scholarly journals Identification of a Mid-anaphase Checkpoint in Budding Yeast

1997 ◽  
Vol 136 (2) ◽  
pp. 345-354 ◽  
Author(s):  
Sam S. Yang ◽  
Elaine Yeh ◽  
E.D. Salmon ◽  
Kerry Bloom
Keyword(s):  
Mitotic Spindle ◽  
Cellular Response ◽  
Cell Cycle Progression ◽  
Time Lapse ◽  
Physical Distance ◽  
Cycle Progression ◽  
Contrast Analysis ◽  
Spindle Elongation ◽  
Chromosome Integrity ◽  
Anaphase B

Activation of a facultative, dicentric chromosome provides a unique opportunity to introduce a double strand DNA break into a chromosome at mitosis. Time lapse video enhanced-differential interference contrast analysis of the cellular response upon dicentric activation reveals that the majority of cells initiates anaphase B, characterized by pole–pole separation, and pauses in mid-anaphase for 30–120 min with spindles spanning the neck of the bud before completing spindle elongation and cytokinesis. The length of the spindle at the delay point (3–4 μm) is not dependent on the physical distance between the two centromeres, indicating that the arrest represents surveillance of a dicentric induced aberration. No mid-anaphase delay is observed in the absence of the RAD9 checkpoint gene, which prevents cell cycle progression in the presence of damaged DNA. These observations reveal RAD9- dependent events well past the G2/M boundary and have considerable implications in understanding how chromosome integrity and the position and state of the mitotic spindle are monitored before cytokinesis.

scholarly journals Key phosphorylation events in Spc29 and Spc42 guide multiple steps of yeast centrosome duplication

2018 ◽  
Vol 29 (19) ◽  
pp. 2280-2291 ◽  
Author(s):  
Michele Haltiner Jones ◽  
Eileen T. O’Toole ◽  
Amy S. Fabritius ◽  
Eric G. Muller ◽  
Janet B. Meehl ◽  
...  
Keyword(s):  
Cell Cycle Progression ◽  
Spindle Pole Body ◽  
Spindle Pole ◽  
Centrosome Duplication ◽  
Phosphorylation Sites ◽  
Cycle Progression ◽  
Cellular Processes ◽  
Pole Body ◽  
Core Components ◽  
Spindle Elongation

Phosphorylation modulates many cellular processes during cell cycle progression. The yeast centrosome (called the spindle pole body, SPB) is regulated by the protein kinases Mps1 and Cdc28/Cdk1 as it nucleates microtubules to separate chromosomes during mitosis. Previously we completed an SPB phosphoproteome, identifying 297 sites on 17 of the 18 SPB components. Here we describe mutagenic analysis of phosphorylation events on Spc29 and Spc42, two SPB core components that were shown in the phosphoproteome to be heavily phosphorylated. Mutagenesis at multiple sites in Spc29 and Spc42 suggests that much of the phosphorylation on these two proteins is not essential but enhances several steps of mitosis. Of the 65 sites examined on both proteins, phosphorylation of the Mps1 sites Spc29-T18 and Spc29-T240 was shown to be critical for function. Interestingly, these two sites primarily influence distinct successive steps; Spc29-T240 is important for the interaction of Spc29 with Spc42, likely during satellite formation, and Spc29-T18 facilitates insertion of the new SPB into the nuclear envelope and promotes anaphase spindle elongation. Phosphorylation sites within Cdk1 motifs affect function to varying degrees, but mutations only have significant effects in the presence of an MPS1 mutation, supporting a theme of coregulation by these two kinases.

