Hypoxia induces major effects on cell cycle kinetics and protein expression in Drosophila melanogaster embryos

2005 ◽  
Vol 288 (2) ◽  
pp. R511-R521 ◽  
Author(s):  
R. M. Douglas ◽  
R. Farahani ◽  
P. Morcillo ◽  
A. Kanaan ◽  
T. Xu ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Drosophila Melanogaster ◽  
Cycle Length ◽  
Fluorescent Protein ◽  
Fruit Fly ◽  
Cyclin A ◽  
S Phase ◽  
Green Fluorescent ◽  
Cell Cycle Length

Hypoxia induces a stereotypic response in Drosophila melanogaster embryos: depending on the time of hypoxia, embryos arrest cell cycle activity either at metaphase or just before S phase. To understand the mechanisms underlying hypoxia-induced arrest, two kinds of experiments were conducted. First, embryos carrying a kinesin-green fluorescent protein construct, which permits in vivo confocal microscopic visualization of the cell cycle, showed a dose-response relation between O2 level and cell cycle length. For example, mild hypoxia (Po2 ∼55 Torr) had no apparent effect on cell cycle length, whereas severe hypoxia (Po2 ∼25–35 Torr) or anoxia (Po2 = 0 Torr) arrested the cell cycle. Second, we utilized Drosophila embryos carrying a heat shock promoter driving the string ( cdc25) gene (HS-STG3), which permits synchronization of embryos before the start of mitosis. Under conditions of anoxia, we induced a stabilization or an increase in the expression of several G1/S (e.g., dE2F1, RBF2) and G2/M (e.g., cyclin A, cyclin B, dWee1) proteins. This study suggests that, in fruit fly embryos, 1) there is a dose-dependent relationship between cell cycle length and O2 levels in fruit fly embryos, and 2) stabilized cyclin A and E2F1 are likely to be the mediators of hypoxia-induced arrest at metaphase and pre-S phase.

scholarly journals Flagellar Morphogenesis: Protein Targeting and Assembly in the Paraflagellar Rod of Trypanosomes

1999 ◽  
Vol 19 (12) ◽  
pp. 8191-8200 ◽  
Author(s):  
Philippe Bastin ◽  
Thomas H. MacRae ◽  
Susan B. Francis ◽  
Keith R. Matthews ◽  
Keith Gull
Keyword(s):  
Cell Cycle ◽  
Trypanosoma Brucei ◽  
Protein Targeting ◽  
Fluorescent Protein ◽  
Green Fluorescent Protein Reporter ◽  
Mutant Proteins ◽  
Green Fluorescent ◽  
A Minor ◽  
Fluorescent Protein Reporter

ABSTRACT The paraflagellar rod (PFR) of the African trypanosomeTrypanosoma brucei represents an excellent model to study flagellum assembly. The PFR is an intraflagellar structure present alongside the axoneme and is composed of two major proteins, PFRA and PFRC. By inducible expression of a functional epitope-tagged PFRA protein, we have been able to monitor PFR assembly in vivo. As T. brucei cells progress through their cell cycle, they possess both an old and a new flagellum. The induction of expression of tagged PFRA in trypanosomes growing a new flagellum provided an excellent marker of newly synthesized subunits. This procedure showed two different sites of addition: a major, polar site at the distal tip of the flagellum and a minor, nonpolar site along the length of the partially assembled PFR. Moreover, we have observed turnover of epitope-tagged PFRA in old flagella that takes place throughout the length of the PFR structure. Expression of truncated PFRA mutant proteins identified a sequence necessary for flagellum localization by import or binding. This sequence was not sufficient to confer full flagellum localization to a green fluorescent protein reporter. A second sequence, necessary for the addition of PFRA protein to the distal tip, was also identified. In the absence of this sequence, the mutant PFRA proteins were localized both in the cytosol and in the flagellum where they could still be added along the length of the PFR. This seven-amino-acid sequence is conserved in all PFRA and PFRC proteins and shows homology to a sequence in the flagellar dynein heavy chain of Chlamydomonas reinhardtii.

scholarly journals Replication fork rate and origin activation during the S phase of Saccharomyces cerevisiae.

