Division Delay Research Articles

Natural transformation and cell division delay in competent Staphylococcus aureus.

Genetic competence for natural transformation, considered one of the three main mechanisms leading to horizontal gene transfer in bacteria, is able to promote evolution, through genomic plasticity, and foster antibiotic resistance and virulence factors spreading. Conserved machinery and actors required to perform natural transformation have been shown to accumulate at different cellular localizations depending on the model organism considered. In this study, we investigate the transformation apparatus composition, localization, and dynamics in the human pathogen Staphylococcus aureus. We particularly show that most of the natural transformation actors co-localize in clusters. We also reveal that the localization of natural transformation proteins is dynamic, following the cell cycle. Ultimately, the natural transformation apparatus is preferentially established in the vicinity of the division septum. All these results demonstrate that DNA binding, uptake, and recombination are spatially and temporally coordinated to ensure S. aureus natural transformation. Finally, we hypothesize that S. aureus competent cells would initiate and then block cell division to ensure the success of natural transformation before the final constriction of the cytokinetic ring. IMPORTANCE Natural transformation, considered one of the three main mechanisms leading to horizontal gene transfer in bacteria, is able to promote genomic plasticity and foster antibiotic resistance spreading. Conserved machinery and actors required to perform natural transformation have been shown to accumulate at different cellular localizations depending on the model organism considered. Here, we show in the human pathogen Staphylococcus aureus that DNA binding, uptake, and recombination are spatially and temporally coordinated to ensure S. aureus natural transformation. We also reveal that localization of natural transformation proteins occurs in the vicinity of the division septum allowing S. aureus competent cells to block cell division to ensure the success of natural transformation before the final constriction of the cytokinetic ring.

Cell size homeostasis under the circadian regulation of cell division in cyanobacteria

Bacterial cells maintain their characteristic cell size over many generations. Several rod-shaped bacteria, such as Escherichia coli and the cyanobacteria Synechococcus elongatus, divide after adding a constant length to their length at birth. Through this division control known as the adder mechanism, perturbation in cell length due to physiological fluctuation decays over generations at a rate of 2−1 per cell division. However, previous experiments have shown that the circadian clock in cyanobacteria reduces cell division frequency at a specific time of day under constant light. This circadian gating should modulate the division control by the adder mechanism, but its significance remains unknown. Here we address how the circadian gating affects cell length, doubling time, and cell length stability in cyanobacteria by using mathematical models. We show that a cell subject to circadian gating grows for a long time, and gives birth to elongated daughter cells. These elongated daughter cells grow faster than the previous generation, as elongation speed is proportional to cell length and divide in a short time before the next gating. Hence, the distributions of doubling time and cell length become bimodal, as observed in experimental data. Interestingly, the average doubling time over the population of cells is independent of gating because the extension of doubling time by gating is compensated by its reduction in the subsequent generation. On the other hand, average cell length is increased by gating, suggesting that the circadian clock controls cell length. We then show that the decay rate of perturbation in cell length depends on the ratio of delay in division by the gating τG to the average doubling time τ0 as 2τG/τ0−1. We estimated τG≈2.5, τ0≈13.6 hours, and τG/τ0≈0.18 from experimental data, indicating that a long doubling time in cyanobacteria maintains the decay rate similar to that of the adder mechanism. Thus, our analysis suggests that the acquisition of the circadian clock during evolution did not impose a constraint on cell size homeostasis in cyanobacteria.

A Versatile Strategy to Coat Individual Cell with Fully/Partially Covered Shell for Preparation of Self-Propelling Living Cells.