Cell cycle progression, growth and patterning in imaginal discs despite inhibition of cell division after inactivation of Drosophila Cdc2 kinase

Development ◽  
1997 ◽  
Vol 124 (18) ◽  
pp. 3555-3563 ◽  
Author(s):  
K. Weigmann ◽  
S.M. Cohen ◽  
C.F. Lehner
Keyword(s):  
Cell Division ◽  
Cell Cycle Progression ◽  
Positional Information ◽  
Imaginal Discs ◽  
Physical Distance ◽  
Cdc2 Kinase ◽  
Cycle Progression ◽  
Mitotic Kinase ◽  
Control Growth ◽  
Local Cell

During larval development, Drosophila imaginal discs increase in size about 1000-fold and cells are instructed to acquire distinct fates as a function of their position. The secreted signaling molecules Wingless and Decapentaplegic have been implicated as sources of positional information that globally control growth and patterning. Evidence has also been presented that local cell interactions play an important role in controlling cell proliferation in imaginal discs. As a first step to understanding how patterning cues influence growth we investigated the effects of blocking cell division at different times and in spatially controlled manner by inactivation of the mitotic kinase Cdc2 in developing imaginal discs. We find that cell growth continues after inactivation of Cdc2, with little effect on overall patterning. The mechanisms that regulate size of the disc therefore do not function by regulating cell division, but appear to act primarily by regulating size in terms of physical distance or tissue volume.

The regulation of the DNA damage response at telomeres: focus on kinases

2021 ◽  
Author(s):  
Michela Galli ◽  
Chiara Frigerio ◽  
Maria Pia Longhese ◽  
Michela Clerici
Keyword(s):  
Cell Cycle ◽  
Cellular Response ◽  
Cell Cycle Progression ◽  
Binding Proteins ◽  
Replicative Senescence ◽  
Genome Instability ◽  
Cycle Progression ◽  
Strand Breaks ◽  
Cycle Arrest ◽  
Linear Chromosomes

The natural ends of linear chromosomes resemble those of accidental double-strand breaks (DSBs). DSBs induce a multifaceted cellular response that promotes the repair of lesions and slows down cell cycle progression. This response is not elicited at chromosome ends, which are organized in nucleoprotein structures called telomeres. Besides counteracting DSB response through specialized telomere-binding proteins, telomeres also prevent chromosome shortening. Despite of the different fate of telomeres and DSBs, many proteins involved in the DSB response also localize at telomeres and participate in telomere homeostasis. In particular, the DSB master regulators Tel1/ATM and Mec1/ATR contribute to telomere length maintenance and arrest cell cycle progression when chromosome ends shorten, thus promoting a tumor-suppressive process known as replicative senescence. During senescence, the actions of both these apical kinases and telomere-binding proteins allow checkpoint activation while bulk DNA repair activities at telomeres are still inhibited. Checkpoint-mediated cell cycle arrest also prevents further telomere erosion and deprotection that would favor chromosome rearrangements, which are known to increase cancer-associated genome instability. This review summarizes recent insights into functions and regulation of Tel1/ATM and Mec1/ATR at telomeres both in the presence and in the absence of telomerase, focusing mainly on discoveries in budding yeast.

The role of gamma-tubulin in mitotic spindle formation and cell cycle progression in Aspergillus nidulans

1997 ◽  
Vol 110 (5) ◽  
pp. 623-633 ◽  
Author(s):  
M.A. Martin ◽  
S.A. Osmani ◽  
B.R. Oakley
Keyword(s):  
Cell Cycle ◽  
Mitotic Spindle ◽  
Cell Cycle Progression ◽  
Spindle Pole ◽  
Microtubule Assembly ◽  
Cytoplasmic Microtubule ◽  
Cycle Progression ◽  
Spindle Microtubules ◽  
Gamma Tubulin

gamma-Tubulin has been hypothesized to be essential for the nucleation of the assembly of mitotic spindle microtubules, but some recent results suggest that this may not be the case. To clarify the role of gamma-tubulin in microtubule assembly and cell-cycle progression, we have developed a novel variation of the gene disruption/heterokaryon rescue technique of Aspergillus nidulans. We have used temperature-sensitive cell-cycle mutations to synchronize germlings carrying a gamma-tubulin disruption and observe the phenotypes caused by the disruption in the first cell cycle after germination. Our results indicate that gamma-tubulin is absolutely required for the assembly of mitotic spindle microtubules, a finding that supports the hypothesis that gamma-tubulin is involved in spindle microtubule nucleation. In the absence of functional gamma-tubulin, nuclei are blocked with condensed chromosomes for about the length of one cell cycle before chromatin decondenses without nuclear division. Our results indicate that gamma-tubulin is not essential for progression from G1 to G2, for entry into mitosis nor for spindle pole body replication. It is also not required for reactivity of spindle pole bodies with the MPM-2 antibody which recognizes a phosphoepitope important to mitotic spindle formation. Finally, it does not appear to be absolutely required for cytoplasmic microtubule assembly but may play a role in the formation of normal cytoplasmic microtubule arrays.

