Synchronized Cell Populations Research Articles

Death patterns resulting from cell cycle-independent cell death.

Drug-induced cell death on the one hand, and dynamics of the cell cycle on the other hand, represent two well-investigated biological principles. As the cell death machinery is highly regulated and depends on a large number of signaling proteins, a connection to the cell cycle is standing to reason. In recent studies, fluorescent cell cycle marker techniques have been combined with longterm microscopy approaches in order to shed light upon a possible cell cycle/cell death dependency for different death-inducing drugs. Here, we present a simple mechanistic model of the null hypothesis where a cell’s cycle does not influence cell death, and calculate death events in different phases of the cell cycle. In a first sample calculation, cells located according to a steady state distribution within the cell cycle are exposed to three different drugs. Dependent on the drug’s mode of action, different death patterns are observed. The second sample calculation considers the exposure of synchronized cell populations to one particular drug. Results show that an increase of cell death in G1 can be obtained although cells are initially synchronized in different phases. We conclude that cumulated cell death in one phase can be explained by the stated null hypothesis. Last, the empirical mean and standard deviation are calculated for different sample sizes. Given experimental data, the presented results may help to find out whether the null hypothesis can be rejected.

Temporal fluxomics reveals oscillations in TCA cycle flux throughout the mammalian cell cycle

Cellular metabolic demands change throughout the cell cycle. Nevertheless, a characterization of how metabolic fluxes adapt to the changing demands throughout the cell cycle is lacking. Here, we developed a temporal‐fluxomics approach to derive a comprehensive and quantitative view of alterations in metabolic fluxes throughout the mammalian cell cycle. This is achieved by combining pulse‐chase LC‐MS‐based isotope tracing in synchronized cell populations with computational deconvolution and metabolic flux modeling. We find that TCA cycle fluxes are rewired as cells progress through the cell cycle with complementary oscillations of glucose versus glutamine‐derived fluxes: Oxidation of glucose‐derived flux peaks in late G1 phase, while oxidative and reductive glutamine metabolism dominates S phase. These complementary flux oscillations maintain a constant production rate of reducing equivalents and oxidative phosphorylation flux throughout the cell cycle. The shift from glucose to glutamine oxidation in S phase plays an important role in cell cycle progression and cell proliferation.

Ribosome profiling the cell cycle: lessons and challenges.

Understanding the causes and consequences of dynamic changes in the abundance and activity of cellular components during cell division is what most cell cycle studies are about. Here we focus on control of gene expression in the cell cycle at the level of translation. The advent of deep sequencing methodologies led to technologies that quantify the levels of all mRNAs that are bound by ribosomes and engaged in translation in the cell (Ingolia et al. Science 324:218-223, 2009). This approach has been applied recently to synchronous cell populations to find transcripts under translational control at different cell cycle phases (Blank et al. EMBO J 36:487-502, 2017; Stumpf et al. Mol Cell 52:574-582, 2013; Tanenbaum et al. Elife 4:e07957, 2015). These studies revealed new biology, but they also have limitations, pointing to challenges that need to be addressed in the future.

Synchronization of Bacillus subtilis Cells by Spore Germination and Outgrowth.

This protocol defines conditions under which the germination of spores can be used to synchronize Bacillus subtilis cells, utilizing the time-ordered sequence of events taking place during the transition from spore to vegetative cells. The transition stages involve: phase change, swelling, emergence, initial division, and elongation. By using this method we have obtained two distinctive synchronized cell cycles, while the synchrony faded away in the third cycle. The advantage of using spore outgrowth and germination is that a highly synchronized population of bacterial cells can be obtained. Non-dividing spores stay synchronized, while synchrony rapidly decays during a few divisions. The limitations of this method are that it can be applied only for sporulating bacteria and synchrony lasts for only a limited period of time exceeding not more than two cycles.

