GCN2/eIF2αK4 is exclusively seen as an eIF2α kinase, which regulates reprogramming of protein translation in response to stress. Here, we show that GCN2 has an unexpected role in unstressed cells as a regulator of mitosis. This function is not through its canonical role in translation reprogramming, but through the regulation of two previously unidentified substrates, PP1α and γ.
View Article and Find Full Text PDFMany patients with locally advanced cervical cancer experience recurrence within the radiation field after chemoradiotherapy. Biomarkers of tumor radioresistance are required to identify patients in need of intensified treatment. Here, the biomarker potential of miR-200 family members was investigated in this disease.
View Article and Find Full Text PDFCellular asymmetry plays a major role in the ageing and evolution of multicellular organisms. However, it remains unknown how the cell distinguishes 'old' from 'new' and whether asymmetry is an attribute of highly specialized cells or a feature inherent in all cells. Here, we investigate the segregation of three asymmetric features: old and new DNA, the spindle pole body (SPB, the centrosome analogue) and the old and new cell ends, using a simple unicellular eukaryote, .
View Article and Find Full Text PDFWe discuss novel insight into the role and consequences of the phosphorylation of the translation initiation factor eIF2α in the context of stress responses and cell-cycle regulation. eIF2α is centrally located to regulate translation and its phosphorylation in response to different environmental challenges is one of the best characterized stress-response pathways. In addition to its role in stress management, eIF2α phosphorylation is also linked to cell-cycle progression and memory consolidation in the nervous system.
View Article and Find Full Text PDFIt is a long-standing view that global translation varies during the cell cycle and is much lower in mitosis than in other cell-cycle phases. However, the central papers in the literature are not in agreement about the extent of downregulation in mitosis, ranging from a dramatic decrease to only a marginal reduction. Herein, it is argued that the discrepancy derives from technical challenges.
View Article and Find Full Text PDFCurrently the greatest challenge in oncology is the lack of homogeneity of the lesions where different cell components respond differently to treatment. There is growing consensus that monotherapies are insufficient to eradicate the disease and there is an unmet need for more potent combinatorial treatments. We have previously shown that hypericin photodynamic therapy (HYP-PDT) triggers electron transport chain (ETC) inhibition in cell mitochondria.
View Article and Find Full Text PDFFor optimal exploitation of nanoparticles (NPs) in biomedicine, and to predict nanotoxicity, detailed knowledge of the cellular responses to cell-bound or internalized NPs is imperative. The final outcome of NP-cell interaction is dictated by the type and magnitude of the NP insult and the cellular response. Here, this has been systematically studied by using poly(alkylcyanoacrylate) (PACA) particles differing only in their alkyl side chains; butyl (PBCA), ethylbutyl (PEBCA), or octyl (POCA), respectively.
View Article and Find Full Text PDFAtaxia telangiectasia mutated and Rad3-related (ATR) kinase is a key factor activated by DNA damage and replication stress. An alternative pathway for ATR activation has been proposed to occur via stalled RNA polymerase II (RNAPII). However, how RNAPII might signal to activate ATR remains unknown.
View Article and Find Full Text PDFIt is generally accepted that global translation varies during the cell cycle and is low during mitosis. However, addressing this issue is challenging because it involves cell synchronization, which evokes stress responses that, in turn, affect translation rates. Here, we have used two approaches to measure global translation rates in different cell-cycle phases.
View Article and Find Full Text PDFFission yeast 'cut' mutants show defects in temporal coordination of nuclear division with cytokinesis, resulting in aberrant mitosis and lethality. Among other causes, the 'cut' phenotype can be triggered by genetic or chemical perturbation of lipid metabolism, supposedly resulting in shortage of membrane phospholipids and insufficient nuclear envelope expansion during anaphase. Interestingly, penetrance of the 'cut' phenotype in mutants of the transcription factor cbf11 and acetyl-coenzyme A carboxylase cut6, both related to lipid metabolism, is highly dependent on growth media, although the specific nutrient(s) affecting 'cut' occurrence is not known.
View Article and Find Full Text PDFCheckpoint kinases are important in cellular surveillance pathways that help cells to cope with DNA damage and protect their genomes. In cycling cells, DNA replication is one of the most sensitive processes and therefore all organisms carefully regulate replication initiation and progression. The checkpoint kinase ATR plays important roles both in response to DNA damage and replication stress, and ATR inhibitors are currently in clinical trials for cancer treatment.
View Article and Find Full Text PDFWhen cells are exposed to stress they delay entry into mitosis. The most extensively studied mechanism behind this delay is the DNA-damage-induced G2/M checkpoint. Here, we show the existence of an additional stress-response pathway in that is independent of the classic ATR/Rad3-dependent checkpoint.
