Synchronization of Mycobacterium life cycle: A possible novel mechanism of antimycobacterial drug resistance evolution and its manipulation.

Mycobacterium Tuberculosis (Mtb) causing Tuberculosis (TB) is a widespread disease infecting millions of people worldwide. Additionally, emergence of drug resistant tuberculosis is a major challenge and concern in high TB burden countries. Most of the drug resistance in mycobacteria is attributed to developing acquired resistance due to spontaneous mutations or intrinsic resistance mechanisms. In this review, we emphasize on the role of bacterial cell cycle synchronization as one of the intrinsic mechanisms used by the bacteria to cope with stress response and perhaps involved in evolution of its drug resistance. The importance of cell cycle synchronization and its function in drug resistance in cancer cells, malarial and viral pathogens is well understood, but its role in bacterial pathogens has yet to be established. From the extensive literature survey, we could collect information regarding how mycobacteria use synchronization to overcome the stress response. Additionally, it has been observed that most of the microbial pathogens including mycobacteria are responsive to drugs predominantly in their logarithmic phase, while they show resistance to antibiotics when they are in the lag or stationary phase. Therefore, we speculate that Mtb might use this novel strategy wherein they regulate their cell cycle upon antibiotic pressure such that they either enter in their low metabolic phase i.e., either the lag or stationary phase to overcome the antibiotic pressure and function as persister cells. Thus, we propose that manipulating the mycobacterial drug resistance could be possible by fine-tuning its cell cycle.

Modulation of FDG Uptake by Cell Cycle Synchronization Using a T-Type Calcium Channel Inhibitor.

BACKGROUND: We investigated whether cell cycle synchronization induced by the T-type calcium channel inhibitor mibefradil could increase tumoral 2-[18F] fluoro-2-deoxy-d-glucose (FDG) uptake in vitro and in vivo. METHODS: Human prostate cancer cells (PC-3) were treated with 10 µM mibefradil for 24, 48, and 72 h to induce G1 arrest. Cell cycle distribution was analyzed at 0, 4, 8, 12, 15, 18, and 24 h after mibefradil withdrawal. Cellular uptake was measured after incubating cells with [3H] Deoxy-d-Glucose (DDG) for 1 h at the same time points used in the cell cycle analysis. The correlation between [3H] DDG uptake and each cell cycle phase was evaluated in the early (0-12 h) and late phases (15-24 h) of synchronization. In vivo FDG PET imaging was performed in PC-3-bearing mice at baseline, 24 h, and 48 h after mibefradil treatment. RESULTS: The G0/G1 fraction of PC-3 cells was significantly increased from 33.1% ± 0.2% to 60.9% ± 0.8% after 24 h mibefradil treatment, whereas the S and G2/M fractions were decreased from 36.3% ± 1.4% to 23.2% ± 1.1% and from 29.7% ± 1.3% to 14.9% ± 0.9%, respectively, which were similar to the results by serum starvation. Mibefradil treatment for 24, 48, and 72 h increased the number of cells in S phase at 18-24 h after withdrawal; however, only the 72 h treatment increased [3H] DDG uptake (145.8 ± 5.8% of control at 24 h after withdrawal). [3H] DDG uptake was positively correlated with the size of the S phase fraction and negatively correlated with the size of the G0/G1 fraction in the late phase of synchronization. DDG uptake was significantly increased by mibefradil-induced cell cycle synchronization and correlated with the sizes of cell cycle fractions. In vivo FDG PET imaging also demonstrated a significant increase in tumor uptake after mibefradil treatment. Quantified tumor FDG uptake (%ID/g) increased from 4.13 ± 2.10 to 4.7 ± 2.16 at 24 h, and 5.95 ± 2.57 at 48 h (p < 0.05). CONCLUSION: Cell cycle synchronization could be used to increase the diagnostic sensitivity of clinical FDG positron emission tomography.

Cell Cycle Regulation by NF-YC in Drosophila Eye Imaginal Disc: Implications for Synchronization in the Non-Proliferative Region.