1980 ◽  
Vol 85 (1) ◽  
pp. 108-115 ◽  
Author(s):  
C J Rivin ◽  
W L Fangman
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Cycle ◽  
Cycle Length ◽  
Replication Fork ◽  
Nitrogen Sources ◽  
S Phase ◽  
Phase Duration ◽  
Yeast Saccharomyces Cerevisiae ◽  
Origin Activation ◽  
Cell Cycle Length

When the growth rate of the yeast Saccharomyces cerevisiae is limited with various nitrogen sources, the duration of the S phase is proportional to cell cycle length over a fourfold range of growth rates (C.J. Rivin and W. L. Fangman, 1980, J. Cell Biol. 85:96-107). Molecular parameters of the S phases of these cells were examined by DNA fiber autoradiography. Changes in replication fork rate account completely for the changes in S-phase duration. No changes in origin-to-origin distances were detected. In addition, it was found that while most adjacent replication origins are activated within a few minutes of each other, new activations occur throughout the S phase.

435 REDUCTION OF CELL CYCLE LENGTH AND INCREASE IN S-PHASE BY GROWTH FACTORS IN PIG FETAL FIBROBLASTS

2010 ◽  
Vol 22 (1) ◽  
pp. 374
Author(s):  
S. Waghmare ◽  
B. Mir
Keyword(s):  
Cell Cycle ◽  
Cell Proliferation ◽  
Growth Factors ◽  
Growth Factor ◽  
Cycle Length ◽  
S Phase ◽  
Proliferation Rate ◽  
Fetal Fibroblasts ◽  
Cell Cycle Length ◽  
Number Of Cells

Gene targeting in primary somatic cells is inefficient compared with embryonic stem cells. This is because of a slow rate of cell proliferation, fewer cells in S-phase at a given time point under normal culture conditions, and low rate of homologous recombination. Homologous recombination occurs mainly in late S-phase and increase in gene targeting efficiency has been reported in S-phase synchronized cells in bovine and rhesus macaque fetal fibroblasts. In this study we tested several growth factors: platelet-derived growth factor (PDGF), tumor necrosis factor a (TNFα), epidermal growth factor (EGF), fibroblast growth factor (FGF), transforming growth factor β1 (TGFβ1), insulin-like growth factor 1 (ILGF-1) and insulin-like growth factor II (ILGF-II) individually and in various combinations to see the effect on cell proliferation rate. Each experimental set consisted of 3 replicates. TGFβ1-, ILGF1-, ILGFII-, and FGF-treated cells grew very slowly compared with untreated cells. However, a combination of 3 growth factors: PDGF (15 ng mL-1), EGF (50 ng mL-1) and TNFa (100 pg mL-1), herein referred to as the cocktail, accelerated cell proliferation rate and reduced cell cycle length on average from 24.5 ± 0.2 to 20.4 ± 0.5 h with no significant change in number of cells in S-phase. Further, cells grown in the presence of the cocktail showed changes in morphology. The cells became spindle-shaped and occupied less surface area per cell compared with untreated cells. Importantly, cocktail-treated cells maintained a normal karyotype without any chromosomal abnormality. Thymidine has been used successfully to block various cell types in S-phase but it failed to synchronize these cells in S-phase in the concentration range of 2 to 10 mM for 24 to 48 h. However, serum starvation (0.2% fetal bovine serum) for 48 h blocked the cell proliferation rate effectively and synchronized cells in G0 phase (80-82% cells). After releasing from the block, cells were grown in the absence or presence of cocktail and cell cycle analysis was done at different time points by flow cytometry. Each time point was repeated 3 times. We observed the maximum number of cells in S-phase at 22 to 23 h (61.33% ± 7.77 in cocktail-treated cells v. 41.7% ± 3.28 in untreated cells). In summary, the cocktail-treated cells showed changes in cell morphology, higher proliferation rate, reduction in cell cycle length by 16.7%, and maximum percentage of cells in S-phase following serum starvation but maintained normal karyotypes. This high proliferation rate, reduction in cell cycle length, and maximum number of cells in S-phase should be very helpful in increasing the efficiency of gene-targeting in pig fetal fibroblasts.

