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Search results for: Recombinant Human GMNN Proteins

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#34085231   // To Up

Live Imaging and Analysis of Cilia and Cell Cycle Dynamics with the Arl13bCerulean-Fucci2a Biosensor and Fucci Tools.

The cell and cilia cycles are inextricably linked through the dual functions of the centrioles at both the basal body of cilia and at mitotic centrosomes. How cilia assembly and disassembly, either through slow resorption or rapid deciliation, are coordinated with cell cycle progression remains unclear in many cell types and developmental paradigms. Moreover, little is known about how additional cilia parameters including changes in ciliary length or frequency of distal tip shedding change with cell cycle stage. In order to explore these questions, we have developed the Arl13bCerulean-Fucci2a tricistronic cilia and cell cycle biosensor (Ford et al., Dev Cell 47:509-523.e7, 2018). This reporter allowed us to document the heterogeneity in ciliary behaviors during the cell cycle at a population level. Without the need for external stimuli, it revealed that in several cell types and in the developing embryo cilia persist beyond the G1/S checkpoint. Here, we describe the generation of stable cell lines expressing Arl13bCerulean-Fucci2a and open-source software to aid morphometric profiling of the primary cilium with cell cycle phases, including changes in cilium length. This resource will allow the investigation of multiple morphometric questions relating to cilia and cell cycle biology.
Melinda Van Kerckvoorde, Matthew J Ford, Patricia L Yeyati, Pleasantine Mill, Richard L Mort

2150 related Products with: Live Imaging and Analysis of Cilia and Cell Cycle Dynamics with the Arl13bCerulean-Fucci2a Biosensor and Fucci Tools.

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#27195810   2016/05/19 To Up

Manipulation of Cell Cycle and Chromatin Configuration by Means of Cell-Penetrating Geminin.

Geminin regulates chromatin remodeling and DNA replication licensing which play an important role in regulating cellular proliferation and differentiation. Transcription of the Geminin gene is regulated via an E2F-responsive region, while the protein is being closely regulated by the ubiquitin-proteasome system. Our objective was to directly transduce Geminin protein into cells. Recombinant cell-penetrating Geminin (CP-Geminin) was generated by fusing Geminin with a membrane translocating motif from FGF4 and was efficiently incorporated into NIH 3T3 cells and mouse embryonic fibroblasts. The withdrawal study indicated that incorporated CP-Geminin was quickly reduced after removal from medium. We confirmed CP-Geminin was imported into the nucleus after incorporation and also that the incorporated CP-Geminin directly interacted with Cdt1 or Brahma/Brg1 as the same manner as Geminin. We further demonstrated that incorporated CP-Geminin suppressed S-phase progression of the cell cycle and reduced nuclease accessibility in the chromatin, probably through suppression of chromatin remodeling, indicating that CP-Geminin constitutes a novel tool for controlling chromatin configuration and the cell cycle. Since Geminin has been shown to be involved in regulation of stem cells and cancer cells, CP-Geminin is expected to be useful for elucidating the role of Geminin in stem cells and cancer cells, and for manipulating their activity.
Yoshinori Ohno, Kyoko Suzuki-Takedachi, Shin'ichiro Yasunaga, Toshiaki Kurogi, Mimoko Santo, Yoshikazu Masuhiro, Shigemasa Hanazawa, Motoaki Ohtsubo, Kazuhito Naka, Yoshihiro Takihara

1246 related Products with: Manipulation of Cell Cycle and Chromatin Configuration by Means of Cell-Penetrating Geminin.

1 kit2 Pieces/Box 100ul2 Pieces/Box2 Pieces/Box1 kit400 ug10 Plates 25 ml 100 TestsOne 96-Well Microplate Ki

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#26854237   2016/02/04 To Up

Post-translational Regulation of Cas9 during G1 Enhances Homology-Directed Repair.

