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1.
J Cell Biol ; 216(11): 3571-3590, 2017 11 06.
Article in English | MEDLINE | ID: mdl-28972102

ABSTRACT

Establishing the bipolar spindle in mammalian oocytes after their prolonged arrest is crucial for meiotic fidelity and subsequent development. In contrast to somatic cells, the first meiotic spindle assembles in the absence of centriole-containing centrosomes. Ran-GTP can promote microtubule nucleation near chromatin, but additional unidentified factors are postulated for the activity of multiple acentriolar microtubule organizing centers in the oocyte. We now demonstrate that partially overlapping, nonredundant functions of Aurora A and Plk4 kinases contribute to initiate acentriolar meiosis I spindle formation. Loss of microtubule nucleation after simultaneous chemical inhibition of both kinases can be significantly rescued by drug-resistant Aurora A alone. Drug-resistant Plk4 can enhance Aurora A-mediated rescue, and, accordingly, Plk4 can phosphorylate and potentiate the activity of Aurora A in vitro. Both kinases function distinctly from Ran, which amplifies microtubule growth. We conclude that Aurora A and Plk4 are rate-limiting factors contributing to microtubule growth as the acentriolar oocyte resumes meiosis.


Subject(s)
Aurora Kinase A/metabolism , Centrioles/enzymology , Meiosis , Microtubules/enzymology , Oocytes/enzymology , Protein Serine-Threonine Kinases/metabolism , Animals , Aurora Kinase A/antagonists & inhibitors , Aurora Kinase A/genetics , Cells, Cultured , Centrioles/drug effects , Embryo Culture Techniques , Female , Kinetics , Meiosis/drug effects , Mice, Inbred C57BL , Mice, Inbred CBA , Microtubules/drug effects , Oocytes/drug effects , Phosphorylation , Protein Kinase Inhibitors/pharmacology , Protein Serine-Threonine Kinases/antagonists & inhibitors , Protein Serine-Threonine Kinases/genetics , Signal Transduction , ran GTP-Binding Protein/metabolism
2.
Open Biol ; 5(12): 150209, 2015 Dec.
Article in English | MEDLINE | ID: mdl-26701933

ABSTRACT

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 ß-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.


Subject(s)
Centrosome/metabolism , Cilia/metabolism , Hyperplasia/metabolism , Protein Serine-Threonine Kinases/metabolism , Animals , Cell Proliferation , Cells, Cultured , Centrioles/metabolism , Filaggrin Proteins , Intermediate Filament Proteins/metabolism , Islets of Langerhans/metabolism , Membrane Proteins/metabolism , Mice , Protein Precursors/metabolism , Protein Serine-Threonine Kinases/genetics
3.
Dev Cell ; 27(5): 586-97, 2013 Dec 09.
Article in English | MEDLINE | ID: mdl-24268700

ABSTRACT

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.


Subject(s)
Centrioles/metabolism , Microtubules/metabolism , Protein Serine-Threonine Kinases/metabolism , Spindle Apparatus/metabolism , Animals , Cell Cycle Proteins/metabolism , Cell Division/physiology , Female , Fetus/cytology , Male , Meiosis/physiology , Mice , Mice, Inbred C57BL , Mice, Inbred CBA , Mitosis/physiology , Pregnancy , Protein Serine-Threonine Kinases/genetics , RNA, Messenger/metabolism
4.
EMBO J ; 30(12): 2431-44, 2011 May 20.
Article in English | MEDLINE | ID: mdl-21602789

ABSTRACT

Regulated alternative polyadenylation is an important feature of gene expression, but how gene transcription rate affects this process remains to be investigated. polo is a cell-cycle gene that uses two poly(A) signals in the 3' untranslated region (UTR) to produce alternative messenger RNAs that differ in their 3'UTR length. Using a mutant Drosophila strain that has a lower transcriptional elongation rate, we show that transcription kinetics can determine alternative poly(A) site selection. The physiological consequences of incorrect polo poly(A) site choice are of vital importance; transgenic flies lacking the distal poly(A) signal cannot produce the longer transcript and die at the pupa stage due to a failure in the proliferation of the precursor cells of the abdomen, the histoblasts. This is due to the low translation efficiency of the shorter transcript produced by proximal poly(A) site usage. Our results show that correct polo poly(A) site selection functions to provide the correct levels of protein expression necessary for histoblast proliferation, and that the kinetics of RNA polymerase II have an important role in the mechanism of alternative polyadenylation.