Cell cycle progression during development is meticulously coordinated with differentiation. This is particularly evident in the Drosophila 3rd instar eye imaginal disc, where the cell cycle is synchronized and arrests at the G1 phase in the non-proliferative region (NPR), setting the stage for photoreceptor cell differentiation. Here, we identify the transcription factor Nuclear Factor-YC (NF-YC) as a crucial player in this finely tuned progression, elucidating its specific role in the synchronized movement of the morphogenetic furrow. Depletion of NF-YC leads to extended expression of Cyclin A (CycA) and Cyclin B (CycB) from the FMW to the NPR. Notably, NF-YC knockdown resulted in decreased expression of Eyes absent (Eya) but did not affect Decapentaplegic (Dpp) and Hedgehog (Hh). Our findings highlight the role of NF-YC in restricting the expression of CycA and CycB in the NPR, thereby facilitating cell-cycle synchronization. Moreover, we identify the transcriptional cofactor Eya as a downstream target of NF-YC, revealing a new regulatory pathway in Drosophila eye development. This study expands our understanding of NF-YC's role from cell cycle control to encompass developmental processes.

Can roscovitine and trichostatin A be alternatives to standard protocols for cell cycle synchronization of ovine adult and foetal fibroblast cells?

Synchronization of donor cells is an important step for the success of somatic cell nuclear transfer application and facilitates the development of embryos. Contact inhibition, serum starvation and different chemical agents are used in synchronizing different types of somatic cells. In this study, to synchronize the primary ovine adult (POF) and foetal (POFF) fibroblast cells to G0/G1 phases, the contact inhibition, the serum starvation, roscovitine and trichostatin A (TSA) methods were used. In the first part of the study, roscovitine (10, 15, 20 and 30 µM) and TSA (25, 50, 75 and 100 nM) were applied for 24 h to determine the optimal concentration for POF and POFF cells. In the second part, optimal concentrations of roscovitine and TSA for these cells were compared with contact inhibition and serum starvation methods. Cell cycle distribution and apoptotic activity analysis were performed by flow cytometry to compare this synchronization methods. Serum starvation method resulted in higher cell synchronization rate in both cells compared to other groups. Although contact inhibition and TSA also achieved high success rates of synchronized cell value, it was observed that the difference between serum starvation and these groups was significant (p < .05). When the apoptosis rates of the two cell types were examined, it was observed that the early apoptotic cells in contact inhibition and late apoptotic cells in the serum starvation were higher than the other groups (p < .05). Although the 10 and 15 µM concentrations of roscovitine gave the lowest apoptosis rates, it was observed that it failed to synchronize both the ovine fibroblast cells to G0/G1 phase. As a result, it was concluded that while roscovitine was not successful to synchronize both the POFF and POF cell lines, TSA (50 nM for POF cells and 100 nM for POFF cells) can be used efficiently as an alternative to the contact inhibition and the serum starvation methods.

Full confluency, serum starvation, and roscovitine for inducing arrest in the G0/G1 phase of the cell cycle in puma skin-derived fibroblast lines.

The puma population is constantly decreasing, and cloning by somatic cell nuclear transfer can be used to conserve the species. One of the factors determining the success of the development of cloned embryos is the cell cycle stage of the donor cells. We evaluated the effects of full confluency (~100%), serum starvation (0.5% serum), and roscovitine (15 µM) treatments on the cell cycle synchronization in G0/G1 of puma skin-derived fibroblasts by flow cytometric analysis. Also, we assessed the effects of these synchronization methods on morphology, viability, and apoptosis levels using microscopy tools. The results showed that culturing the cells to confluence for 24 h (84.0%), 48 h (84.6%), and 72 h (84.2%) and serum starvation for 96 h (85.4%) yielded a significantly higher percentage of cells arrested in the G0/G1 (P 0.05) phase than cells not subjected to any cell cycle synchronization method (73.9%). Nevertheless, while serum starvation reduced the percentage of viable cells, no difference was observed for the full confluence and roscovitine treatments (P 0.05). Moreover, roscovitine for 12 h (78.6%) and 24 h (82.1%) was unable to synchronize cells in G0/G1 (P 0.05). In summary, full confluency induces puma fibroblast cell cycle synchronization at the G0/G1 stage without affecting cell viability. These outcomes may be valuable for planning donor cells for somatic cell nuclear transfer in pumas.

