Kisielewska J and Blow JJ (2012), Dynamic interactions of high Cdt1 and geminin l...

XB-ART-44478

Development 2012 Jan 01;1391:63-74. doi: 10.1242/dev.068676.

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Dynamic interactions of high Cdt1 and geminin levels regulate S phase in early Xenopus embryos.

Kisielewska J , Blow JJ .

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Cdt1 plays a key role in licensing DNA for replication. In the somatic cells of metazoans, both Cdt1 and its natural inhibitor geminin show reciprocal fluctuations in their protein levels owing to cell cycle-dependent proteolysis. Here, we show that the protein levels of Cdt1 and geminin are persistently high during the rapid cell cycles of the early Xenopus embryo. Immunoprecipitation of Cdt1 and geminin complexes, together with their cell cycle spatiotemporal dynamics, strongly supports the hypothesis that Cdt1 licensing activity is regulated by periodic interaction with geminin rather than its proteolysis. Overexpression of ectopic geminin slows down, but neither arrests early embryonic cell cycles nor affects endogenous geminin levels; apparent embryonic lethality is observed around 3-4 hours after mid-blastula transition. However, functional knockdown of geminin by ΔCdt1_193-447, which lacks licensing activity and degradation sequences, causes cell cycle arrest and DNA damage in affected cells. This contributes to subsequent developmental defects in treated embryos. Our results clearly show that rapidly proliferating early Xenopus embryonic cells are able to regulate replication licensing in the persistent presence of high levels of licensing proteins by relying on changing interactions between Cdt1 and geminin during the cell cycle, but not their degradation.

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Species referenced: Xenopus laevis
Genes referenced: actl6a cdc6 cdt1 gmnn mcm2 orc1 orc2 pcna rad51 tp53 tubg1
???displayArticle.antibodies??? Cdc6 Ab2 Mcm2 Ab3 Orc1 Ab1 Orc2 Ab1

