A Genetic Screen for Suppressors and Enhancers of the Drosophila Cdk1-Cyclin B Identifies Maternal Factors That Regulate Microtubule and Microfilament Stability

Abstract Coordination between cell-cycle progression and cytoskeletal dynamics is important for faithful transmission of genetic information. In early Drosophila embryos, increasing maternal cyclin B leads to higher Cdk1-CycB activity, shorter microtubules, and slower nuclear movement during cycles 5-7 and delays in nuclear migration to the cortex at cycle 10. Later during cycle 14 interphase of six cycB embryos, we observed patches of mitotic nuclei, chromosome bridges, abnormal nuclear distribution, and small and large nuclei. These phenotypes indicate disrupted coordination between the cell-cycle machinery and cytoskeletal function. Using these sensitized phenotypes, we performed a dosage-sensitive genetic screen to identify maternal proteins involved in this process. We identified 10 suppressors classified into three groups: (1) gene products regulating Cdk1 activities, cdk1 and cyclin A; (2) gene products interacting with both microtubules and microfilaments, Actin-related protein 87C; and (3) gene products interacting with microfilaments, chickadee, diaphanous, Cdc42, quail, spaghetti-squash, zipper, and scrambled. Interestingly, most of the suppressors that rescue the astral microtubule phenotype also reduce Cdk1-CycB activities and are microfilament-related genes. This suggests that the major mechanism of suppression relies on the interactions among Cdk1-CycB, microtubule, and microfilament networks. Our results indicate that the balance among these different components is vital for normal early cell cycles and for embryonic development. Our observations also indicate that microtubules and cortical microfilaments antagonize each other during the preblastoderm stage.

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A Genetic Screen for Suppressors and Enhancers of the Drosophila PAN GU Cell Cycle Kinase Identifies Cyclin B as a Target

Genetics ◽

10.1093/genetics/158.4.1545 ◽

2001 ◽

Vol 158 (4) ◽

pp. 1545-1556 ◽

Cited By ~ 3

Author(s):

Laura A Lee ◽

Lisa K Elfring ◽

Giovanni Bosco ◽

Terry L Orr-Weaver

Keyword(s):

Cell Cycle ◽

Dna Replication ◽

Genetic Screen ◽

S Phase ◽

Cyclin B ◽

Cell Cycles ◽

Protein Levels ◽

Drosophila Embryogenesis ◽

Giant Nuclei ◽

Cell Cycle Kinase

Abstract The early cell cycles of Drosophila embryogenesis involve rapid oscillations between S phase and mitosis. These unique S-M cycles are driven by maternal stockpiles of components necessary for DNA replication and mitosis. Three genes, pan gu (png), plutonium (plu), and giant nuclei (gnu) are required to control the cell cycle specifically at the onset of Drosophila development by inhibiting DNA replication and promoting mitosis. PNG is a protein kinase that is in a complex with PLU. We employed a sensitized png mutant phenotype to screen for genes that when reduced in dosage would dominantly suppress or enhance png. We screened deficiencies covering over 50% of the autosomes and identified both enhancers and suppressors. Mutations in eIF-5A and PP1 87B dominantly suppress png. Cyclin B was shown to be a key PNG target. Mutations in cyclin B dominantly enhance png, whereas png is suppressed by cyclin B overexpression. Suppression occurs via restoration of Cyclin B protein levels that are decreased in png mutants. The plu and gnu phenotypes are also suppressed by cyclin B overexpression. These studies demonstrate that a crucial function of PNG in controlling the cell cycle is to permit the accumulation of adequate levels of Cyclin B protein.