Subject(s)
Drosophila Proteins/metabolism , Drosophila melanogaster/genetics , Poly A/metabolism , Polyadenylation/genetics , Protein Serine-Threonine Kinases/metabolism , RNA Polymerase II/metabolism , Signal Transduction/genetics , 3' Untranslated Regions/genetics , Animals , Animals, Genetically Modified , Cell Proliferation , Cell Survival/genetics , Drosophila Proteins/genetics , Drosophila Proteins/physiology , Drosophila melanogaster/embryology , Genetic Variation/genetics , Kinetics , Poly A/genetics , Protein Serine-Threonine Kinases/genetics , Protein Serine-Threonine Kinases/physiology , RNA Polymerase II/biosynthesis , RNA Polymerase II/genetics
5.
PLoS Biol ; 6(8): e207, 2008 Aug 26.
Article in English | MEDLINE | ID: mdl-18752348

ABSTRACT

Chromosome segregation requires sister chromatid resolution. Condensins are essential for this process since they organize an axial structure where topoisomerase II can work. How sister chromatid separation is coordinated with chromosome condensation and decatenation activity remains unknown. We combined four-dimensional (4D) microscopy, RNA interference (RNAi), and biochemical analyses to show that topoisomerase II plays an essential role in this process. Either depletion of topoisomerase II or exposure to specific anti-topoisomerase II inhibitors causes centromere nondisjunction, associated with syntelic chromosome attachments. However, cells degrade cohesins and timely exit mitosis after satisfying the spindle assembly checkpoint. Moreover, in topoisomerase II-depleted cells, Aurora B and INCENP fail to transfer to the central spindle in late mitosis and remain tightly associated with centromeres of nondisjoined sister chromatids. Also, in topoisomerase II-depleted cells, Aurora B shows significantly reduced kinase activity both in S2 and HeLa cells. Codepletion of BubR1 in S2 cells restores Aurora B kinase activity, and consequently, most syntelic attachments are released. Taken together, our results support that topoisomerase II ensures proper sister chromatid separation through a direct role in centromere resolution and prevents incorrect microtubule-kinetochore attachments by allowing proper activation of Aurora B kinase.


Subject(s)
Centromere/physiology , DNA Topoisomerases, Type II/physiology , Mitosis/physiology , Protein Serine-Threonine Kinases/physiology , Animals , Aurora Kinase B , Aurora Kinases , Cell Cycle Proteins/metabolism , Cells, Cultured , Chromosomal Proteins, Non-Histone/metabolism , Chromosome Segregation/physiology , DNA Topoisomerases, Type II/genetics , Drosophila Proteins/metabolism , Drosophila melanogaster , Enzyme Activation , HeLa Cells , Humans , Kinetochores/physiology , Microtubules/physiology , RNA Interference/physiology , Sister Chromatid Exchange/physiology , Spindle Apparatus/physiology , Topoisomerase II Inhibitors
6.
Mol Cell Biol ; 25(20): 8971-84, 2005 Oct.
Article in English | MEDLINE | ID: mdl-16199875

ABSTRACT

During cell division, chromatin undergoes structural changes essential to ensure faithful segregation of the genome. Condensins, abundant components of mitotic chromosomes, are known to form two different complexes, condensins I and II. To further examine the role of condensin I in chromosome structure and in particular in centromere organization, we depleted from S2 cells the Drosophila CAP-H homologue Barren, a subunit exclusively associated with condensin I. In the absence of Barren/CAP-H the condensin core subunits DmSMC4/2 still associate with chromatin, while the other condensin I non-structural maintenance of chromosomes family proteins do not. Immunofluorescence and in vivo analysis of Barren/CAP-H-depleted cells showed that mitotic chromosomes are able to condense but fail to resolve sister chromatids. Additionally, Barren/CAP-H-depleted cells show chromosome congression defects that do not appear to be due to abnormal kinetochore-microtubule interaction. Instead, the centromeric and pericentromeric heterochromatin of Barren/CAP-H-depleted chromosomes shows structural problems. After bipolar attachment, the centromeric heterochromatin organized in the absence of Barren/CAP-H cannot withstand the forces exerted by the mitotic spindle and undergoes irreversible distortion. Taken together, our data suggest that the condensin I complex is required not only to promote sister chromatid resolution but also to maintain the structural integrity of centromeric heterochromatin during mitosis.


Subject(s)
Adenosine Triphosphatases/chemistry , Adenosine Triphosphatases/metabolism , Cell Cycle Proteins/chemistry , Cell Cycle Proteins/metabolism , Centromere/metabolism , DNA-Binding Proteins/chemistry , DNA-Binding Proteins/metabolism , Drosophila Proteins/chemistry , Drosophila Proteins/metabolism , Heterochromatin/metabolism , Mitosis/physiology , Multiprotein Complexes/chemistry , Multiprotein Complexes/metabolism , Adenosine Triphosphatases/genetics , Animals , Cell Cycle Proteins/genetics , Cell Line , DNA-Binding Proteins/genetics , Drosophila/cytology , Drosophila/genetics , Drosophila/metabolism , Drosophila Proteins/genetics , Genes, Insect , Kinetochores/metabolism , Mitosis/genetics , Multiprotein Complexes/genetics , Protein Subunits , RNA Interference
7.
Cell Cycle ; 3(5): 538-40, 2004 May.
Article in English | MEDLINE | ID: mdl-15020843

ABSTRACT

One of the most remarkable and yet poorly understood events during the cell cycle is how dispersed chromatin fragments are transformed into chromosomes every time cells undergo mitosis. It has been postulated that mitotic chromosomes might contain an axial scaffold that is involved in condensation but its molecules and structure have remained elusive. Recent data suggests that the condensin complex might indeed be an essential part of the scaffold that provides a platform for other proteins to localize and promote different aspects of chromosome condensation.