In Vitro Methods to Investigate the Disassembly of the Golgi Ribbon During the G2-M Transition of the Cell Cycle.

The Golgi complex is the central hub of the secretory pathway. In mammalian cells, it is formed by stacks of flattened cisternae organized in a continuous membrane system, the Golgi ribbon, located near the centrosome. During G2, the Golgi ribbon is disassembled into isolated stacks that, at the onset of mitosis, are further fragmented into small tubular-vesicular clusters that disperse throughout the cytoplasm. Here, we describe a set of methods to study the Golgi complex in different phases of the cell cycle, drawing attention to reproducing the mitotic Golgi fragmentation to gain knowledge and acquire the skills to study the mechanisms that regulate mitotic Golgi reorganization as well as its biological significance. The investigations based on these assays have been instrumental in understanding that Golgi disassembly is not only a consequence of mitosis but is also required for mitotic entry and cell division.

Comprehensive mapping of alternative polyadenylation site usage and its dynamics at single-cell resolution.

Alternative polyadenylation (APA) plays an important role in posttranscriptional gene regulation such as transcript stability and translation efficiency. However, our knowledge about APA dynamics at the single-cell level is largely unexplored. Here, we developed single-cell polyadenylation sequencing, a strand-specific approach for sequencing the 3' end of transcripts, to investigate the landscape of APA at the single-cell level. By analyzing several cell lines, we found many genes using multiple polyA sites in bulk data are prone to use only one polyA site in each single cell. Interestingly, cell cycle genes were significantly enriched in genes with high variation in polyA site usages. Furthermore, the 414 genes showing a polyA site usage switch after cell synchronization enriched cell cycle genes, while the differentially expressed genes after cell synchronization did not enrich cell cycle genes. We further identified 812 genes showing polyA site usage changes between neighboring cell cycles, which were grouped into six clusters, with cell phase-specific functional categories enriched in each cluster. Deletion of one polyA site in MSL1 and SCCPDH results in slower and faster cell cycle progression, respectively, supporting polyA site usage switch played an important role in cell cycle. These results indicate that APA is an important layer for cell cycle regulation.

Cell Synchronization Techniques for Studying Mitosis.

Cell synchronization allows the examination of cell cycle progression. Nocodazole and other microtubule poisons have been used extensively to interfere with microtubule function and arrest cells in mitosis. Since microtubules are important for many cellular functions, alternative cell cycle synchronization techniques independent of microtubule inhibition are also used for synchronizing cells in mitosis. Here we describe using nocodazole, STLC, and combining thymidine block with MG132 to synchronize cells in mitosis. These inhibitors are reversible and mitotic cells can be released into the G1 phase synchronously. These techniques can be applied to both Western blot and timelapse imaging to study mitotic progression.

Synchronization of HeLa Cells to Various Interphases Including G1, S, and G2 Phases.

The typical cell cycle in eukaryotes is composed of four phases including the G1, S, G2, and M phases. G1, S, and G2 together are called interphase. Cell synchronization is a process that brings cultured cells at different stages of the cell cycle to the same phase, which allows the study of phase-specific cellular events. While interphase cells can be easily distinguished from mitotic cells by examining their chromosome morphology, it is much more difficult to separate and distinguish the interphases from each other. Here, we describe drug-derived protocols for synchronizing HeLa cells to various interphases of the cell cycle: G1 phase, S phase, and G2 phase. G1 phase synchronization is achieved through serum starvation, S phase synchronization is achieved through a double thymidine block, and G2 phase synchronization is achieved through the release of the double thymidine block followed by roscovitine treatment. Successful synchronization can be assessed using flow cytometry to examine the DNA content and Western blot to examine the expression of various cyclins.

Synchronization of Leishmania amazonensis Cell Cycle Using Hydroxyurea.