Coating living cells with a functional shell has been regarded as an effective way to protect them against environmental stress, regulate their biological behaviors, or extend their functionalities. Here, we reported a facile method to prepare fully or partially coated shells on an individual yeast cell surface by visible light-induced graft polymerization. In this strategy, yeast cells that were surface-absorbed with polyethylenimine (PEI) were deposited on the negatively charged glass slide to form a single layer by electrostatic interaction. Then, surface-initiated graft polymerization of poly(ethylene glycol) diacrylate (PEGDA) on yeast cells under visible light irradiation was carried out to generate cross-linked shells on the cells. The process of surface modification had negligible influence on the viability of yeast cells due to the mild reaction condition. Additionally, compared to the native yeast cells, a 17.5 h of delay in division was observed when the graft polymerization was performed under 15 mW/cm2 irradiation for 30 min. Introducing artificial shell endowed yeast cells with significant resistance against lyticase, and the protection can be enhanced by increasing the thickness of shell. Moreover, the partially coated yeast cells would be prepared by simply adjusting the reaction condition such as irradiation density and time. By immobilizing urease on the functional patch, the asymmetrically modified yeast cells exhibited self-propelling capability, and the speed of directional movement reached 4 μm/s in the presence of 200 mM urea. This tunable coating individual cell strategy with varying functionality has great potential applications in fields of cell-based drug delivery, cell therapy, biocatalysis, and tissue engineering.

Changes in the Distribution of Cell Contacts and Mitotic Cycle Disturbances in Cells of the Allograft of Rat Embryonic Neocortex.

Morphological changes in the allograft of rat anterior cerebral vesicle at the early stages after transplantation into the peripheral nerve of an adult rat were studied by immunohistochemical methods. Immunohistochemical reaction to bromodeoxyuridine showed that the delay of mitotic division in neural stem/progenitor cells in the grafts occurred during S/G2 stage. In transplants of rat embryonic neocortex (E13), changes in the cell cycle of neural stem/progenitor cells in 3 h after transplantation into the nerve correlated with abnormal distribution of adherens junctions and interkinetic nuclear migration.

EzrA, a cell shape regulator contributing to biofilm formation and competitiveness in Streptococcus mutans

Bacterial cell division is initiated by tubulin homologue FtsZ that assembles into a ring structure at mid-cell to facilitate cytokinesis. EzrA has been identified to be implicated in FtsZ-ring dynamics and cell wall biosynthesis during cell division of Bacillus subtilis and Staphylococcus aureus, the model rod and cocci. However, its role in pathogenic streptococci remains largely unknown. Here, the role of EzrA was investigated in Streptococcus mutans, the primary etiological agent of human dental caries, by constructing an ezrA in-frame deletion mutant. Our data showed that the ezrA mutant was slow-growing with a shortened length and extended width round cell shape compared to the wild type, indicating a delay in cell division with abnormalities of peptidoglycan biosynthesis. Additionally, FtsZ irregularly localized in dividing ezrA mutant cells forming angled division planes, potentially contributing to an aberrant cell shape. Furthermore, investigation using single-species cariogenic biofilm model revealed that deletion of ezrA resulted in defective biofilm formation with less extracellular polysaccharides and altered three-dimensional biofilm architecture. Unexpectedly, in a dual-species ecological model, the ezrA mutant exhibited substantially lower tolerance for H2 O2 and reduced competitiveness against one commensal species, Streptococcus sanguinis. Taken together, these results demonstrate that EzrA plays a key role in regulating cell division and maintaining a normal morphology in S.mutans and is required for its robust biofilm formation/interspecies competition. Therefore, EzrA protein represents a potential therapeutic target in the development of drugs controlling dental caries and other biofilm-related diseases.

Prolonged blastomere movement induced by the delay of pronuclear fading and first cell division adversely affects pregnancy outcomes after fresh embryo transfer on Day 2: a time-lapse study.

What is the incidence, origin and clinical significance of blastomere movement after the first cell division in the human embryo? A total of 1096 embryos, cultured in the EmbryoScope+ ® time-lapse system and subjected to a single fresh cleaved embryo transfer, were retrospectively analysed. Type and duration of blastomere movement (dBMov) between the first (t2) and second cell division (t3) was monitored, and the ratio of dBMov during the 2-cell stage [dBMov/(t3-t2)] was calculated. Morphological evaluation of embryos was performed by referring to the size of the blastomere and fragmentation after first division in addition to Veeck's criteria on Day 2. The correlation between dBMov and ongoing pregnancy was evaluated and the association of dBMov with patient and embryonic characteristics was determined. Both movement type and the value of dBMov/(t3-t2) were significantly associated with asymmetrical first division, fragment formation and morphological grade on Day 2. Multivariate logistic regression analysis revealed that a higher value of dBMov/(t3-t2) significantly correlated with a decreased ongoing pregnancy rate, even after adjustment for co-founders (odds ratio 0.399, P = 0.0419). The time intervals of pronuclear (PN) alignment and PN fading were significantly correlated with the dBMov/(t3-t2) value. Embryos with extended blastomere movement after the first cell division, which is associated with the delay of PN fading and first cell division, have a lower competence to initiate an ongoing pregnancy after fresh embryo transfer on Day 2. Thus, blastomere movement could be a useful predictive parameter for selecting embryos at the early cleavage stage.