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	Fig. 1. Cdt1 and geminin cellular levels. (A,B) Synchronised embryos from stage 1 (A) and stage 7 (B) were used. Extract from one oocyte, egg or embryo was loaded per time point onto a gel and immunobloted for Orc1 or Orc2, Cdt1, Cdc6 and geminin. The experiment was repeated with different batches of embryos derived from different frogs with similar results (n=5 for A and B separately). β-Actin is a loading control; asterisks indicate unspecific binding. (C) Isolated chromatin from synchronised embryos stage 7 was immunoblotted for Orc1, Cdc6, PCNA, Cdt1 and geminin. One full cell cycle can be detected between 4.15 and 4.40 hours. Accumulation of Cdc6 and PCNA indicates that S phase occurs between 4.25 and 4.30 hours. (D) DNAse-treated and control chromatin samples from 4.30 hours AF.
	Fig. 2. Cdt1:geminin interaction during early cell cycles. (A) Cdt1:geminin complex in unfertilised eggs compared with IgG mock antibody. (B) Cdt1 and geminin immunoprecipitation showing mutual interactions during the first 20 minutes. The complex is still visible within the first 10 minutes followed by weaker interaction during the licensing at 20 minutes marked by arrows. (C) Cdt1 immunoprecipitation showing 120 minutes after fertilisation covering the first and second cell cycle, as indicated by cyclin B1 in the same cell cycle (lower panel). Black arrow indicates first decline in geminin binding. Red arrowheads indicate cytokinesis at 80 and 120 minutes. Asterisks indicate unspecific binding.
	Fig. 3. Cellular distribution of Cdt1 and geminin in early embryos. (A,B) Immunofluorescence of geminin (A) and Cdt1 (B) using embryonic slices from stage 7. (1,2) Anaphase from two different angles. Nuclear fluorescence of geminin and Cdt1 is detected during S phase. (B) Cdt1, together with Mcm2, bind strongly to chromosomes at the end of anaphase. Scale bars: 20 μm. (C) Control for polyclonal anti-rabbit antibody with rabbit IgG. Merge between DAPI (blue) and IgG (red) is shown. Scale bar: 20 μm. (D) GFP-Cdt1 distribution in Xenopus egg extract depends on sperm DNA concentration. GFP-Cdt1 (6 μg/ml) was added to the extract supplemented either with 20 or 0.3 ng DNA/μl. The extract was fixed at indicated time points. White outline indicates nuclear border, arrows show GFP-Cdt1 in nucleoplasm. Scale bars: 20 μm.
	Fig. 4. Overexpression of geminin slows down but does not prevent cell cycle progression before MBT. (A) Geminin-RFP mRNA was injected into stage 1 and stage 2 embryos. Control embryos were injected with ×0.5 PBS. Embryonic development was followed by live macroimaging. (B,C) Immunodetection of RFP and geminin in extracts from embryos at different times after injection. RFP was detected at ∼72 kDa, suggesting that expression of mRNA occurred. Geminin antibody recognised both geminin-RFP at 72 kDa and endogenous protein at 36 kDa. (D) Injected embryos were fixed at 6 hours AF and geminin-RFP expression was detected by confocal/two-photon single cell imaging. Scale bar: 20 μm. (E) Geminin-RFP was injected into one cell of the four-cell embryo and post-MBT embryonic progression was monitored. Arrows show an injected region that does not develop further after 24 hours.
	Fig. 5. Neutralisation of Geminin exposes the role of Cdt1 in early cleavages. (A) Schematic diagram of δCdt1_193-447 protein with activity to neutralise geminin. (B) Recombinant δCdt1_193-447 activity was measured in Xenopus egg extract. GemininDEL (1.8 μg/ml) blocked DNA replication. Different concentrations of δCdt1_193-447 (2-12 μg/ml) were used to restore DNA synthesis. (C) Recombinant δCdt1_193-447 was injected into stage 1 and 2 embryos. Arrows show injected cells at stage 6 and 7. For a positive control, embryos were injected either with gemininDEL (1.8 ng/μl) or RFP-PCNA (1.6 pM) recombinant protein into stage 1. Scale bar: 500 μm. (D) δCdt1_193-447 injected into animal pole triggers their arrest during the following cell cycle. Arrows indicate three affected cells on the surface of an embryo. (E) δCdt1_193-447 mRNA (14 ng) was injected into animal pole at 4.3 hours AF and embryos were imaged at 5.3 hours AF. Arrow shows a significant region of arrested cells. Scale bar: 500 μm. The control embryos were injected with ∼15 ng of RFP-PCNA mRNA (i) and mRNA expression confirmed by confocal microscopy at 5 hours (ii). (F) Neutralisation of geminin in a number of cells before MBT results in deformation of post-MBT embryos. Scale bar: 500 μm.
	Fig. 6. Disruption of the Cdt1 protein level triggers cell cycle arrest. (A) Increase in average DAPI fluorescence intensity between arrested and pre-MBT cells. A total of 12 nuclei from three independent experiments were used. Data are mean±s.e.m. (B,C) Arrested cells after δCdt1_193-447 injection at 4 hours AF were subjected to whole-mount immunofluorescence. (Bc) Nuclei of arrested cells contain particulate DNA distribution, unlike the uniform decondensed chromatin during typical S phase of early embryos at the same time (Ba), or organised replication factories (arrows) in post-MBT middle S-phase nuclei (Bb). Scale bars: 10 μm. (C) Pre-MBT control cells oscillate between S phase and mitosis. Cell treated with δCdt1_193-447 (a and b) are arrested in replication-like stage with Cdt1, geminin, Mcm2 and PCNA on chromatin. Scale bars: 20 μm. (D) Graphical comparison of control cycling cells (4.3 and 5 hours, S phase; 4.4-4.5, mitosis) and geminin-depleted cells (arrested cells).
	Fig. 7. Functional downregulation of geminin results in significant increase in p53 and RAD51 level on chromatin. Embryos treated with δCdt1_193-447 were fixed and subjected to immunofluorescence with anti-p53 and anti-RAD51. Confocal/two-photon microscopy was performed with the same detection settings for both control and arrested cells. (A,C) Accumulation of p53 and RAD51 on chromatin compared with control by average fluorescent intensity or confocal/two-photon single cell imaging. Graphs represent mean±s.e.m. of two separate experiments with 6-8 embryos for each protein used for quantification, ***P<0.001. (B,D) Confocal/two-photon images comparing the amount of p53 (B) and RAD51 (D) between control cycling cells and cells arrested using δCdt1_193-447. Scale bars: 20 μm.

References [+] :

Arias, PCNA functions as a molecular platform to trigger Cdt1 destruction and prevent re-replication. 2006, Pubmed, Xenbase