Interaction between the plant ApDef1 defensin and Saccharomyces cerevisiae results in yeast death through a cell cycle- and caspase-dependent process occurring via uncontrolled oxidative stress

BackgroundPlant defensins were discovered at beginning of the 90s'; however, their precise mechanism of action is still unknown. Herein, we studied ApDef1-Saccharomyces cerevisiae interaction. MethodsApDef1-S. cerevisiae interaction was studied by determining the MIC, viability and death kinetic assays. Viability assay was repeated with hydroxyurea synchronized-yeast and pretreated with CCCP. Plasma membrane permeabilization, ROS induction, chromatin condensation, and caspase activation analyses were assessed through Sytox green, DAB, DAPI and FITC-VAD-FMK, respectively. Viability assay was done in presence of ascorbic acid and Z-VAD-FMK. Ultrastructural analysis was done by electron microscopy. ResultsApDef1 caused S. cerevisiae cell death and MIC was 7.8μM. Whole cell population died after 18h of ApDef1 interaction. After 3h, 98.76% of synchronized cell population died. Pretreatment with CCCP protected yeast from ApDef1 induced death. ApDef1-S. cerevisiae interaction resulted in membrane permeabilization, H2O2 increased production, chromatin condensation and caspase activation. Ascorbic acid prevented yeast cell death and membrane permeabilization. Z-VAD-FMK prevented yeast cell death. ConclusionsApDef1-S. cerevisiae interaction caused cell death through cell cycle dependentprocess which requires preserved membrane potential. After interaction, yeast went through uncontrolled ROS production and accumulation, which led to plasma membrane permeabilization, chromatin condensation and, ultimately, cell death by activation of caspase-dependent apoptosis via. General significanceWe show novel requirements for the interaction between plant defensin and fungi cells, i.e. cell cycle phase and membrane potential, and we indicate that membrane permeabilization is probably caused by ROS and therefore, it would be an indirect event of the ApDef1-S. cerevisiae interaction.

Synchronization of Green Algae by Light and Dark Regimes for Cell Cycle and Cell Division Studies.

A synchronous population of cells is one of the prerequisites for studying cell cycle processes such as DNA replication, nuclear and cellular division. Green algae dividing by multiple fission represent a unique single cell system enabling the preparation of highly synchronous cultures by application of a light-dark regime similar to what they experience in nature. This chapter provides detailed protocols for synchronization of different algal species by alternating light-dark cycles; all critical points are discussed extensively. Moreover, detailed information on basic analysis of cell cycle progression in such cultures is presented, including analyses of nuclear, cellular, and chloroplast divisions. Modifications of basic protocols that enable changes in cell cycle progression are also suggested so that nuclear or chloroplast divisions can be followed separately.

Cell Cycle Synchronization Using a Microfluidic Synchronizer for Fission Yeast Cells.

To produce synchronized cell colonies, many cell cycle synchronization technologies have been developed, among which the baby machine may be considered the most artifact-free. Baby machines incubate "mother cells" under normal conditions and collects their "babies," producing cell cultures that are similar not only in cell cycle phase but also in age. Several macroscale and microfluidic baby machines have been applied to synchronized cell research. However, for rod-shaped cells like fission yeast (Schizosaccharomyces pombe), it is still a challenge to immobilize only the mother cells in a microfluidic device. Here, we present a new baby machine suitable for fission yeast. The device fixes one end of the cell and releases the free-end daughter cell every time the cell finishes cytokinesis. A variety of structures for cell immobilization were attempted to find the optimal design. For the convenience of collection and to enable further assays, we integrated a cell screener into the baby machine, which exploits the deformation of polymer material to switch between open and closed states. The device, producing synchronous populations of fission yeast cells, provides a new on-chip tool for cell biology studies.

Isolation of Plant Nuclei at Defined Cell Cycle Stages Using EdU Labeling and Flow Cytometry.