Leishmania spp. comprises a group of protozoan parasites that affect millions of people around the world. Understanding the main cell cycle-dependent events could provide an important route for developing specific therapies since some factors involved in cell cycle control may have low similarity relative to their homologs in mammals. Furthermore, accurate cell cycle-dependent analyses often require many cells, which can be achieved through cell cycle synchronization. Here, we described a useful method to synchronize procyclic promastigote forms of Leishmania amazonensis using hydroxyurea (HU) and the analysis of its DNA content profile. This approach can be extended to other trypanosomatids, such as Trypanosoma cruzi or Trypanosoma brucei, and provides an effective method for arresting more than 80% of cells at the G1/S phase transition.

Cell Cycle Synchronization and Time-Lapse Imaging of Cytokinetic Tobacco BY-2 Cells.

Transgenic tobacco BY-2 cell lines stably expressing fluorescent protein-tagged marker proteins have been used to visualize the dynamic behaviors of cytoskeletons and organelles during plant cell division. Using time-lapse confocal imaging, we recently revealed that the pharmacological disruption of actin filaments results in the abnormal organization of phragmoplast microtubules during the early phase of cytokinesis in cell cycle-synchronized BY-2 cells. Additionally, disrupting the actin filaments shortens the time from cell plate emergence to the accumulation of green fluorescent protein-tagged NACK1 kinesin on the cell plate, suggesting that there are two functionally diverse types of microtubules in the phragmoplast. We herein describe a protocol for the cell cycle synchronization of BY-2 cells and the time-lapse confocal imaging of cytokinesis combined with a treatment with an actin polymerization inhibitor and the visualization of an emerging cell plate with a vital stain. This protocol is useful for examining the dynamic changes in protein localization or the intracellular architecture and the effects of actin disruption during plant cell division.

Chromosome analysis and sorting.

Flow cytometric analysis and sorting of plant mitotic chromosomes has been mastered by only a few laboratories worldwide. Yet, it has been contributing significantly to progress in plant genetics, including the production of genome assemblies and the cloning of important genes. The dissection of complex genomes by flow sorting into the individual chromosomes that represent small parts of the genome reduces DNA sample complexity and streamlines projects relying on molecular and genomic techniques. Whereas flow cytometric analysis, that is, chromosome classification according to fluorescence and light scatter properties, is an integral part of any chromosome sorting project, it has rarely been used on its own due to lower resolution and sensitivity as compared to other cytogenetic methods. To perform chromosome analysis and sorting, commercially available electrostatic droplet sorters are suitable. However, in order to resolve and purify chromosomes of interest the instrument must offer high resolution of optical signals as well as stability during long runs. The challenge is thus not the instrumentation, but the adequate sample preparation. The sample must be a suspension of intact mitotic metaphase chromosomes and the protocol, which includes the induction of cell cycle synchrony, accumulation of dividing cells at metaphase, and release of undamaged chromosomes, is time consuming and laborious and needs to be performed very carefully. Moreover, in addition to fluorescent staining chromosomal DNA, the protocol may include specific labelling of DNA repeats to facilitate discrimination of particular chromosomes. This review introduces the applications of chromosome sorting in plants, and discusses in detail sample preparation, chromosome analysis and sorting to achieve the highest purity in flow-sorted fractions, and their suitability for downstream applications.

Effect of cell cycle synchronization on cadmium-induced apoptosis and necrosis in NRK-52E cells.

Heavy metal pollution is a problem that cannot be ignored. Due to the prevalence of cadmium in the environment and its harmful effects on humans, cadmium pollution has become a research hotspot recently. The mechanism of cadmium-induced toxicity has also drawn much attention and most studies have been conducted using whole cells, but the toxicological mechanism of cadmium remains unclear. In this study, we aimed to obtain NRK-52E cells at different growth stages by various methods and analyze the differences in cadmium toxicity. The results show that the cadmium sensitivity of cells in each phase was different and the late apoptotic rate was increased significantly after 5 µM Cd treatment. In addition, cadmium easily induces apoptosis of G0- and S-phase cells, as well as necrosis of S- and M-phase cells, but has no significant effect on G1-phase cells. Overall, we first explored the differences in the effects of cadmium on NRK-52E cells at various growth phases. Besides, the findings of this study might provide a theoretical basis for further exploration of the toxicological mechanism of cadmium.Abbreviations Cd: cadmium; CDK: cyclin-dependent kinases; DAPI 2-(4-amidinophenyl)-1H-indole-6-carboxamidine; TBST: Tris-buffered saline with Tween-20; PI: propidium iodide; DMEM: Dulbecco's Modified Eagle Medium; BCA: bicinchoninic acid.

