Apical migration of nuclei during G2 is a prerequisite for all nuclear motion in zebrafish neuroepithelia

Nuclei in the proliferative pseudostratified epithelia of vastly different organisms exhibit a characteristic dynamics - the so-called interkinetic nuclear migration (IKNM). Although these movements are thought to be intimately tied to the cell cycle, little is known about the relationship between IKNM and distinct phases of the cell cycle and the role that this association plays in ensuring balanced proliferation and subsequent differentiation. Here, we perform a quantitative analysis of modes of nuclear migration during the cell cycle using a marker that enables the first unequivocal differentiation of all four phases in proliferating neuroepithelial cells in vivo. In zebrafish neuroepithelia, nuclei spend the majority of the cell cycle in S phase, less time in G1, with G2 and M being noticeably shorter still in comparison. Correlating cell cycle phases with nuclear movements shows that IKNM comprises rapid apical nuclear migration during G2 phase and stochastic nuclear motion during G1 and S phases. The rapid apical migration coincides with the onset of G2, during which we find basal actomyosin accumulation. Inhibiting the transition from G2 to M phase induces a complete stalling of nuclei, indicating that IKNM and cell cycle continuation cannot be uncoupled and that progression from G2 to M is a prerequisite for rapid apical migration. Taken together, these results suggest that IKNM involves an actomyosin-driven contraction of cytoplasm basal to the nucleus during G2, and that the stochastic nuclear movements observed in other phases arise passively due to apical migration in neighboring cells.     

Loss of Llgl1 in retinal neuroepithelia reveals links between apical domain size, Notch activity and neurogenesis

To gain insights into the cellular mechanisms of neurogenesis, we analyzed retinal neuroepithelia deficient for Llgl1, a protein implicated in apicobasal cell polarity, asymmetric cell division, cell shape and cell cycle exit. We found that vertebrate retinal neuroepithelia deficient for Llgl1 retained overt apicobasal polarity, but had expanded apical domains. Llgl1 retinal progenitors also had increased Notch activity and reduced rates of neurogenesis. Blocking Notch function by depleting Rbpj restored normal neurogenesis. Experimental expansion of the apical domain, through inhibition of Shroom3, also increased Notch activity and reduced neurogenesis. Significantly, in wild-type retina, neurogenic retinal progenitors had smaller apical domains compared with proliferative neuroepithelia. As nuclear position during interkinetic nuclear migration (IKNM) has been previously linked with cell cycle exit, we analyzed this phenomenon in cells depleted of Llgl1. We found that although IKNM was normal, the relationship between nuclear position and neurogenesis was shifted away from the apical surface, consistent with increased pro-proliferative and/or anti-neurogenic signals associated with the apical domain. These data, in conjunction with other findings, suggest that, in retinal neuroepithelia, the size of the apical domain modulates the strength of polarized signals that influence neurogenesis.     

The terminal basal mitosis of chicken retinal Lim1 horizontal cells is not sensitive to cisplatin-induced cell cycle arrest

For proper development, cells need to coordinate proliferation and cell cycle-exit. This is mediated by a cascade of proteins making sure that each phase of the cell cycle is controlled before the initiation of the next. Retinal progenitor cells divide during the process of interkinetic nuclear migration, where they undergo S-phase on the basal side, followed by mitoses on the apical side of the neuroepithelium. The final cell cycle of chicken retinal horizontal cells (HCs) is an exception to this general cell cycle behavior. Lim1 expressing (+) horizontal progenitor cells (HPCs) have a heterogenic final cell cycle, with some cells undergoing a terminal mitosis on the basal side of the retina. The results in this study show that this terminal basal mitosis of Lim1+ HPCs is not dependent on Chk1/2 for its regulation compared to retinal cells undergoing interkinetic nuclear migration. Neither activating nor blocking Chk1 had an effect on the basal mitosis of Lim1+ HPCs. Furthermore, the Lim1+ HPCs were not sensitive to cisplatin-induced DNA damage and were able to continue into mitosis in the presence of γ-H2AX without activation of caspase-3. However, Nutlin3a-induced expression of p21 did reduce the mitoses, suggesting the presence of a functional p53/p21 response in HPCs. In contrast, the apical mitoses were blocked upon activation of either Chk1/2 or p21, indicating the importance of these proteins during the process of interkinetic nuclear migration. Inhibiting Cdk1 blocked M-phase transition both for apical and basal mitoses. This confirmed that the cyclin B1-Cdk1 complex was active and functional during the basal mitosis of Lim1+ HPCs. The regulation of the final cell cycle of Lim1+ HPCs is of particular interest since it has been shown that the HCs are able to sustain persistent DNA damage, remain in the cell cycle for an extended period of time and, consequently, survive for months.     