View Article and Find Full Text PDFAll organisms have evolved pathways to respond to different forms of cellular stress. The Gcn2 kinase is best known as a regulator of translation initiation in response to starvation for amino acids. Work in budding yeast has showed that the molecular mechanism of GCN2 activation involves the binding of uncharged tRNAs, which results in a conformational change and GCN2 activation.
View Article and Find Full Text PDFCold Spring Harb Protoc
June 2016
Flow cytometry can be used to measure the DNA content of individual cells. The data are usually presented as DNA histograms that can be used to examine the cells' progression through the cell cycle. Under standard growth conditions, fission yeast cells do not complete cytokinesis until after G1 phase; therefore, DNA histograms show one major peak representing cells in G1 (2×1C DNA) and G2 phase (1×2C DNA).
View Article and Find Full Text PDFCyclin Dependent Kinases (CDKs) are important regulators of DNA replication. In this work we have investigated the consequences of increasing or decreasing the CDK activity in S phase. To this end we identified S-phase regulators of the fission yeast CDK, Cdc2, and used appropriate mutants to modulate Cdc2 activity.
View Article and Find Full Text PDFExposure of fission yeast cells to ultraviolet (UV) light leads to inhibition of translation and phosphorylation of the eukaryotic initiation factor-2α (eIF2α). This phosphorylation is a common response to stress in all eukaryotes. It leads to inhibition of translation at the initiation stage and is thought to be the main reason why stressed cells dramatically reduce protein synthesis.
View Article and Find Full Text PDFThymidine analogues are powerful tools when studying DNA synthesis including DNA replication, repair and recombination. However, these analogues have been reported to have severe effects on cell-cycle progression and growth, the very processes being investigated in most of these studies. Here, we have analyzed the effects of 5-ethynyl-2'-deoxyuridine (EdU) and 5-Chloro-2'-deoxyuridine (CldU) using fission yeast cells and optimized the labelling procedure.
View Article and Find Full Text PDFRegulating growth and the cell cycle in response to environmental fluctuations is important for all organisms in order to maintain viability. Two major pathways for translational regulation are found in higher eukaryotes: the Tor signaling pathway and those operating through the eIF2α kinases. Studies from several organisms indicate that the two pathways are interlinked, in that Tor complex 1 (TORC1) negatively regulates the Gcn2 kinase.
View Article and Find Full Text PDFGcn2 was first described in budding yeast as a serine/threonine protein kinase involved in the response to amino acid starvation and this is its best characterized role to date. Recent work has revealed new and exciting roles for Gcn2, which affect many aspects of cellular physiology in response to a number of stresses in addition to starvation. Furthermore, the Gcn2 pathway has been implicated in diseases such as cancer and Alzheimer's disease, and therefore elucidating the new roles of Gcn2 seems ever more important.
View Article and Find Full Text PDFHere we characterize a novel protein in S. pombe. It has a high degree of homology with the Zn-finger domain of the human Poly(ADP-ribose) polymerase (PARP).
View Article and Find Full Text PDFEntry into S phase is carefully regulated and, in most organisms, under the control of a G(1)-S checkpoint. We have previously described a G(1)-S checkpoint in fission yeast that delays formation of the prereplicative complex at chromosomal replication origins after exposure to UV light (UVC). This checkpoint absolutely depends on the Gcn2 kinase.
View Article and Find Full Text PDFThe cell cycle of the fission yeast, Schizosaccharomyces pombe, does not easily lend itself to analysis by flow cytometry, mainly because cells in G(1) and G(2) phase contain the same amount of DNA. This occurs because fission yeast cells under standard growth conditions do not complete cytokinesis until after G(1) phase. We have devised a flow cytometric method exploiting the fact that cells in G(1) phase contain two nuclei, whereas cells in G(2) are mononuclear.
View Article and Find Full Text PDFBackground: In many cell types, including the fission yeast Schizosaccharomyces pombe, a set of checkpoints are induced by perturbations of the cell cycle or by DNA damage. Many of the checkpoint responses include a substantial change of the transcriptional pattern. As part of characterising a novel G1/S checkpoint in fission yeast we have investigated whether a transcriptional response is induced after irradiation with ultraviolet light.
View Article and Find Full Text PDFWe discuss the mechanisms regulating entry into and progression through S phase in eukaryotic cells. Methods to study the G1/S transition are briefly reviewed and an overview of G1/S-checkpoints is given, with particular emphasis on fission yeast. Thereafter we discuss different aspects of the intra-S checkpoint and introduce the main molecular players and mechanisms.
View Article and Find Full Text PDFThe initiation of DNA replication is a complex, multistep process with important implications for genomic stability. In this issue, Wu and Nurse (2009) find that initiation factors are differentially recruited to replication origins. They uncover evidence suggesting that the efficiency of this recruitment may determine whether and when an origin is used to initiate DNA replication in S phase.
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