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Association of cyclin-bound p34cdc2 with subcellular structures in xenopus eggs

Journal of Cell Science ◽

10.1242/jcs.102.2.285 ◽

1992 ◽

Vol 102 (2) ◽

pp. 285-297 ◽

Cited By ~ 1

Author(s):

D. Leiss ◽

M.A. Felix ◽

E. Karsenti

Keyword(s):

Cell Cycle ◽

Ribosomal Proteins ◽

Cell Cycle Progression ◽

Gradient Centrifugation ◽

Preliminary Evidence ◽

Embryonic Cell ◽

Cytoskeletal Proteins ◽

Cyclin B ◽

Post Translational Modifications ◽

Egg Extracts

Cell cycle progression is controlled by changes in kinase activity of homologs of the fission yeast protein p34cdc2. The p34cdc2 kinase is activated by its association with a cyclin subunit, followed by post-translational modifications. Here, we show that in Xenopus eggs stimulated to enter the early embryonic cell cycle by an electric shock, part of the p34cdc2 becomes associated with subcellular fractions as the eggs progress towards mitosis. This occurs as a result of cyclin accumulation because most of the B-type cyclins and some of the A-type cyclins are found in the particulate fraction. Moreover, as soon as cyclins are degraded, p34cdc2 is released in the soluble fraction. The p34cdc2-cyclin complex can be solubilised by 80 mM beta-glycerophosphate (in the standard MPF extraction buffer) or by high salt concentrations. The post-translational modifications leading to cdc2 kinase activation by cyclin occur in the insoluble form. Following fractionation of egg extracts by sucrose gradient centrifugation, the p34cdc2-cyclin B complex is found in several fractions, but especially in two discrete peaks. We present evidence that in the slow-sedimenting peak the p34cdc2-cyclin B complex is associated with the 60 S subunit of monoribosomes. It could be targeted in this fashion to substrates such as ribosomal proteins and maybe to cytoskeletal proteins, since ribosomes bind to microtubules and are present in the spindle. The p34cdc2-cyclin B complex is also found in a faster-migrating fraction containing various membranous structures, including Golgi stacks. Therefore, as observed by immunofluorescence in other systems, it seems that cyclin subunits target p34cdc2 to specific cellular sites and this is certainly important for its function. In addition, we present preliminary evidence suggesting that some component present in the ribosome-containing fraction is required for activation of the p34cdc2-cyclin B complex.

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The interplay between cyclin-B-Cdc2 kinase (MPF) and MAP kinase during maturation of oocytes

Journal of Cell Science ◽

10.1242/jcs.114.2.257 ◽

2001 ◽

Vol 114 (2) ◽

pp. 257-267 ◽

Cited By ~ 7

Author(s):

A. Abrieu ◽

M. Doree ◽

D. Fisher

Keyword(s):

Cell Cycle ◽

Map Kinase ◽

Cell Cycle Progression ◽

Mitotic Cell ◽

Model Systems ◽

Cyclin B ◽

Cdc2 Kinase ◽

Meiotic Chromosomes ◽

Map Kinase Cascade ◽

Kinase Cascade

Throughout oocyte maturation, and subsequently during the first mitotic cell cycle, the MAP kinase cascade and cyclin-B-Cdc2 kinase are associated with the control of cell cycle progression. Many roles have been directly or indirectly attributed to MAP kinase and its influence on cyclin-B-Cdc2 kinase in different model systems; yet a principle theme does not emerge from the published literature, some of which is apparently contradictory. Interplay between these two kinases affects the major events of meiotic maturation throughout the animal kingdom, including the suppression of DNA replication, the segregation of meiotic chromosomes, and the prevention of parthenogenetic activation. Central to many of these events appears to be the control by MAP kinase of cyclin translation and degradation.

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The decision to enter mitosis: feedback and redundancy in the mitotic entry network

The Journal of Cell Biology ◽

10.1083/jcb.200812045 ◽

2009 ◽

Vol 185 (2) ◽

pp. 193-202 ◽

Cited By ~ 357

Author(s):

Arne Lindqvist ◽

Verónica Rodríguez-Bravo ◽

René H. Medema

Keyword(s):