5-Ethynyl-2'-deoxyuridine (EdU) is a nucleoside analog of thymidine that can be rapidly incorporated into replicating DNA in vivo and, subsequently, detected by using "click" chemistry to couple its terminal alkyne group to fluorescent azides such as Alexa Fluor 488. Recently, EdU incorporation followed by coupling with a fluorophore has been used to visualize newly synthesized DNA in a wide range of plant species. One particularly useful application is in flow cytometry, where two-parameter sorting can be employed to analyze different phases of the cell cycle, as defined both by total DNA content and the amount of EdU pulse-labeled DNA. This approach allows analysis of the cell cycle without the need for synchronous cell populations, which can be difficult to obtain in many plant systems. The approach presented here, which was developed for fixed, EdU-labeled nuclei, can be used to prepare analytical profiles as well as to make highly purified preparations of G1, S, or G2/M phase nuclei for molecular or biochemical analysis. We present protocols for EdU pulse labeling, tissue fixation and harvesting, nuclei preparation, and flow sorting. Although developed for Arabidopsis suspension cells and maize root tips, these protocols should be modifiable to many other plant systems.

Deep Proteomic Analysis of Human Erythropoiesis

*the first two authors are co-first authorsIntroduction. Erythropoiesis is a complex process starting from pluripotent medullary progenitors and leading to the production of highly specialized and enucleated erythrocytes. Two successive phases are generally distinguished: an amplification phase with intense proliferation of morphologically similar progenitors and a terminal differentiation phase with few cell divisions and strong cellular modifications. Although erythropoiesis is a continuous process, these modifications allow the identification of specific maturation stages and the passage from one stage to the following one seems to correlate with a cell division. Several transcriptomic analyses of erythroid differentiation have been published but only few and very limited proteomic studies have been reported. Since post transcriptomic modifications are responsible for a large part of the proteome variations, a direct proteomic analysis of the erythroid differentiation is required to accurately assess the modifications that occur during this process.Results. For this study, we used CD34+ cord blood progenitors and an optimized three step cell culture method allowing the production of highly synchronized cell populations of erythroid cells at various differentiation stages. Several cellular populations from erythroid progenitors up to reticulocytes were analyzed by a label-free analysis and mass spectrometry that led to the absolute quantification of more than 6000 proteins with a false discovery rate of less than 1% (n=3). Moreover, the relative expression of well-known stage-specific erythroid markers such as TFRC, BAND3 or GLUT1, transcription factors, heme biosynthesis enzymes followed the expected pattern.To complete this study, we performed a quantitative analysis of the repartition of proteins between the generated reticulocytes and the expelled nucleus (pyrenocyte). To do that, pyrenocytes and reticulocytes were sorted by FACS according to size, Hoechst 33342 and glycophorin A labelling. Equal numbers of reticulocytes and pyrenocytes were used to prepare peptides that were analyzed by mass spectrometry after iTRAQ labelling. These experiments allowed the quantitative repartition of 1153 proteins including most erythrocyte-specific membrane proteins.Conclusion. All these results significantly increase our knowledge of the protein expression pattern during erythropoiesis and should constitute a valuable data base for subsequent studies regarding both physiological and disordered erythropoiesis. DisclosuresNo relevant conflicts of interest to declare.

Human Papillomavirus Infectious Entry and Trafficking Is a Rapid Process.

Previous studies have indicated that human papillomavirus (HPV) infectious entry is slow, requiring many hours after initial infection for the virus to gain entry into the nucleus. However, intracellular transport pathways typically are very rapid, and in the context of a natural HPV infection in a wounded epithelium, such slow intracellular transport would seem to be at odds with a normal viral infection. Using synchronized cell populations, we show that HPV trafficking can be a rapid process. In cells that are infected in the late S-early G₂/M phase of the cell cycle, HPV16 pseudovirion (PsV) reporter DNA gene expression is detectable by 8 h postinfection. Likewise, reporter DNA can be visualized within the nucleus in conjunction with PML nuclear bodies 1 h to 2 h postinfection in cells that are infected with PsVs just prior to mitotic entry. This demonstrates that endosomal trafficking of HPV is rapid, with mitosis being the main restriction on nuclear entry. HPV infectious entry appears to be slow and requires mitosis to occur before the incoming viral DNA can access the nucleus. In this study, we show that HPV trafficking in the cell actually is very rapid. This demonstrates that in the context of a normal virus infection, the cell cycle state will have a major influence on the time it takes for an incoming virus to enter the nucleus and initiate viral gene expression.

