15 research outputs found
Role of PIASγ in chromosome segregation
<p>A model illustrating the role of PIASγ in mitosis and the relationships between cohesin complexes and DNA catenations. In theory, sister chromatids remain cohered when either cohesin or DNA catenations are removed. Faithful chromosome segregation therefore relies on the concerted action of separase (which removes centromeric cohesin), PIASγ (which helps localize Topo II to centromere regions) and Topo II (which resolves DNA catenations at the centromere).</p
Normal spindle morphologies and reduced CENP-E staining at the kinetochores of aligned chromosomes in PIASγ-depleted cells.
<div><p>(<i>A,G</i>) Mitotic spindles (stained with anti-alpha-tubulin antibody) were indistinguishable in control and PIASγ-depleted metaphase cells, except that a small % at late time-points possessed extra poles (arrow in <i>A</i>): (<i>A,G</i>) Red = alpha-tubulin, Blue = DAPI.</p>
<p>(<i>B–F,H–K</i>) CENP-E staining (Red) is detected strongly on kinetochores of chromosomes away from the metaphase plate in both control-treated and PIASγ-depleted cells (<i>B,H,I</i>), but was much reduced in chromosomes at the plate (<i>C,J,K</i>).</p>
<p>After prolonged arrest in metaphase some of PIASγ-depleted cells had one or two chromosomes that left the plate and regained strong CENP-E staining at their kinetochores (<i>D–F</i> and see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0000053#pone.0000053.s002" target="_blank">Fig. S1<i>N,O</i></a>).</p>
<p>The same cell is shown in <i>D</i> (merged image), <i>E</i> (CENP-E straining) and <i>F</i> (DAPI straining).</p></div
Live-cell time-lapse analysis of PIASγ-depleted cells.
<div><p>Selected frames of cells extracted from two fields of control and two fields of PIASγ-depleted cells that were filmed over a 16 (<i>A,C</i>) or 20 hour (<i>B,D</i>) period after release from double thymidine synchrony.</p>
<p>(Supporting Information includes movies of the entire fields from which series <i>B</i> and <i>D</i> were taken and also the movies corresponding to <i>A</i> and <i>C</i>, and a detailed analysis of the cell cycle progression of each cell in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0000053#pone.0000053.s004" target="_blank">Fig. S3</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0000053#pone.0000053.s001" target="_blank">Table S1</a>.) (<i>A,B</i>) Control, (<i>C,D</i>) PIASγ-depleted cells. Numbers in the right top corner indicate time after the release of the second thymidine block.</p>
<p>(<i>A</i>) Two control cells dividing. Length of mitosis (M) was 1 hour 14 minutes in both cases (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0000053#pone.0000053.s005" target="_blank">Movie S1</a>).</p>
<p>(<i>B</i>) Six control cells dividing.</p>
<p>The two cells with longer mitoses are indicated by arrows and the total mitotic length is indicated; cells are selected from a field shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0000053#pone.0000053.s006" target="_blank">Movie S2</a>.</p>
<p>(<i>C</i>) Two PIASγ-depleted cells entering mitosis (10 hours 07 minutes and 10 hours 28 minutes, respectively).</p>
<p>They reached metaphase 18 minutes and 15 minutes after nuclear envelope breakdown, and remained in metaphase for 3 hours 26 minutes and 4 hours 18 minutes before undergoing anaphase.</p>
<p>The cell at the bottom displayed some lagging chromosomes (arrows) during anaphase that finally incorporated into one of the daughter nuclei.</p>
<p>These cells are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0000053#pone.0000053.s007" target="_blank">Movie S3</a>. (<i>D</i>) PIASγ-depleted cells dividing.</p>
<p>The cell at the bottom reached metaphase, delayed in metaphase, several chromosomes departed from the plate (de-congressed metaphase), recovered a complete metaphase plate, then returned to de-congressed metaphase and finally initiated anaphase just after re-achieving metaphase alignment once more.</p>
<p>The cell at the top spent most of the time in de-congressed metaphase after having spent more than one hour in metaphase. These cells are selected from a wider field shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0000053#pone.0000053.s008" target="_blank">Movie S4</a>.</p></div
Sister chromatids cannot separate in PIASγ-depleted cells lacking the cohesin protector hSgo1.