Release from cell cycle arrest with Cdk4/6 inhibitors generates highly synchronized cell cycle progression in human cell culture.

Each approach used to synchronize cell cycle progression of human cell lines presents a unique set of challenges. Induction synchrony with agents that transiently block progression through key cell cycle stages are popular, but change stoichiometries of cell cycle regulators, invoke compensatory changes in growth rate and, for DNA replication inhibitors, damage DNA. The production, replacement or manipulation of a target molecule must be exceptionally rapid if the interpretation of phenotypes in the cycle under study is to remain independent of impacts upon progression through the preceding cycle. We show how these challenges are avoided by exploiting the ability of the Cdk4/6 inhibitors, palbociclib, ribociclib and abemaciclib to arrest cell cycle progression at the natural control point for cell cycle commitment: the restriction point. After previous work found no change in the coupling of growth and division during recovery from CDK4/6 inhibition, we find high degrees of synchrony in cell cycle progression. Although we validate CDK4/6 induction synchronization with hTERT-RPE-1, A549, THP1 and H1299, it is effective in other lines and avoids the DNA damage that accompanies synchronization by thymidine block/release. Competence to return to cycle after 72 h arrest enables out of cycle target induction/manipulation, without impacting upon preceding cycles.

Cell-Cycle-Dependent Regulation of Translation: New Interpretations of Old Observations in Light of New Approaches.

It 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. Cell-cycle-dependent variations are most conveniently studied in synchronized cells, but the synchronization methods by themselves often evoke stress responses that, in turn, affect translation rates. Further, it is argued that previously reported cell-cycle-dependent changes in the global translation rate to a large extent reflect responses to the synchronization methods. Recent findings strongly suggest that the global translation rate is not regulated in a cell-cycle-dependent manner. Novel techniques allowing a genome-wide analysis of translational profiles suggest that the extent and importance of selective translational regulation associated with cell-cycle transitions have been underestimated. Therefore, the main question is which messenger RNAs (mRNAs) are translated, rather than whether the global translation rate is decreased.

Preparation of Cell Cycle-Synchronized Saccharomyces cerevisiae Cells for Hi-C.

The chromosome organization activities of SMC complexes are crucial for correct gene expression and genetic inheritance in cells. Hi-C assays have revealed previously unsuspected levels of chromosome structure, with different types of chromosome structure facilitating function at different stages of the cell cycle. Elucidating how SMC complexes regulate these distinct types of organization is currently a key question in molecular biology.The range of genetic tools and the small genome size of the budding yeast Saccharomyces cerevisiae make it an ideal tool for studying how SMC complexes control chromosome structure in eukaryotic cells. A crucial advantage of S. cerevisiae over other systems is that large populations of cells can be easily arrested at distinct stages of the cell cycle and SMC gene function specifically ablated in the synchronized cells. Here we describe methods to prepare synchronously cell cycle-arrested populations of genetically modified S. cerevisiae cells for Hi-C analysis.

Cell-cycle-dependences of membrane permeability and viability observed for HeLa cells undergoing multi-bubble-cell interactions.

Microbubble-mediated sonoporation is a promising strategy for intracellular gene/drug delivery, but the biophysical mechanisms involved in the interactions between microbubbles and cells are not well understood. Here, HeLa cells were synchronized in individual cycle phases, then the cell-cycle-dependences of the membrane permeability and viability of HeLa cells undergoing multi-bubble sonoporation were evaluated using focused ultrasound exposure apparatus coupled passive cavitation detection system. The results indicated that: (1) the microbubble cavitation activity should be independent on cell cycle phases; (2) G1-phase cells with the largest Young's modulus were the most robust against microbubble-mediated sonoporation; (3) G2/M-phase cells exhibited the greatest accumulated FITC uptake with the lowest viability, which should be mainly attributed to the chemical effect of synchronization drugs; and (4) more important, S-phase cells with the lowest stiffness seemed to be the most susceptible to the mechanical effect generated by microbubble cavitation activity, which resulted in the greatest enhancement in sonoporation-facilitated membrane permeabilization without further scarifying their viability. The current findings may benefit ongoing efforts aiming to pursue rational utilization of microbubble-mediated sonoporation in cell-cycle-targeted gene/drug delivery for cancer therapy.

