Strict regulation of DNA replication is essential to ensure proper duplication and segregation of chromosomes during the cell cycle, as its deregulation can lead to genomic instability and cancer. Thus, eukaryotic organisms have evolved multiple mechanisms to restrict DNA replication to once per cell cycle. Here, we show that inactivation of Geminin, an inhibitor of origin licensing, leads to rereplication in human normal and tumor cells within the same cell cycle. We found a CHK1-dependent checkpoint to be activated in rereplicating cells accompanied by formation of γH2AX and RAD51 nuclear foci. Abrogation of the checkpoint leads to abortive mitosis and death of rereplicated cells. In addition, we demonstrate that the induction of rereplication is dependent on the replication initiation factors CDT1 and CDC6, and independent of the functional status of p53. These data show that Geminin is required for maintaining genomic stability in human cells.

Geminin is an unstable inhibitor of DNA replication that negatively regulates the licensing factor CDT1 and inhibits pre-replicative complex (pre-RC) formation in Xenopus egg extracts. Here we describe a novel function of Geminin. We demonstrate that human Geminin protects CDT1 from proteasome-mediated degradation by inhibiting its ubiquitination. In particular, Geminin ensures basal levels of CDT1 during S phase and its accumulation during mitosis. Consistently, inhibition of Geminin synthesis during M phase leads to impairment of pre-RC formation and DNA replication during the following cell cycle. Moreover, we show that inhibition of CDK1 during mitosis, and not Geminin depletion, is sufficient for premature formation of pre-RCs, indicating that CDK activity is the major mitotic inhibitor of licensing in human cells. Taken together with recent data from our laboratory, our results demonstrate that Geminin is both a negative and positive regulator of pre-RC formation in human cells, playing a positive role in allowing CDT1 accumulation in G2-M, and preventing relicensing of origins in S-G2.

ES cells proliferate with very short gap phases yet maintain their capacity to differentiate. It had been thought that the levels of cyclins and other substrates of ubiquitin ligase APC/C remain nearly constant and Cdk activity remains constitutively high in mouse ES cells. Here we demonstrate that APC/C (anaphase-promoting complex/cyclosome) enzyme is active in ES cells but attenuated by high levels of the Emi1 (early mitotic inhibitor-1) protein. Despite the presence of high Cdk activity during the G1 phase, chromatin can be effectively licensed for DNA replication and fast entry into the S phase can still occur. High Cdk activity during S-G2-M phases produces high levels of the DNA replication factor Cdt1, and this leads to efficient Mcm proteins loading on chromatin after mitotic exit. Although disturbing the usual balance between Cdk activity and APC/C activity found in somatic cells, a few key adaptations allow normal progression of a very rapid cell cycle.E mbryonic stem cells show unusual cell-cycle features: the duration of the S phase is comparable to somatic cells but they have remarkably short G1 and G2 phases (1-3). In somatic cells, the duration of G1 and G2 is determined by relative levels of Cdk kinase activity and other cell cycle-related proteins (4). Many of these proteins, including Cyclin A, Cyclin B, Cdt1, Cdc6, and Geminin fluctuate along the cell cycle because of degradation mediated by E3 ubiquitin ligase APC/C (anaphase-promoting complex/ cyclosome) together with E2 enzymes, such as UbcH10 and UBE2S (5-8). APC/C is activated at the end of mitosis by interaction with Cdc20 and Cdh1 proteins and inactivated just before the S phase by the pseudosubstrate inhibitor Emi1 (early mitotic inhibitor-1) and by the phosphorylation and degradation of Cdh1 (6, 9, 10). Cdk kinases are activated by Cyclins and phosphorylate a number of cell-cycle proteins important for mitotic and S phase progression. Cdk activity is inhibited during G1 in somatic cells because of degradation of Cyclins and presence of inhibitor proteins, like p21 (11). Inhibition of Cdk activity in the G1 phase allows the replication factors Cdt1 and Cdc6 to recruit Mcm proteins on chromatin, form prereplicative complexes (pre-RCs), and license DNA for replication (12)(13)(14). Geminin protein inhibits Cdt1 during the S phase and promotes its stabilization during mitosis (3,13,(15)(16)(17)(18)(19)(20). A puzzling feature of ES cells is that APC/C substrates were shown to be constant and Cdk activity to be high throughout the ES cell cycle (1,3,21), raising the question of whether the APC/C complex is functional and how ES cells regulate pre-RC assembly at G1. Remarkably, APC/C substrates and other positive cell-cycle regulators decrease after differentiation (1,3,22). We carefully reinvestigated cell-cycle dynamics in ES cells. Contrary to previous conclusions, APC/C substrate levels and Cdk activity both oscillate, although in a more muted manner compared with most studied somatic models. A few key adaptations promote an abbreviated...