A programmed cell division delay preserves genome integrity during natural genetic transformation in Streptococcus pneumoniae

Competence for genetic transformation is a differentiation program during which exogenous DNA is imported into the cell and integrated into the chromosome. In Streptococcus pneumoniae, competence develops transiently and synchronously in all cells during exponential phase, and is accompanied by a pause in growth. Here, we reveal that this pause is linked to the cell cycle. At least two parallel pathways impair peptidoglycan synthesis in competent cells. Single-cell analyses demonstrate that ComM, a membrane protein induced during competence, inhibits both initiation of cell division and final constriction of the cytokinetic ring. Competence also interferes with the activity of the serine/threonine kinase StkP, the central regulator of pneumococcal cell division. We further present evidence that the ComM-mediated delay in division preserves genomic integrity during transformation. We propose that cell division arrest is programmed in competent pneumococcal cells to ensure that transformation is complete before resumption of cell division, to provide this pathogen with the maximum potential for genetic diversity and adaptation.

Multi-parameter calibration of streak/framing camera based on integrated optical system with delay and attenuation modules

We demonstrate a multi-parameter calibration method of the streak camera and the framing camera with the help of beam division delay and attenuation devices in an integrated optical system based on sub-picosecond short-pulse UV laser. In the designed optical scheme, the four operating modes employed the optical delay, beam division delay and attenuation, and etalon modules can be switched to synchronize the UV laser pulse with the trigger signal of diagnosis instruments, calibrate the temporal resolution and dynamic range of framing cameras, and correct the sweep speed of streak cameras, respectively. The results show that the delay time error of ±0.1ns in the optical delay module, the uniform distribution of sub-beams with the delay time precision of ±0.03ps and isocon descending energy in the beam division delay and attenuation modules, and pulse broadening of ∼22% in the whole system are achieved. In the meantime, the experiment results display the sweep speed of 1.2ps/pixel in a streak camera and the dynamic range of 131 in a framing camera. The proposed folding optical system with high-precision, low-loss and multi-function can be extensively applied in the temporal calibration of diagnosis instruments such as streak cameras and framing cameras.

Ploidy Manipulation of Zebrafish Embryos with Heat Shock 2 Treatment.

Manipulation of ploidy allows for useful transformations, such as diploids to tetraploids, or haploids to diploids. In the zebrafish Danio rerio, specifically the generation of homozygous gynogenetic diploids is useful in genetic analysis because it allows the direct production of homozygotes from a single heterozygous mother. This article describes a modified protocol for ploidy duplication based on a heat pulse during the first cell cycle, Heat Shock 2 (HS2). Through inhibition of centriole duplication, this method results in a precise cell division stall during the second cell cycle. The precise one-cycle division stall, coupled to unaffected DNA duplication, results in whole genome duplication. Protocols associated with this method include egg and sperm collection, UV treatment of sperm, in vitro fertilization and heat pulse to cause a one-cell cycle division delay and ploidy duplication. A modified version of this protocol could be applied to induce ploidy changes in other animal species.

Casiopeina III-Ea, a copper-containing small molecule, inhibits the in vitro growth of primitive hematopoietic cells from chronic myeloid leukemia

Several novel compounds have been developed for the treatment of different types of leukemia. In the present study, we have assessed the in vitro effects of Casiopeina III-Ea, a copper-containing small molecule, on cells from patients with Chronic Myeloid Leukemia (CML). We included primary CD34+ Lineage-negative (Lin−) cells selected from CML bone marrow, as well as the K562 and MEG01 cell lines. Bone marrow cells obtained from normal individuals – both total mononuclear cells as well as CD34+ Lin− cells- were used as controls. IC50 corresponded to 0.5μM for K562 cells, 0.63μM for MEG01 cells, 0.38μM for CML CD34+ lin− cells, and 1.0μM for normal CD34+ lin− cells. Proliferation and expansion were also inhibited to significantly higher extents in cultures of CML cells as compared to their normal counterparts. All these effects seemed to occur via a bcr-abl transcription-independent mechanism that involved a delay in cell division, an increase in cell death, generation of Reactive Oxygen Species and changes in cell cycle. Our results demonstrate that Casiopeina III-Ea possesses strong antileukemic activity in vitro, and warrant further preclinical (animal) studies to assess such effects in vivo.