scholarly journals Cellular Ras and Cyclin D1 Are Required during Different Cell Cycle Periods in Cycling NIH 3T3 Cells

1999 ◽  
Vol 19 (7) ◽  
pp. 4623-4632 ◽  
Author(s):  
Masahiro Hitomi ◽  
Dennis W. Stacey
Keyword(s):  
Cell Cycle ◽  
Dna Synthesis ◽  
Cyclin D1 ◽  
Cell Cycle Progression ◽  
Time Lapse ◽  
S Phase ◽  
3T3 Cells ◽  
Cycle Progression ◽  
Nih 3T3 ◽  
Nih 3T3 Cells

ABSTRACT Novel techniques were used to determine when in the cell cycle of proliferating NIH 3T3 cells cellular Ras and cyclin D1 are required. For comparison, in quiescent cells, all four of the inhibitors of cell cycle progression tested (anti-Ras, anti-cyclin D1, serum removal, and cycloheximide) became ineffective at essentially the same point in G1 phase, approximately 4 h prior to the beginning of DNA synthesis. To extend these studies to cycling cells, a time-lapse approach was used to determine the approximate cell cycle position of individual cells in an asynchronous culture at the time of inhibitor treatment and then to determine the effects of the inhibitor upon recipient cells. With this approach, anti-Ras antibody efficiently inhibited entry into S phase only when introduced into cells prior to the preceding mitosis, several hours before the beginning of S phase. Anti-cyclin D1, on the other hand, was an efficient inhibitor when introduced up until just before the initiation of DNA synthesis. Cycloheximide treatment, like anti-cyclin D1 microinjection, was inhibitory throughout G1 phase (which lasts a total of 4 to 5 h in these cells). Finally, serum removal blocked entry into S phase only during the first hour following mitosis. Kinetic analysis and a novel dual-labeling technique were used to confirm the differences in cell cycle requirements for Ras, cyclin D1, and cycloheximide. These studies demonstrate a fundamental difference in mitogenic signal transduction between quiescent and cycling NIH 3T3 cells and reveal a sequence of signaling events required for cell cycle progression in proliferating NIH 3T3 cells.

scholarly journals Mitotic spindle disassembly occurs via distinct subprocesses driven by the anaphase-promoting complex, Aurora B kinase, and kinesin-8

2010 ◽  
Vol 191 (4) ◽  
pp. 795-808 ◽  
Author(s):  
Jeffrey B. Woodruff ◽  
David G. Drubin ◽  
Georjana Barnes
Keyword(s):  
Mitotic Spindle ◽  
Cell Cycle Progression ◽  
Dynamic Structure ◽  
Aurora B ◽  
Anaphase Promoting Complex ◽  
Microtubule Depolymerization ◽  
Aurora B Kinase ◽  
Synthetic Lethal ◽  
Spindle Disassembly ◽  
Spindle Elongation

The mitotic spindle is a complex and dynamic structure. Although much has been learned about how spindles assemble and mediate chromosome segregation, how spindles rapidly and irreversibly disassemble during telophase is less clear. We used synthetic lethal screens in budding yeast to identify mutants defective in spindle disassembly. Real-time, live cell imaging analysis of spindle disassembly was performed on nine mutants defective in this process. Results of this analysis suggest that spindle disassembly is achieved by mechanistically distinct but functionally overlapping subprocesses: disengagement of the spindle halves, arrest of spindle elongation, and initiation of interpolar microtubule depolymerization. These subprocesses are largely governed by the anaphase-promoting complex, Aurora B kinase, and kinesin-8. Combinatorial inhibition of these subprocesses yielded cells with hyperstable spindle remnants and dramatic defects in cell cycle progression, establishing that rapid spindle disassembly is crucial for cell proliferation.

