Journal: Molecular Cell

Article Title: DNA Replication Determines Timing of Mitosis by Restricting CDK1 and PLK1 Activation

doi: 10.1016/j.molcel.2018.05.026

Figure Lengend Snippet: Plk1 Activation Correlates to Completion of DNA Replication (A) Schematic of hypothesis. (B) Example of RPE cell expressing PLK1-FRET and PCNA-cb in S phase, G2 phase, and mitosis. Time between images is 20 min. Please note negative correlation between nuclear PLK1 activity and presence of PCNA-cb foci. (C) S phase cells expressing PCNA-cb foci were imaged every 20 min and either mock treated or exposed to 2.5 mM thymidine. (Top) Single-cell examples of PLK1 activity and PCNA foci quantifications are shown. (Bottom) Color-coded heatmap of PLK1 activity and PCNA-cb quantifications of multiple single cells are shown. Dotted line highlights temporal correlation between DNA replication completion and PLK1 activation. Further characterization of thymidine-induced S phase arrest is described in Figure S1 . (D) U2OS, RPE, or BJ cells were fixed after a 1-hr EdU pulse and monitored by high-content microscopy. Cells were sorted based on cyclin A2 levels and nuclear size and plotted versus estimated time ( Akopyan et al., 2016 ). Graphs show moving median and SD of EdU signal and pTCTP signal from >1,600 single cells. EdU incorporation is used to measure DNA replication in single cells. pTCTP signal is corrected by treating a control population with the Plk1 inhibitor BI2536. A stepwise scheme of simultaneous cell cycle and TCTP phosphorylation analysis is described in Figure S2 .

Article Snippet: RPE and U2OS cells were a kind gift from Dr. René Medema.

Techniques: Activation Assay, Expressing, Activity Assay, Microscopy, Control, Phospho-proteomics

Journal: Molecular Cell

Article Title: DNA Replication Determines Timing of Mitosis by Restricting CDK1 and PLK1 Activation

doi: 10.1016/j.molcel.2018.05.026

Figure Lengend Snippet: DNA Replication Restricts Activation of PLK1 (A) Examples of U2OS cells expressing PLK1-FRET and PCNA-cb followed in the absence or presence of combined CDC6/CDT1 RNAi. Time between images is 45 min. Please note absence of PCNA foci despite proficient PCNA-cb expression and nuclear PLK1 activation within 5 hr. (B) U2OS cells expressing PLK1-FRET were treated as outlined in left panel and fixed after a 2-hr pulse of EdU or followed by time-lapse FRET microscopy. (Top) EdU quantification of >600 single cells; nuclear EdU intensity was plotted versus nuclear cyclin A2 levels to monitor DNA replication during cell cycle progression. Outlined boxes indicate EdU-negative cells; please note that the three most right conditions generate cells that have intermediate cyclin A2 levels but fail to incorporate EdU, which corresponds to the conditions that have premature PLK1 activation. (Bottom) Live-cell traces of single cells that entered mitosis within 2 hr upon addition of CDC7i were followed for 16 hr. Graphs show quantified PLK1 activity of 25 single cells. (C) Representative single-cell traces of the different classes observed upon Cdc6/Cdt1 co-depletion. U2OS cells expressing PLK1-FRET and PCNA-cb were transfected with control or Cdc6/Cdt1 small interfering RNA (siRNA) for 24 hr and mitotic cells were followed by live-cell imaging. The distinct classes include (class I) cells similar to controls, having discrete PCNA foci and PLK1-FRET activity only upon PCNA foci resolution, (class II) cells maintaining dim PCNA foci and no PLK1-FRET activity, and (classes III and IV) cells with no or very few PCNA foci and premature PLK1-FRET activity (4–16 hr post), which either undergo S/G2 arrest (class III) or premature mitosis (class IV). (D) Pie charts depict distribution of classes I–IV among cells depicted in (B). Whereas targeting licensing or firing can cause premature mitosis, we find the latter approach to be most effective.

Article Snippet: RPE and U2OS cells were a kind gift from Dr. René Medema.