<div><p>(<i>A,B</i>) HeLa cells arrest in mitosis with separated sisters when an essential component of the APC/C (Apc2) is depleted together with the cohesin protector hSgo1.</p>
<p>RNAi was performed as previously described <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0000053#pone.0000053-GimenezAbian1" target="_blank">[13]</a> and cells allowed to reach mitosis after early S-phase synchrony in the presence of nocodazole: (<i>A</i>) c-mitosis arrest with nocodazole after Apc2 depletion; (<i>B</i>) complete sister chromatid separation in the presence of nocodazole after hSgo1 and Apc2 co-depletion. (<i>C,D</i>) hSgo1 is required for sister cohesion when a persistent spindle checkpoint is induced by Hec1-depletion (cells were synchronized and Hec1/hSgo1 depleted as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0000053#pone-0000053-g003" target="_blank">Figure 3</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0000053#pone.0000053-GimenezAbian1" target="_blank">[13]</a>): (<i>C</i>) Prometaphase arrest after Hec1-depletion; (<i>D</i>) complete sister separation after hSgo1 and Hec1 co-depletion. Numbers on these micrographs (<i>A–D</i>) indicate % cells that arrested with these phenotypes 20 hours after release from S-phase. (<i>E–N</i>) hSgo1 depletion does not result in sister separation when PIASγ is co-depleted, either in the absence (<i>E–J</i>) or presence of nocodazole (<i>K–N</i>): (<i>E,F</i>) metaphase arrest after PIASγ-depletion; (<i>K</i>) c-mitosis arrest in nocodazole after PIASγ-depletion; (<i>G,L</i>) complete sister separation after hSgo1-depletion, with or without nocodazole; (<i>H,I</i>) metaphase arrest after hSgo1 and PIASγ co-depletion; (<i>M</i>) c-mitosis arrest in nocodazole after hSgo1 and PIASγ co-depletion. (<i>J</i>) After release from early S-phase, hSgo1-depleted cells arrest in mitosis with separated sisters, while almost all PIASγ-depleted and hSgo1/PIASγ co-depleted cells arrest with cohered sisters. Similar results were obtained in cells treated with nocodazole upon release from early S-phase (<i>N</i>). Nocodazole used at 0.25 µM. (<i>O–R′</i>) Immunostaining of myc-tagged Rad21 in HeLa cells. (<i>O–R</i>) Merge of DAPI (blue), CREST (green), myc-Rad21 (red). (<i>O′–R′</i>) myc-Rad21 staining only.</p>
<p>(<i>O,O′</i>) Control cell. Rad21 localizes strongly between kinetochores, revealed by CREST signals, and weakly between chromosome arms. The term “metaphase-like”, as in the other panels, refers to the fact that metaphases are not unequivocally identifiable after the pre-extraction and fixation procedures required before immunostaining. (<i>P, P′</i>) PIASγ-depleted cell at metaphase displaying a pattern of myc-Rad21 localization similar to that of control cells. (<i>Q–Q′</i>) Sgo1-depleted cells. The cell on the left is most likely an early prophase cell (judging by the closely paired sister kinetochores) and shows high myc-Rad21 staining throughout the nucleus, while the cell on the right does not have any detectable myc-Rad21 (most chromosomes have paired kinetochores while several have already separated, typical of precocious sister separation in prometaphase after hSgo1 depletion). (<i>R,R′</i>) Metaphase cell after PIASγ and hSgo1 co-depletion. No detectable myc-Rad21 staining is observed though sister centromere cohesion is maintained (kinetochores are paired).</p></div
Sister chromatids cannot separate in PIASγ-depleted cells lacking Topoisomerase II activity.