scholarly journals Budding Yeast Bub2 Is Localized at Spindle Pole Bodies and Activates the Mitotic Checkpoint via a Different Pathway from Mad2

1999 ◽  
Vol 145 (5) ◽  
pp. 979-991 ◽  
Author(s):  
Roberta Fraschini ◽  
Elisa Formenti ◽  
Giovanna Lucchini ◽  
Simonetta Piatti
Keyword(s):  
Cell Cycle ◽  
Mitotic Spindle ◽  
Cell Cycle Progression ◽  
Budding Yeast ◽  
Spindle Pole Body ◽  
Mitotic Checkpoint ◽  
Spindle Pole ◽  
Cycle Progression ◽  
Spindle Pole Bodies

The mitotic checkpoint blocks cell cycle progression before anaphase in case of mistakes in the alignment of chromosomes on the mitotic spindle. In budding yeast, the Mad1, 2, 3, and Bub1, 2, 3 proteins mediate this arrest. Vertebrate homologues of Mad1, 2, 3, and Bub1, 3 bind to unattached kinetochores and prevent progression through mitosis by inhibiting Cdc20/APC-mediated proteolysis of anaphase inhibitors, like Pds1 and B-type cyclins. We investigated the role of Bub2 in budding yeast mitotic checkpoint. The following observations indicate that Bub2 and Mad1, 2 probably activate the checkpoint via different pathways: (a) unlike the other Mad and Bub proteins, Bub2 localizes at the spindle pole body (SPB) throughout the cell cycle; (b) the effect of concomitant lack of Mad1 or Mad2 and Bub2 is additive, since nocodazole-treated mad1 bub2 and mad2 bub2 double mutants rereplicate DNA more rapidly and efficiently than either single mutant; (c) cell cycle progression of bub2 cells in the presence of nocodazole requires the Cdc26 APC subunit, which, conversely, is not required for mad2 cells in the same conditions. Altogether, our data suggest that activation of the mitotic checkpoint blocks progression through mitosis by independent and partially redundant mechanisms.

scholarly journals Alpha-Tubulin Acetylation in Trypanosoma cruzi: A Dynamic Instability of Microtubules Is Required for Replication and Cell Cycle Progression

2021 ◽  
Vol 11 ◽  
Author(s):  
Victoria Lucia Alonso ◽  
Mara Emilia Carloni ◽  
Camila Silva Gonçalves ◽  
Gonzalo Martinez Peralta ◽  
Maria Eugenia Chesta ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Trypanosoma Cruzi ◽  
Basal Body ◽  
Mitotic Spindle ◽  
Cell Cycle Progression ◽  
Dynamic Instability ◽  
Cycle Progression ◽  
Alpha Tubulin ◽  
Over Expression ◽  
Tubulin Acetylation

Trypanosomatids have a cytoskeleton arrangement that is simpler than what is found in most eukaryotic cells. However, it is precisely organized and constituted by stable microtubules. Such microtubules compose the mitotic spindle during mitosis, the basal body, the flagellar axoneme and the subpellicular microtubules, which are connected to each other and also to the plasma membrane forming a helical arrangement along the central axis of the parasite cell body. Subpellicular, mitotic and axonemal microtubules are extensively acetylated inTrypanosoma cruzi. Acetylation on lysine (K) 40 of α-tubulin is conserved from lower eukaryotes to mammals and is associated with microtubule stability. It is also known that K40 acetylation occurs significantly on flagella, centrioles, cilia, basal body and the mitotic spindle in eukaryotes. Several tubulin posttranslational modifications, including acetylation of K40, have been cataloged in trypanosomatids, but the functional importance of these modifications for microtubule dynamics and parasite biology remains largely undefined. The primary tubulin acetyltransferase was recently identified in several eukaryotes as Mec-17/ATAT, a Gcn5-related N-acetyltransferase. Here, we report thatT. cruziATAT acetylates α-tubulinin vivoand is capable of auto-acetylation.TcATAT is located in the cytoskeleton and flagella of epimastigotes and colocalizes with acetylated α-tubulin in these structures. We have expressedTcATAT with an HA tag using the inducible vector pTcINDEX-GW inT. cruzi. Over-expression ofTcATAT causes increased levels of the alpha tubulin acetylated species, induces morphological and ultrastructural defects, especially in the mitochondrion, and causes a halt in the cell cycle progression of epimastigotes, which is related to an impairment of the kinetoplast division. Finally, as a result ofTcATAT over-expression we observed that parasites became more resistant to microtubule depolymerizing drugs. These results support the idea that α-tubulin acetylation levels are finely regulated for the normal progression ofT. cruzicell cycle.

