Revisiting the Role of the Mother Centriole in Centriole Biogenesis

Centrioles duplicate once in each cell division cycle through so-called templated or canonical duplication. SAK, also called PLK4 (SAK/PLK4), a kinase implicated in tumor development, is an upstream regulator of canonical biogenesis necessary for centriole formation. We found that overexpression of SAK/PLK4 could induce amplification of centrioles in Drosophila embryos and their de novo formation in unfertilized eggs. Both processes required the activity of DSAS-6 and DSAS-4, two molecules required for canonical duplication. Thus, centriole biogenesis is a template-free self-assembly process triggered and regulated by molecules that ordinarily associate with the existing centriole. The mother centriole is not a bona fide template but a platform for a set of regulatory molecules that catalyzes and regulates daughter centriole assembly

Heterogeneous proliferative potential in regenerative adult newt cardiomyocytes.

Adult newt cardiomyocytes, in contrast to their mammalian counterparts, can proliferate after injury and contribute to the functional regeneration of the heart. In order to understand the mechanisms underlying this plasticity we performed longitudinal studies on single cardiomyocytes in culture. We find that the majority of cardiomyocytes can enter S phase, a process that occurs in response to serum-activated pathways and is dependent on the phosphorylation of the retinoblastoma protein. However, more than half of these cells stably arrest at either entry to mitosis or during cytokinesis, thus resembling the behaviour observed in mammalian cardiomyocytes. Approximately a third of the cells progress through mitosis and may enter successive cell divisions. When cardiomyocytes divided more than once, the proliferative behaviour of sister cells was significantly correlated, in terms of whether they underwent a subsequent cell cycle, and if so, the duration of that cycle. These observations suggest a mechanism whereby newt heart regeneration depends on the retention of proliferative potential in a subset of cardiomyocytes. The regulation of the remaining newt cardiomyocytes is similar to that described for their mammalian counterparts, as they arrest during mitosis or cytokinesis. Understanding the nature of this block and why it arises in some but not other newt cardiomyocytes may lead to an augmentation of the regenerative potential in the mammalian heart

Polo-like kinase 4 controls centriole duplication but does not directly regulate cytokinesis.

Centrioles organize the centrosome, and accurate control of their number is critical for the maintenance of genomic integrity. Centrioles duplicate once per cell cycle, and duplication is coordinated by Polo-like kinase 4 (Plk4). We previously demonstrated that Plk4 accumulation is autoregulated by its own kinase activity. However, loss of heterozygosity of Plk4 in mouse embryonic fibroblasts has been proposed to cause cytokinesis failure as a primary event, leading to centrosome amplification and gross chromosomal abnormalities. Using targeted gene disruption, we show that human epithelial cells with one inactivated Plk4 allele undergo neither cytokinesis failure nor increase in centrosome amplification. Plk4 is shown to localize exclusively at the centrosome, with none in the spindle midbody. Substantial depletion of Plk4 by small interfering RNA leads to loss of centrioles and subsequent spindle defects that lead to a modest increase in the rate of cytokinesis failure. Therefore, Plk4 is a centriole-localized kinase that does not directly regulate cytokinesis

DSAS-6 Organizes a Tube-like Centriole Precursor, and Its Absence Suggests Modularity in Centriole Assembly

Centrioles are microtubule-based cylindrical structures that exhibit 9-fold symmetry and facilitate the organization of centrosomes, flagella, and cilia [1]. Abnormalities in centrosome structure and number occur in many cancers 1, 2. Despite its importance, very little is known about centriole biogenesis. Recent studies in C. elegans have highlighted a group of molecules necessary for centriole assembly 1, 3. ZYG-1 kinase recruits a complex of two coiled-coil proteins, SAS-6 and SAS-5, which are necessary to form the C. elegans centriolar tube, a scaffold in centriole formation 4, 5. This complex also recruits SAS-4, which is required for the assembly of the centriolar microtubules that decorate that tube 4, 5. Here we show that Drosophila SAS-6 is involved in centriole assembly and cohesion. Overexpression of DSAS-6 in syncitial embryos led to the de novo formation of multiple microtubule-organizing centers (MTOCs). Strikingly, the center of these MTOCs did not contain centrioles, as described previously for SAK/PLK4 overexpression [6]. Instead, tube-like structures were present, supporting the idea that centriolar assembly starts with the formation of a tube-like scaffold, dependent on DSAS-6 [5]. In DSAS-6 loss-of-function mutants, centrioles failed to close and to elongate the structure along all axes of the 9-fold symmetry, suggesting modularity in centriole assembly. We propose that the tube is built from nine subunits fitting together laterally and longitudinally in a modular and sequential fashion, like pieces of a layered “hollow” cake