Techniques: Activation Assay, Expressing, Microscopy, Activity Assay, Transfection, Control, Small Interfering RNA, Live Cell Imaging

Journal: Molecular Cell

Article Title: DNA Replication Determines Timing of Mitosis by Restricting CDK1 and PLK1 Activation

doi: 10.1016/j.molcel.2018.05.026

Figure Lengend Snippet: DNA Replication Restricts PLK1 and CDK1/2 Activity to Prevent Replication Stress (A) Asynchronous U2OS cells expressing PLK1-FRET and APC/C Cdh1 substrate reporter were mock treated or treated with 50 nM CHIR-124 at mitosis (±1 hr) and followed by time-lapse microscopy for 24 hr. Each line represents a single cell showing expression of APC/C Cdh1 substrate probe (light blue) and/or PLK1 activity (red). Visual appearance of nuclear GEM-RED signal (i.e., stabilization of the APC/C Cdh1 substrate probe) was used to assess APC/C inactivation at the G1/S transition (APC OFF ). Detection of nuclear PLK1 FRET signal was used to assess PLK1 activation at the S/G2 transition (PLK1 ON ). (B) Boxplot depicts G1 phase duration of 50 single cells, as determined by the time observed between mitosis and APC/C Cdh1 substrate appearance at the G1/S transition. Boxplots indicate 10, 25, 50, 75, and 90 th percentiles. n.s. indicates p > 0.5; Student’s t test. (C) As in (B), yet boxplot depicts S phase duration as determined by the time observed between APC/C Cdh1 substrate appearance and the detection of nuclear PLK1 activity. Boxplots indicate 10, 25, 50, 75, and 90 th percentiles. ∗ indicates p < 0.01; Student’s t test. (D) Images illustrate representative cells in S phase (13 hr post-mitosis) or G2 phase (17 hr post-mitosis). (E) Asynchronous RPE cells were treated with EdU, CHIR-124 (Chk1i), and SB202190 (p38i) for 2 hr and during the last hour with RO-3306 (CDK1i) and NU6140 (CDK2i) as indicated. EdU is used to mark S phase cells and directly correlate the presence of DNA replication with CHK1i-induced CDK activation and DNA damage. Graphs show DAPI, lamin A/C pS22, and H2AX pS139 for >800 cells measured by high-content microscopy. Single-cell illustrations below the graph indicate cell cycle phases and presence of DNA replication stress. (F) RPE cells were treated with CHIR-124 (CHK1i) and SB202190 (p38i) as indicated. To define S phase, cells were incubated with EdU 1 hr prior to fixation. Panels show quantifications of high-content microscopy for H2AX pS139 in single cells. Cell populations were separated according to DAPI and EdU intensity. (G) RPE cells were treated with CHIR-124 (CHK1i), SB202190 (p38i), NU6140 (CDK2i), RO3306 (CDK1i), CDK1/2 inhibitor III (CDK1/2i), and Roscovitine as indicated for 4 hr. Before fixation, cells were incubated for 1 hr with EdU. Boxplots show 90, 75, 50, 25, and 10 th percentiles of EdU signal or H2AX pS139 signal of EdU positive cells, as assessed by high-content microscopy. ∗∗∗ indicates p < 0.001, and ns indicates p > 0.1; Student’s t test.

Article Snippet: RPE and U2OS cells were a kind gift from Dr. René Medema.

Techniques: Activity Assay, Expressing, Time-lapse Microscopy, Activation Assay, Microscopy, Incubation