Cell cycle progression and cell division are sensitive to hypoxia in Drosophila melanogaster embryos

2001 ◽  
Vol 280 (5) ◽  
pp. R1555-R1563 ◽  
Author(s):  
Robert M. Douglas ◽  
Tian Xu ◽  
Gabriel G. Haddad
Keyword(s):  
Cell Cycle ◽  
Drosophila Melanogaster ◽  
Mammalian Cells ◽  
Fluorescent Protein ◽  
Immunoblot Analysis ◽  
Embryonic Cell ◽  
Cell Cycle Checkpoints ◽  
Cycle Phase ◽  
Embryonic Cells

We and others recently demonstrated that Drosophila melanogaster embryos arrest development and embryonic cells cease dividing when they are deprived of O2. To further characterize the behavior of these embryos in response to O2 deprivation and to define the O2-sensitive checkpoints in the cell cycle, embryos undergoing nuclear cycles 3–13 were subjected to O2deprivation and examined by confocal microscopy under control, hypoxic, and reoxygenation conditions. In vivo, real-time analysis of embryos carrying green fluorescent protein-kinesin demonstrated that cells arrest at two major points of the cell cycle, either at the interphase (before DNA duplication) or at metaphase, depending on the cell cycle phase at which O2 deprivation was induced. Immunoblot analysis of embryos whose cell divisions are synchronized by inducible String (cdc25 homolog) demonstrated that cyclin B was degraded during low O2 conditions in interphase-arrested embryos but not in those arrested in metaphase. Embryos resumed cell cycle activity within ∼20 min of reoxygenation, with very little apparent change in cell cycle kinetics. We conclude that there are specific points during the embryonic cell cycle that are sensitive to the O2 level in D. melanogaster. Given the fact that O2deprivation also influences the growth and development of other species, we suggest that similar hypoxia-sensitive cell cycle checkpoints may also exist in mammalian cells.

scholarly journals The Sudden Recruitment of γ-Tubulin to the Centrosome at the Onset of Mitosis and Its Dynamic Exchange Throughout the Cell Cycle, Do Not Require Microtubules

1999 ◽  
Vol 146 (3) ◽  
pp. 585-596 ◽  
Author(s):  
Alexey Khodjakov ◽  
Conly L. Rieder
Keyword(s):  
Cell Cycle ◽  
Green Fluorescent Protein ◽  
Fluorescence Recovery After Photobleaching ◽  
Fluorescent Protein ◽  
Green Fluorescent Protein Fusion ◽  
Protein Fusion ◽  
Green Fluorescent ◽  
Dynamic Exchange ◽  
Two Populations

γ-Tubulin is a centrosomal component involved in microtubule nucleation. To determine how this molecule behaves during the cell cycle, we have established several vertebrate somatic cell lines that constitutively express a γ-tubulin/green fluorescent protein fusion protein. Near simultaneous fluorescence and DIC light microscopy reveals that the amount of γ-tubulin associated with the centrosome remains relatively constant throughout interphase, suddenly increases during prophase, and then decreases to interphase levels as the cell exits mitosis. This mitosis-specific recruitment of γ-tubulin does not require microtubules. Fluorescence recovery after photobleaching (FRAP) studies reveal that the centrosome possesses two populations of γ-tubulin: one that turns over rapidly and another that is more tightly bound. The dynamic exchange of centrosome-associated γ-tubulin occurs throughout the cell cycle, including mitosis, and it does not require microtubules. These data are the first to characterize the dynamics of centrosome-associated γ-tubulin in vertebrate cells in vivo and to demonstrate the microtubule-independent nature of these dynamics. They reveal that the additional γ-tubulin required for spindle formation does not accumulate progressively at the centrosome during interphase. Rather, at the onset of mitosis, the centrosome suddenly gains the ability to bind greater than three times the amount of γ-tubulin than during interphase.