Timing of Tissue-specific Cell Division Requires a Differential Onset of Zygotic Transcription during Metazoan Embryogenesis

Metazoan development demands not only precise cell fate differentiation but also accurate timing of cell division to ensure proper development. How cell divisions are temporally coordinated during development is poorly understood. Caenorhabditis elegans embryogenesis provides an excellent opportunity to study this coordination due to its invariant development and widespread division asynchronies. One of the most pronounced asynchronies is a significant delay of cell division in two endoderm progenitor cells, Ea and Ep, hereafter referred to as E2, relative to its cousins that mainly develop into mesoderm organs and tissues. To unravel the genetic control over the endoderm-specific E2 division timing, a total of 822 essential and conserved genes were knocked down using RNAi followed by quantification of cell cycle lengths using in toto imaging of C. elegans embryogenesis and automated lineage. Intriguingly, knockdown of numerous genes encoding the components of general transcription pathway or its regulatory factors leads to a significant reduction in the E2 cell cycle length but an increase in cell cycle length of the remaining cells, indicating a differential requirement of transcription for division timing between the two. Analysis of lineage-specific RNA-seq data demonstrates an earlier onset of transcription in endoderm than in other germ layers, the timing of which coincides with the birth of E2, supporting the notion that the endoderm-specific delay in E2 division timing demands robust zygotic transcription. The reduction in E2 cell cycle length is frequently associated with cell migration defect and gastrulation failure. The results suggest that a tissue-specific transcriptional activation is required to coordinate fate differentiation, division timing, and cell migration to ensure proper development.

Dynamically-enhanced retention of gold nanoclusters in HeLa cells following X-rays exposure: A cell cycle phase-dependent targeting approach

Background and purposeCell cycle phase could affect the cellular uptake of nanoparticles. Based on the fact that ionizing radiation exposure can delay cell cycle progression including inducing G2/M phase arrest, we propose that ionizing radiation exposure is a cell cycle phase-dependent targeting approach for intracellular delivery of nano-agents in tumor cells. Materials and methodsWe synthesized luminescent gold nanoclusters (AuNCs) using a one-pot green synthetic method. Subsequently, we used the as-prepared AuNCs as both “nano-agents” and fluorescent trafficking probes for our study using human cervical carcinoma HeLa cells. Estimating the cellular uptake of AuNCs and cell cycle analysis were performed following X-rays irradiation and cell synchronization. ResultsOur work showed that X-rays irradiation could delay the division of HeLa cells and thereby enhance the retention of AuNCs in HeLa cells, which is a reverse strategy compared with other studies on synergistic nano-radiotherapy. Our results demonstrated that the cell cycle synchronization influenced the cellular uptake processes of AuNCs, suggesting that dynamic cell cycle progression could affect the cellular uptake kinetics of AuNCs. ConclusionWe consider that the radiation-induced cell division delay might provide a possible mechanism underlying the enhanced effect for the cellular uptake of AuNCs in irradiated HeLa cells.

Glutathione reductase gsr-1 is an essential gene required for Caenorhabditis elegans early embryonic development

Glutathione is the most abundant thiol in the vast majority of organisms and is maintained in its reduced form by the flavoenzyme glutathione reductase. In this work, we describe the genetic and functional analysis of the Caenorhabditis elegans gsr-1 gene that encodes the only glutathione reductase protein in this model organism. By using green fluorescent protein reporters we demonstrate that gsr-1 produces two GSR-1 isoforms, one located in the cytoplasm and one in the mitochondria. gsr-1 loss of function mutants display a fully penetrant embryonic lethal phenotype characterized by a progressive and robust cell division delay accompanied by an aberrant distribution of interphasic chromatin in the periphery of the cell nucleus. Maternally expressed GSR-1 is sufficient to support embryonic development but these animals are short-lived, sensitized to chemical stress, have increased mitochondrial fragmentation and lower mitochondrial DNA content. Furthermore, the embryonic lethality of gsr-1 worms is prevented by restoring GSR-1 activity in the cytoplasm but not in mitochondria. Given the fact that the thioredoxin redox systems are dispensable in C. elegans, our data support a prominent role of the glutathione reductase/glutathione pathway in maintaining redox homeostasis in the nematode.

