associated with it until the next phase of splitting and duplication occurs. The results are consistent with what is termed a "generative" model of the self-reproduction of an intracellular body. According to this, the body does not immediately produce a full-fledged copy of itself, with simultaneous fission, but the primary duplication event involves only a part of the parent structure. This gives rise to a "germ" or "seed" which then grows to be equivalent to the parent body, and finally splits from it. PMID:19866563

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

PubMed Central

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

Electrophilic aldehyde products of lipid peroxidation selectively adduct to heat shock protein 90 and arylsulfatase A in stallion spermatozoa.

PubMed

Hall, Sally E; Aitken, R John; Nixon, Brett; Smith, Nathan D; Gibb, Zamira

2017-01-01

Oxidative stress is a major determinant of mammalian sperm function stimulating lipid peroxidation cascades that culminate in the generation of potentially cytotoxic aldehydes. The aim of this study was to assess the impact of such aldehydes on the functionality of stallion spermatozoa. The impact of exposure to exogenous acrolein (ACR) and 4-hydroxynonenal (4HNE) was manifested in a highly significant dose- and time-dependent increase in mitochondrial reactive oxygen species (ROS), total cellular ROS, a decrease in sperm motility, and a time-dependent increase in lipid peroxidation. Notably, low doses of ACR and 4HNE also caused a significant decrease in zona binding. In contrast, exogenous malondialdehyde, a commonly used marker of oxidative stress, had little impact on the various sperm parameters assessed. In accounting for the negative physiological impact of ACR and 4HNE, it was noted that both aldehydes readily adducted to sperm proteins located predominantly within the head, proximal centriole, and tail. The detoxifying activity of mitochondrial aldehyde dehydrogenase 2 appeared responsible for a lack of adduction in the midpiece; however, this activity was overwhelmed by 24 h of electrophilic aldehyde exposure. Sequencing of the dominant proteins targeted for ACR and 4HNE covalent modification identified heat shock protein 90 alpha (cytosolic) class A member 1 and arylsulfatase A, respectively. These collective findings may prove useful in the identification of diagnostic biomarkers of stallion fertility and resolving the mechanistic basis of sperm dysfunction in this species. © The Authors 2016. Published by Oxford University Press on behalf of Society for the Study of Reproduction. All rights reserved. For permissions, please journals.permissions@oup.com.

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

PubMed

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.

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

NASA Astrophysics Data System (ADS)

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 Petalomera lateralis in the large size of the sperm nucleus relative to the acrosome compared with D. edwardsi and homolids.

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.

PubMed

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.

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

PubMed

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

2017-01-01

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.

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

Fitzgerald, Kent, E-mail: Kent.fitzgerald@elan.com; Bergeron, Marcelle, E-mail: Marcelle.bergeron@elan.com; Willits, Christopher, E-mail: Chris.willits@elan.com

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 numbermore » 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 phosphorylation of alpha-synuclein at non-genotoxic doses.« less

Biallelic Variants in TTLL5, Encoding a Tubulin Glutamylase, Cause Retinal Dystrophy

PubMed Central

Sergouniotis, Panagiotis I.; Chakarova, Christina; Murphy, Cian; Becker, Mirjana; Lenassi, Eva; Arno, Gavin; Lek, Monkol; MacArthur, Daniel G.; Bhattacharya, Shomi S.; Moore, Anthony T.; Holder, Graham E.; Robson, Anthony G.; Wolfrum, Uwe; Webster, Andrew R.; Plagnol, Vincent

2014-01-01

In a subset of inherited retinal degenerations (including cone, cone-rod, and macular dystrophies), cone photoreceptors are more severely affected than rods; ABCA4 mutations are the most common cause of this heterogeneous class of disorders. To identify retinal-disease-associated genes, we performed exome sequencing in 28 individuals with “cone-first” retinal disease and clinical features atypical for ABCA4 retinopathy. We then conducted a gene-based case-control association study with an internal exome data set as the control group. TTLL5, encoding a tubulin glutamylase, was highlighted as the most likely disease-associated gene; 2 of 28 affected subjects harbored presumed loss-of-function variants: c.[1586_1589delAGAG];[1586_1589delAGAG], p.[Glu529Valfs∗2];[Glu529Valfs∗2], and c.[401delT(;)3354G>A], p.[Leu134Argfs∗45(;)Trp1118∗]. We then inspected previously collected exome sequence data from individuals with related phenotypes and found two siblings with homozygous nonsense variant c.1627G>T (p.Glu543∗) in TTLL5. Subsequently, we tested a panel of 55 probands with retinal dystrophy for TTLL5 mutations; one proband had a homozygous missense change (c.1627G>A [p.Glu543Lys]). The retinal phenotype was highly similar in three of four families; the sibling pair had a more severe, early-onset disease. In human and murine retinae, TTLL5 localized to the centrioles at the base of the connecting cilium. TTLL5 has been previously reported to be essential for the correct function of sperm flagella in mice and play a role in polyglutamylation of primary cilia in vitro. Notably, genes involved in the polyglutamylation and deglutamylation of tubulin have been associated with photoreceptor degeneration in mice. The electrophysiological and fundus autofluorescence imaging presented here should facilitate the molecular diagnosis in further families. PMID:24791901