SAK/PLK4 Is Required for Centriole Duplication and Flagella Development

Background. SAK/PLK4 is a distinct member of the polo-like kinase family. SAK−/− mice die during embryogenesis, whereas SAK+/− mice develop liver and lung tumors and SAK+/− MEFs show mitotic abnormalities. However, the mechanism underlying these phenotypes is still not known. Results. Here, we show that downregulation of SAK in Drosophila cells, by mutation or RNAi, leads to loss of centrioles, the core structures of centrosomes. Such cells are able to undergo repeated rounds of cell division, but display broad disorganized mitotic spindle poles. We also show that SAK mutants lose their centrioles during the mitotic divisions preceding male meiosis but still produce cysts of 16 primary spermatocytes as in the wild-type. Mathematical modeling of the stereotyped cell divisions of spermatogenesis can account for such loss by defective centriole duplication. The majority of spermatids in SAK mutants lack centrioles and so are unable to make sperm axonemes. Finally, we show that depletion of SAK in human cells also prevents centriole duplication and gives rise to mitotic abnormalities. Conclusions: SAK/PLK4 is necessary for centriole duplication both in Drosophila and human cells. Drosophila cells tolerate the lack of centrioles and undertake mitosis but cannot form basal bodies and hence flagella. Human cells depleted of SAK show error-prone mitosis, likely to underlie its tumor-suppressor role

Cereal landraces genetic resources in worldwide GeneBanks. A review

Since the dawn of agriculture, cereal landraces have been the staples for food production worldwide, but their use dramatically declined in the 2nd half of the last century, replaced by modern cultivars. In most parts of the world, landraces are one of the most threatened components of agrobiodiversity, facing the risk of genetic erosion and extinction. Since landraces have a tremendous potential in the development of new cultivars adapted to changing environmental conditions, GeneBanks holding their genetic resources potentially play an important role in supporting sustainable agriculture. This work reviews the current knowledge on cereal landraces maintained in GeneBanks and highlights the strengths and weaknesses of existing information about their taxonomy, origin, structure, threats, sampling methodologies and conservation and GeneBanks’ documentation and management. An overview of major collections of cereal landraces is presented, using the information available in global metadatabase systems. This review on winter cereal landrace conservation focuses on: (1) traditional role of GeneBanks is evolving beyond their original purpose to conserve plant materials for breeding programmes. Today’s GeneBank users are interested in landraces’ history, agro-ecology and traditional knowledge associated with their use, in addition to germplasm traits. (2) GeneBanks therefore need to actively share their germplasm collections’ information using different channels, to promote unlimited and effective use of these materials for the further development of sustainable agriculture. (3) Access to information on the 7.4 million accessions conserved in GeneBanks worldwide, of which cereal accessions account for nearly 45 %, particularly information on cereal landraces (24 % of wheat, 23 % of barley, 14 % of oats and 29 % of rye accessions), is often not easily available to potential users, mainly due to the lack of consistent or compatible documentation systems, their structure and registration. (4) Enhancing the sustainable use of landraces maintained in germplasm collections through the effective application of recent advances in landrace knowledge (origin, structure and traits) and documentation using the internet tools and data providing networks, including the use of molecular and biotechnological tools for the material screening and detection of agronomic traits. (5) Cereal landraces cannot be exclusively conserved as seed samples maintained under ex situ conditions in GeneBanks. The enormous contribution of farmers in maintaining the crop and landraces diversity is recognised. Sharing of benefits and raising awareness of the value of cereal landraces are the most effective ways to promote their conservation and to ensure their continued availability and sustainable use. (6) Evaluation of costs and economic benefits attributed to sustainable use of cereal landraces conserved in the GeneBanks requires comprehensive studies conducted on a case-by-case basis, that take into consideration species/crop resources, conservation conditions and quality and GeneBank location and functions.This work was support by the European Community, through the INTERREG IIIB and MAC programmes, research projects Germobanco Agrícola da Macaronesia and AGRICOMAC. This paper was edited by Olga Spellman (Bioversity International)info:eu-repo/semantics/publishedVersio

