2015 Abstracts

Coelho, PA; Bury, L; Shahbazi, MN; Liakath-Ali, K; Tate, PH; Wormald, S; Hindley, CJ; Huch, M; Archer, J; Skarnes, WC; Zernicka-Goetz, M; Glover, DM

Over-expression of Plk4 induces centrosome amplification, loss of primary cilia and associated tissue hyperplasia in the mouse

To address the long-known relationship between supernumerary centrosomes and cancer, we have generated a transgenic mouse that permits inducible expression of the master regulator of centriole duplication, Polo-like-kinase-4 (Plk4). Over-expression of Plk4 from this transgene advances the onset of tumour formation that occurs in the absence of the tumour suppressor p53. Plk4 over-expression also leads to hyperproliferation of cells in the pancreas and skin that is enhanced in a p53 null background. Pancreatic islets become enlarged following Plk4 over-expression as a result of equal expansion of α- and β-cells, which exhibit centrosome amplification. Mice overexpressing Plk4 develop grey hair due to a loss of differentiated melanocytes and bald patches of skin associated with a thickening of the epidermis. This reflects an increase in proliferating cells expressing keratin 5 in the basal epidermal layer and the expansion of these cells into suprabasal layers. Such cells also express keratin 6, a marker for hyperplasia. This is paralleled by a decreased expression of later differentiation markers, involucrin, filaggrin and loricrin. Proliferating cells showed an increase in centrosome number and a loss of primary cilia, events that were mirrored in primary cultures of keratinocytes established from these animals. We discuss how repeated duplication of centrioles appears to prevent the formation of basal bodies leading to loss of primary cilia, disruption of signalling and thereby aberrant differentiation of cells within the epidermis. The absence of p53 permits cells with increased centrosomes to continue dividing, thus setting up a neoplastic state of error prone mitoses, a prerequisite for cancer development.

Open Biology vol. 5(12)

Fu, J; Lipinszki, Z; Rangone, H; Min, M; Mykura, C; Chao-Chu, J; Schneider, S; Dzhindzhev, NS; Gottardo, M; Riparbelli, MG; Callaini, G; Glover, DM

Conserved molecular interactions in centriole-to-centrosome conversion.

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.

Nature Cell Biology PMID: 26595382 URL - http://www.ncbi.nlm.nih.gov/pubmed/26595382?dopt=Citation

Carmena, M; Earnshaw, WC; Glover, DM

The Dawn of Aurora Kinase Research: From Fly Genetics to the Clinic

Aurora kinases comprise a family of highly conserved serine-threonine protein kinases that play a pivotal role in the regulation of cell cycle. Aurora kinases are not only involved in the control of multiple processes during cell division but also coordinate chromosomal and cytoskeletal events, contributing to the regulation of checkpoints and ensuring the smooth progression of the cell cycle. Because of their fundamental contribution to cell cycle regulation, Aurora kinases were originally identified in independent genetic screens designed to find genes involved in the regulation of cell division. The first aurora mutant was part of a collection of mutants isolated in C. Nusslein-Volhard's laboratory. This collection was screened in D. M. Glover's laboratory in search for mutations disrupting the centrosome cycle in embryos derived from homozygous mutant mothers. The mutants identified were given names related to the "polar regions," and included not only aurora but also the equally famous polo. Ipl1, the only Aurora in yeast, was identified in a genetic screen looking for mutations that caused chromosome segregation defects. The discovery of a second Aurora-like kinase in mammals opened a new chapter in the research of Aurora kinases. The rat kinase AIM was found to be highly homologous to the fly and yeast proteins, but localized at the midzone and midbody and was proposed to have a role in cytokinesis. Homologs of the equatorial Aurora (Aurora B) were identified in metazoans ranging from flies to humans. Xenopus Aurora B was found to be in a complex with the chromosomal passenger INCENP, and both proteins were shown to be essential in flies for chromosome structure, segregation, central spindle formation and cytokinesis. Fifteen years on, Aurora kinase research is an active field of research. After the successful introduction of the first anti-mitotic agents in cancer therapy, both Auroras have become the focus of attention as targets for the development of new anti-cancer drugs. In this review we will aim to give a historical overview of the research on Aurora kinases, highlighting the most relevant milestones in the advance of the field.

Frontiers in Cell and Developmental Biology vol. 3 pp. 73 PMID: 26636082

Haider, S; Lipinszki, Z; Przewloka, MR; Ladak, Y; D'Avino, PP; Kimata, Y; Lio', P; Glover, DM

DAPPER: a data-mining resource for protein-protein interactions

BACKGROUND: The identification of interaction networks between proteins and complexes holds the promise of offering novel insights into the molecular mechanisms that regulate many biological processes. With increasing volumes of such datasets, especially in model organisms such as Drosophila melanogaster, there exists a pressing need for specialised tools, which can seamlessly collect, integrate and analyse these data. Here we describe a database coupled with a mining tool for protein-protein interactions (DAPPER), developed as a rich resource for studying multi-protein complexes in Drosophila melanogaster. RESULTS: This proteomics database is compiled through mass spectrometric analyses of many protein complexes affinity purified from Drosophila tissues and cultured cells. The web access to DAPPER is provided via an accelerated version of BioMart software enabling data-mining through customised querying and output formats. The protein-protein interaction dataset is annotated with FlyBase identifiers, and further linked to the Ensembl database using BioMart's data-federation model, thereby enabling complex multi-dataset queries. DAPPER is open source, with all its contents and source code are freely available. CONCLUSIONS: DAPPER offers an easy-to-navigate and extensible platform for real-time integration of diverse resources containing new and existing protein-protein interaction datasets of Drosophila melanogaster.

