. 2021 Jan 5;4(3):e202000980. Show doi: 10.26508/lsa.202000980. Print 2021 Mar. Affiliations
Free PMC article Cyclin A2 localises in the cytoplasm at the S/G2 transition to activate PLK1Helena Silva Cascales et al. Life Sci Alliance. 2021. Free PMC article AbstractCyclin A2 is a key regulator of the cell cycle, implicated both in DNA replication and mitotic entry. Cyclin A2 participates in feedback loops that activate mitotic kinases in G2 phase, but why active Cyclin A2-CDK2 during the S phase does not trigger mitotic kinase activation remains unclear. Here, we describe a change in localisation of Cyclin A2 from being only nuclear to both nuclear and cytoplasmic at the S/G2 border. We find that Cyclin A2-CDK2 can activate the mitotic kinase PLK1 through phosphorylation of Bora, and that only cytoplasmic Cyclin A2 interacts with Bora and PLK1. Expression of predominately cytoplasmic Cyclin A2 or phospho-mimicking PLK1 T210D can partially rescue a G2 arrest caused by Cyclin A2 depletion. Cytoplasmic presence of Cyclin A2 is restricted by p21, in particular after DNA damage. Cyclin A2 chromatin association during DNA replication and additional mechanisms contribute to Cyclin A2 localisation change in the G2 phase. We find no evidence that such mechanisms involve G2 feedback loops and suggest that cytoplasmic appearance of Cyclin A2 at the S/G2 transition functions as a trigger for mitotic kinase activation. © 2021 Silva Cascales et al. Conflict of interest statementE Müllers is an employee of AstraZeneca. Other authors declare no competing interests. Figures
 (A) Time-lapse imaging through mitosis of a single RPE cell gene-targeted to express CycA2-eYFP. Time between images is 20 min. (B) Quantification of mean fluorescence intensity in the nucleus (top) and in the cytoplasm (middle) of 50 individual RPE CycA2-eYFP cells over time. Cells were synchronised in silico to set t = 0 at mitosis. Nuclear and cytoplasmic fluorescence in mitosis is approximated to central and peripheral regions of the mitotic cell to show a continuous graph. Please note that the increase in mean intensity during mitosis largely is due to cell rounding. Bottom graph shows the average of nuclear and cytoplasmic mean intensities. (C) Time-course estimate from fixed cells, pulse-labelled with EdU. 2,100 RPE-CycA2-eYFP cells were sorted based on increasing anti-GFP and DAPI staining (left) and 1,567 RPE cells were sorted based on increasing anti-CycA2 and DAPI staining (right). Graphs focus on area where CycA2 expression is apparent. Lower graphs show running median of 100 (left) or 50 (right) cells. (D) Western blot of nuclear and cytoplasmic fractions of unsynchronised RPE-CycA2-eYFP cells using the indicated antibodies. (E) Quantification of the percentage of RPE-CycA2-eYFP cells accumulating CycA2-eYFP in the cytoplasm after different treatments. Cells were treated with DMSO (control), Thymidine or Hydroxyurea and immediately imaged. The number of cells accumulating CycA2 in the cytoplasm was recorded and plotted as a percentage of the total number of cells tracked. All experiments were repeated at least three times. (A) Western blot of cell lines indicated. S-trityl-L-cysteine (Eg5 inhibitor) is added to arrest cells in mitosis, etoposide (topoisomerase 2 inhibitor) is added to create DNA damage. GAPDH is used as loading control. (B) Time-lapse images of U2OS CycA2-eYFP cell. Time between images is 20 min. (C) RPE CycA2-eYFP cells were incubated for 20 min with EdU and fixed. Left graphs shows quantification of nuclear and cytoplasmic integrated intensity of GFP staining versus nuclear DAPI intensity in at least 1,500 cells; each dot represents one cell. Middle graph shows nuclear versus cytoplasmic integrated intensities of GFP staining; the grey square indicates the gating for expressors of both nuclear and cytoplasmic CycA2-eYFP. Right graphs show integrated EdU intensity versus integrated DAPI intensity, with or without gating for expressors of both nuclear and cytoplasmic CycA2-eYFP (bottom right). (D) RPE cells were treated as in (C) and stained with CycA2 antibodies. At least 1,200 cells were quantified. (A) Time-lapse sequence of U2OS cells expressing polo-like kinase 1 FRET reporter and PCNA chromobody. Time points (h) are indicated in figure. Top Ctrl siRNA, bottom CycA2 siRNA. Scale bar 10 μm. (B) Representative quantifications of individual cells, imaged as in (A). Red line shows 1/FRET and blue line shows PCNA foci. Please note that both pattern of polo-like kinase 1 activation and pattern of PCNA foci are altered after CycA2 siRNA. PCNA foci fluorescence is normalised on the maximal value, and 1/FRET is normalised on the minimal value. (C) Schematic of siRNA-non-targetable CycA2 expression constructs. (D) Images show RPE p53−/− TetON OsTIR, co-transfected with Piggy-BAC transposase, GFP flanked by Piggy-BAC integration sites (to mark transfected cells) and the constructs outlined in (C) after 18 h Doxycycline addition. Scale bar 20 μm. (E) Quantification of mitotic entry in GFP-expressing cells. Unsynchronised polyclonal RPE p53−/− TetON OsTIR populations treated as in (D) were transfected with CycA2 siRNA. 