CRISPR/Cas9 induces DNA double-strand breaks that are repaired by cell-autonomous repair pathways, namely, non-homologous end-joining (NHEJ), or homology-directed repair (HDR). While HDR is absent in G1, NHEJ is active throughout the cell cycle and, thus, is largely favored over HDR. We devised a strategy to increase HDR by directly synchronizing the expression of Cas9 with cell-cycle progression. Fusion of Cas9 to the N-terminal region of human Geminin converted this gene-editing protein into a substrate for the E3 ubiquitin ligase complex APC/Cdh1, resulting in a cell-cycle-tailored expression with low levels in G1 but high expression in S/G2/M. Importantly, Cas9-hGem(1/110) increased the rate of HDR by up to 87% compared to wild-type Cas9. Future developments may enable high-resolution expression of genome engineering proteins, which might increase HDR rates further, and may contribute to a better understanding of DNA repair pathways due to spatiotemporal control of DNA damage induction.
Tony Gutschner, Monika Haemmerle, Giannicola Genovese, Giulio F Draetta, Lynda Chin

2798 related Products with: Post-translational Regulation of Cas9 during G1 Enhances Homology-Directed Repair.

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#23814078   2013/06/29 To Up

Cell cycle-dependent subcellular translocation of the human DNA licensing inhibitor geminin.

Once per cell cycle replication is crucial for maintaining genome integrity. Geminin interacts with the licensing factor Cdt1 to prevent untimely replication and is controlled by APC/C-dependent cell cycle specific proteolysis during mitosis and in G1. We show here that human geminin, when expressed in human cells in culture under a constitutive promoter, is excluded from the nucleus during part of the G1 phase and at the transition from G0 to G1. The N-terminal 30 amino acids of geminin, which contain its destruction box, are essential for nuclear exclusion. In addition, 30 amino acids within the central domain of geminin are required for both nuclear exclusion and nuclear accumulation. Cdt1 overexpression targets geminin to the nucleus, while reducing Cdt1 levels by RNAi leads to the appearance of endogenous geminin in the cytoplasm. Our data propose a novel means of regulating the balance of Cdt1/geminin in human cells, at the level of the subcellular localization of geminin.
Maria Dimaki, Georgia Xouri, Ioanna-Eleni Symeonidou, Chaido Sirinian, Hideo Nishitani, Stavros Taraviras, Zoi Lygerou

2380 related Products with: Cell cycle-dependent subcellular translocation of the human DNA licensing inhibitor geminin.

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#21382497   2011/03/05 To Up

Expression of protein complexes using multiple Escherichia coli protein co-expression systems: a benchmarking study.

Escherichia coli (E. coli) remains the most commonly used host for recombinant protein expression. It is well known that a variety of experimental factors influence the protein production level as well as the solubility profile of over-expressed proteins. This becomes increasingly important for optimizing production of protein complexes using co-expression strategies. In this study, we focus on the effect of the choice of the expression vector system: by standardizing experimental factors including bacterial strain, cultivation temperature and growth medium composition, we compare the effectiveness of expression technologies used by the partners of the Structural Proteomics in Europe 2 (SPINE2-complexes) consortium. Four different protein complexes, including three binary and one ternary complex, all known to be produced in the soluble form in E. coli, are used as the benchmark targets. The respective genes were cloned by each partner into their preferred set of vectors. The resulting constructs were then used for comparative co-expression analysis done in parallel and under identical conditions at a single site. Our data show that multiple strategies can be applied for the expression of protein complexes in high yield. While there is no 'silver bullet' approach that was infallible even for this small test set, our observations are useful as a guideline to delineate co-expression strategies for particular protein complexes.
Didier Busso, Yoav Peleg, Tatjana Heidebrecht, Christophe Romier, Yossi Jacobovitch, Ada Dantes, Loubna Salim, Edouard Troesch, Anja Schuetz, Udo Heinemann, Gert E Folkers, Arie Geerlof, Matthias Wilmanns, Andrea Polewacz, Claudia Quedenau, Konrad Büssow, Rachel Adamson, Elena Blagova, Julia Walton, Jared L Cartwright, Louise E Bird, Raymond J Owens, Nick S Berrow, Keith S Wilson, Joel L Sussman, Anastassis Perrakis, Patrick H N Celie

1135 related Products with: Expression of protein complexes using multiple Escherichia coli protein co-expression systems: a benchmarking study.