scholarly journals Acute effects of wheel running on adult hippocampal precursor cells in mice are not caused by changes in cell cycle length or S phase length

2014 ◽  
Vol 8 ◽  
Author(s):  
Tim J. Fischer ◽  
Tara L. Walker ◽  
Rupert W. Overall ◽  
Moritz D. Brandt ◽  
Gerd Kempermann
Keyword(s):  
Cell Cycle ◽  
Cycle Length ◽  
Wheel Running ◽  
Precursor Cells ◽  
S Phase ◽  
Acute Effects ◽  
Cell Cycle Length ◽  
Phase Length

scholarly journals ZFP100, a Component of the Active U7 snRNP Limiting for Histone Pre-mRNA Processing, Is Required for Entry into S Phase

2006 ◽  
Vol 26 (17) ◽  
pp. 6702-6712 ◽  
Author(s):  
Eric J. Wagner ◽  
William F. Marzluff
Keyword(s):  
Fluorescent Protein ◽  
S Phase ◽  
Mrna Processing ◽  
Limiting Factor ◽  
Stem Loop ◽  
Histone Mrnas ◽  
U7 Snrnp ◽  
Eukaryotic Mrnas ◽  
Green Fluorescent

ABSTRACT Metazoan replication-dependent histone mRNAs are the only eukaryotic mRNAs that are not polyadenylated. The cleavage of histone pre-mRNA to form the unique 3′ end requires the U7 snRNP and the stem-loop binding protein (SLBP) that binds the 3′ end of histone mRNA. U7 snRNP contains three novel proteins, Lsm10 and Lsm11, which are part of the core U7 Sm complex, and ZFP100, a Zn finger protein that helps stabilize binding of the U7 snRNP to the histone pre-mRNA by interacting with the SLBP/pre-mRNA complex. Using a reporter gene that encodes a green fluorescent protein mRNA ending in a histone 3′ end and mimics histone gene expression, we demonstrate that ZFP100 is the limiting factor for histone pre-mRNA processing in vivo. The overexpression of Lsm10 and Lsm11 increases the cellular levels of U7 snRNP but has no effect on histone pre-mRNA processing, while increasing the amount of ZFP100 increases histone pre-mRNA processing but has no effect on U7 snRNP levels. We also show that knocking down the known components of U7 snRNP by RNA interference results in a reduction in cell growth and an unsuspected cell cycle arrest in early G1, suggesting that active U7 snRNP is necessary to allow progression through G1 phase to S phase.

scholarly journals SCAPER, a novel cyclin A–interacting protein that regulates cell cycle progression

2007 ◽  
Vol 178 (4) ◽  
pp. 621-633 ◽  
Author(s):  
William Y. Tsang ◽  
Leyu Wang ◽  
Zhihong Chen ◽  
Irma Sánchez ◽  
Brian David Dynlacht
Keyword(s):  
Cell Cycle ◽  
Cell Cycle Progression ◽  
Regulatory Protein ◽  
Ectopic Expression ◽  
Cyclin A ◽  
Eukaryotic Cell ◽  
S Phase ◽  
Interacting Protein ◽  
M Phase

Cyclin A/Cdk2 plays an important role during S and G2/M phases of the eukaryotic cell cycle, but the mechanisms by which it regulates cell cycle events are not fully understood. We have biochemically purified and identified SCAPER, a novel protein that specifically interacts with cyclin A/Cdk2 in vivo. Its expression is cell cycle independent, and it associates with cyclin A/Cdk2 at multiple phases of the cell cycle. SCAPER localizes primarily to the endoplasmic reticulum. Ectopic expression of SCAPER sequesters cyclin A from the nucleus and results specifically in an accumulation of cells in M phase of the cell cycle. RNAi-mediated depletion of SCAPER decreases the cytoplasmic pool of cyclin A and delays the G1/S phase transition upon cell cycle re-entry from quiescence. We propose that SCAPER represents a novel cyclin A/Cdk2 regulatory protein that transiently maintains this kinase in the cytoplasm. SCAPER could play a role in distinguishing S phase– from M phase–specific functions of cyclin A/Cdk2.