Application of cell sorting for enhancing the performance of the cytokinesis-block micronucleus assay

Among the numerous methods available to assess genotoxicity, the cytokinesis-block micronucleus (CBMN) assay is very popular due its relative simplicity and power to detect both clastogenic and aneugenic compounds. A problem with the CBMN assay is that all DNA damaging agents also inhibit the ability of cells to progress through mitosis, leading to a low number of binucleated cells (BNCs). One method to resolve this issue is to ensure a sufficient proportion of BNCs in the samples. In the current study, the applicability of a cell sorting system capable of isolating cell fractions containing abundant BNCs was investigated. Furthermore, to investigate the relationship between the cell division delay due to radiation exposure and the generation of BNCs and micronuclei (MN), we assessed a series of lag times between radiation exposure and addition of cytochalasin-B (Cyt-B). Cells from the human chronic myelogenous leukemia cell line K562 were exposed to X-rays (2 Gy and 4 Gy), and Cyt-B was subsequently added at 0, 6 and 12 h following irradiation. After treatment with Cyt-B for 24 h, the percentage of BNCs, the MN frequency and the cell cycle distribution were analyzed. In addition, cells displaying the DNA contents corresponding to BNCs were isolated and analyzed. The results indicate that applying the cell sorter to the CBMN assay increased the percentage of BNCs compared with the standard method. Thus, this technique is a promising way of enhancing the capacity of the CBMN assay.

High levels of inorganic nutrients affect fertilization kinetics, early development and settlement of the scleractinian coral Platygyra acuta

Dose–response experiments were conducted to investigate the effects of ammonia nitrogen (NH3/NH4+) and orthophosphate (PO43−) on four stages of larval development in Platygyra acuta, including fertilization, embryonic development and the survival, motility, and settlement of planula larvae. Fertilization success was reduced significantly under 200 μM NH3/NH4+ or PO43−. These high doses of NH3/NH4+ and PO4− affected egg viability (or sperm viability and polyspermic block simultaneously) and polyspermic block, respectively. These results provide the first evidence to indicate the mechanisms of how inorganic nutrients might affect coral fertilization processes. For embryonic development, NH3/NH4+ at 25–200 μM caused delay in cell division after 2-h exposure and NH3/NH4+ at 100–200 μM resulted in larval death after 72 h. However, no significant differences were observed in the mobility and survivorship of either planula or competent larvae under different levels of NH3/NH4+ or PO43−. There was a significant (~30 %) drop in the settlement of competent larvae under the combined effect of 100 μM NH3/NH4+ and PO43−. The effects of elevated nutrients appeared to become more significant only on gametes or larvae undergoing active cellular activities at fertilization, early development, and settlement.

Stella preserves maternal chromosome integrity by inhibiting 5hmC-induced γH2AX accumulation.

In the mouse zygote, Stella/PGC7 protects 5-methylcytosine (5mC) of the maternal genome from Tet3-mediated oxidation to 5-hydroxymethylcytosine (5hmC). Although ablation of Stella causes early embryonic lethality, the underlying molecular mechanisms remain unknown. In this study, we report impaired DNA replication and abnormal chromosome segregation (ACS) of maternal chromosomes in Stella-null embryos. In addition, phosphorylation of H2AX (γH2AX), which has been reported to inhibit DNA replication, accumulates in the maternal chromatin of Stella-null zygotes in a Tet3-dependent manner. Cell culture assays verified that ectopic appearance of 5hmC induces abnormal accumulation of γH2AX and subsequent growth retardation. Thus, Stella protects maternal chromosomes from aberrant epigenetic modifications to ensure early embryogenesis.

Induction of sister chromatid exchanges and cell division delays by clomiphene citrate in human lymphocytes.