<p>Metaphase arrested cells depleted of PIASγ were collected as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0000053#pone-0000053-g004" target="_blank">Figure 4</a>, then the Aurora B inhibitor ZM447439 and the Topoisomerase II inhibitor ICRF-193 were added and samples taken for cytological analysis. (<i>A</i>) metaphase; (<i>B,C,D</i>) failed sister disjunction during anaphase and subsequent exit from mitosis. In <i>B</i> and <i>C</i> sister chromatids can still be observed to be cohered while the chromatin is largely decondensing (double arrows). For comparison, small inserted panels show metaphase (left-most panel) and anaphase (two panels on the right) in the presence of ZM447439 alone. (<i>E</i>) Frequency of cells in which sisters were able to disjoin upon addition of ZM447439, with or without ICRF-193. (<i>F–I</i>) Immunostaining of Topoisomerase IIα in HeLa cells expressing H2B-GFP (inserts show a slight enlargement of a selected region from the same micrograph). (<i>F–I</i>) Merge - Topoisomerase IIα (red) and H2B-GFP (green). (<i>F′–I′</i>) Topoisomerase IIα staining only. (<i>F′′–I′′</i>) H2B-GFP only. (<i>F–G</i>) Control mitotic cells showing Topoisomerase IIα localized to cores and concentrated at centromere regions; (<i>H</i>) PIASγ-depleted mitotic cell with diffusely localized Topoisomerase IIα on the chromatin; (<i>I</i>) PIASγ-depleted mitotic cell with Topoisomerase IIα localized to cores but not strongly enriched at centromere regions. (<i>J</i>) Classification of Topoisomerase IIα and INCENP immunostaining patterns in control and PIASγ-depleted mitotic cells. The “Kinetochore” and “Core & Kinetochore” categories required that Topoisomerase IIα was strongly concentrated at the centromere regions.</p
Prolonged metaphase arrest and proper chromosome alignment in PIASγ-depleted cells.
<div><p>Synchronous time-course experiment after release from early S-phase arrest in PIASγ-depleted HeLa cells (RNA-interference).</p>
<p>(<i>A,B</i>) Protocol for depletion of PIASγ and Western blot analysis (mit = mitotic shake-off; int = remaining interphase cells; Tub = alpha-tubulin). (<i>C</i>) Cyclin B and Phospho-H3 transiently peaked as control-treated cells passed through mitosis, while they accumulated in PIASγ-B treated cells (these blots are from mit + int cells).</p>
<p>Apc2 and Smc3 are loading controls. (<i>D–G</i>) Most PIASγ-depleted cells appeared to arrest in metaphase until the end of the experiment (∼12 hours after reaching mitosis).</p>
<p>Accordingly, chromosomes became progressively over-condensed (from <i>D</i> to <i>G</i>); arrow indicates the largest metacentric chromosome in each spread.</p>
<p>Unlike cells arrested in c-mitosis with nocodazole, PIASγ-depleted cells did not open chromosome arms. Proper chromosome alignment is evident in the side views of metaphase plates (<i>D,F</i>) and in the polar views (<i>E,G</i>).</p>
<p>In some cells one or two chromosomes lay off the metaphase plate (<i>H</i>); since these cells usually displayed overcondensed chromosomes and because this stage followed formation of a complete metaphase plate (see time-lapse material), we describe these as de-congressed metaphases.</p>
<p>For a detailed cytological comparison of PIASγ and control-treated cells, see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0000053#pone.0000053.s002" target="_blank">Figure S1</a>.</p>
<p>(<i>I–K</i>) Cells progressively accumulated in metaphase (<i>I</i>) after PIASγ depletion (1000 cells scored per time-point).</p>
<p>Shaded areas in <i>I,J</i> show control mitotic wave. (<i>J</i>) Anaphase/telophase index – Most PIASγ-depleted cells failed to initiate anaphase. (<i>K</i>) Accumulation in c-mitosis in the presence of nocodazole after control treatment or PIASγ-depletion and cell cycle synchrony in early S phase.</p>
<p>Some PIASγ-depleted cells failed to reach mitosis as indicated by the accumulation of only ∼50% c-mitotics after 16 hours compared with over 90% in control-treated samples, indicating that PIASγ might also have roles in interphase (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0000053#pone.0000053.s003" target="_blank">Fig. S2</a>).</p></div
