scholarly journals Chk1 and Wee1 kinases coordinate DNA replication, chromosome condensation, and anaphase entry

2012 ◽  
Vol 23 (6) ◽  
pp. 1047-1057 ◽  
Author(s):  
Barbara Fasulo ◽  
Carol Koyama ◽  
Kristina R. Yu ◽  
Ellen M. Homola ◽  
Tao S. Hsieh ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Dna Replication ◽  
Anticancer Agents ◽  
Spindle Assembly Checkpoint ◽  
Metaphase Plate ◽  
Chromosome Condensation ◽  
Drosophila Embryo ◽  
X Rays ◽  
Checkpoint Activation ◽  
Early Drosophila Embryo

Defects in DNA replication and chromosome condensation are common phenotypes in cancer cells. A link between replication and condensation has been established, but little is known about the role of checkpoints in monitoring chromosome condensation. We investigate this function by live analysis, using the rapid division cycles in the early Drosophila embryo. We find that S-phase and topoisomerase inhibitors delay both the initiation and the rate of chromosome condensation. These cell cycle delays are mediated by the cell cycle kinases chk1 and wee1. Inhibitors that cause severe defects in chromosome condensation and congression on the metaphase plate result in delayed anaphase entry. These delays are mediated by wee1 and are not the result of spindle assembly checkpoint activation. In addition, we provide the first detailed live analysis of the direct effect of widely used anticancer agents (aclarubicin, ICRF-193, VM26, doxorubicin, camptothecin, aphidicolin, hydroxyurea, cisplatin, mechlorethamine and x-rays) on key nuclear and cytoplasmic cell cycle events.

scholarly journals The Concentration of Nuf, a Rab11 Effector, at the Microtubule-organizing Center Is Cell Cycle–regulated, Dynein-dependent, and Coincides with Furrow Formation

2007 ◽  
Vol 18 (9) ◽  
pp. 3313-3322 ◽  
Author(s):  
Blake Riggs ◽  
Barbara Fasulo ◽  
Anne Royou ◽  
Sarah Mische ◽  
Jian Cao ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Small Gtpase ◽  
Drosophila Embryo ◽  
Microtubule Organizing Center ◽  
Recycling Endosome ◽  
Actin Organization ◽  
Protein Levels ◽  
Nuclear Fallout ◽  
Early Drosophila Embryo ◽  
Organizing Center

Animal cytokinesis relies on membrane addition as well as acto-myosin–based constriction. Recycling endosome (RE)-derived vesicles are a key source of this membrane. Rab11, a small GTPase associated with the RE and involved in vesicle targeting, is required for elongation of the cytokinetic furrow. In the early Drosophila embryo, Nuclear-fallout (Nuf), a Rab11 effector, promotes vesicle-mediated membrane delivery and actin organization at the invaginating furrow. Although Rab11 maintains a relatively constant localization at the microtubule-organizing center (MTOC), Nuf is present at the MTOC only during the phases of the cell cycle in which furrow invagination occurs. We demonstrate that Nuf protein levels remain relatively constant throughout the cell cycle, suggesting that Nuf is undergoing cycles of concentration and dispersion from the MTOC. Microtubules, but not microfilaments, are required for proper MTOC localization of Nuf and Rab11. The MTOC localization of Nuf also relies on Dynein. Immunoprecipitation experiments demonstrate that Nuf and Dynein physically interact. In accord with these findings, and in contrast to previous reports, we demonstrate that microtubules are required for proper metaphase furrow formation. We propose that the cell cycle–regulated, Dynein-dependent recruitment of Nuf to the MTOC influences the timing of RE-based vesicle delivery to the invaginating furrows.

scholarly journals LIN-5 Is a Novel Component of the Spindle Apparatus Required for Chromosome Segregation and Cleavage Plane Specification in Caenorhabditis elegans

2000 ◽  
Vol 148 (1) ◽  
pp. 73-86 ◽  
Author(s):  
Monique A. Lorson ◽  
H. Robert Horvitz ◽  
Sander van den Heuvel
Keyword(s):  
Cell Cycle ◽  
Caenorhabditis Elegans ◽  
Dna Replication ◽  
Chromosome Segregation ◽  
Cleavage Plane ◽  
Coiled Coil ◽  
Metaphase Plate ◽  
Dependent Manner ◽  
Spindle Apparatus ◽  
Cytoplasmic Cleavage

