Kinetoplast morphology and segregation pattern as a marker for cell cycle progression in Leishmania donovani

Trypanosomatids are typified by uniquely configured mitochondrial DNA--the kinetoplast. The replication timing of kinetoplast DNA (kDNA) is closely linked to nuclear S phase, but nuclear and kinetoplast compartments display staggered timing of segregation, post-replication. Kinetoplast division is completed before nuclear division in Trypanosoma species while nuclear division is completed first in Crithidia species. Leishmania donovani is the causative agent of visceral leishmaniasis, a form of leishmanial infection that is often fatal. Cell cycle related studies in Leishmania are hampered by difficulties in synchronizing these cells. This report examines the replication/segregation pattern and morphology of the kinetoplast in L. donovani with the aim of determining if these traits can be used to assign cell cycle stage to individual cells. By labeling replicating cells with bromodeoxyuridine after synchronization with hydroxyurea, we find that although both nuclear and kDNA initiate replication in early S phase, nuclear division precedes kinetoplast segregation in 80% of the cells. The kinetoplast is roundish/short rod-like in G1 and in early to mid-S phase, but prominently elongated/bilobed in late S phase and early G2/M. These morphological traits and segregation pattern of the kinetoplast can be used as a marker for cell cycle stage in a population of asynchronously growing L. donovani promastigotes, in place of cell synchronization procedures or instead of using antibody staining for cell cycle stage marker proteins.     

Biochemical and morphological identification of ceramide-induced cell cycle arrest and apoptosis in cultured granulosa cells

We have investigated the effects of ceramide on the progression of cell cycle and on apoptotic cell death in ovarian cultured granulosa cells. Rates of cellular proliferation were measured by immunocytochemical staining for proliferating cell nuclear antigen (PCNA) and flow cytometric cell cycle analysis. We also examined for morphological and biochemical signs of apoptosis. The PCNA expression was downregulated in a dose-dependent manner after treatment with C6-ceramide. Flow cytometric analysis demonstrated that the exposure of granulosa cells to C6-ceramide markedly decreased the population associated with G0/G1 DNA content and the reduction of cell numbers in G0/G1 phase was accompanied by the elevation of the A0 phase. The exposure of granulosa cells to exogenous C6-ceramide induced drastic morphological changes including cytoplasmic- or nuclear condensation and typical apoptotic DNA degradation. We also observed that phorbol 12-myristate 13-acetate, a protein kinase C (PKC) activator, significantly inhibited the ceramide-induced apoptosis. These results suggested that ceramide might block the progression of cell cycle at G0/G1 phase and as a consequence, granulosa cells would be committed to apoptosis. Our findings also indicated that down-regulation of the PKC activity might be involved in the ceramide-induced apoptosis in cultured granulosa cells.     

A genome–wide screen to identify genes controlling the rate of entry into mitosis in fission yeast

We have carried out a haploinsufficiency (HI) screen in fission yeast using heterozygous deletion diploid mutants of a genome-wide set of cell cycle genes to identify genes encoding products whose level determines the rate of progression through the cell cycle. Cell size at division was used as a measure of advancement or delay of the G2-M transition of rod-shaped fission yeast cells. We found that 13 mutants were significantly longer or shorter (greater than 10%) than control cells at cell division. These included mutants of the cdc2, cdc25, wee1 and pom1 genes, which have previously been shown to play a role in the timing of entry into mitosis, and which validate this approach. Seven of these genes are involved in regulation of the G2-M transition, 5 for nuclear transport and one for nucleotide metabolism. In addition we identified 4 more genes that were 8–10% longer or shorter than the control that also had roles in regulation of the G2-M transition or in nuclear transport. The genes identified here are all conserved in human cells, suggesting that this dataset will be useful as a basis for further studies to identify rate-limiting steps for progression through the cell cycle in other eukaryotes.     