Direct Monitoring of the Strand Passage Reaction of DNA Topoisomerase II Triggers Checkpoint Activation
<div><p>By necessity, the ancient activity of type II topoisomerases co-evolved with the double-helical structure of DNA, at least in organisms with circular genomes. In humans, the strand passage reaction of DNA topoisomerase II (Topo II) is the target of several major classes of cancer drugs which both poison Topo II and activate cell cycle checkpoint controls. It is important to know the cellular effects of molecules that target Topo II, but the mechanisms of checkpoint activation that respond to Topo II dysfunction are not well understood. Here, we provide evidence that a checkpoint mechanism monitors the strand passage reaction of Topo II. In contrast, cells do not become checkpoint arrested in the presence of the aberrant DNA topologies, such as hyper-catenation, that arise in the absence of Topo II activity. An overall reduction in Topo II activity (i.e. slow strand passage cycles) does not activate the checkpoint, but specific defects in the T-segment transit step of the strand passage reaction do induce a cell cycle delay. Furthermore, the cell cycle delay depends on the divergent and catalytically inert C-terminal region of Topo II, indicating that transmission of a checkpoint signal may occur via the C-terminus. Other, well characterized, mitotic checkpoints detect DNA lesions or monitor unattached kinetochores; these defects arise <i>via</i> failures in a variety of cell processes. In contrast, we have described the first example of a distinct category of checkpoint mechanism that monitors the catalytic cycle of a single specific enzyme in order to determine when chromosome segregation can proceed faithfully.</p></div
Top2-B44 is Defective in ATP Hydrolysis.
<p><b>a</b>, Cleavage activity of Top2-B44. Agarose gel electrophoresis of supercoiled (SC) plasmid DNA either untreated (C) or after incubation with purified wild type Top2 or Top2-B44 enzymes in the presence of increasing concentrations of etoposide (Etop). N = nicked forms, L = linear form. The linear form indicates plasmid that was cut by Top2 and re-ligation blocked by binding of etoposide to the Top2-DNA complex. <b>b</b>, Relaxation activity of Top2-B44. <i>Left panel</i>, Agarose gel electrophoresis of supercoiled (SC) plasmid DNA either untreated (C) or after 0–15 min. incubated with purified wild type Top2 or Top2-B44 enzymes. L = Linear form, R = relaxed topoisomers. <i>Right panel</i>, Quantification of relaxation activity of purified wild type (WT) Top2 or Top2-B44 (B44) enzymes versus time at 37°C and 28°C. <b>c</b>, Rate of ATP hydrolysis by wild type (WT) Top2 and Top2-B44 (B44) enzymes at 37°C and 28°C (measured by the release of free phosphate).</p
Topo II Strand Passage Reaction (SPR) and Mutants Analyzed in this Study.
<p><i>Left column</i>, Main features of the SPR of the wild type Top2 enzyme. The cartoons and description are based on Dong and Berger 2007 and Wang 2002 <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003832#pgen.1003832-Wang1" target="_blank">[3]</a>, <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003832#pgen.1003832-Dong1" target="_blank">[27]</a>. <b>a</b>, Top2 homodimer bound to G-segment DNA (see Key). N-Gate is open in the absence of bound nucleotide. <b>b</b>, Binding of one molecule of ATP to each monomer is required for N-Gate closure. If a T-segment is captured, then G-segment cleavage is thought to be coupled with N-Gate closure, in order for the T-segment to be accommodated. <b>c</b>–<b>d</b>, hydrolysis of one ATP promotes conformation changes that swivel the Transducer domain, opening the DNA-Gate and leading to T-transport. <b>e</b>, DNA-Gate closure leads to G-segment re-ligation. <b>f</b>, C-Gate opening allows T-segment release followed by hydrolysis of the second ATP and N-Gate opening after release of the hydrolysis products. <i>Mutants</i>: <u>K651A</u> has greatly reduced affinity for G-segment DNA and thus does not perform appreciable strand passage reactions <i>in vivo</i> and cannot support viability <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003832#pgen.1003832-Liu3" target="_blank">[30]</a>. <i>In vitro</i>, relaxation of supercoiled DNA can be