An IPTG‐inducible derivative of the fission yeast nmt promoter

We here describe an IPTG-inducible system that reveals that the lac repressor alone can function as a potent transmodulator to regulate gene expression in the fission yeast, Schizosaccharomyces pombe. This expression system is a derivative of the Sz. pombe nmt promoter, which normally is strongly repressed by thiamine. With appropriate positioning of a lac operator site (lacO) downstream of the TATA-box, we show that gene expression from a chimeric nmt::lacO promoter can be regulated by the lac repressor up to two orders of magnitude in response to IPTG. The chimeric nmt::lacO promoter is rapidly induced and when GFP is used as a reporter; almost full induction is achieved 40 min after the addition of IPTG. Like the wild-type nmt promoter, the chimeric nmt::lacO is repressed by thiamine. This allows expression in a short and defined window, e.g. the S-phase of a synchronized cell population, by first adding IPTG to turn on expression, followed by addition of thiamine to switch off expression.

Weak correlation of starch and volume in synchronized photosynthetic cells.

In cultures of unicellular algae, features of single cells, such as cellular volume and starch content, are thought to be the result of carefully balanced growth and division processes. Single-cell analyses of synchronized photoautotrophic cultures of the unicellular alga Chlamydomonas reinhardtii reveal, however, that the cellular volume and starch content are only weakly correlated. Likewise, other cell parameters, e.g., the chlorophyll content per cell, are only weakly correlated with cell size. We derive the cell size distributions at the beginning of each synchronization cycle considering growth, timing of cell division and daughter cell release, and the uneven division of cell volume. Furthermore, we investigate the link between cell volume growth and starch accumulation. This work presents evidence that, under the experimental conditions of light-dark synchronized cultures, the weak correlation between both cell features is a result of a cumulative process rather than due to asymmetric partition of biomolecules during cell division. This cumulative process necessarily limits cellular similarities within a synchronized cell population.

Using in Vivo Biotinylated Ubiquitin to Describe a Mitotic Exit Ubiquitome from Human Cells

Mitotic division requires highly regulated morphological and biochemical changes to the cell. Upon commitment to exit mitosis, cells begin to remove mitotic regulators in a temporally and spatially controlled manner to bring about the changes that reestablish interphase. Ubiquitin-dependent pathways target these regulators to generate polyubiquitin-tagged substrates for degradation by the 26S proteasome. However, the lack of cell-based assays to investigate in vivo ubiquitination limits our knowledge of the identity of substrates of ubiquitin-mediated regulation in mitosis. Here we report an in vivo ubiquitin tagging system used in human cells that allows efficient purification of ubiquitin conjugates from synchronized cell populations. Coupling purification with mass spectrometry, we have identified a series of mitotic regulators targeted for polyubiquitination in mitotic exit. We show that some are new substrates of the anaphase-promoting complex/cyclosome and validate KIFC1 and RacGAP1/Cyk4 as two such targets involved respectively in timely mitotic spindle disassembly and cell spreading. We conclude that in vivo biotin tagging of ubiquitin can provide valuable information about the role of ubiquitin-mediated regulation in processes required for rebuilding interphase cells.

Activation of the Prereplication Complex Is Blocked by Mimosine through Reactive Oxygen Species-activated Ataxia Telangiectasia Mutated (ATM) Protein without DNA Damage