Journal: Molecular Cell

Article Title: DNA Replication Determines Timing of Mitosis by Restricting CDK1 and PLK1 Activation

doi: 10.1016/j.molcel.2018.05.026

Figure Lengend Snippet: DNA Replication Limits CDK1/2 Activation upon S Phase Entry (A) Single U2OS cells were filmed upon mitotic exit, and cells that were ±1 hr from mitosis upon addition of CHIR-124 (CHK1i) were selected for analysis. Montages depict an example cell expressing CDK1/2 activity sensor and PCNA-cb followed by time-lapse microscopy. Time between images is 45 min. Note the rapid cytoplasmic translocation of the CDK1/2 sensor at the G1/S transition in the presence of CHK1i. Please see Figure S6 C for readout details. (B) Quantification of single cells imaged as in (A). Red line indicates relative CDK1/2 activity, and blue line indicates PCNA-cb intensity variation as a surrogate measurement for DNA replication. Grey lines show average CDK1/2 activity of control cells. To illustrate concurrence at G1/S, three single cells with different G1 lengths are depicted. (C) U2OS cells expressing CDK1/2 activity sensor and APC/C Cdh1 substrate (GEM-RED) after CHIR-124 treatment and followed as in (A). To illustrate concurrence at G1/S, two single cells with different G1 lengths are depicted. (D) Quantification of single cells imaged as in (A) yet 24 hr after transfection of Cdc6 and Cdt1 siRNA. Red line indicates relative CDK1/2 activity, and blue line indicates PCNA-cb intensity variation as a surrogate measurement for DNA replication. Grey lines show average CDK1/2 activity of control cells. (E) U2OS cells expressing CDK1/2 activity sensor and APC/C Cdh1 substrate (GEM-RED) treated and followed as in (D). To illustrate concurrence at G1/S, two single cells with different G1 lengths are depicted per condition. (F) Quantification of CDK1/2 reporter of cells monitored as in (C) and (E). G1 is defined as 1 hr before and S phase as 3 hr after appearance of APC/C Cdh1 substrate reporter. Graph shows mean and SD of 14 cells per condition. ∗ p < 0.04; Student’s t test.

Article Snippet: RPE and U2OS cells were a kind gift from Dr. René Medema.

Techniques: Activation Assay, Expressing, Activity Assay, Time-lapse Microscopy, Translocation Assay, Control, Transfection

Journal: Molecular Cell

Article Title: DNA Replication Determines Timing of Mitosis by Restricting CDK1 and PLK1 Activation

doi: 10.1016/j.molcel.2018.05.026

Figure Lengend Snippet:

Article Snippet: RPE and U2OS cells were a kind gift from Dr. René Medema.

Techniques: Recombinant, Software

Journal: Cell Research

Article Title: Molecular basis of vasohibins-mediated detyrosination and its impact on spindle function and mitosis

doi: 10.1038/s41422-019-0187-y

Figure Lengend Snippet: Molecular mechanism of tubulin detysosination by VASH1. a The structure of VASH157–306 (red) in complex with SVBP1–52 (green) and epoY (cyan). epoY and epoY-interacting residues of VASH1 are shown in sticks. The |Fo|–|Fc| difference electron density map of epoY is contoured at 3.0 σ and is shown as blue mesh with epoY deleted prior to one round of refinement. b The structure of VASH170–306 (red) in complex with SVBP3–49 (green) and −7GEEEGECY0 peptide (yellow). Peptide and peptide-interacting residues of VASH1 are shown in sticks. Only five residues of the peptide (−4EGECY0) are visible in the structure. The |Fo|–|Fc| difference electron density map of the peptide is contoured at 3.0 σ and is shown as blue mesh with the peptide deleted prior to one round of refinement. c Superposition of epoY-bound and −7GEEEGECY0-bound SVBP-VASH1 structures. epoY, −4EGECY0, and VASH1 residues involved in peptide binding are shown in cyan, yellow, and gray sticks, respectively. d Allosteric regulatory role of SVBP. SVBP, VASH1 and the peptide are colored in the same way as shown in b. The 133QYNH136 motif, Ile167 and Cys169 of VASH1, Asn43 of SVBP and peptide residues are shown in sticks.

Article Snippet: Parental U2OS, U2OS cell lines stably expressing H2B-GFP and mCherry-α tubulin (gift from S. Geley, Innsbruck Medical University, Austria), 65 photoactivatable GFP (PA-GFP) and mCherry-α tubulin (gift from R. Medema, NKI, Amsterdam, The Netherlands), and EB1-GFP 66 (gift from P. Draber, IMG ASCR, Prague, Czech Republic) were grown in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS; Invitrogen) at 37 °C with 5% CO2.