Nuclear translocation of Cyclin B1 marks the restriction point for terminal cell cycle exit in G2 phase

Upon DNA damage, cell cycle progression is temporally blocked to avoid propagation of mutations. While transformed cells largely maintain the competence to recover from a cell cycle arrest, untransformed cells past the G1/S transition lose mitotic inducers, and thus the ability to resume cell division. This permanent cell cycle exit depends on p21, p53, and APC/C^Cdh1. However, when and how permanent cell cycle exit occurs remains unclear. Here, we have investigated the cell cycle response to DNA damage in single cells that express Cyclin B1 fused to eYFP at the endogenous locus. We find that upon DNA damage Cyclin B1-eYFP continues to accumulate up to a threshold level, which is reached only in G2 phase. Above this threshold, a p21 and p53-dependent nuclear translocation required for APC/C^Cdh1-mediated Cyclin B1-eYFP degradation is initiated. Thus, cell cycle exit is decoupled from activation of the DNA damage response in a manner that correlates to Cyclin B1 levels, suggesting that G2 activities directly feed into the decision for cell cycle exit. Once Cyclin B1-eYFP nuclear translocation occurs, checkpoint inhibition can no longer promote mitotic entry or re-expression of mitotic inducers, suggesting that nuclear translocation of Cyclin B1 marks the restriction point for permanent cell cycle exit in G2 phase.     

Independence of centriole formation and initiation of DNA synthesis in Chinese hamster ovary cells

The relationship between centriole formation and DNA synthesis was investigated by examining the effect of taxol on the centriole cycle and the initiation of DNA synthesis in synchronized cells. The centriole cycle was monitored by electron microscopy of whole-mount preparations [Kuriyama and Borisy, J. Cell Biol., 1981, 91:814-821]. A short daughter centriole appeared in perpendicular orientation to each parent during late G1 or early S and elongated slowly during S to G2. Addition of 5-20 micrograms/ml taxol to a synchronous population of cells in S phase did not inhibit centriole elongation; rather, elongation was accelerated. In contrast, when taxol was added to M phase or early G1 cells, centriole duplication was completely inhibited. The taxol block was reversible since nucleation and elongation of centrioles resumed as soon as the drug was removed. Cells exposed to taxol progressed through the cell cycle and became blocked in mitosis, as indicated by an increase in the mitotic index, but eventually the mitotic arrest was overcome, resulting in formation of multinucleated cells. A peak in mitotic index was seen in the following generation, indicating that chromosomes duplicated in the presence of taxol. Incorporation of 3H-thymidine followed by autoradiography confirmed that DNA synthesis was initiated in the presence of taxol even though formation of daughter centrioles was inhibited. It seems, therefore, that centriole duplication is not a prerequisite for entry into S phase. Since DNA synthesis has already been demonstrated not to be necessary for centriole duplication, these two events, normally coordinated in time, appear to be independent of each other.     

Isolation and partial characterization of conditional cell division cycle mutants inChlamydomonas

Summary We have isolated a number of temperature conditional cell division cycle mutants of the unicellular plantChlamydomonas reinhardtii that are defective in single nuclear genes. Cells grow and divide normally at the permissive temperature (21 °C), but arrest in division at the restrictive temperature (33 °C). We have characterized these mutants using DNA probes and immunofluorescence techniques to localize cytoskeletal and microtubule organizing centre proteins. We describe here 3 broad classes of cell cycle mutation which result in cell cycle arrest with: unreplicated DNA (G1 arrest), duplicated DNA (G2 arrest) and multiple nuclei due to defective cytokinesis (cytokinesis arrest). The continuation of nuclear division in mutants blocked in cytokinesis provides support of an earlier hypothesis that stage specific events in theChlamydomonas cell cycle are arranged in separate dependent sequences. The mutants isolated in the present study provide insights into the role of cytoskeletal proteins in the coordination of plant cell division and the means to investigate the molecular mechanisms whereby division by multiple fission is controlled in the unicellular plantChlamydomonas.Abbreviations BB basal bodies - EMS ethylmethane sulphonate - MT microtubule - MTOC Microtubule organizing centre - NBBC nucleus-basal body connector - PAR photosynthetically active radiation     

Leishmania amazonensis Promastigotes Present Two Distinct Modes of Nucleus and Kinetoplast Segregation during Cell Cycle