detected at very low levels, indicating that Top2<sup>K651A</sup> can, albeit with a very limited capacity, perform the SPR and therefore overall folding of the enzyme is not abolished. <u>Y782F</u> lacks the active site tyrosine and therefore cannot cut G-segment DNA <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003832#pgen.1003832-Liu2" target="_blank">[29]</a>. It can bind to the G-segment and undergo rounds of ATP binding/hydrolysis as well as N-gate opening and closure. It is predicted to lack the ability to capture a T-segment due to space constraints within the N-terminal orifice of the enzyme in the absence of G-segment cleavage. <u>G144I</u> cannot bind nucleotide and therefore cannot lock the N-Gate closed <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003832#pgen.1003832-Lindsley1" target="_blank">[28]</a>. For this reason it is unlikely to capture a T-segment and since T-segment capture stimulates G-segment cleavage, it has much reduced cleavage activity. <i>In vitro</i>, however, cleavage activity has been measured and given this event, the enzyme may sample conformations normally associated with T-transport even in the absence of ATP hydrolysis and T-segment capture. <u>E66Q</u> has 200-fold reduced ATP hydrolysis activity and therefore inefficiently performs conformation changes that promote T-transport, including DNA-Gate opening <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003832#pgen.1003832-Baird1" target="_blank">[26]</a>. <i>In vitro</i> studies indicate that inefficient T-transport is followed by SPR arrest after release of the T-segment. A second SPR cycle is not possible because the N-Gate cannot open without release of the hydrolysis products. <u>Top2-B44</u> is predicted to be defective in T-transport. The mutated residue is positioned where the TOPRIM-fold lies adjacent to the DNA binding domain. The mutant has a reduced rate of ATP hydrolysis (this study). <u>L475A/L480P</u> has wild type ATP hydrolysis activity but T-transport occurs at a much reduced rate <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003832#pgen.1003832-Wasserman1" target="_blank">[32]</a>. <u>G738D and P824S</u> are positioned in the C-gate portion of the enzyme. These mutants have a much reduced rate of the SPR but are predicted to not affect the T-transport steps associated with DNA-gate opening, but rather would affect a later step of the SPR <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003832#pgen.1003832-Liu4" target="_blank">[31]</a>. <i>Right column</i>, Indicates if checkpoint activation occurs upon expression of each mutant at endogenous levels in yeast cells depleted of Top2<sup>deg</sup> during G1 (this study).</p
Characterization of a Yeast <i>top2</i> Degron Strain.
<p><b>a</b>, Schematic of the chromosomal <i>MET3</i>-<i>top2</i><sup>deg</sup> gene encoding thermo-labile Top2<sup>deg</sup> protein and controlled transcriptionally <i>via</i> the presence or absence of methionine in the growth medium. <b>b</b>, Western blot of Top2<sup>deg</sup>. Temperature and carbon-source shifts (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003832#pgen.1003832.s002" target="_blank">Figure S2</a>) promote efficient degradation (Tub1-GFP, loading-control). <b>c</b>, Micrographs (left) and quantification (right) of failed nucleus segregation (DAPI) in anaphase cells with elongated spindles (Tub1-GFP) after Top2<sup>deg</sup> was degraded in G1 and the subsequent cell cycle analyzed. Shaded region on graphs indicates the fraction of anaphase nuclei that were not segregated and the top left insets show expanded budding curves. <b>d</b>, CHEF gel analysis of separated chromosomes after Southern blotting to detect catenated topoisomers of the endogenous 2-micron plasmid. Wild type (WT), <i>top2-4</i> and <i>top2<sup>deg</sup></i> strains were initially arrested in G1 (alpha-factor) at 26°C (37°C in the case of <i>top2<sup>deg</sup></i>) or subsequently allowed 2 hours to reach G2/M at 37°C in the presence of nocodazole to prevent anaphase onset (G2/M, 37°C). For <i>top2<sup>deg</sup></i>, additional samples were taken at 45 min and 70 min following alpha factor release at 37°C with nocodazole (Noc. 45 min, Noc. 70 min).</p