48 h later, cells were transferred to L15 medium with or without Doxycycline and GFP-expressing cells were followed by time-lapse microscopy. At least 275 cells were followed per condition. Data from Fig 2E, with the addition of control siRNA treated cells (200 cells counted per condition). Please note that CycA2 siRNA leads to accumulation of G2 cells and that magnitude of rescue therefore cannot be determined by comparing control siRNA and CycA2 siRNA–treated cells. (A) Inhibition of cyclin dependent kinase (CDK) activity in U2OS cells expressing PLK1 FRET reporter. Cells with intermediate PLK1 FRET signal, indicative of G2 phase, were followed after addition of indicated inhibitors. Graph shows average and s.e.m of at least 10 cells per condition. (B) Inhibition of CDK activity in RPE cells synchronised in G2 decreases the level of PLK1 phosphorylation at T210. PLK1 blot is shown twice using a high and a low exposure. (C) Phosphorylation of Bora by CycA2-CDK2 promotes modification of PLK1 at T210 mediated by Aurora-A. Empty arrowhead indicates position of the kinase dead PLK1, full arrowhead indicates position of Bora. (D) CycA2-CDK2 can phosphorylate Bora on SP/TP sites. (E, F) Cytosolic and nuclear extracts from RPE cells synchronised in G2 phase were subjected to immunoprecipitation with anti-CycA2 (E) or anti-PLK1 (F) antibodies and bound proteins were probed with indicated antibodies. For (E), data using additional antibodies can be found in Fig 4C. (G) Expression of PLK1 T210D can partially rescue CycA2 siRNA-mediated cell cycle arrest. U2TR cells with inducible expression of PLK1 T210D were transfected with CycA2 siRNA. 20 h later, Doxycycline was added and cells were followed using phase contrast time-lapse microscopy. 300 cells per condition were followed manually over time. All experiments were repeated at least three times. (A) Quantification of nuclear (left) and cytoplasmic (right) accumulation of CycA2-eYFP in at least 17 single RPE-CycA2-eYFP cells treated with DMSO (top), RO-3306 (middle), or Nu6140 (bottom) for 4 h; each track represents a single cell. Cells were selected based on clear but low cytoplasmic CycA2-eYFP signal at time point of inhibitor addition, and synchronised in silico at the time point when cells reached a threshold of cytoplasmic CycA2-eYFP (indicated with the grey dotted line). Average intensities (below). Error bars show SD. Please note that differences after 2 h involve that DMSO-treated cells enter mitosis, giving an increase in average fluorescence upon cell rounding followed by a drop in fluorescence upon mitotic CycA2-eYFP degradation. (B) Quantification of at least 95 single RPE-CycA2-eYFP cells upon treatment with either DMSO (control) or Wee1 inhibitor (MK-1775). Cells were treated with the drugs and tracked using live-cell imaging. Each track represents one single cell, the black dot represents the first time point when cytoplasmic CycA2 can be detected and the red dot indicates mitotic entry. Overlap of the cumulative appearance of cytoplasmic CycA2 in the two different treatments (right). (C) Western blot of indicated cell lines transfected with CDK1, CDK2, or scrambled (Control) siRNAs for 48 h. Samples were prepared from 4 wells to 96-well plate to mimic conditions used for microscopy. (D) Proportion of RPE cells passing through mitosis during 16 h after knockdown of CDK1 or CDK2 for 48 h (left). Duration of mitosis in the indicated knockdown conditions (right). Source data are available for this figure. (A) RPE cells were transfected with siRNAs for either CDK1 or CDK2 for 48 h, incubated with EdU for 20 min and fixed. Arrows indicate G2 cells with low cytoplasmic CycA2. Scale bar 20 μm. (B) Quantification of cytoplasmic and nuclear integrated intensities of CycA2 in at least 500 RPE cells imaged as in (A). Cells were gated for DAPI and EdU levels and assigned to the S phase (red