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

PubMed

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 .

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

PubMed

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 centrosomin interactions that support the expansion into the metaphase matrix.

The phagotrophic origin of eukaryotes and phylogenetic classification of Protozoa.

PubMed

Cavalier-Smith, T

2002-03-01

Eukaryotes and archaebacteria form the clade neomura and are sisters, as shown decisively by genes fragmented only in archaebacteria and by many sequence trees. This sisterhood refutes all theories that eukaryotes originated by merging an archaebacterium and an alpha-proteobacterium, which also fail to account for numerous features shared specifically by eukaryotes and actinobacteria. I revise the phagotrophy theory of eukaryote origins by arguing that the essentially autogenous origins of most eukaryotic cell properties (phagotrophy, endomembrane system including peroxisomes, cytoskeleton, nucleus, mitosis and sex) partially overlapped and were synergistic with the symbiogenetic origin of mitochondria from an alpha-proteobacterium. These radical innovations occurred in a derivative of the neomuran common ancestor, which itself had evolved immediately prior to the divergence of eukaryotes and archaebacteria by drastic alterations to its eubacterial ancestor, an actinobacterial posibacterium able to make sterols, by replacing murein peptidoglycan by N-linked glycoproteins and a multitude of other shared neomuran novelties. The conversion of the rigid neomuran wall into a flexible surface coat and the associated origin of phagotrophy were instrumental in the evolution of the endomembrane system, cytoskeleton, nuclear organization and division and sexual life-cycles. Cilia evolved not by symbiogenesis but by autogenous specialization of the cytoskeleton. I argue that the ancestral eukaryote was uniciliate with a single centriole (unikont) and a simple centrosomal cone of microtubules, as in the aerobic amoebozoan zooflagellate Phalansterium. I infer the root of the eukaryote tree at the divergence between opisthokonts (animals, Choanozoa, fungi) with a single posterior cilium and all other eukaryotes, designated 'anterokonts' because of the ancestral presence of an anterior cilium. Anterokonts comprise the Amoebozoa, which may be ancestrally unikont, and a vast ancestrally biciliate clade, named 'bikonts'. The apparently conflicting rRNA and protein trees can be reconciled with each other and this ultrastructural interpretation if long-branch distortions, some mechanistically explicable, are allowed for. Bikonts comprise two groups: corticoflagellates, with a younger anterior cilium, no centrosomal cone and ancestrally a semi-rigid cell cortex with a microtubular band on either side of the posterior mature centriole; and Rhizaria [a new infrakingdom comprising Cercozoa (now including Ascetosporea classis nov.), Retaria phylum nov., Heliozoa and Apusozoa phylum nov.], having a centrosomal cone or radiating microtubules and two microtubular roots and a soft surface, frequently with reticulopodia. Corticoflagellates comprise photokaryotes (Plantae and chromalveolates, both ancestrally with cortical alveoli) and Excavata (a new protozoan infrakingdom comprising Loukozoa, Discicristata and Archezoa, ancestrally with three microtubular roots). All basal eukaryotic radiations were of mitochondrial aerobes; hydrogenosomes evolved polyphyletically from mitochondria long afterwards, the persistence of their double envelope long after their genomes disappeared being a striking instance of membrane heredity. I discuss the relationship between the 13 protozoan phyla recognized here and revise higher protozoan classification by updating as subkingdoms Lankester's 1878 division of Protozoa into Corticata (Excavata, Alveolata; with prominent cortical microtubules and ancestrally localized cytostome--the Parabasalia probably secondarily internalized the cytoskeleton) and Gymnomyxa [infrakingdoms Sarcomastigota (Choanozoa, Amoebozoa) and Rhizaria; both ancestrally with a non-cortical cytoskeleton of radiating singlet microtubules and a relatively soft cell surface with diffused feeding]. As the eukaryote root almost certainly lies within Gymnomyxa, probably among the Sarcomastigota, Corticata are derived. Following the single symbiogenetic origin of chloroplasts in a corticoflagellate host with cortical alveoli, this ancestral plant radiated rapidly into glaucophytes, green plants and red algae. Secondary symbiogeneses subsequently transferred plastids laterally into different hosts, making yet more complex cell chimaeras--probably only thrice: from a red alga to the corticoflagellate ancestor of chromalveolates (Chromista plus Alveolata), from green algae to a secondarily uniciliate cercozoan to form chlorarachneans and independently to a biciliate excavate to yield photosynthetic euglenoids. Tertiary symbiogenesis involving eukaryotic algal symbionts replaced peridinin-containing plastids in two or three dinoflagellate lineages, but yielded no major novel groups. The origin and well-resolved primary bifurcation of eukaryotes probably occurred in the Cryogenian Period, about 850 million years ago, much more recently than suggested by unwarranted backward extrapolations of molecular 'clocks' or dubious interpretations as 'eukaryotic' of earlier large microbial fossils or still more ancient steranes. The origin of chloroplasts and the symbiogenetic incorporation of a red alga into a corticoflagellate to create chromalveolates may both have occurred in a big bang after the Varangerian snowball Earth melted about 580 million years ago, thereby stimulating the ensuing Cambrian explosion of animals and protists in the form of simultaneous, poorly resolved opisthokont and anterokont radiations.