Techniques: Binding Assay

Journal: Cell Research

Article Title: Molecular basis of vasohibins-mediated detyrosination and its impact on spindle function and mitosis

doi: 10.1038/s41422-019-0187-y

Figure Lengend Snippet: Analysis of the mutants linked to the structural features of SVBP-VASH1 complex. a Influence of VASH1 mutations on its activity in the presence and absence of SVBP. Immunoblot of lysates from cells expressing VASH1-GFP mutants with/without SVBP-myc. The activity of VASH1 was assessed with the antibody against detyrosinated tubulin pool. Expression levels of VASH1 and SVBP were observed using anti-GFP and anti-myc antibodies respectively with GAPDH as loading control. b Immunoblotting analysis of detyrosination levels of U2OS cells expressing GFP-tagged VASH1 mutants treated with control or VASH1/2 siRNAs. Expression of VASH1-GFP constructs was detected using anti-GFP antibody, with GAPDH serving as a loading control. Indicated are relative levels of detyrosinated α-tubulin normalized to GAPDH. c Effect of SVBP mutation on activity of SVBP-VASH1 complex was detected by immunoblotting. Levels of detyrosinated tubulin were analyzed in cells overexpressing VASH1-myc with or without overexpression of SVBP-myc constructs. Expression of VASH1 and SVBP constructs were monitored using an anti-myc antibody. d Quantification of the impact of VASH1 activity on preservation of proper spindle length in metaphase U2OS cells treated with control and VASH1/2 siRNAs in presence or absence of VASH1 constructs. N (number of cells, number of independent experiments): siCTRL (94, 6); siVASH1/2 (82, 5); V1 (49, 3); H204A (45, 3); C169A (45, 3); K146A/R222A (45, 3). P-values were calculated using Mann–Whitney U test. n.s.—not significant, ****p < 0.0001. e Polar distribution plots of spindle angle deviation of the same conditions as presented in d

Article Snippet: Parental U2OS, U2OS cell lines stably expressing H2B-GFP and mCherry-α tubulin (gift from S. Geley, Innsbruck Medical University, Austria), 65 photoactivatable GFP (PA-GFP) and mCherry-α tubulin (gift from R. Medema, NKI, Amsterdam, The Netherlands), and EB1-GFP 66 (gift from P. Draber, IMG ASCR, Prague, Czech Republic) were grown in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS; Invitrogen) at 37 °C with 5% CO2.

Techniques: Activity Assay, Western Blot, Expressing, Control, Construct, Mutagenesis, Over Expression, Preserving, MANN-WHITNEY

Journal: Cell Research

Article Title: Molecular basis of vasohibins-mediated detyrosination and its impact on spindle function and mitosis

doi: 10.1038/s41422-019-0187-y

Figure Lengend Snippet: The impact of vasohibins and SVBP on tubulin detyrosination levels and mitosis. a Immunoblot analysis of detyrosinated tubulin levels upon RNAi-mediated knockdown of the components of vasohibin-SVBP complex. Protein extracts from U2OS cells transfected with target specific siRNAs against VASH1, VASH2 and SVBP were probed with α-tubulin antibody and an antibody that recognizes detyrosinated form of α-tubulin, with GAPDH serving as a loading control. Relative levels of detyrosinated α-tubulin normalized to GAPDH are indicated. b Effect of VASH1 and SVBP overexpression on detyrosinated tubulin levels. Immunoblot of cell lysates from U2OS cells transfected with plasmids expressing VASH1-GFP and/or SVBP-myc. The expression of VASH1 and SVBP were observed using anti-GFP and anti-myc antibodies, respectively. The levels of detyrosinated and total α-tubulin were assessed using the respective antibodies, with GAPDH serving as the loading control. c Spinning-disk confocal imaging of U2OS cells stably expressing H2B-GFP (cyan) and mCherry-α-tubulin (red) transfected with control and VASH1/2 siRNAs. Time in hour:min. Scale bar: 10 µm. d Duration of different phases of mitosis in control versus VASH1/2 siRNAs-treated cells. N (number of cells, number of independent experiments): NEB-metaphase—siCTRL (56, 3), siVASH1/2 (28, 5); Metaphase-AO—siCTRL (78, 3), siVASH1/2 (62, 5); NEB-AO—siCTRL (56, 3); siVASH1/2 (31, 5), NEB represents nuclear envelope breakdown, AO represents anaphase onset. e Quantification of the spindle length from live-cell imaging presented in c. N (number of cells, number of independent experiments): siCTRL (63, 3); siVASH1/2 (68, 5). f Percentage of cells with spindle positioning defect obtained from live-cell imaging presented in c. N (number of cells, number of independent experiments): siCTRL (88,3); siVASH1/2 (96, 5). P-values were calculated using Mann–Whitney U test. **p < 0.01, ****p < 0.0001