A role for CK2 upon interkinetic nuclear migration in the cell cycle of retinal progenitor cells

In developing retina, the nucleus of the elongated neuroepithelial cells undergoes interkinetic nuclear migration (INM), that is it migrates back and forth across the proliferative layer during the cell cycle. S-phase occurs at the basal side, while M-phase occurs at the apical margin of the retinal progenitors. G1 and G2-phases occur along the nuclear migration pathway. We tested whether this feature of the retinal cell cycle is controlled by CK2, which, among its many substrates, phosphorylates both molecular motors and cytoskeletal components. Double immunolabeling showed that CK2 is contained in BrdU-labeled retinal progenitors. INM was examined after pulse labeling the retina of newborn rats with BrdU, by plotting nuclear movement from basal to apical sides of the retinal progenitors during G2. The CK2 specific inhibitor 4,5,6,7-tetrabromobenzotriazole inhibited the activity of rat retinal CK2, and blocked nuclear movement proper in a dose-dependent way. No apoptosis was detected, and total numbers of BrdU-labeled nuclei remained constant following treatment. Immunohistochemistry showed that, following inhibition of CK2, the tubulin cytoskeleton is disorganized, with reduced acetylated and increased tyrosinated tubulin. This indicates a reduction in stable microtubules, with accumulation of free tubulin dimers. The results show that CK2 activity is required for INM in retinal progenitor cells.     

Regulation of interkinetic nuclear migration by cell cycle-coupled active and passive mechanisms in the developing brain

A hallmark of neurogenesis in the vertebrate brain is the apical-basal nuclear oscillation in polarized neural progenitor cells. Known as interkinetic nuclear migration (INM), these movements are synchronized with the cell cycle such that nuclei move basally during G1-phase and apically during G2-phase. However, it is unknown how the direction of movement and the cell cycle are tightly coupled. Here, we show that INM proceeds through the cell cycle-dependent linkage of cell-autonomous and non-autonomous mechanisms. During S to G2 progression, the microtubule-associated protein Tpx2 redistributes from the nucleus to the apical process, and promotes nuclear migration during G2-phase by altering microtubule organization. Thus, Tpx2 links cell-cycle progression and autonomous apical nuclear migration. In contrast, in vivo observations of implanted microbeads, acute S-phase arrest of surrounding cells and computational modelling suggest that the basal migration of G1-phase nuclei depends on a displacement effect by G2-phase nuclei migrating apically. Our model for INM explains how the dynamics of neural progenitors harmonize their extensive proliferation with the epithelial architecture in the developing brain.     

Interkinetic nuclear migration is a broadly conserved feature of cell division in pseudostratified epithelia

Animal development requires tight integration between the processes of proliferative growth and epithelial morphogenesis, both of which play out at the level of individual cells. In this respect, not only must polarized epithelial cells assume complex morphologies, these distinct forms must be radically and repeatedly transformed to permit mitosis. A dramatic illustration of this integration between epithelial morphogenesis and cell proliferation is interkinetic nuclear migration (IKNM), wherein the nuclei of pseudostratified epithelial cells translocate to the apical epithelial surface to execute cell division. IKNM is widely considered a hallmark of pseudostratified vertebrate neuroepithelia, and prior investigations have proposed both actomyosin- and microtubule-dependent mechanisms for apical localization of the mitotic nucleus. Here, using comparative functional analysis in arthropod and cnidarian systems (Drosophila melanogaster and Nematostella vectensis), we show that actomyosin-dependent IKNM is likely to be a general feature of mitosis in pseudostratified epithelia throughout Eumetazoa. Furthermore, our studies suggest a mechanistic link between IKNM and the fundamental process of mitotic cell rounding.     