Here, we show the morphological events associated with organelle segregation and their timing in the cell cycle of a reference strain of Leishmania (L.) amazonensis promastigotes, the main causative agent of Tegumentary leishmaniasis in the Americas. We show evidences that during the cell cycle, L. amazonensis promastigotes present two distinct modes of nucleus and kinetoplast segregation, which occur in different temporal order in different proportions of cells. We used DAPI-staining and EdU-labeling to monitor the segregation of DNA-containing organelles and DNA replication in wild-type parasites. The emergence of a new flagellum was observed using a specific monoclonal antibody. The results show that L. amazonensis cell cycle division is peculiar, with 65% of the dividing cells duplicating the kinetoplast before the nucleus, and the remaining 35% doing the opposite or duplicating both organelles concomitantly. In both cases, the new flagellum appeared during S to G2 phase in 1N1K cells and thus before the segregation of both DNA-containing organelles; however, we could not determine the exact timing of flagellar synthesis. Most of these results were confirmed by the synchronization of parasites using hydroxyurea. Altogether, our data show that during the cell cycle of L. amazonensis promastigotes, similarly to L. donovani, the segregation of nucleus and kinetoplast do not follow a specific order, especially when compared to other trypanosomatids, reinforcing the idea that this characteristic seems to be species-specific and may represent differences in cellular biology among members of the Leishmania genus.     

Androgen and taxol cause cell type-specific alterations of centrosome and DNA organization in androgen-responsive LNCaP and androgen-independent DU145 prostate cancer cells

We investigated the effects of androgen and taxol on the androgen-responsive LNCaP and androgen-independent DU145 prostate cancer cell lines. Cells were treated for 48 and 72 h with 0.05-1 nM of the synthetic androgen R1881 and with 100 nM taxol. Treatment of LNCaP cells with 0.05 nM R1881 led to increased cell proliferation, whereas treatment with 1 nM R1881 resulted in inhibited cell division, DNA cycle arrest, and altered centrosome organization. After treatment with 1 nM R1881, chromatin became clustered, nuclear envelopes convoluted, and mitochondria accumulated around the nucleus. Immunofluorescence microscopy with antibodies to centrosomes showed altered centrosome structure. Although centrosomes were closely associated with the nucleus in untreated cells, they dispersed into the cytoplasm after treatment with 1 nM R1881. Microtubules were only faintly detected in 1 nM R1881-treated LNCaP cells. The effects of taxol included microtubule bundling and altered mitochondria morphology, but not DNA organization. As expected, the androgen-independent prostate cancer cell line DU145 was not affected by R1881. Treatment with taxol resulted in bundling of microtubules in both cell lines. Additional taxol effects were seen in DU145 cells with micronucleation of DNA, an indication of apoptosis. Simultaneous treatment with R1881 and taxol had no additional effects on LNCaP or DU145 cells. These results suggest that LNCaP and DU145 prostate cancer cells show differences not only in androgen responsiveness but in sensitivity to taxol as well.     

Microtubule-interacting drugs induce moderate and reversible damage to human bone marrow mesenchymal stem cells

Objectives:  This study aimed to investigate molecular and cellular changes induced in human bone marrow mesenchymal stem cells (hMSCs) after treatment with microtubule-interacting agents and to estimate damage to the bone marrow microenvironment caused by chemotherapy.
Materials and methods:  Using an in vitro hMSC culture system and biochemical and morphological approaches, we studied the effect of nocodazole and taxol® on microtubule and nuclear envelope organization, tubulin and p53 synthesis, cell cycle progression and proliferation and death of hMSCs isolated from healthy donors.
Results and conclusions:  Both nocodazole and taxol reduced hMSC proliferation and induced changes in the microtubular network and nuclear envelope morphology and organization. However, they exhibited only a moderate effect on cell death and partial arrest of hMSCs at G₂ but not at M phase of the cell cycle. Both agents induced expression of p53, exclusively localized in abnormally shaped nuclei, while taxol, but not nocodazole, increased synthesis of β-tubulin isoforms. Cell growth rates and microtubule and nuclear envelope organization gradually normalized after transfer, in drug-free medium. Our data indicate that microtubule-interacting drugs reversibly inhibit proliferation of hMSCs; additionally, their cytotoxic action and effect on microtubule and nuclear envelope organization are moderate and reversible. We conclude that alterations in human bone marrow cells of patients under taxol chemotherapy are transient and reversible.     