Mimosine is an effective cell synchronization reagent used for arresting cells in late G1 phase. However, the mechanism underlying mimosine-induced G1 cell cycle arrest remains unclear. Using highly synchronous cell populations, we show here that mimosine blocks S phase entry through ATM activation. HeLa S3 cells are exposed to thymidine for 15 h, released for 9 h by washing out the thymidine, and subsequently treated with 1 mM mimosine for a further 15 h (thymidine → mimosine). In contrast to thymidine-induced S phase arrest, mimosine treatment synchronizes >90% of cells at the G1-S phase boundary by inhibiting the transition of the prereplication complex to the preinitiation complex. Mimosine treatment activates ataxia telangiectasia mutated (ATM)/ataxia telangiectasia and Rad3-related (ATR)-mediated checkpoint signaling without inducing DNA damage. Inhibition of ATM activity is found to induce mimosine-arrested cells to enter S phase. In addition, ATM activation by mimosine treatment is mediated by reactive oxygen species (ROS). These results suggest that, upon mimosine treatment, ATM blocks S phase entry in response to ROS, which prevents replication fork stalling-induced DNA damage.

A Novel Function of CRL4Cdt2: REGULATION OF THE SUBUNIT STRUCTURE OF DNA POLYMERASE δ IN RESPONSE TO DNA DAMAGE AND DURING THE S PHASE

DNA polymerase δ (Pol δ4) is a heterotetrameric enzyme, whose p12 subunit is degraded in response to DNA damage, leaving behind a trimer (Pol δ3) with altered enzymatic characteristics that participate in gap filling during DNA repair. We demonstrate that CRL4(Cdt2), a key regulator of cell cycle progression that targets replication licensing factors, also targets the p12 subunit of Pol δ4 in response to DNA damage and on entry into S phase. Evidence for the involvement of CRL4(Cdt2) included demonstration that p12 possesses a proliferating cell nuclear antigen-interacting protein-degron (PIP-degron) and that knockdown of the components of the CRL4(Cdt2) complex inhibited the degradation of p12 in response to DNA damage. Analysis of p12 levels in synchronized cell populations showed that p12 is partially degraded in S phase and that this is affected by knockdowns of CUL4A or CUL4B. Laser scanning cytometry of overexpressed wild type p12 and a mutant resistant to degradation showed that the reduction in p12 levels during S phase was prevented by mutation of p12. Thus, CRL4(Cdt2) also regulates the subunit composition of Pol δ during the cell cycle. These studies reveal a novel function of CRL4(Cdt2), i.e. the direct regulation of DNA polymerase δ, adding to its known functions in the regulation of the licensing of replication origins and expanding the scope of its overall control of DNA replication. The formation of Pol δ3 in S phase as a normal aspect of cell cycle progression leads to the novel implications that it is involved in DNA replication as well as DNA repair.

Age-dependent stochastic models for understanding population fluctuations in continuously cultured cells

For symmetrically dividing cells, large variations in the cell cycle time are typical, even among clonal cells. The consequence of this variation is important in stem cell differentiation, tissue and organ size control, and cancer development, where cell division rates ultimately determine the cell population. We explore the connection between cell cycle time variation and population-level fluctuations using simple stochastic models. We find that standard population models with constant division and death rates fail to predict the level of population fluctuation. Instead, variations in the cell division time contribute to population fluctuations. An age-dependent birth and death model allows us to compute the mean squared fluctuation or the population dispersion as a function of time. This dispersion grows exponentially with time, but scales with the population. We also find a relationship between the dispersion and the cell cycle time distribution for synchronized cell populations. The model can easily be generalized to study populations involving cell differentiation and competitive growth situations.

DNA repair choice defines a common pathway for recruitment of chromatin regulators

DNA double‐strand break (DSB) repair is essential for maintenance of genome stability. Recent work has implicated a host of chromatin regulators in the DNA damage response, but their functional roles remain unclear. In budding yeast, DSB recruitment of chromatin regulators has been monitored primarily in asynchronous cell populations, and thus it is unclear if these events are linked to non‐homologous end joining (NHEJ) or homologous recombination (HR). By using synchronized cell populations, we surprisingly find that subunits of the INO80, SWR‐C, NuA4, SWI/SNF, and RSC enzymes, as monitored by chromatin immunoprecipitation (ChIP), are primarily recruited outside of the G1 cell phase. Furthermore, recruitment is inhibited by the NHEJ machinery, and requires early steps of HR, strongly supporting a role for these enzymes in the HR repair pathway. Strikingly, and in contrast to previous reports, we also find no significant role for H2A.X phosphorylation (γH2AX) in the recruitment of chromatin regulators, but rather their recruitment coincides with reduced levels of γH2AX.This work was supported by a grant from the NIH (GM54096).