Heterogenic Final Cell Cycle by Chicken Retinal Lim1 Horizontal Progenitor Cells Leads to Heteroploid Cells with a Remaining Replicated Genome

Retinal progenitor cells undergo apical mitoses during the process of interkinetic nuclear migration and newly generated post-mitotic neurons migrate to their prospective retinal layer. Whereas this is valid for most types of retinal neurons, chicken horizontal cells are generated by delayed non-apical mitoses from dedicated progenitors. The regulation of such final cell cycle is not well understood and we have studied how Lim1 expressing horizontal progenitor cells (HPCs) exit the cell cycle. We have used markers for S- and G2/M-phase in combination with markers for cell cycle regulators Rb1, cyclin B1, cdc25C and p27Kip1 to characterise the final cell cycle of HPCs. The results show that Lim1+ HPCs are heterogenic with regards to when and during what phase they leave the final cell cycle. Not all horizontal cells were generated by a non-apical (basal) mitosis; instead, the HPCs exhibited three different behaviours during the final cell cycle. Thirty-five percent of the Lim1+ horizontal cells was estimated to be generated by non-apical mitoses. The other horizontal cells were either generated by an interkinetic nuclear migration with an apical mitosis or by a cell cycle with an S-phase that was not followed by any mitosis. Such cells remain with replicated DNA and may be regarded as somatic heteroploids. The observed heterogeneity of the final cell cycle was also seen in the expression of Rb1, cyclin B1, cdc25C and p27Kip1. Phosphorylated Rb1-Ser608 was restricted to the Lim1+ cells that entered S-phase while cyclin B1 and cdc25C were exclusively expressed in HPCs having a basal mitosis. Only HPCs that leave the cell cycle after an apical mitosis expressed p27Kip1. We speculate that the cell cycle heterogeneity with formation of heteroploid cells may present a cellular context that contributes to the suggested propensity of these cells to generate cancer when the retinoblastoma gene is mutated.     

Live imaging of the Dictyostelium cell cycle reveals widespread S phase during development, a G2 bias in spore differentiation and a premitotic checkpoint

The regulation of the Dictyostelium cell cycle has remained ambiguous owing to difficulties in long-term imaging of motile cells and a lack of markers for defining cell cycle phases. There is controversy over whether cells replicate their DNA during development, and whether spores are in G1 or G2 of the cell cycle. We have introduced a live-cell S-phase marker into Dictyostelium cells that allows us to precisely define cycle phase. We show that during multicellular development, a large proportion of cells undergo nuclear DNA synthesis. Germinating spores enter S phase only after their first mitosis, indicating that spores are in G2. In addition, we demonstrate that Dictyostelium heterochromatin is copied late in S phase and replicates via accumulation of replication factors, rather than recruitment of DNA to pre-existing factories. Analysis of variability in cycle times indicates that regulation of the cycle manifests at a single random transition in G2, and we present the first identified checkpoint in Dictyostelium, which operates at the G2-M transition in response to DNA damage.     

Nuclear export of cyclin B1 and its possible role in the DNA damage-induced G2 checkpoint.

M-phase-promoting factor (MPF), a complex of cdc2 and a B-type cyclin, is a key regulator of the G2/M cell cycle transition. Cyclin B1 accumulates in the cytoplasm through S and G2 phases and translocates to the nucleus during prophase. We show here that cytoplasmic localization of cyclin B1 during interphase is directed by its nuclear export signal (NES)-dependent transport mechanism. Treatment of HeLa cells with leptomycin B (LMB), a specific inhibitor of the NES-dependent transport, resulted in nuclear accumulation of cyclin B1 in G2 phase. Disruption of an NES which has been identified in cyclin B1 here abolished the nuclear export of this protein, and consequently the NES-disrupted cyclin B1 when expressed in cells accumulated in the nucleus. Moreover, we show that expression of the NES-disrupted cyclin B1 or LMB treatment of the cells is able to override the DNA damage-induced G2 checkpoint when combined with caffeine treatment. These results suggest a role of nuclear exclusion of cyclin B1 in the DNA damage-induced G2 checkpoint.     