Successful divisions of eukaryotic cells require accurate and coordinated cycles of DNA replication, spindle formation, chromosome segregation, and cytoplasmic cleavage. The Caenorhabditis elegans gene lin-5 is essential for multiple aspects of cell division. Cells in lin-5 null mutants enter mitosis at the normal time and form bipolar spindles, but fail chromosome alignment at the metaphase plate, sister chromatid separation, and cytokinesis. Despite these defects, cells exit from mitosis without delay and progress through subsequent rounds of DNA replication, centrosome duplication, and abortive mitoses. In addition, early embryos that lack lin-5 function show defects in spindle positioning and cleavage plane specification. The lin-5 gene encodes a novel protein with a central coiled-coil domain. This protein localizes to the spindle apparatus in a cell cycle- and microtubule-dependent manner. The LIN-5 protein is located at the centrosomes throughout mitosis, at the kinetochore microtubules in metaphase cells, and at the spindle during meiosis. Our results show that LIN-5 is a novel component of the spindle apparatus required for chromosome and spindle movements, cytoplasmic cleavage, and correct alternation of the S and M phases of the cell cycle.

scholarly journals Onset of the DNA Replication Checkpoint in the Early Drosophila Embryo

Genetics ◽  
2006 ◽  
Vol 175 (2) ◽  
pp. 567-584 ◽  
Author(s):  
Justin Crest ◽  
Nathan Oxnard ◽  
Jun-Yuan Ji ◽  
Gerold Schubiger
Keyword(s):  
Dna Replication ◽  
Drosophila Embryo ◽  
Replication Checkpoint ◽  
Early Drosophila Embryo ◽  
Dna Replication Checkpoint

scholarly journals The cell cycle-dependent localization of the CP190 centrosomal protein is determined by the coordinate action of two separable domains.

1995 ◽  
Vol 131 (5) ◽  
pp. 1261-1273 ◽  
Author(s):  
K Oegema ◽  
W G Whitfield ◽  
B Alberts
Keyword(s):  
Cell Cycle ◽  
Amino Acids ◽  
Nuclear Localization ◽  
Fusion Proteins ◽  
Drosophila Embryo ◽  
Dependent Manner ◽  
Fluorescence Confocal Microscopy ◽  
Early Drosophila Embryo ◽  
A Cell ◽  
Cycle Dependent

CP190, a protein of 1,096 amino acids from Drosophila melanogaster, oscillates in a cell cycle-specific manner between the nucleus during interphase, and the centrosome during mitosis. To characterize the regions of CP190 responsible for its dynamic behavior, we injected rhodamine-labeled fusion proteins spanning most of CP190 into early Drosophila embryos, where their localizations were characterized using time-lapse fluorescence confocal microscopy. A single bipartite 19-amino acid nuclear localization signal was detected that causes nuclear localization. Robust centrosomal localization is conferred by a separate region of 124 amino acids; two adjacent, nonoverlapping fusion proteins containing distinct portions of this region show weaker centrosomal localization. Fusion proteins that contain both nuclear and centrosomal localization sequences oscillate between the nucleus and the centrosome in a manner identical to native CP190. Fusion proteins containing only the centrosome localization sequence are found at centrosomes throughout the cell cycle, suggesting that CP190 is actively recruited away from the centrosome by its movement into the nucleus during interphase. Both native and bacterially expressed CP190 cosediment with microtubules in vitro. Tests with fusion proteins show that the domain responsible for microtubule binding overlaps the domain required for centrosomal localization. CP60, a protein identified by its association with CP190, also localizes to centrosomes and to nuclei in a cell cycle-dependent manner. Experiments in which colchicine is used to depolymerize microtubules in the early Drosophila embryo demonstrate that both CP190 and CP60 are able to attain and maintain their centrosomal localization in the absence of microtubules.

scholarly journals Delays in anaphase initiation occur in individual nuclei of the syncytial Drosophila embryo.

1993 ◽  
Vol 4 (9) ◽  
pp. 885-896 ◽  
Author(s):  
W Sullivan ◽  
D R Daily ◽  
P Fogarty ◽  
K J Yook ◽  
S Pimpinelli
Keyword(s):  
Cell Cycle ◽  
Metaphase Plate ◽  
Normal Karyotype ◽  
Fold Increase ◽  
Cell Cycle Checkpoints ◽  
Drosophila Embryo ◽  
Mitotic Apparatus ◽  
Late Anaphase ◽  
Error Frequency ◽  
Established Cell