Subject(s)
Adenosine Triphosphatases/metabolism , Chromosomes/metabolism , DNA-Binding Proteins/metabolism , Mitosis/physiology , Animals , Chromosome Segregation , Macromolecular Substances , Multiprotein Complexes , Nucleic Acid Conformation
8.
J Cell Sci ; 116(Pt 23): 4763-76, 2003 Dec 01.
Article in English | MEDLINE | ID: mdl-14600262

ABSTRACT

Assembly of compact mitotic chromosomes and resolution of sister chromatids are two essential processes for the correct segregation of the genome during mitosis. Condensin, a five-subunit protein complex, is thought to be required for chromosome condensation. However, recent genetic analysis suggests that condensin is only essential to resolve sister chromatids. To study further the function of condensin we have depleted DmSMC4, a subunit of the complex, from Drosophila S2 cells by dsRNA-mediated interference. Cells lacking DmSMC4 assemble short mitotic chromosomes with unresolved sister chromatids where Barren, a non-SMC subunit of the complex is unable to localise. Topoisomerase II, however, binds mitotic chromatin after depletion of DmSMC4 but it is no longer confined to a central axial structure and becomes diffusely distributed all over the chromatin. Furthermore, cell extracts from DmSMC4 dsRNA-treated cells show significantly reduced topoisomerase II-dependent DNA decatenation activity in vitro. Nevertheless, DmSMC4-depleted chromosomes have centromeres and kinetochores that are able to segregate, although sister chromatid arms form extensive chromatin bridges during anaphase. These chromatin bridges do not result from inappropriate maintenance of sister chromatid cohesion by DRAD21, a subunit of the cohesin complex. Moreover, depletion of DmSMC4 prevents premature sister chromatid separation, caused by removal of DRAD21, allowing cells to exit mitosis with chromatin bridges. Our results suggest that condensin is required so that an axial chromatid structure can be organised where topoisomerase II can effectively promote sister chromatid resolution.


Subject(s)
Adenosine Triphosphatases/metabolism , Chromosomal Proteins, Non-Histone/metabolism , Chromosomes/metabolism , DNA Topoisomerases, Type II/metabolism , DNA-Binding Proteins/metabolism , Adenosine Triphosphatases/antagonists & inhibitors , Animals , Cell Cycle Proteins/metabolism , Cells, Cultured , Chromosomal Proteins, Non-Histone/genetics , DNA-Binding Proteins/antagonists & inhibitors , Drosophila/metabolism , Drosophila Proteins/metabolism , Kinetochores/metabolism , Microscopy, Fluorescence , Mitosis/physiology , Multiprotein Complexes , Protein Subunits/metabolism , RNA, Small Interfering/metabolism , Spindle Apparatus/metabolism
9.
Genetics ; 164(4): 1459-69, 2003 Aug.
Article in English | MEDLINE | ID: mdl-12930752

ABSTRACT

In Drosophila there is limited evidence on the nature of evolutionary forces affecting chromosomal arrangements other than inversions. The study of the X/4 fusion polymorphism of Drosophila americana is thus of interest. Polymorphism patterns at the paralytic (para) gene, located at the base of the X chromosome, suggest that there is suppressed crossing over in this region between fusion and nonfusion chromosomes but not within fusion and nonfusion chromosomes. These data are thus compatible with previous claims that within fusion chromosomes the amino acid clines found at fused1 (also located at the base of the X chromosome) are likely maintained by local selection. The para data set also suggests a young age of the X/4 fusion. Polymorphism data on para and elav (located at the middle region of the X chromosome) suggest that there is no population structure other than that caused by the X/4 fusion itself. These findings are therefore compatible with previous claims that selection maintains the strong association observed between the methionine/threonine variants at fused1 and the status of the X chromosome as fused or unfused to the fourth chromosome.


Subject(s)
Biological Evolution , Chromosomes , Drosophila Proteins/genetics , Drosophila/genetics , Polymorphism, Genetic , Ribonucleoproteins/genetics , Sodium Channels/genetics , X Chromosome , Aldehyde Dehydrogenase/genetics , Animals , Base Sequence , ELAV Proteins , Genes, Insect , Genetic Linkage , Haplotypes , Heterochromatin/genetics , In Situ Hybridization , Introns , Male , Molecular Sequence Data
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