Apical movement during interkinetic nuclear migration is a two-step process

Neural progenitor cells in the pseudostratified neuroepithelium in vertebrates undergo interkinetic nuclear migration, which results in mitotic cells localized to the apical surface. Interphase nuclei are distributed throughout the rest of the epithelium. How mitosis is coordinated with nuclear movement is unknown, and the mechanism by which the nucleus migrates apically is controversial. Using time-lapse confocal microscopy, we show that nuclei migrate apically in G2 phase via microtubules. However, late in G2, centrosomes leave the apical surface after cilia are disassembled, and mitosis initiates away from the apical surface. The mitotic cell then rounds up to the apical surface, which is an actin-dependent process. This behavior is observed in both chicken neural-tube-slice preparations and in mouse cortical slices, and therefore is likely to be a general feature of interkinetic nuclear migration. We propose a new model for interkinetic nuclear migration in which actin and microtubules are used to position the mitotic cell at the apical surface.     

An overactivated ATR/CHK1 pathway is responsible for the prolonged G2 accumulation in irradiated AT cells

Induction of checkpoint responses in G1, S, and G2 phases of the cell cycle after exposure of cells to ionizing radiation (IR) is essential for maintaining genomic integrity. Ataxia telangiectasia mutated (ATM) plays a key role in initiating this response in all three phases of the cell cycle. However, cells lacking functional ATM exhibit a prolonged G2 arrest after IR, suggesting regulation by an ATM-independent checkpoint response. The mechanism for this ataxia telangiectasia (AT)-independent G2-checkpoint response remains unknown. We report here that the G2 checkpoint in irradiated human AT cells derives from an overactivation of the ATR/CHK1 pathway. Chk1 small interfering RNA abolishes the IR-induced prolonged G2 checkpoint and radiosensitizes AT cells to killing. These results link the activation of ATR/CHK1 with the prolonged G2 arrest in AT cells and show that activation of this G2 checkpoint contributes to the survival of AT cells.     

Human cytomegalovirus infection inhibits G1/S transition.

Cell cycle progression during cytomegalovirus infection was investigated by fluorescence-activated cell sorter (FACS) analysis of the DNA content in growth-arrested as well as serum-stimulated human fibroblasts. Virus-infected cells maintained in either low (0.2%) or high (10%) serum failed to progress into S phase and failed to divide. DNA content analysis in the presence of G1/S (hydroxyurea and mimosine) and G2/M (nocodazole and colcemid) inhibitors demonstrated that upon virus infection of quiescent (G0) cells, the cell cycle did not progress beyond the G1/S border even after serum stimulation. Proteins which normally indicate G1/S transition (proliferating cell nuclear antigen [PCNA]) or G2/M transition (cyclin B1) were elevated by virus infection. PCNA levels were induced in infected cells and exhibited a punctate pattern of nuclear staining instead of the diffuse pattern observed in mock-infected cells. Cyclin B1 was induced in infected cells which exhibited a G1/S DNA content by FACS analysis, suggesting that expression of this key cell cycle function was dramatically altered by viral functions. These data demonstrate that contrary to expectations, cytomegalovirus inhibits normal cell cycle progression. The host cell is blocked prior to S phase to provide a favorable environment for viral replication.     

Midblastula transition (MBT) of the cell cycles in the yolk and pigment granule-free translucent blastomeres obtained from centrifuged Xenopus embryos

We obtained translucent blastomeres free of yolk and pigment granules from Xenopus embryos which had been centrifuged at the beginning of the 8-cell stage with cellular integrity. They divided synchronously regardless of their cell size until they had decreased to 37.5 microm in radius; those smaller than this critical size, however, divided asynchronously with cell cycle times inversely proportional to the square of the cell radius after midblastula transition (MBT). The length of the S phase was determined as the time during which nuclear DNA fluorescence increased in Hoechst-stained blastomeres. When the cell cycle time exceeded 45 min, S and M phases were lengthened; when the cell cycle times exceeded 70 min, the G2 phase appeared; and after cell cycle times became longer than 150 min, the G1 phase appeared. Lengths of G1, S and M phases increased linearly with increasing cell cycle time. Enhanced green fluorescent protein (EGFP)-tagged proliferating cell nuclear antigen (PCNA) expressed in the blastomeres appeared in the S phase nucleus, but suddenly dispersed into the cytoplasm at the M phase. The system developed in this study is useful for examining the cell cycle behavior of the cell cycle-regulating molecules in living Xenopus blastomeres by fluorescence microscopy in real time.     