BLD10/CEP135 Is a Microtubule-Associated Protein that Controls the Formation of the Flagellum Central Microtubule Pair

The deposited article is a post-print version and has been submitted to peer review.The deposited article is a pre-print versionThis deposit is composed by the main article and the supplementary materials are present in the publisher's page in the following link: https://ars.els-cdn.com/content/image/1-s2.0-S1534580712002511-mmc1.pdfCilia and flagella are involved in a variety of processes and human diseases, including ciliopathies and sterility. Their motility is often controlled by a central microtubule (MT) pair localized within the ciliary MT-based skeleton, the axoneme. We characterized the formation of the motility apparatus in detail in Drosophila spermatogenesis. We show that assembly of the central MT pair starts prior to the meiotic divisions, with nucleation of a singlet MT within the basal body of a small cilium, and that the second MT of the pair only assembles much later, upon flagella formation. BLD10/CEP135, a conserved player in centriole and flagella biogenesis, can bind and stabilize MTs and is required for the early steps of central MT pair formation. This work describes a genetically tractable system to study motile cilia formation and provides an explanation for BLD10/CEP135's role in assembling highly stable MT-based structures, such as motile axonemes and centrioles.Fundação para a Ciência e Tecnologia grants: (PTDC/BIA-BCM/105602/2008); EMBO Installation Grant; Instituto Gulbenkian de Ciência; EMBO YIP Program; European Research Council grant: ([FP7/2010]/ERC Grant “261344-CentrioleStructNumber.”); Ciência 2007; EMBO, Marie Curie Actions.info:eu-repo/semantics/publishedVersio

Stepwise evolution of the centriole-assembly pathway

The centriole and basal body (CBB) structure nucleates cilia and flagella, and is an essential component of the centrosome, underlying eukaryotic microtubule-based motility, cell division and polarity. In recent years, components of the CBB-assembly machinery have been identified, but little is known about their regulation and evolution. Given the diversity of cellular contexts encountered in eukaryotes, but the remarkable conservation of CBB morphology, we asked whether general mechanistic principles could explain CBB assembly. We analysed the distribution of each component of the human CBB-assembly machinery across eukaryotes as a strategy to generate testable hypotheses. We found an evolutionarily cohesive and ancestral module, which we term UNIMOD and is defined by three components (SAS6, SAS4/CPAP and BLD10/CEP135), that correlates with the occurrence of CBBs. Unexpectedly, other players (SAK/PLK4, SPD2/CEP192 and CP110) emerged in a taxon-specific manner. We report that gene duplication plays an important role in the evolution of CBB components and show that, in the case of BLD10/CEP135, this is a source of tissue specificity in CBB and flagella biogenesis. Moreover, we observe extreme protein divergence amongst CBB components and show experimentally that there is loss of cross-species complementation among SAK/PLK4 family members, suggesting species-specific adaptations in CBB assembly. We propose that the UNIMOD theory explains the conservation of CBB architecture and that taxon- and tissue-specific molecular innovations, gained through emergence, duplication and divergence, play important roles in coordinating CBB biogenesis and function in different cellular contexts.Fundação Calouste Gulbenkian; Fundação para a Ciência e Tecnologia scholarships and grants: (POCI2010); Câmara Municipal de Oeiras; EMBO Installation Grant

IFT88 transports Gucy2d, a guanylyl cyclase, to maintain sensory cilia function in Drosophila

Cilia are involved in a plethora of motility and sensory-related functions. Ciliary defects cause several ciliopathies, some of which with late-onset, suggesting cilia are actively maintained. While much is known about cilia assembly, little is understood about the mechanisms of their maintenance. Given that intraflagellar transport (IFT) is essential for cilium assembly, we investigated the role of one of its main players, IFT88, in ciliary maintenance. We show that DmIFT88, the Drosophila melanogaster orthologue of IFT88, continues to move along fully formed sensory cilia, and that its acute knockdown in the ciliated neurons of the adult affects sensory behaviour. We further identify DmGucy2d, the Drosophila guanylyl cyclase 2d, as a DmIFT88 cargo, whose loss also leads to defects in sensory behaviour maintenance. DmIFT88 binds to the intracellular part of DmGucy2d, a highly, evolutionarily conserved and mutated in several degenerative retina diseases, taking the cyclase into the cilia. Our results offer a novel mechanism for the maintenance of sensory cilia function and its potential role in human diseases