Cell Cycle ◽

Normal Cell ◽

Cell Cycle Progression ◽

Feedback Loops ◽

Cyclin B ◽

Cycle Progression ◽

Mitotic Entry ◽

Different Levels ◽

Normal Cell Cycle

The decision to enter mitosis is mediated by a network of proteins that regulate activation of the cyclin B–Cdk1 complex. Within this network, several positive feedback loops can amplify cyclin B–Cdk1 activation to ensure complete commitment to a mitotic state once the decision to enter mitosis has been made. However, evidence is accumulating that several components of the feedback loops are redundant for cyclin B–Cdk1 activation during normal cell division. Nonetheless, defined feedback loops become essential to promote mitotic entry when normal cell cycle progression is perturbed. Recent data has demonstrated that at least three Plk1-dependent feedback loops exist that enhance cyclin B–Cdk1 activation at different levels. In this review, we discuss the role of various feedback loops that regulate cyclin B–Cdk1 activation under different conditions, the timing of their activation, and the possible identity of the elusive trigger that controls mitotic entry in human cells.

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Chromosome Association of Minichromosome Maintenance Proteins in Drosophila Mitotic Cycles

The Journal of Cell Biology ◽

10.1083/jcb.139.1.13 ◽

1997 ◽

Vol 139 (1) ◽

pp. 13-21 ◽

Cited By ~ 31

Author(s):

Tin Tin Su ◽

Patrick H. O'Farrell

Keyword(s):

Cell Cycle ◽

Cyclin E ◽

Embryonic Cell ◽

Cyclin B ◽

Chromatin Interaction ◽

G1 Phase ◽

Minichromosome Maintenance ◽

Cell Cycles ◽

Mcm Proteins ◽

Replication Factors

Minichromosome maintenance (MCM) proteins are essential DNA replication factors conserved among eukaryotes. MCMs cycle between chromatin bound and dissociated states during each cell cycle. Their absence on chromatin is thought to contribute to the inability of a G2 nucleus to replicate DNA. Passage through mitosis restores the ability of MCMs to bind chromatin and the ability to replicate DNA. In Drosophila early embryonic cell cycles, which lack a G1 phase, MCMs reassociate with condensed chromosomes toward the end of mitosis. To explore the coupling between mitosis and MCM–chromatin interaction, we tested whether this reassociation requires mitotic degradation of cyclins. Arrest of mitosis by induced expression of nondegradable forms of cyclins A and/or B showed that reassociation of MCMs to chromatin requires cyclin A destruction but not cyclin B destruction. In contrast to the earlier mitoses, mitosis 16 (M16) is followed by G1, and MCMs do not reassociate with chromatin at the end of M16. dacapo mutant embryos lack an inhibitor of cyclin E, do not enter G1 quiescence after M16, and show mitotic reassociation of MCM proteins. We propose that cyclin E, inhibited by Dacapo in M16, promotes chromosome binding of MCMs. We suggest that cyclins have both positive and negative roles in controlling MCM–chromatin association.

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Phosphatidylinositol-4-phosphate 5-Kinase 1α Modulates Ribosomal RNA Gene Silencing through Its Interaction with Histone H3 Lysine 9 Trimethylation and Heterochromatin Protein HP1-α

Journal of Biological Chemistry ◽

10.1074/jbc.m114.633727 ◽

2015 ◽

Vol 290 (34) ◽

pp. 20893-20903 ◽

Cited By ~ 15

Author(s):

Rajarshi Chakrabarti ◽

Sulagna Sanyal ◽

Amit Ghosh ◽

Kaushik Bhar ◽

Chandrima Das ◽

...

Keyword(s):