Cell cycle-related transformation of the E2F4-p130 repressor complex

During G0 phase the p130, member of the pRb tumor suppressor protein family, forms a repressor complex with E2F4 which is inactivated in G1/S by hyperphosphorylation of the p130. The role of p130 after G1/S remains poorly investigated. We found that in nuclear extracts of T98G cells, the p130-E2F4-DNA (pp-E2F4) complex does not dissociate at G1/S transition, but instead reverts to the p130-E2F4-cyclin E/A-cdk2 (cyc/cdk-pp-E2F4) complex, which is detected in S and G2/M phases of the cell cycle. Hyperphosphorylation of the p130 at G1/S transition is associated with a decrease of its total amount; however, this protein is still detected during the rest of the cell cycle, and it is increasingly hyperphosphorylated in the cytosol, but continuously dephosphorylated in the nucleus. Both nuclear and cytosol cell fractions in T98G cells contain a hyperphosphorylated form of p130 in complex with E2F4 at S and G2/M cell cycle phases. In contrast to T98G cells, transformation of the p130 containing cyc/cdk-pp-E2F4 complex into the p130-pp-E2F4 repressor does not occur in HeLa cells under growth restriction conditions.     

Cell cycle-related proteins: a flow cytofluorometric study in human tumors

We used 2-parameter flow cytometry (FCM) to investigate the relationship between the cell cycle phases and 3 proteins whose expression is known to increase in proliferating cells: the surface antigen transferrin receptor (Trf-r), the "cyclin" (a proliferating cell nuclear antigen, PCNA), and the nuclear antigen recognized by the monoclonal antibody (MoAb) Ki-67. FITC-labeled antibodies against Trf-r, PCNA, and the Ki-67-reactive antigen, as well as propidium iodide-DNA distribution, were simultaneously measured on human leukemia HL-60 and K562, and breast carcinoma MCF-7 cell lines and on fresh human leukemic and glioblastoma cells. The 70% ethanol fixation for Trf-r and PCNA and the 95% acetone fixation for Ki-67 plus permeabilization (with 0.1% and 1% Triton X100, respectively, for the surface and the nuclear antigens) produced cell suspensions with negligible cell clumping, high-quality DNA profiles, and bright specific immunofluorescent staining. The investigated proteins have different relationships with the proliferative state of the cell. Trf-r is expressed mainly at the transition from G0/G1 to S-phase. PCNA expression is prominent in late G1 and through S-phase and decreases in G2-M. The Ki-67-reactive antigen is widely distributed in G1, S, and G2-M phases. Knowledge regarding the relationships between proliferation-associated antigens and cell cycle phase in normal and neoplastic cells could improve our understanding of the mechanisms underlying growth regulation and neoplastic transformation. Bivariate FCM is an easy method for obtaining these data from large numbers of cells.     

Role of p38 MAPK and RNA-dependent protein kinase (PKR) in hepatitis C virus core-dependent nuclear delocalization of cyclin B1

Some hepatitis C virus (HCV) proteins, including core protein, deregulate the cell cycle of infected cells, thereby playing an important role in the viral pathogenesis of HCC. Thus far, there are only few studies that have deeply investigated in depth the effects of the HCV core protein expression on the progression through the G1/S and G2/M phases of the cell cycle. To shed light on the molecular mechanisms by which the HCV core protein modulates cell proliferation, we have examined its effects on cell cycle in hepatocarcinoma cells. We show here that HCV core protein perturbs progression through both the G1/S and the G2/M phases, by modulating the expression and the activity of several cell cycle regulatory proteins. In particular, our data provided evidence that core-dependent deregulation of the G1/S phase and its related cyclin-CDK complexes depends upon the ERK1/2 pathway. On the other hand, the viral protein also increases the activity of the cyclin B1-CDK1 complex via the p38 MAPK and JNK pathways. Moreover, we show that HCV core protein promotes nuclear import of cyclin B1, which is affected by the inhibition of both the p38 and the RNA-dependent protein kinase (PKR) activities. The important role of p38 MAPK in regulating G2/M phase transition has been previously documented. It is becoming clear that PKR has an important role in regulating both the G1/S and the G2/M phase, in which it induces M phase arrest. Based on our model, we now show, for the first time, that HCV core expression leads to deregulation of the mitotic checkpoint via a p38/PKR-dependent pathway.     