BioData Min vol. 8 pp. 30, PMID: 26405458

Chen, CC; Bowers, S; Lipinszki, Z; Palladino, J; Trusiak, S; Bettini, E; Rosin, L; Przewloka, MR; Glover, DM; O'Neill, RJ; Mellone, BG

Establishment of Centromeric Chromatin by the CENP-A Assembly Factor CAL1 Requires FACT-Mediated Transcription.

Centromeres are essential chromosomal structures that mediate accurate chromosome segregation during cell division. Centromeres are specified epigenetically by the heritable incorporation of the centromeric histone H3 variant CENP-A. While many of the primary factors that mediate centromeric deposition of CENP-A are known, the chromatin and DNA requirements of this process have remained elusive. Here, we uncover a role for transcription in Drosophila CENP-A deposition. Using an inducible ectopic centromere system that uncouples CENP-A deposition from endogenous centromere function and cell-cycle progression, we demonstrate that CENP-A assembly by its loading factor, CAL1, requires RNAPII-mediated transcription of the underlying DNA. This transcription depends on the CAL1 binding partner FACT, but not on CENP-A incorporation. Our work establishes RNAPII passage as a key step in chaperone-mediated CENP-A chromatin establishment and propagation. Copyright © 2015 Elsevier Inc. All rights reserved.

Dev. Cell 34(1) pp. 73-84

Edlich-Muth, C; Artero, JB; Callow, P; Przewloka, MR; Watson, AA; Zhang, W; Glover, DM; Debski, J; Dadlez, M; Round, AR; Trevor Forsyth, V; Laue, ED

The pentameric nucleoplasmin fold is present in Drosophila FKBP39 and a large number of chromatin-related proteins

Nucleoplasmin is a histone chaperone that consists of a pentameric N-terminal domain and an unstructured C-terminal tail. The pentameric core domain, a doughnut-like structure with a central pore, is only found in the nucleoplasmin family. Here, we report the first structure of a nucleoplasmin-like (NPL) domain from the unrelated Drosophila protein, FKBP39, and we present evidence that this protein associates with chromatin. Furthermore, we show that two other chromatin proteins, A. thaliana histone deacetylase type 2 (HD2) and S. cerevisiae Fpr4, share the NPL fold and form pentamers, in the case of HD2 a dimer of pentamers. Thus we propose a new family of proteins that share the pentameric nucleoplasmin-like NPL domain and are found in protists, fungi, plants and animals. Copyright © 2015. Published by Elsevier Ltd.

J. Mol. Biol. PMID: 25813344

Fu, J; Hagan, IM; Glover, DM

The Centrosome and Its Duplication Cycle

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. Copyright © 2015 Cold Spring Harbor Laboratory Press; all rights reserve

Cold Spring Harb Perspect Biol 2015;7:a015800

Lipinszki, Z; Lefevre, S; Savoian, MS; Singleton, MR; Glover, DM; Przewloka, MR

Centromeric binding and activity of Protein Phosphatase 4

The cell division cycle requires tight coupling between protein phosphorylation and dephosphorylation. However, understanding the cell cycle roles of multimeric protein phosphatases has been limited by the lack of knowledge of how their diverse regulatory subunits target highly conserved catalytic subunits to their sites of action. Phosphoprotein phosphatase 4 (PP4) has been recently shown to participate in the regulation of cell cycle progression. We now find that the EVH1 domain of the regulatory subunit 3 of Drosophila PP4, Falafel (Flfl), directly interacts with the centromeric protein C (CENP-C). Unlike other EVH1 domains that interact with proline-rich ligands, the crystal structure of the Flfl amino-terminal EVH1 domain bound to a CENP-C peptide reveals a new target-recognition mode for the phosphatase subunit. We also show that binding of Flfl to CENP-C is required to bring PP4 activity to centromeres to maintain CENP-C and attached core kinetochore proteins at chromosomes during mitosis.

Nature Communications 2015 vol. 6 pp. 5894

Jedrusik, A; Cox, A; Wicher, K; Glover, DM; Zernicka-Goetz, M

Maternal-zygotic knockout reveals a critical role of Cdx2 in the morula to blastocyst transition

The first lineage segregation in the mouse embryo generates the inner cell mass (ICM), which gives rise to the pluripotent epiblast and therefore the future embryo, and the trophectoderm (TE), which will build the placenta. The TE lineage depends on the transcription factor Cdx2. However, when Cdx2 first starts to act remains unclear. Embryos with zygotic deletion of Cdx2 develop normally until the late blastocyst stage leading to the conclusion that Cdx2 is important for the maintenance but not specification of the TE. In contrast, down-regulation of Cdx2 transcripts from the early embryo stage results in defects in TE specification before the blastocyst stage. Here, to unambiguously address at which developmental stage Cdx2 becomes first required, we genetically deleted Cdx2 from the oocyte stage using a Zp3-Cre/loxP strategy. Careful assessment of a large cohort of Cdx2 maternal-zygotic null embryos, all individually filmed, examined and genotyped, reveals an earlier lethal phenotype than observed in Cdx2 zygotic null embryos that develop until the late blastocyst stage. The developmental failure of Cdx2 maternal-zygotic null embryos is associated with cell death and failure of TE specification, starting at the morula stage. These results indicate that Cdx2 is important for the correct specification of TE from the morula stage onwards and that both maternal and zygotic pools of Cdx2 are required for correct pre-implantation embryogenesis. Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.

Developmental Biology 398(2):147-152 PMID: 25512302

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