Branching process deconvolution algorithm reveals a detailed cell-cycle transcription program

Due to cell-to-cell variability and asymmetric cell division, cells in a synchronized population lose synchrony over time. As a result, time-series measurements from synchronized cell populations do not reflect the underlying dynamics of cell-cycle processes. Here, we present a branching process deconvolution algorithm that learns a more accurate view of dynamic cell-cycle processes, free from the convolution effects associated with imperfect cell synchronization. Through wavelet-basis regularization, our method sharpens signal without sharpening noise and can remarkably increase both the dynamic range and the temporal resolution of time-series data. Although applicable to any such data, we demonstrate the utility of our method by applying it to a recent cell-cycle transcription time course in the eukaryote Saccharomyces cerevisiae. Our method more sensitively detects cell-cycle-regulated transcription and reveals subtle timing differences that are masked in the original population measurements. Our algorithm also explicitly learns distinct transcription programs for mother and daughter cells, enabling us to identify 82 genes transcribed almost entirely in early G1 in a daughter-specific manner.

A microfluidic synchronizer for fission yeast cells

Among all the cell cycle synchronization technologies, the baby machine may be considered as the most artifact-free method. A baby machine incubates "mother cells" under normal conditions and collects their "babies", producing cell cultures that are similar not only in cell cycle phase but also in age. Unlike many other synchronization methods, no cell-cycle-blocking agent or metabolic stress is introduced in this method. Several macroscale and microfluidic baby machines have been developed for producing synchronized cell colonies. However, for rod-shaped cells like fission yeast (Schizosaccharomyces pombe), it is still a challenge to immobilize only the mother cells in a microfluidic device. Here we presented a new baby machine suitable for fission yeast. The device is fixed one end of the cell and releases the free-end daughter cell every time the cell finishes cytokinesis. A variety of structures for cell immobilization were attempted to find the optimal design. For the convenience of collection and further assay, we integrated into our baby machine chip a cell screener, which exploited the deformation of polymer material to switch between opening and closing states. Synchronous populations of fission yeast cells were produced with this device, its working detail was analyzed and performance was evaluated. The device provides a new on-chip tool for cell biology studies.

X-Ray Sensitivity during the Cell Generation Cycle of Cultured Chinese Hamster Cells1

Our earlier reports (1–3) have provided a general, qualitative description of the X-ray sensitivity of Chinese hamster cells as a function of their age within the generation cycle. With a partially synchronized cell population and colony survival used as an assay, it was established that cells were least sensitive to X-rays in the latter part of the DNA synthetic period (S period) and appeared to be about equally sensitive in the pre-DNA synthetic period (G1) and post-DNA synthetic period (G2). The variations in sensitivity throughout the cycle seemed mainly but not exclusively due to variations in the threshold or shoulder of the survival curve. We were, however, aware of the fact that even a few resistant S cells present during stages we had designated as G1 or G2 could significantly alter the survival curve for G1 and G2 cells. Such S cells may be present not only because of inadequacies in the synchronization procedure but also because of differences in the rate of progression of cells through the cycle which tends to desynchronize the population. Even if the number of S cells present were known, their exact contribution to the survival curve would be difficult to estimate, especially if the sensitivity of S cells varied throughout the S period. To determine X-ray sensitivity more accurately at all stages, we have used lethal amounts of tritiated thymidine (HTdR) to inactivate the S cells. Furthermore, since the previous technique could not be used to obtain the sensitivity of mitotic (M) cells, we have modified our procedure so that this may be accomplished. Finally, in view of the possible association between X-ray resistance and DNA synthesis, it was important to find out whether cells in S had a constant or variable sensitivity throughout the synthetic period. MATERIALS AND METHODS