Cell cycle traverse and protein metabolism in human NHIK 3025 cells: the role of anchorage

We have studied the effect of cell anchorage on the human cell line NHIK 3025 in vitro, to see whether the growth regulating effect of cell anchorage primarily affected DNA division cycle or mass growth cycle. It was found that cell to cell anchorage had the same effect on cell cycle progression as anchorage to a solid surface, which indicates that it is anchorage per se and not cell shape that is important for growth control in NHIK 3025 cells. When NHIK 3025 cells were grown without attachment to a solid surface, both G1 and cell cycle duration was prolonged by 6 h, which means that the prolonged cell cycle was due to a prolonged G1. During the first part of the cell cycle the rate of protein synthesis and degradation was constant, and at the same level in cells grown with and without attachment. This means that the prolonged G1 was not due to a reduced protein accumulation or mass growth. Towards the end of the cell cycle protein accumulation was reduced. This effect was either due to a size control before cell division or a secondary effect of the prolonged G1. We therefore conclude that cell anchorage as a growth regulator primarily affects the DNA/cell division cycle.     

Effect of n-butyrate on cell cycle progression and in situ chromatin structure of L1210 cells

In the presence of 1–5 mM n-butyrate, murine leukemic L1210 cells cease proliferation and become arrested in the G1A compartment of the G1 phase. Cells in this compartment, in comparison with the remaining cells of the G1 phase (G1B), are characterized by low RNA content and more condensed chromatin. During unperturbed growth the cell residence times in G1A are of indeterminate duration (exponentially distributed); the half-time of L1210 cell residence in G1A is about 1.4 h. The effect of n-butyrate in arresting cells in G1A was concentration-dependent. However, the sensitivity of L1210 cells to this drug was markedly enhanced when cells were treated for longer than one generation (12 h). Cells arrested in G1A remained viable and when n-butyrate was removed, after a lag period, they resumed progression through the cycle.The effect of n-butyrate on cell progression through various parts of the cycle was studied in a stathmokinetic experiment. The rate of cell entrance into mitosis was decreased by 30, 60 and 110%, in the presence of 1, 2.5 and 5 mM n-butyrate respectively, thus indicating a slowdown in cell progression through G2 and S. The duration of G2 was prolonged by 20, 70 and 140% at 1, 2.5 and 5 mM n-butyrate respectively. The half-time of cell residence in G1A was increased by as much as 1.5-, 6.3- and 15.6-fold by 1, 2.5 and 5 mM n-butyrate. Progression through late G1 (G1B) was not affected at 1 mM, and could not be estimated at higher drug concentrations. The effects on cell cycle progression were evident 1 h after addition of n-butyrate.DNA in situ in nuclei of n-butyrate-treated cells had lowered (by 2–8 °C) stability to thermal denaturation and increased (by 15%) accessibility to DNase I. The decrease in DNA stability to heat was more pronounced when permealized cells were heated in the presence of 1 mM MgCl₂ rather than EDTA. DNA in situ in the nuclei of n-butyrate-treated cells also showed decreased sensitivity to acid-induced denaturation. Changes in chromatin were seen in all cells, regardless of cell cycle phase, within the first hours after addition of n-butyrate. Mitotic cells, however, reacted to n-butyrate more rapidly than interphase cells. The observed changes in L1210 cells are most likely a consequence of histone modifications (acetylation of inner histones, dephosphorylation of histone H1) induced by n-butyrate.     

A dual‐color marker system for in vivo visualization of cell cycle progression in Arabidopsis

Visualization of the spatiotemporal pattern of cell division is crucial to understand how multicellular organisms develop and how they modify their growth in response to varying environmental conditions. The mitotic cell cycle consists of four phases: S (DNA replication), M (mitosis and cytokinesis), and the intervening G1 and G2 phases; however, only G2/M‐specific markers are currently available in plants, making it difficult to measure cell cycle duration and to analyze changes in cell cycle progression in living tissues. Here, we developed another cell cycle marker that labels S‐phase cells by manipulating Arabidopsis CDT1a, which functions in DNA replication origin licensing. Truncations of the CDT1a coding sequence revealed that its carboxy‐terminal region is responsible for proteasome‐mediated degradation at late G2 or in early mitosis. We therefore expressed this region as a red fluorescent protein fusion protein under the S‐specific promoter of a histone 3.1‐type gene, HISTONE THREE RELATED2 (HTR2), to generate an S/G2 marker. Combining this marker with the G2/M‐specific CYCB1‐GFP marker enabled us to visualize both S to G2 and G2 to M cell cycle stages, and thus yielded an essential tool for time‐lapse imaging of cell cycle progression. The resultant dual‐color marker system, Cell Cycle Tracking in Plant Cells (Cytrap), also allowed us to identify root cells in the last mitotic cell cycle before they entered the endocycle. Our results demonstrate that Cytrap is a powerful tool for in vivo monitoring of the plant cell cycle, and thus for deepening our understanding of cell cycle regulation in particular cell types during organ development.     