Cell Cycle ◽

Gene Transcription ◽

Cell Cycle Progression ◽

Gene Activation ◽

Cellular Motility ◽

Heterochromatin Protein ◽

Cyclical Pattern ◽

Rdna Loci ◽

Cytoskeletal Dynamics ◽

Phosphatidylinositol 4

Phosphoinositide signaling has been implicated in the regulation of numerous cellular processes including cytoskeletal dynamics, cellular motility, vesicle trafficking, and gene transcription. Studies have also shown that nuclear phosphoinositide(s) regulates processes such as mRNA export, cell cycle progression, gene transcription, and DNA repair. We have shown previously that the nuclear form of phosphatidylinositol-4-phosphate 5-kinase 1α (PIP5K), the enzyme responsible for phosphatidylinositol 4,5-bisphosphate synthesis, is modified by small ubiquitin-like modifier (SUMO)-1. In this study, we have shown that due to the site-specific Lys to Ala mutations of PIP5K at Lys-244 and Lys-490, it is unable to localize in the nucleus and nucleolus, respectively. Furthermore, by using chromatin immunoprecipitation assays, we have observed that PIP5K associates with the chromatin silencing complex constituted of H3K9me3 and heterochromatin protein 1α at multiple ribosomal DNA (rDNA) loci. These interactions followed a definite cyclical pattern of occupancy (mostly G1) and release from the rDNA loci (G1/S) throughout the cell cycle. Moreover, the immunoprecipitation results clearly demonstrate that PIP5K SUMOylated at Lys-490 interacts with components of the chromatin silencing machinery, H3K9me3 and heterochromatin protein 1α. However, PIP5K does not interact with the gene activation signature protein H3K4me3. This study, for the first time, demonstrates that PIP5K, an enzyme actively associated with lipid modification pathway, has additional roles in rDNA silencing.

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TPR proteins required for anaphase progression mediate ubiquitination of mitotic B-type cyclins in yeast.

Molecular Biology of the Cell ◽

10.1091/mbc.7.5.791 ◽

1996 ◽

Vol 7 (5) ◽

pp. 791-801 ◽

Cited By ~ 74

Author(s):

W Zachariae ◽

K Nasmyth

Keyword(s):

Cell Cycle ◽

Cyclin B ◽

Yeast Cells ◽

Tetratricopeptide Repeat ◽

Gene Products ◽

Ubiquitin Pathway ◽

Ubiquitin Protein Ligase ◽

A Cell ◽

Synthesis And Degradation

The abundance of B-type cyclin-CDK complexes is determined by regulated synthesis and degradation of cyclin subunits. Cyclin proteolysis is required for the final exit from mitosis and for the initiation of a new cell cycle. In extracts from frog or clam eggs, degradation is accompanied by ubiquitination of cyclin. Three genes, CDC16, CDC23, and CSE1 have recently been shown to be required specifically for cyclin B proteolysis in yeast. To test whether these genes are required for cyclin ubiquitination, we prepared extracts from G1-arrested yeast cells capable of conjugating ubiquitin to the B-type cyclin Clb2. The ubiquitination activity was cell cycle regulated, required Clb2's destruction box, and was low if not absent in cdc16, cdc23, cdc27, and cse1 mutants. Furthermore all these mutants were also defective in ubiquitination of another mitotic B-type cyclin, Clb3. The Cdc16, Cdc23, and Cdc27 proteins all contain several copies of the tetratricopeptide repeat and are subunits of a complex that is required for the onset of anaphase. The finding that gene products that are required for ubiquitination of Clb2 and Clb3 are also required for cyclin proteolysis in vivo provides the best evidence so far that cyclin B is degraded via the ubiquitin pathway in living cells. Xenopus homologues of Cdc16 and Cdc27 have meanwhile been shown to be associated with a 20S particle that appears to function as a cell cycle-regulated ubiquitin-protein ligase.

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Microtubule-associated protein/microtubule affinity-regulating kinase 4 (MARK4) plays a role in cell cycle progression and cytoskeletal dynamics

European Journal of Cell Biology ◽

10.1016/j.ejcb.2014.07.004 ◽

2014 ◽

Vol 93 (8-9) ◽

pp. 355-365 ◽

Cited By ~ 25

Author(s):

Davide Rovina ◽

Laura Fontana ◽

Laura Monti ◽

Chiara Novielli ◽

Nicolò Panini ◽

...