dots) or G2 phase (green dots). Each dot represents one cell; the percentages indicate the proportion of the S- and G2 phase cells in each condition. Number of cells within indicated gate and total amount of G2 cells are shown to the right. (C) RPE (RPE) or RPE CycA2-eYFP (CycA2) cells were synchronised in G2, separated to cytosolic and nuclear fractions and immunoprecipitated with GFP Trap (left) or with control IgG and CycA2 antibody (right). Proteins bound to the carrier were probed with indicated antibodies. For (C), data using additional antibodies can be found in Fig 3E. All experiments were repeated at least three times. (A) Chromatin immunoprecipitation (ChIP) in RPE-CycA2-eYFP cells, synchronised with hydroxyurea (HU, 2 mM in PBS) for 18 h and harvested after HU washout at different time points (0, 4 and 8 h). The enrichment of CycA2-eYFP and IgG, determined by ChIP-qPCR is plotted as percentage of input (ratio of immunoprecipitated DNA to the total amount of DNA). Genomic locations are indicated by alphabetical letters A–E (details in Table S1). Graph shows one representative experiment. Mean value and SD are based on duplicate samples, each with duplicate qPCR reactions. Mean relative recovery for non-immune IgG are 0.0072. Asterisks indicate two-tailed paired t test; *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. (B) FRAP in the nucleus of RPE-CycA2-eYFP cells. Graph shows normalised average of cells with (G2 phase) and without (S phase) detectable cytoplasmic CycA2-eYFP. N = 2. (C) RPE-CycA2-eYFP cells were synchronised with hydroxyurea for 18 h and harvested after 4 and 10 h in HU-free media. Cytoplasmic proteins (Cyto), soluble nuclear proteins (N.sol), and chromatin bound proteins (Chr) were isolated and detection of Cyclin A2 and PCNA was performed by Western blot. 5 μg of protein was loaded for each fraction. β-Tubulin, KAP1, and Histone H3 were used as controls of cytosolic, nuclear soluble and chromatin fraction, respectively. N = 2. (A) Time-lapse images of RPE CycA2-eYFP cells treated with 2 nM Neocarzinostatin (NCS). Asterisk indicates a cell exposed to NCS in the S phase, and arrow indicates a cell exposed to NCS in the G2 phase. Scale bar 10 μm. (B) Quantification of average nuclear and cytoplasmic CycA2-eYFP intensity after NCS addition. RPE CycA2-eYFP cells were imaged as in (A). Graphs show quantification over time in single cells, separated in cells showing cytoplasmic CycA2-eYFP at time of NCS addition (left, G2 phase, n = 10) or not (right, S phase, n = 10). No cell entered mitosis. (C) Quantification of average nuclear and cytoplasmic CycA2-eYFP intensity after etoposide and MG132 addition. RPE CycA2-eYFP cells that showed both nuclear and cytoplasmic fluorescence (G2 cells) at start of experiment were followed over time. Graphs show quantification over time in single cells, separated in cells entering mitosis before etoposide addition (left, n = 9, plotted until time point before mitosis), etoposide addition alone (middle, n = 8), etoposide, and MG132 addition (n = 9). (D) RPE CycA2-eYFP cells were transfected with p21, p53 or control siRNAs for 48 h and treated with etoposide at t = 0. Single cells were tracked over time and the time point of loss of CycA2-eYFP was determined visually. Graph shows cytoplasmic and nuclear loss of CycA2-eYFP of at least 100 cells that contained cytoplasmic CycA2-eYFP at time point of etoposide addition. (E) WT (ctrl), p21−/− or p53−/− RPE cells were incubated for 20 min with EdU and fixed. Graph to left shows quantification of integrated intensity of EdU staining versus nuclear DAPI intensity in at least 1,500 wt cells; each circle represents one cell. The large grey rectangle indicates gating for EdU positive cells (S phase) and the small grey rectangle indicates gating for EdU-negative 4N cells (G2). Box plots to right show 90, 75, 50, 25, and 10 percentiles of cells in S phase (top) and in G2 phase (bottom). Squares indicate average value. * indicates P < 0.05, *** indicates P < 0.0001, t test. (A) Time-lapse microscopy of a G2 phase RPE CycA2-eYFP cell after etoposide addition. (B) Western blot of indicated cell lines (two different clones of p21−/− and p53−/− RPE cell lines). KAP1 and TFIIH are used as loading controls. CycA2 is restricted to the nucleus during the S phase. At the S/G2 transition, part of CycA2 localises to the cytoplasm, where it can phosphorylate Bora, resulting in activation of polo-like kinase 1. Similar articles
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