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#19906994   2009/11/11 To Up

Quaternary structure of the human Cdt1-Geminin complex regulates DNA replication licensing.

All organisms need to ensure that no DNA segments are rereplicated in a single cell cycle. Eukaryotes achieve this through a process called origin licensing, which involves tight spatiotemporal control of the assembly of prereplicative complexes (pre-RCs) onto chromatin. Cdt1 is a key component and crucial regulator of pre-RC assembly. In higher eukaryotes, timely inhibition of Cdt1 by Geminin is essential to prevent DNA rereplication. Here, we address the mechanism of DNA licensing inhibition by Geminin, by combining X-ray crystallography, small-angle X-ray scattering, and functional studies in Xenopus and mammalian cells. Our findings show that the Cdt1:Geminin complex can exist in two distinct forms, a "permissive" heterotrimer and an "inhibitory" heterohexamer. Specific Cdt1 residues, buried in the heterohexamer, are important for licensing. We postulate that the transition between the heterotrimer and the heterohexamer represents a molecular switch between licensing-competent and licensing-defective states.
V De Marco, P J Gillespie, A Li, N Karantzelis, E Christodoulou, R Klompmaker, S van Gerwen, A Fish, M V Petoukhov, M S Iliou, Z Lygerou, R H Medema, J J Blow, D I Svergun, S Taraviras, A Perrakis

2664 related Products with: Quaternary structure of the human Cdt1-Geminin complex regulates DNA replication licensing.

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#19732753   2009/09/02 To Up

Visualization of radiation-induced cell cycle-associated events in tumor cells expressing the fusion protein of Azami Green and the destruction box of human Geminin.

Ionizing radiation (IR) influences cell cycle-associated events in tumor cells. We expressed the fusion protein of Azami Green (AG) and the destruction box plus nuclear localization signal of human Geminin, an inhibitor of DNA replication licensing factor, in oral tumor cells. This approach allowed us to visualize G2 arrest in living cells following irradiation. The combination of time-lapse imaging analysis allowed us to observe the nuclear envelope break down (NEBD) at early M phase, and disappearance of fluorescence (DF) at the end of M phase. The duration from NEBD to DF was not much affected in irradiated cells; however, most of daughter cells harbored double-strand breaks. Complete DF was also observed in cells exhibiting abnormal mitosis or cytokinesis. We conclude that the fluorescent Geminin probe could function as a stable cell cycle indicator irrespective of genome integrity.
Mayuko Ishikawa, Yusuke Ogihara, Masahiko Miura

2601 related Products with: Visualization of radiation-induced cell cycle-associated events in tumor cells expressing the fusion protein of Azami Green and the destruction box of human Geminin.

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#18650381   2008/07/23 To Up

Polycomb-group complex 1 acts as an E3 ubiquitin ligase for Geminin to sustain hematopoietic stem cell activity.