Clomiphene citrate (CC) is a selective estrogen receptor modulator and is used for the treatment of in vitro fertilization, intracytoplasmic sperm injection, intrauterine insemination, and so on. In this study, sister chromatid exchanges (SCEs) and cell cycle delays were analyzed to investigate genotoxicity and cytotoxicity of CC in peripheral blood lymphocytes of healthy women. Human peripheral blood lymphocytes obtained from two donors were used to detect genotoxicity and cytotoxicity of CC. Lymphocytes were treated with various concentrations (0.40, 0.80, 1.60, and 3.20 µg/ml) of CC. A negative (distilled water) and a positive control (mitomycin-C = 0.20 µg/ml) were also used simultaneously with test substance-treated cultures. SCEs and cell division delays were measured from 25 cells and 100 cells perdonor, respectively. CC significantly increased the mean SCE value at all concentrations compared with the negative control. This increase was found to be dose dependent (r = 0.83) and at the highest concentration, nearly two times higher increase was observed than the negative control. However, replication index was not affected by the CC treatment. The present study shows that CC is genotoxic for human lymphocytes in vitro. Further investigations, especially in vivo are now needed in different test organisms to clarify the genotoxic activity of CC, which should also help to better understand genotoxic mechanism of this ovulation-stimulating drug.

Damage to mitotic telomeres results in DNA damage response activation, cell division delay, and micronucleation (548.1)

Telomeres are nucleoprotein structures required to protect chromosome ends. Uncapping of telomeres during interphase elicits a DNA damage response causing cell cycle arrest, aberrant DNA damage repair resulting in, chromosomal end fusion, chromosome instability and eventually lead to tumorigenesis. In the present study we investigate the consequences of DNA damage occurring at telomeres during the mitotic phase of the cell cycle using laser microirradiation. Microirradiation was used in combination with GFP live‐cell imaging and dual‐labeling to study double‐strand break (DSB) repair by monitoring the co‐localization of γH2AX with DNA damage response (DDR) factors. We found DDR proteins such as ATM and WRN localize to damaged telomeres but not to other chromosomal sites of focal laser damage. Interestingly, damage at mitotic telomeres results in a delay in anaphase onset, which is dependent on the activity of ATM, Chk1 and the spindle assembly checkpoint kinase, Mps1. Telomere‐damaged cells exited mitosis with unresolved telomere repair and increased occurrence of micronuclei. Telomeric damage is therefore distinct from damage at other chromosomal loci during mitosis and elicits an intra‐mitotic delay that may permit timely repair.

Potentiation by caffeine of cytogenetic damage induced by steroidal derivatives in human lymphocytes in vitro

We studied the effects of three newly synthesized steroidal derivatives of nitrogen mustards, alone or in combination with caffeine, on sister chromatid exchange (SCE) frequencies and on human lymphocyte proliferation kinetics. The agents have as alkylator functionalities either P-N,N-bis(2-chloroethyl)aminophenyl-buturate (CHL) or P-N,N-bis(2-chloroethyl)aminophenyl-acetate (PHE), esterified with a modified steroidal nucleus. An enhancement of SCE frequency was seen with compounds which contain either PHE or CHL as alkylators and are esterified with a steroidal nucleus having added a cholestene group in the 17-position of the D-ring. The exocyclic insertion of an –NHCO– group in the D-ring of the steroidal nucleus esterified with PHE (amide ester of PHE) gave a compound showing increased SCE frequency. Enhanced cytogenetic damage was observed when lymphocytes were exposed in vitro to caffeine. The compounds, alone or in combination with caffeine, caused a concentration-dependent increase in SCE frequencies and cell division delays, and caffeine was found to act synergistically with the steroidal alkylators.

Effects of elongation delay in transcription dynamics.

In the transcription process, elongation delay is induced by the movement of RNA polymerases (RNAP) along the DNA sequence, and can result in changes in the transcription dynamics. This paper studies the transcription dynamics that involved the elongation delay and effects of cell division and DNA replication. The stochastic process of gene expression is modeled with delay chemical master equation with periodic coefficients, and is studied numerically through the stochastic simulation algorithm with delay. We show that the average transcription level approaches to a periodic dynamics over cell cycles at homeostasis, and the elongation delay can reduce the transcription level and increase the transcription noise. Moreover, the transcription elongation can induce bimodal distribution of mRNA levels that can be measured by the techniques of flow cytometry.