scholarly journals Association of human ubiquitin-conjugating enzyme CDC34 with the mitotic spindle in anaphase

2000 ◽  
Vol 113 (10) ◽  
pp. 1687-1694 ◽  
Author(s):  
F. Reymond ◽  
C. Wirbelauer ◽  
W. Krek
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Cycle ◽  
Mitotic Spindle ◽  
Mammalian Cells ◽  
Cell Cycle Progression ◽  
Intracellular Localization ◽  
Cycle Progression ◽  
Ubiquitin Conjugating Enzyme ◽  
Regulation Of Cell Cycle ◽  
Conjugating Enzyme

Present in organisms ranging from yeast to man, homologues of the Saccharomyces cerevisiae ubiquitin-conjugating enzyme CDC34 have been shown to play important roles in the regulation of cell cycle progression and checkpoint function. Here we analyze the expression and intracellular localization of endogenous CDC34 during mammalian cell cycle progression. We find that CDC34 protein is constitutively expressed during all stages of the cell cycle. Immunofluorescence experiments reveal that during interphase, endogenous CDC34 is localized to distinct speckles in both the nucleus and the cytoplasm. The presence of CDC34 in these compartments has also been established by biochemical fractionation experiments. Interestingly, nuclear localization depends on the presence of specific carboxy-terminal CDC34 sequences that have previously been shown to be required for CDC34's cell cycle function in Saccharomyces cerevisiae. Finally, we find that in anaphase and not during early stages of mitosis, CDC34 colocalizes with (beta)-tubulin at the mitotic spindle, implying that it may contribute to spindle function at later stages of mitosis. Taken together, these results support a model in which CDC34 ubiquitin-conjugating enzyme functions in the regulation of nuclear and cytoplasmic activities as well as in the process of chromosome segregation at the onset of anaphase in mammalian cells.

scholarly journals The CWI Pathway: A Versatile Toolbox to Arrest Cell-Cycle Progression

2021 ◽  
Vol 7 (12) ◽  
pp. 1041
Author(s):  
Inma Quilis ◽  
Mercè Gomar-Alba ◽  
Juan Carlos Igual
Keyword(s):  
Cell Cycle ◽  
Cell Wall ◽  
Cellular Response ◽  
Cell Cycle Progression ◽  
Genetic Material ◽  
Regulatory System ◽  
Genotoxic Stress ◽  
Dna Integrity ◽  
Cycle Progression ◽  
Reciprocal Regulation

Cell-signaling pathways are essential for cells to respond and adapt to changes in their environmental conditions. The cell-wall integrity (CWI) pathway of Saccharomyces cerevisiae is activated by environmental stresses, compounds, and morphogenetic processes that compromise the cell wall, orchestrating the appropriate cellular response to cope with these adverse conditions. During cell-cycle progression, the CWI pathway is activated in periods of polarized growth, such as budding or cytokinesis, regulating cell-wall biosynthesis and the actin cytoskeleton. Importantly, accumulated evidence has indicated a reciprocal regulation of the cell-cycle regulatory system by the CWI pathway. In this paper, we describe how the CWI pathway regulates the main cell-cycle transitions in response to cell-surface perturbance to delay cell-cycle progression. In particular, it affects the Start transcriptional program and the initiation of DNA replication at the G1/S transition, and entry and progression through mitosis. We also describe the involvement of the CWI pathway in the response to genotoxic stress and its connection with the DNA integrity checkpoint, the mechanism that ensures the correct transmission of genetic material and cell survival. Thus, the CWI pathway emerges as a master brake that stops cell-cycle progression when cells are coping with distinct unfavorable conditions.

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