In somatic cells, the length of the G1 phase of the cell cycle is tightly linked to differentiation, and its elongation can drive differentiation in many cases. Although it has been suggested that the situation is very similar in embryonic stem cells (ESCs), where a rapid cell cycle and a short G1 phase maintain the pluripotent state, evidence has been contradictory. Here we show that, in murine ESCs, elongation of the cell cycle and elongation of G1 are compatible with their pluripotent state. Multiple methods that lengthen the cell cycle and that target cyclin-dependent kinase, retinoblastoma protein, and E2F activity all fail to induce differentiation on their own or even to facilitate differentiation. The resistance of murine ESCs to differentiation induced by lengthening G1 and/or the cell cycle could allow for separate control of these events and provide new opportunities for investigation and application.stemness | proliferation | decoupling

Rapid progression through the cell cycle and a very short G1 phase are defining characteristics of embryonic stem cells. This distinct cell cycle is driven by a positive feedback loop involving Rb inactivation and reduced oscillations of cyclins and cyclin-dependent kinase (Cdk) activity. In this setting, we inquired how ES cells avoid the potentially deleterious consequences of premature mitotic entry. We found that the pluripotency transcription factor Oct4 (octamer-binding transcription factor 4) plays an unappreciated role in the ES cell cycle by forming a complex with cyclin-Cdk1 and inhibiting Cdk1 activation. Ectopic expression of Oct4 or a mutant lacking transcriptional activity recapitulated delayed mitotic entry in HeLa cells. Reduction of Oct4 levels in ES cells accelerated G2 progression, which led to increased chromosomal missegregation and apoptosis. Our data demonstrate an unexpected nontranscriptional function of Oct4 in the regulation of mitotic entry. Cell cycle progression is driven by the activation and inactivation of cyclin-dependent kinases (Cdks). The G1-to-S phase transition is regulated by cyclins E and A, both of which activate Cdk2 (5-8). The retinoblastoma protein (Rb) maintains the G1 phase by inhibiting the E2F transcription factors. In somatic cells, increased Cdk activity at the end of G1 leads to phosphorylation and inactivation of Rb and increased expression of E2F targets, including cyclins E and A2 (hereafter A) (9, 10). This feedback, in which rising cyclin levels inactivate Rb to promote further increases in cyclins E and A, mediates the transition to S phase. The G2 to mitosis (M) phase transition is governed by activation of Cdk1, primarily through cyclins A and B1 (hereafter B). Cells synthesize cyclin B in interphase, but cyclin B-bound Cdk1 remains inactive due to phosphorylation on residues T14 and Y15 by Myt1/Wee1 kinases (11-13). Once cyclin B reaches a threshold concentration, the Myt1/Wee1 kinases are inhibited, and the CDC25 phosphatases are activated (14), which leads to cyclin B-bound Cdk1 activation and mitotic entry. Cyclin A promotes the early stage of mitosis in particular, and a critical part of this role is activating cyclin B-Cdk1 by weakening the Wee1-mediated inhibition of cyclin B-Cdk1 and promoting the nuclear localization of cyclin B (7,(15)(16)(17)(18)(19)(20).Cell cycle regulation is noticeably altered in ES cells to allow for more rapid cell proliferation. In contrast to somatic cells, Rb is inactive throughout the ES cell cycle due to constitutive phosphorylation (3), which allows for increased expression of cyclins E and A and extended cyclin E/A-Cdk2 activity (4, 21, 22). Moreover, cyclin-dependent kinase inhibitors (CKIs) such as p16, p21, and p27 are absent or expressed at low abundance in ES cells, which further unleash Cdk activity (4, 22-24). The dampened oscillation of Cdk activity is at least partially responsible for the rapid cell cycle progression by reducing the time needed for Cdk activation.Although the features producing a shorte...

Automated image-based 3D reconstruction methods are more and more flooding our 3D modeling applications. Fully automated solutions give the impression that from a sample of randomly acquired images we can derive quite impressive visual 3D models. Although the level of automation is reaching very high standards, image quality is a fundamental pre-requisite to produce successful and photo-realistic 3D products, in particular when dealing with large datasets of images. This article presents an efficient pipeline based on color enhancement, image denoising, color-to-gray conversion and image content enrichment. The pipeline stems from an analysis of various state-of-the-art algorithms and aims to adjust the most promising methods, giving solutions to typical failure causes. The assessment evaluation proves how an effective image pre-processing, which considers the entire image dataset, can improve the automated orientation procedure and dense 3D point cloud reconstruction, even in the case of poor texture scenarios.

This paper reports the latest developments for the photogrammetric open‐source tool called GRAPHOS (inteGRAted PHOtogrammetric Suite). GRAPHOS includes some recent innovations in the image‐based 3D reconstruction pipeline, from automatic feature detection/description and network orientation to dense image matching and quality control. GRAPHOS also has a strong educational component beyond its automated processing functions, reinforced with tutorials and didactic explanations about algorithms and performance. The paper highlights recent developments carried out at different levels: graphical user interface (GUI), didactic simulators for image processing, photogrammetric processing with weight parameters, dataset creation and system evaluation.

Every day new tools and algorithms for automated image processing and 3D reconstruction purposes become available, giving the possibility to process large networks of unoriented and markerless images, delivering sparse 3D point clouds at reasonable processing time. In this paper we evaluate some feature-based methods used to automatically extract the tie points necessary for calibration and orientation procedures, in order to better understand their performances for 3D reconstruction purposes. The performed tests -based on the analysis of the SIFT algorithm and its most used variants -processed some datasets and analysed various interesting parameters and outcomes (e.g. number of oriented cameras, average rays per 3D points, average intersection angles per 3D points, theoretical precision of the computed 3D object coordinates, etc.).