The syncytial divisions of the Drosophila melanogaster embryo lack some of the well established cell-cycle checkpoints. It has been suggested that without these checkpoints the divisions would display a reduced fidelity. To test this idea, we examined division error frequencies in individuals bearing an abnormally long and rearranged second chromosome, designated C(2)EN. Relative to a normal chromosome, this chromosome imposes additional structural demands on the mitotic apparatus in both the early syncytial embryonic divisions and the later somatic divisions. We demonstrate that the C(2)EN chromosome does not increase the error frequency of the late larva neuroblast divisions. However, in the syncytial embryonic nuclear divisions, the C(2)EN chromosome produces a 10-fold increase in division errors relative to embryos with a normal karyotype. During late anaphase of the neuroblast divisions, the sister C(2)EN chromosomes cleanly separate from one another. In contrast, during late anaphase of the syncytial divisions in C(2)EN-bearing nuclei, large amounts of chromatin often lag on the metaphase plate. Live analysis of C(2)EN-bearing embryos demonstrates that individual nuclei in the syncytial population of dividing nuclei often delay in their initiation of anaphase. These delays frequently lead to division errors. Eventually the products of the nuclei delayed in anaphase sink inward and are removed from the dividing population of syncytial nuclei. These results suggest that the Drosophila embryo may be equipped with mechanisms that monitor the fidelity of the syncytial nuclear divisions. Unlike checkpoints that rely on cell cycle delays to identify and correct division errors, these embryonic mechanisms rely on cell cycle delays to identify and discard the products of division errors.

scholarly journals Interphase Cytogenetic Analysis of Micronucleated and Multinucleated Cells Supports the Premature Chromosome Condensation Hypothesis as the Mechanistic Origin of Chromothripsis

Cancers ◽  
2019 ◽  
Vol 11 (8) ◽  
pp. 1123 ◽  
Author(s):  
Pantelias ◽  
Karachristou ◽  
Georgakilas ◽  
Terzoudi
Keyword(s):  
Cell Cycle ◽  
Dna Replication ◽  
Cytogenetic Analysis ◽  
Alternative Hypothesis ◽  
Chromatin Condensation ◽  
Human Lymphocytes ◽  
Chinese Hamster ◽  
Chromosome Condensation ◽  
Premature Chromosome Condensation ◽  
Multinucleated Cells

The discovery of chromothripsis in cancer genomes challenges the long-standing concept of carcinogenesis as the result of progressive genetic events. Despite recent advances in describing chromothripsis, its mechanistic origin remains elusive. The prevailing conception is that it arises from a massive accumulation of fragmented DNA inside micronuclei (MN), whose defective nuclear envelope ruptures or leads to aberrant DNA replication, before main nuclei enter mitosis. An alternative hypothesis is that the premature chromosome condensation (PCC) dynamics in asynchronous micronucleated cells underlie chromosome shattering in a single catastrophic event, a hallmark of chromothripsis. Specifically, when main nuclei enter mitosis, premature chromatin condensation provokes the shattering of chromosomes entrapped inside MN, if they are still undergoing DNA replication. To test this hypothesis, the agent RO-3306, a selective ATP-competitive inhibitor of CDK1 that promotes cell cycle arrest at the G2/M boundary, was used in this study to control the degree of cell cycle asynchrony between main nuclei and MN. By delaying the entrance of main nuclei into mitosis, additional time was allowed for the completion of DNA replication and duplication of chromosomes inside MN. We performed interphase cytogenetic analysis using asynchronous micronucleated cells generated by exposure of human lymphocytes to γ-rays, and heterophasic multinucleated Chinese hamster ovary (CHO) cells generated by cell fusion procedures. Our results demonstrate that the PCC dynamics during asynchronous mitosis in micronucleated or multinucleated cells are an important determinant of chromosome shattering and may underlie the mechanistic origin of chromothripsis.

scholarly journals Influence of cyclin type and dose on mitotic entry and progression in the early Drosophila embryo

2009 ◽  
Vol 184 (5) ◽  
pp. 639-646 ◽  
Author(s):  
Mark L. McCleland ◽  
Jeffrey A. Farrell ◽  
Patrick H. O'Farrell
Keyword(s):  
Cell Cycle ◽  
Drosophila Melanogaster ◽  
Drosophila Embryo ◽  
Cell Cycle Regulators ◽  
Mitotic Progression ◽  
Mitotic Entry ◽  
Early Drosophila Embryo ◽  
Real Time Visualization ◽  
The One

Cyclins are key cell cycle regulators, yet few analyses test their role in timing the events that they regulate. We used RNA interference and real-time visualization in embryos to define the events regulated by each of the three mitotic cyclins of Drosophila melanogaster, CycA, CycB, and CycB3. Each individual and pairwise knockdown results in distinct mitotic phenotypes. For example, mitosis without metaphase occurs upon knockdown of CycA and CycB. To separate the role of cyclin levels from the influences of cyclin type, we knocked down two cyclins and reduced the gene dose of the one remaining cyclin. This reduction did not prolong interphase but instead interrupted mitotic progression. Mitotic prophase chromosomes formed, centrosomes divided, and nuclei exited mitosis without executing later events. This prompt but curtailed mitosis shows that accumulation of cyclin function does not directly time mitotic entry in these early embryonic cycles and that cyclin function can be sufficient for some mitotic events although inadequate for others.

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