Polycomb-group (PcG) genes encode multimeric nuclear protein complexes, PcG complex 1 and 2. PcG complex 2 was proved to induce transcription repression and to further methylate histone H3 at lysine-27 (H3K27). Subsequently PcG complex 1 is recruited through recognition of methylated H3K27 and maintains the transcription silencing by mediating monoubiquitination of histone H2A at lysine-119. Genetic evidence demonstrated a crucial role for PcG complex 1 in stem cells, and Bmi1, a member of PcG complex 1, was shown to sustain adult stem cells through direct repression of the INK4a locus encoding cyclin-dependent kinase inhibitor, p16CKI, and p19ARF. The molecular functions of PcG complex 1, however, remain insufficiently understood. In our study, deficiency of Rae28, a member of PcG complex 1, was found to impair ubiquitin-proteasome-mediated degradation of Geminin, an inhibitor of DNA replication licensing factor Cdt1, and to increase protein stability. The resultant accumulation of Geminin, based on evidence from retroviral transduction experiments, presumably eliminated hematopoietic stem cell activity in Rae28-deficient mice. Rae28 mediates recruiting Scmh1, which provides PcG complex 1 an interaction domain for Geminin. Moreover, PcG complex 1 acts as the E3 ubiquitin ligase for Geminin, as we demonstrated in vivo as well as in vitro by using purified recombinant PcG complex 1 reconstituted in insect cells. Our findings suggest that PcG complex 1 supports the activity of hematopoietic stem cells, in which high-level Geminin expression induces quiescence securing genome stability, by enhancing cycling capability and hematopoietic activity through direct regulation of Geminin.
Motoaki Ohtsubo, Shin'ichiro Yasunaga, Yoshinori Ohno, Miyuki Tsumura, Satoshi Okada, Nobutsune Ishikawa, Kenichiro Shirao, Akira Kikuchi, Hideo Nishitani, Masao Kobayashi, Yoshihiro Takihara

2849 related Products with: Polycomb-group complex 1 acts as an E3 ubiquitin ligase for Geminin to sustain hematopoietic stem cell activity.

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#18267078   // To Up

Visualizing spatiotemporal dynamics of multicellular cell-cycle progression.

The cell-cycle transition from G1 to S phase has been difficult to visualize. We have harnessed antiphase oscillating proteins that mark cell-cycle transitions in order to develop genetically encoded fluorescent probes for this purpose. These probes effectively label individual G1 phase nuclei red and those in S/G2/M phases green. We were able to generate cultured cells and transgenic mice constitutively expressing the cell-cycle probes, in which every cell nucleus exhibits either red or green fluorescence. We performed time-lapse imaging to explore the spatiotemporal patterns of cell-cycle dynamics during the epithelial-mesenchymal transition of cultured cells, the migration and differentiation of neural progenitors in brain slices, and the development of tumors across blood vessels in live mice. These mice and cell lines will serve as model systems permitting unprecedented spatial and temporal resolution to help us better understand how the cell cycle is coordinated with various biological events.
Asako Sakaue-Sawano, Hiroshi Kurokawa, Toshifumi Morimura, Aki Hanyu, Hiroshi Hama, Hatsuki Osawa, Saori Kashiwagi, Kiyoko Fukami, Takaki Miyata, Hiroyuki Miyoshi, Takeshi Imamura, Masaharu Ogawa, Hisao Masai, Atsushi Miyawaki

1330 related Products with: Visualizing spatiotemporal dynamics of multicellular cell-cycle progression.

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#17318181   2007/02/22 To Up

Cdt1 associates dynamically with chromatin throughout G1 and recruits Geminin onto chromatin.

To maintain genome integrity, eukaryotic cells initiate DNA replication once per cell cycle after assembling prereplicative complexes (preRCs) on chromatin at the end of mitosis and during G1. In S phase, preRCs are disassembled, precluding initiation of another round of replication. Cdt1 is a key member of the preRC and its correct regulation via proteolysis and by its inhibitor Geminin is essential to prevent premature re-replication. Using quantitative fluorescence microscopy, we study the interactions of Cdt1 with chromatin and Geminin in living cells. We find that Cdt1 exhibits dynamic interactions with chromatin throughout G1 phase and that the protein domains responsible for chromatin and Geminin interactions are separable. Contrary to existing in vitro data, we show that Cdt1 simultaneously binds Geminin and chromatin in vivo, thereby recruiting Geminin onto chromatin. We propose that dynamic Cdt1-chromatin associations and the recruitment of Geminin to chromatin provide spatio-temporal control of the licensing process.
Georgia Xouri, Anthony Squire, Maria Dimaki, Bart Geverts, Peter J Verveer, Stavros Taraviras, Hideo Nishitani, Adriaan B Houtsmuller, Philippe I H Bastiaens, Zoi Lygerou

1900 related Products with: Cdt1 associates dynamically with chromatin throughout G1 and recruits Geminin onto chromatin.

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