scholarly journals HIRA, the Human Homologue of Yeast Hir1p and Hir2p, Is a Novel Cyclin-cdk2 Substrate Whose Expression Blocks S-Phase Progression

2001 ◽  
Vol 21 (5) ◽  
pp. 1854-1865 ◽  
Author(s):  
Caitlin Hall ◽  
David M. Nelson ◽  
Xiaofen Ye ◽  
Kayla Baker ◽  
James A. DeCaprio ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Ectopic Expression ◽  
Cyclin A ◽  
S Phase ◽  
Dependent Manner ◽  
Sequence Motifs ◽  
Histone Gene Expression ◽  
Phase Progression

ABSTRACT Substrates of cyclin-cdk2 kinases contain two distinct primary sequence motifs: a cyclin-binding RXL motif and one or more phosphoacceptor sites (consensus S/TPXK/R or S/TP). To identify novel cyclin-cdk2 substrates, we searched the database for proteins containing both of these motifs. One such protein is human HIRA, the homologue of two cell cycle-regulated repressors of histone gene expression in Saccharomyces cerevisiae, Hir1p and Hir2p. Here we demonstrate that human HIRA is an in vivo substrate of a cyclin-cdk2 kinase. First, HIRA bound to and was phosphorylated by cyclin A- and E-cdk2 in vitro in an RXL-dependent manner. Second, HIRA was phosphorylated in vivo on two consensus cyclin-cdk2 phosphoacceptor sites and at least one of these, threonine 555, was phosphorylated by cyclin A-cdk2 in vitro. Third, phosphorylation of HIRA in vivo was blocked by cyclin-cdk2 inhibitor p21cip1. Fourth, HIRA became phosphorylated on threonine 555 in S phase when cyclin-cdk2 kinases are active. Fifth, HIRA was localized preferentially to the nucleus, where active cyclin A- and E-cdk2 are located. Finally, ectopic expression of HIRA in cells caused arrest in S phase and this is consistent with the notion that it is a cyclin-cdk2 substrate that has a role in control of the cell cycle.

scholarly journals Cell cycle phase expansion in nitrogen-limited cultures of Saccharomyces cerevisiae.

1980 ◽  
Vol 85 (1) ◽  
pp. 96-107 ◽  
Author(s):  
C J Rivin ◽  
W L Fangman
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Cycle ◽  
Cycle Length ◽  
Time Lapse ◽  
S Phase ◽  
Cycle Phase ◽  
Yeast Saccharomyces Cerevisiae ◽  
Bud Emergence ◽  
Length Changes ◽  
Cell Cycle Length

The time and coordination of cell cycle events were examined in the budding yeast Saccharomyces cerevisiae. Whole-cell autoradiographic techniques and time-lapse photography were used to measure the duration of the S, G1, and G2 phases, and the cell cycle positions of "start" and bud emergence, in cells whose growth rates were determined by the source of nitrogen. It was observed that the G1, S, and G2 phases underwent a proportional expansion with increasing cell cycle length, with the S phase occupying the middle half of the cell cycle. In each growth condition, start appeared to correspond to the G1 phase/S phase boundary. Bud emergence did not occur until mid S phase. These results show that the rate of transit through all phases of the cell cycle can vary considerably when cell cycle length changes. When cells growing at different rates were arrested in G1, the following synchronous S phase were of the duration expected from the length of S in each asynchronous population. Cells transferred from a poor nitrogen source to a good one after arrest in G1 went through the subsequent S phase at a rate characteristic of the better medium, indicating that cells are not committed in G1 to an S phase of a particular duration.

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