Periodic, metabolic and structural phenomena in a protist, Euglena gracilis]

Sychronous divisions of Euglena gracilis strain Z can be obtained by various methods. When the cells are cultivated in a medium containing lactate as the sole carbon source, synchronous divisions are observed, independent of the conditions of illumination. Nevertheless, there exists a relationship between the phase of cell division and ther periods of light and darkness applied to the culture. During the cell cycle, the synthesis of macromolecules is discontinuous--this is true of nuclear and mitochondrial DNA, ribosomal and nonribosomal RNA, and certain proteins (cytochrome c 558). Cyclic variations in the structure of mitochondria and chloroplasts are observed. In the course of the cell cycle, sequential metabolic processes accompany structural modifications of the organelles. Also, at the beginning of the cycle, at the start of phase G1, the cytoplasmic ribosomes are synthesized, and then, in green euglenids, nonribosomal RNAs are formed. These syntheses of RNA precede enlargement of the chondriome and plastids. In mid-G1 phase, a new synthesis of RNA begins, which precedes synthesis of nuclear and mitochondrial DNA. At the end of G1 phase, division of organelles starts, beginning with the chondriome and plastids, arranged in a network.     

Deciliation interferes with cell-cycle progression in Tetrahymena

The impact of ciliary regeneration upon cell-cycle progression of the ciliate Tetrahymena was studied. It was found that cell division ceases during ciliary regeneration, and starts again about 4 h after deciliation. Deciliation of an asynchronously multiplying culture results in a rapid interruption of DNA synthesis, followed by resumption 1 h later. This was shown by pulse-labelling the cells with [3H]thymidine at various times after deciliation. Cytophotometric determinations of the macronuclear DNA content substantiated these observations, since the average DNA content per cell remained constant within the first hour of regeneration, confirming the labelling experiments, after which it rose. At its maximum, the average DNA content was more than doubled as compared with the beginning of the experiment. This indicates that a substantial proportion of the regenerating cells performed two rounds of DNA replication prior to cell division. The massive drop in the average DNA content during the fifth hour after deciliation indicates that the culture becomes partly synchronized for cell division by the deciliation procedure. The division synchrony results from a greater delay of the next cell division when G2 cells are deciliated than occurs in G1 cells. This was shown by deciliating cultures of Tetrahymena thermophila cells in the respective stages of the cell cycle, which had been partly synchronized by elutriator centrifugation. Thus, deciliation followed by ciliary regeneration causes a varying degree of retardation in progression through the cell cycle, being greatest for G2 cells and least for G1 cells.     

The cell cycle of the planctomycete Gemmata obscuriglobus with respect to cell compartmentalization

Background  

Gemmata obscuriglobus is a distinctive member of the divergent phylum Planctomycetes, all known members of which are peptidoglycan-less bacteria with a shared compartmentalized cell structure and divide by a budding process. G. obscuriglobus in addition shares the unique feature that its nucleoid DNA is surrounded by an envelope consisting of two membranes forming an analogous structure to the membrane-bounded nucleoid of eukaryotes and therefore G. obscuriglobus forms a special model for cell biology. Draft genome data for G. obscuriglobus as well as complete genome sequences available so far for other planctomycetes indicate that the key bacterial cell division protein FtsZ is not present in these planctomycetes, so the cell division process in planctomycetes is of special comparative interest. The membrane-bounded nature of the nucleoid in G. obscuriglobus also suggests that special mechanisms for the distribution of this nuclear body to the bud and for distribution of chromosomal DNA might exist during division. It was therefore of interest to examine the cell division cycle in G. obscuriglobus and the process of nucleoid distribution and nuclear body formation during division in this planctomycete bacterium via light and electron microscopy.     