Cell Motility by Labile Association of Molecules

PubMed Central

Inoué, Shinya; Sato, Hidemi

1967-01-01

This article summarizes our current views on the dynamic structure of the mitotic spindle and its relation to mitotic chromosome movements. The following statements are based on measurements of birefringence of spindle fibers in living cells, normally developing or experimentally modified by various physical and chemical agents, including high and low temperatures, antimitotic drugs, heavy water, and ultraviolet microbeam irradiation. Data were also obtained concomitantly with electron microscopy employing a new fixative and through measurements of isolated spindle protein. Spindle fibers in living cells are labile dynamic structures whose constituent filaments (microtubules) undergo cyclic breakdown and reformation. The dynamic state is maintained by an equilibrium between a pool of protein molecules and their linearly aggregated polymers, which constitute the microtubules or filaments. In living cells under physiological conditions, the association of the molecules into polymers is very weak (absolute value of ΔF 25°C < 1 kcal), and the equilibrium is readily shifted to dissociation by low temperature or by high hydrostatic pressure. The equilibrium is shifted toward formation of polymer by increase in temperature (with a large increase in entropy: ΔS 25°C ≃ 100 eu) or by the addition of heavy water. The spindle proteins tend to polymerize with orienting centers as their geometrical foci. The centrioles, kinetochores, and cell plate act as orienting centers successively during mitosis. Filaments are more concentrated adjacent to an orienting center and yield higher birefringence. Astral rays, continuous fibers, chromosomal fibers, and phragmoplast fibers are thus formed by successive reorganization of the same protein molecules. During late prophase and metaphase, polymerization takes place predominantly at the kinetochores; in metaphase and anaphase, depolymerization is prevalent near the spindle poles. When the concentration of spindle protein is high, fusiform bundles of polymer are precipitated out even in the absence of obvious orienting centers. The shift of equilibrium from free protein molecules to polymer increases the length and number of the spindle microtubules or filaments. Slow depolymerization of the polymers, which can be brought about by low concentrations of colchicine or by gradual cooling, allows the filaments to shorten and perform work. The dynamic equilibrium controlled by orienting centers and other factors provides a plasusible mechanism by which chromosomes and other organelles, as well as the cell surface, are deformed or moved by temporarily organized arrays of microtubules or filaments. PMID:6058222

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

PubMed

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 the secondary analyses. We found weak evidence to suggest that genes previously associated with TD are associated with sun exposure in children of European ancestry from southwest England.