Method to Synchronize Cell Cycle of Human Pluripotent Stem Cells without Affecting Their Fundamental Characteristics.

Cell cycle progression and cell fate decisions are closely linked in human pluripotent stem cells (hPSCs). However, the study of these interplays at the molecular level remains challenging due to the lack of efficient methods allowing cell cycle synchronization of large quantities of cells. Here, we screened inhibitors of cell cycle progression and identified nocodazole as the most efficient small molecule to synchronize hPSCs in the G2/M phase. Following nocodazole treatment, hPSCs remain pluripotent, retain a normal karyotype and can successfully differentiate into the three germ layers and functional cell types. Moreover, genome-wide transcriptomic analyses on single cells synchronized for their cell cycle and differentiated toward the endoderm lineage validated our findings and showed that nocodazole treatment has no effect on gene expression during the differentiation process. Thus, our synchronization method provides a robust approach to study cell cycle mechanisms in hPSCs.

S-phase Synchronization Facilitates the Early Progression of Induced-Cardiomyocyte Reprogramming through Enhanced Cell-Cycle Exit.

Direct reprogramming of fibroblasts into induced cardiomyocytes (iCMs) holds a great promise for regenerative medicine and has been studied in several major directions. However, cell-cycle regulation, a fundamental biological process, has not been investigated during iCM-reprogramming. Here, our time-lapse imaging on iCMs, reprogrammed by Gata4, Mef2c, and Tbx5 (GMT) monocistronic retroviruses, revealed that iCM-reprogramming was majorly initiated at late-G1- or S-phase and nearly half of GMT-reprogrammed iCMs divided soon after reprogramming. iCMs exited cell cycle along the process of reprogramming with decreased percentage of 5-ethynyl-20-deoxyuridine (EdU)⁺/α-myosin heavy chain (αMHC)-GFP⁺ cells. S-phase synchronization post-GMT-infection could enhance cell-cycle exit of reprogrammed iCMs and yield more GFPhigh iCMs, which achieved an advanced reprogramming with more expression of cardiac genes than GFPlow cells. However, S-phase synchronization did not enhance the reprogramming with a polycistronic-viral vector, in which cell-cycle exit had been accelerated. In conclusion, post-infection synchronization of S-phase facilitated the early progression of GMT-reprogramming through a mechanism of enhanced cell-cycle exit.

Effects of Cell Cycle Regulators on the Cell Cycle Synchronization of Porcine induced Pluripotent Stem Cells.

Unlike mouse results, cloning efficiency of nuclear transfer from porcine induced pluripotent stem cells (piPSCs) is very low. The present study was performed to investigate the effect of cell cycle inhibitors on the cell cycle synchronization of piPSCs. piPSCs were generated using combination of six human transcriptional factors under stem cell culture condition. To examine the efficiency of cell cycle synchronization, piPSCs were cultured on a matrigel coated plate with stem cell media and they were treated with staurosporine (STA, 20 nM), daidzein (DAI, 100 µM), roscovitine (ROSC, 10 µM), or olomoucine (OLO, 200 µM) for 12 h. Flow Cytometry (FACs) data showed that piPSCs in control were in G1 (37.5±0.2%), S (34.0±0.6%) and G2/M (28.5±0.4%). The proportion of cells at G1 in DAI group was significantly higher than that in control, while STA, ROSC and OLO treatments could not block the cell cycle of piPSCs. Both of viability and apoptosis were affected by STA and ROSC treatment, but there were no significantly differences between control and DAI groups. Real-Time qPCR and FACs results revealed that DAI treatment did not affect the expression of pluripotent gene, Oct4. In case of OLO, it did not affect both of viability and apoptosis, but Oct4 expression was significantly decreased. Our results suggest that DAI could be used for synchronizing piPSCs at G1 stage and has any deleterious effect on survival and pluripotency sustaining of piPSCs.