Keyword(s):

Cell Cycle ◽

Cell Cycle Progression ◽

Cycle Progression ◽

Cytoskeletal Dynamics

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Genome-Wide Identification and Analysis of Cell Cycle Genes in Betula pendula

10.20944/preprints202112.0149.v2 ◽

2021 ◽

Author(s):

Yijie Li ◽

Song Chen ◽

Yuhang Liu ◽

Haijiao Huang

Keyword(s):

Cell Cycle ◽

Growth And Development ◽

Betula Pendula ◽

Cell Cycle Progression ◽

Transcript Abundance ◽

Cell Cycle Gene ◽

Specific Expression ◽

Cell Cycles ◽

Cell Cycle Genes ◽

Functional Studies

Research Highlights: This study identified the cell cycle genes in birch that likely play important roles during plant growth and development. This analysis provides a basis for understanding the regulatory mechanism of various cell cycles in Betula pendula. Background and Objectives: The cell cycle factors not only influence cell cycle progression together, but also regulate accretion, division and differentiation of cells, and then regulate growth and development of plant. In this study, we identified the putative cell cycle genes in B. pendula genome, based on the annotated cell cycle genes in A. thaliana. It could serve as a foundation for further functional studies. Materials and Methods: The transcript abundance was determined for all the cell cycle genes in xylem, root, leaf and flower tissues using RNA-seq technology. Results: We identified 59 cell cycle gene models in the genome of B. pendula, 17 highly expression genes among them. These genes were BpCDKA.1, BpCDKB1.1, BpCDKB2.1, BpCKS1.2, BpCYCB1.1, BpCYCB1.2, BpCYCB2.1, BpCYCD3.1, BpCYCD3.5, BpDEL1, BpDpa2, BpE2Fa, BpE2Fb, BpKRP1, BpKRP2, BpRb1 and BpWEE1. Conclusions: We identified 17 core cell cycle genes in the genome of birch by combining phylogenetic analysis and tissue specific expression data.

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Restoration of miR-193a-5p and miR-146 a-5p Expression Induces G1 Arrest in Colorectal Cancer through Targeting of MDM2/p53

Advanced Pharmaceutical Bulletin ◽

10.15171/apb.2020.017 ◽

2019 ◽

Vol 10 (1) ◽

pp. 130-134 ◽

Cited By ~ 4

Author(s):

Saeed Noorolyai ◽

Elham Baghbani ◽

Leili Aghebati Maleki ◽

Amir Baghbanzadeh Kojabad ◽

Dariush Shanehbansdi ◽

...

Keyword(s):

Gene Expression ◽

Colorectal Cancer ◽

Cell Cycle ◽

Tumor Suppressor ◽

Cell Cycle Progression ◽

Cyclin B ◽

G1 Arrest ◽

Cycle Progression ◽

Ht 29

Purpose: Colorectal cancer (CRC) remains a universal and lethal cancer owing to metastatic and relapsing disease. Currently, the role of microRNAs has been checked in tumorigeneses. Numerous studies have revealed that between the tumor suppressor miRNAs, the reduced expression of miR-146a-5p and -193a-5p in several cancers including CRC tissues are related with tumor progression and poor prognosis of patients. The purpose of this study is to examine the role of miR-146 a-5p and -193 a-5p in CRC cell cycle progression. Methods: The miR-193a-5p and -146 a-5p mimics were transfected into HT-29 CRC cells via jetPEI transfection reagent and their impact was assessed on p53, cyclin B, and NF-kB gene expression. The inhibitory effect of these miRNAs on cell cycle was assessed by flow cytometry. The consequence of miR-193a-5p and miR-146 a-5p on the protein expression level of Murine double minute 2 (MDM2) was assessed by western blotting. Results: miR193a-5p and -146a-5p regulated the expression of MDM2 protein and p53, cyclin B, and NF-kB gene expression in CRC cells. Treatment of HT-29 cells with miRNA-146a-5p and -193a-5p induced G1 cell cycle arrest. Conclusion: The findings of our study suggest that miR146a-5p and -193a-5p may act as a potential tumor suppressor by their influence on cell cycle progression in CRC cells. Thus, miRNA-146a-5p and -193a-5p restoration may be recommended as a potential therapeutic goal in the treatment of CRC patients.

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