Article Snippet: Parental U2OS, U2OS cell lines stably expressing H2B-GFP and mCherry-α tubulin (gift from S. Geley, Innsbruck Medical University, Austria), 65 photoactivatable GFP (PA-GFP) and mCherry-α tubulin (gift from R. Medema, NKI, Amsterdam, The Netherlands), and EB1-GFP 66 (gift from P. Draber, IMG ASCR, Prague, Czech Republic) were grown in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS; Invitrogen) at 37 °C with 5% CO2.

Techniques: Western Blot, Knockdown, Transfection, Control, Over Expression, Expressing, Imaging, Stable Transfection, Live Cell Imaging, MANN-WHITNEY

Journal: Cell Research

Article Title: Molecular basis of vasohibins-mediated detyrosination and its impact on spindle function and mitosis

doi: 10.1038/s41422-019-0187-y

Figure Lengend Snippet: Vasohibins-mediated MT detyrosination regulates mitotic spindle length and positioning. a Representative images of mitotic spindles in control, VASH1/2- or TTL-depleted U2OS cells, immunostained with antibodies against detyrosinated and total α-tubulin. DNA was counterstained with DAPI. Detyrosinated tubulin in red, total α-tubulin in green, and DNA in blue in merged image. Scale bar: 10 µm. b Quantification of the spindle length from immunostained U2OS cells that were subjected to indicated treatments. c Astral MT intensities quantified as described in methods section. d Temporal color-coded projections of mitotic spindles of U2OS EB1-GFP cells treated with indicated siRNAs. Pronounced effect of the siRNA treatments on growing astral MTs shown in insets. Scale bar: 10 µm (Inset scale bar: 2 µm) e, f Astral MT length and stability quantified by manual tracking of EB1-GFP comet signal. N (number of cells, number of independent experiments): -spindle length: siCTRL (94, 6); siVASH1/2 (82, 5); siTTL (31, 2); siVASH1/2 + siMCAK (50, 3); siMCAK (32, 2); MCAK OX (49, 3); -astral MT intensity: siCTRL (106, 4); siVASH1/2 (73, 4); siTTL (33, 3); siVASH1/2 + siMCAK (51, 3); siMCAK (32, 2); MCAK OX (46, 3), -EB1-GFP comet tracking: siCTRL (29, 3); siVASH1/2 (27, 3); siVASH1/2 + siMCAK (29, 3); siMCAK (28, 3). P-values were calculated using Mann–Whitney U test. n.s. not significant, *p < 0.05, **p < 0.01, ****p < 0.0001

Article Snippet: Parental U2OS, U2OS cell lines stably expressing H2B-GFP and mCherry-α tubulin (gift from S. Geley, Innsbruck Medical University, Austria), 65 photoactivatable GFP (PA-GFP) and mCherry-α tubulin (gift from R. Medema, NKI, Amsterdam, The Netherlands), and EB1-GFP 66 (gift from P. Draber, IMG ASCR, Prague, Czech Republic) were grown in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS; Invitrogen) at 37 °C with 5% CO2.

Techniques: Control, MANN-WHITNEY