Correlation of reduced chilling injury with Increased spermine and spermidine levels in zucchini squash

By means of a biparametric cytofluorimetric analysis it is possible to distribute meristematic plant cells in a variety of cell cycle sub-compartments, unidentifiable by DNA measurements alone. In this work, an asynchronous proliferating cell population of pea root meristems was divided into different sub-compartments of the cell cycle phases, i.e. G1A, G1B, S. G2A and G2B on the basis of their DNA-nuclear protein content. By means of the same biparametric analysis, differentiated mesophyll cells and quiescent cells of embryo roots, indicated as G0 and G2Q, were distinguished from cycling cells by their low nuclear protein content. These results conform to those of some analyses performed on animal cells in culture and show that it is possible to get a major insight into cell cycle kinetics and its control in a natural system such as root meristem.     

Flow cytometric multiparameter analysis of proliferating cell nuclear antigen/cyclin and Ki-67 antigen: a new view of the cell cycle

Flow cytometric multiparameter analysis of two proliferation-associated nuclear antigens (proliferating cell nuclear antigen (PCNA)/cyclin and Ki-67) was performed on seven human hematopoietic cell lines. PCNA/cyclin, an S phase-related antigen, was detected using an autoantibody and a fluorescein isothiocyanate-labeled anti-human antibody. The Ki-67 antigen, which in cycling cells is expressed with increasing levels during the S phase with a maximum in the M phase, was detected using a monoclonal antibody and a phycoerythrin-conjugated anti-mouse antibody. In some experiments the PCNA/Ki-67 staining was combined with a DNA stain, 7-amino actinomycin D, and simultaneous detection of the three stains was performed by a single laser flow cytometer. Using this technique four distinct cell populations, representing G1, S, G2, and M, respectively, could be demonstrated in cycling cells on the basis of their PCNA/cyclin and Ki-67 levels. The cell cycle phase specificity could be verified using metaphase (vinblastine, colcemide) and G2 phase (mitoxantrone) blocking agents, as well as by stainings with a mitosis-specific antibody (MPM-2). Also, G0 cells could be discriminated from G1 cells in analysis of a mixture of resting peripheral mononuclear blood cells and a proliferating cell line. This technique can be valuable in detailed cell cycle analysis, since all cell cycle phases can be visualized and calculated using a simple double staining procedure.     

Two Molecularly Distinct G2/M Checkpoints Are Induced by Ionizing Irradiation

Cell cycle checkpoints are among the multiple mechanisms that eukaryotic cells possess to maintain genomic integrity and minimize tumorigenesis. Ionizing irradiation (IR) induces measurable arrests in the G(1), S, and G(2) phases of the mammalian cell cycle, and the ATM (ataxia telangiectasia mutated) protein plays a role in initiating checkpoint pathways in all three of these cell cycle phases. However, cells lacking ATM function exhibit both a defective G(2) checkpoint and a prolonged G(2) arrest after IR, suggesting the existence of different types of G(2) arrest. Two molecularly distinct G(2)/M checkpoints were identified, and the critical importance of the choice of G(2)/M checkpoint assay was demonstrated. The first of these G(2)/M checkpoints occurs early after IR, is very transient, is ATM dependent and dose independent (between 1 and 10 Gy), and represents the failure of cells which had been in G(2) at the time of irradiation to progress into mitosis. Cell cycle assays that can distinguish mitotic cells from G(2) cells must be used to assess this arrest. In contrast, G(2)/M accumulation, typically assessed by propidium iodide staining, begins to be measurable only several hours after IR, is ATM independent, is dose dependent, and represents the accumulation of cells that had been in earlier phases of the cell cycle at the time of exposure to radiation. G(2)/M accumulation after IR is not affected by the early G(2)/M checkpoint and is enhanced in cells lacking the IR-induced S-phase checkpoint, such as those lacking Nbs1 or Brca1 function, because of a prolonged G(2) arrest of cells that had been in S phase at the time of irradiation. Finally, neither the S-phase checkpoint nor the G(2) checkpoints appear to affect survival following irradiation. Thus, two different G(2) arrest mechanisms are present in mammalian cells, and the type of cell cycle checkpoint assay to be used in experimental investigation must be thoughtfully selected.