Tubulinlike protein from Spirochaeta bajacaliforniensis

NASA Technical Reports Server (NTRS)

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 biophysical 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 anitubulin 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 bacteria 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 angstroms 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 displays antitubulin immunoreactivity in whole-cell preparations. Since preliminary observations suggested that Spirochaeta bajacaliforniensis proteins may be related to eukaryotic tubulins, their characterization was undertaken. Brain tubulin can be purified by utilizing its ability to polymerize at warm temperatures and to depolymerize in the cold. After several cycles of sedimentation and redissolution the microtubule fraction is composed of 75% tubulin and 20% high molecular mass microtubule-associated proteins (MAPs). In this paper we report that components of cell lysates, prepared from a spirochete that contains cytoplasmic fibers (Spirochaeta bajacaliforniensis), also exhibit the property of temperature-dependent cyclical sedimentation. Additionally we report the identification and characterization of the polypeptide responsible for cross-reactivity with antitubulin antiserum.

Tubulinlike protein from Spirochaeta bajacaliforniensis

NASA Technical Reports Server (NTRS)

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 displays antitubulin immunoreactivity in whole-cell preparations. Since preliminary observations suggested that Spirochaeta bajacaliforniensis proteins may be related to eukaryotic tubulins, their characterization was undertaken. Brain tubulin can be purified by utilizing its ability to polymerize at warm temperatures and to depolymerize in the cold. After several cycles of sedimentation and redissolution the microtubule fraction is comprised of 75% tubulin and 20% high molecular mass microtubule-associated proteins (MAPs). In this paper we report that components of cell lysates, prepared from a spirochete that contains cytoplasmic fibers (Spirochaeta bajacaliforniensis), also exhibit the property of temperature-dependent cyclical sedimentation. Additionally we report the identification and characterization of the polypeptide responsible for cross-reactivity with antitubulin antiserum.

Identification of EGFLAM, SPATC1L and RNASE13 as novel susceptibility loci for aortic aneurysm in Japanese individuals by exome-wide association studies

PubMed Central

Yamada, Yoshiji; Sakuma, Jun; Takeuchi, Ichiro; Yasukochi, Yoshiki; Kato, Kimihiko; Oguri, Mitsutoshi; Fujimaki, Tetsuo; Horibe, Hideki; Muramatsu, Masaaki; Sawabe, Motoji; Fujiwara, Yoshinori; Taniguchi, Yu; Obuchi, Shuichi; Kawai, Hisashi; Shinkai, Shoji; Mori, Seijiro; Arai, Tomio; Tanaka, Masashi

2017-01-01

We performed an exome-wide association study (EWAS) to identify genetic variants - in particular, low-frequency or rare variants with a moderate to large effect size - that confer susceptibility to aortic aneurysm with 8,782 Japanese subjects (456 patients with aortic aneurysm, 8,326 control individuals) and with the use of Illumina HumanExome-12 DNA Analysis BeadChip or Infinium Exome-24 BeadChip arrays. The correlation of allele frequencies for 41,432 single nucleotide polymorphisms (SNPs) that passed quality control to aortic aneurysm was examined with Fisher's exact test. Based on Bonferroni's correction, a P-value of <1.21×10−6 was considered statistically significant. The EWAS revealed 59 SNPs that were significantly associated with aortic aneurysm. None of these SNPs was significantly (P<2.12×10−4) associated with aortic aneurysm by multivariable logistic regression analysis with adjustment for age, gender and hypertension, although 8 SNPs were related (P<0.05) to this condition. Examination of the correlation of these latter 8 SNPs to true or dissecting aortic aneurysm separately showed that rs1465567 [T/C (W229R)] of the EGF-like, fibronectin type III, and laminin G domains gene (EGFLAM) (dominant model; P=0.0014; odds ratio, 1.63) was significantly (P<0.0016) associated with true aortic aneurysm. We next performed EWASs for true or dissecting aortic aneurysm separately and found that 45 and 19 SNPs were significantly associated with these conditions, respectively. Multivariable logistic regression analysis with adjustment for covariates revealed that rs113710653 [C/T (E231K)] of the spermatogenesis- and centriole associated 1-like gene (SPATC1L) (dominant model; P=0.0002; odds ratio, 5.32) and rs143881017 [C/T (R140H)] of the ribonuclease A family member 13 gene (RNASE13) (dominant model; P=0.0006; odds ratio, 5.77) were significantly (P<2.78×10−4 or P<6.58×10−4, respectively) associated with true or dissecting aortic aneurysm, respectively. EGFLAM and SPATC1L may thus be susceptibility loci for true aortic aneurysm and RNASE13 may be such a locus for dissecting aneurysm in Japanese individuals. PMID:28339009