Increased tRNA modification and gene-specific codon usage regulate cell cycle progression during the DNA damage response

S-phase and DNA damage promote increased ribonucleotide reductase (RNR) activity. Translation of RNR1 has been linked to the wobble uridine modifying enzyme tRNA methyltransferase 9 (Trm9). We predicted that changes in tRNA modification would translationally regulate RNR1 after DNA damage to promote cell cycle progression. In support, we demonstrate that the Trm9-dependent tRNA modification 5-methoxycarbonylmethyluridine (mcm?U) is increased in hydroxyurea (HU)-induced S-phase cells, relative to G? and G?, and that mcm?U is one of 16 tRNA modifications whose levels oscillate during the cell cycle. Codon-reporter data matches the mcm?U increase to Trm9 and the efficient translation of AGA codons and RNR1. Further, we show that in trm9Δ cells reduced Rnr1 protein levels cause delayed transition into S-phase after damage. Codon re-engineering of RNR1 increased the number of trm9Δ cells that have transitioned into S-phase 1 h after DNA damage and that have increased Rnr1 protein levels, similar to that of wild-type cells expressing native RNR1. Our data supports a model in which codon usage and tRNA modification are regulatory components of the DNA damage response, with both playing vital roles in cell cycle progression.     

Characterization of mec1 kinase-deficient mutants and of new hypomorphic mec1 alleles impairing subsets of the DNA damage response pathway

DNA damage checkpoints lead to the inhibition of cell cycle progression following DNA damage. The Saccharomyces cerevisiae Mec1 checkpoint protein, a phosphatidylinositol kinase-related protein, is required for transient cell cycle arrest in response to DNA damage or DNA replication defects. We show that mec1 kinase-deficient (mec1kd) mutants are indistinguishable from mec1Delta cells, indicating that the Mec1 conserved kinase domain is required for all known Mec1 functions, including cell viability and proper DNA damage response. Mec1kd variants maintain the ability to physically interact with both Ddc2 and wild-type Mec1 and cause dominant checkpoint defects when overproduced in MEC1 cells, impairing the ability of cells to slow down S phase entry and progression after DNA damage in G(1) or during S phase. Conversely, an excess of Mec1kd in MEC1 cells does not abrogate the G(2)/M checkpoint, suggesting that Mec1 functions required for response to aberrant DNA structures during specific cell cycle stages can be separable. In agreement with this hypothesis, we describe two new hypomorphic mec1 mutants that are completely defective in the G(1)/S and intra-S DNA damage checkpoints but properly delay nuclear division after UV irradiation in G(2). The finding that these mutants, although indistinguishable from mec1Delta cells with respect to the ability to replicate a damaged DNA template, do not lose viability after UV light and methyl methanesulfonate treatment suggests that checkpoint impairments do not necessarily result in hypersensitivity to DNA-damaging agents.     

Loading and Unloading: Orchestrating Centrosome Duplication and Spindle Assembly by Ran/Crm1

The cell cycle is an intricate process of DNA replication and cell division thatconcludes with the formation of two genetically equivalent daughter cells. In thisprogression, the centrosome is duplicated once and only once during the G1/S transitionto produce the bipolar spindle and ensure proper chromosome segregation. The presenceof multiple centrosomes in cancer cells suggests that this process is mis-regulated duringcarcinogenesis. This suggests that certain factors exist that license the progression ofcentrosome duplication and serve to inhibit further duplications during a single cell cycle.Recent studies suggest that the Ran/Crm1 complex not only regulates nucleocytoplasmictransport but is also independently involved in mitotic spindle assembly. Factors that arecapable of interacting with Ran/Crm1 through their nuclear export sequences, such ascyclins/cdks, p53 and Brca1/2, may potentially function as centrosome checkpoints akinto the G1/S and G2/M checkpoints of the cell cycle. Our recent findings indicate thatnucleophosmin, a protein whose trafficking is mediated by the Ran/Crm1 network, is oneof such checkpoint factors for maintaining proper centrosome duplication. We proposethat Ran/Crm1 may act as a ‘loading dock’ to coordinate various checkpoint factors inregulating the fidelity of centrosome duplication during cell cycle progression, and thedisruption of these processes may lead to genomic instability and an acceleration ofoncogenesis.