Dynamic Features of Chromosomal Instability during Culture of Induced Pluripotent Stem Cells.

Induced pluripotent stem cells (iPSCs) hold great potential for regenerative medicine. By reprogramming a patient's own cells, immunological rejection can be avoided during transplantation. For expansion and gene editing, iPSCs are grown in artificial culture for extended times. Culture affords potential danger for the accumulation of genetic aberrations. To study these, two induced pluripotent stem (iPS) cell lines were cultured and periodically analyzed using advanced optical mapping to detect and classify chromosome numerical and segmental changes that included deletions, insertions, balanced translocations and inversions. In one of the lines, a population trisomic for chromosome 12 gained dominance over a small number of passages. This appearance and dominance of the culture by chromosome 12 trisomic cells was tracked through intermediate passages by the analysis of chromosome spreads. Mathematical modeling suggested that the proliferation rates of diploid versus trisomic cells could not account for the rapid dominance of the trisomic population. In addition, optical mapping revealed hundreds of structural variations distinct from those generally found within the human population. Many of these structural variants were detected in samples obtained early in the culturing process and were maintained in late passage samples, while others were acquired over the course of culturing.

Developing immortal cell lines from Xenopus embryos, four novel cell lines derived from Xenopus tropicalis.

The diploid anuran Xenopus tropicalis has emerged as a key research model in cell and developmental biology. To enhance the usefulness of this species, we developed methods for generating immortal cell lines from Nigerian strain (NXR_1018, RRID:SCR_013731) X. tropicalis embryos. We generated 14 cell lines that were propagated for several months. We selected four morphologically distinct lines, XTN-6, XTN-8, XTN-10 and XTN-12 for further characterization. Karyotype analysis revealed that three of the lines, XTN-8, XTN-10 and XTN-12 were primarily diploid. XTN-6 cultures showed a consistent mixed population of diploid cells, cells with chromosome 8 trisomy, and cells containing a tetraploid content of chromosomes. The lines were propagated using conventional culture methods as adherent cultures at 30°C in a simple, diluted L-15 medium containing fetal bovine serum without use of a high CO2 incubator. Transcriptome analysis indicated that the four lines were distinct lineages. These methods will be useful in the generation of cell lines from normal and mutant strains of X. tropicalis as well as other species of Xenopus.

Live Fluorescence Imaging of Chromosome Segregation in Cultured Cells.

Live-cell fluorescence microscopy is an effective tool for characterizing aberrant mitotic phenotypes resulting from exposure to chemical inhibitors and after RNA interference-mediated or CRISPR knockout-mediated depletion of protein targets. Live imaging of cultured cells during mitotic progression presents challenges in maintaining optimal health of cells while achieving the temporal and spatial resolution to accomplish the goals of the study. Herein are strategies to monitor and analyze mammalian cell mitosis utilizing either a wide field or a light sheet, inverted, fluorescence microscope.

CDK1-mediated phosphorylation at H2B serine 6 is required for mitotic chromosome segregation.

Faithful mitotic chromosome segregation is required for the maintenance of genomic stability. We discovered the phosphorylation of histone H2B at serine 6 (H2B S6ph) as a new chromatin modification site and found that this modification occurs during the early mitotic phases at inner centromeres and pericentromeric heterochromatin. This modification is directly mediated by cyclin B1-associated CDK1, and indirectly by Aurora B, and is antagonized by PP1-mediated dephosphorylation. H2B S6ph impairs chromatin binding of the histone chaperone SET (I2PP2A), which is important for mitotic fidelity. Injection of phosphorylation-specific H2B S6 antibodies in mitotic cells caused anaphase defects with impaired chromosome segregation and incomplete cytokinesis. As H2B S6ph is important for faithful chromosome separation, this modification may contribute to the prevention chromosomal instability and aneuploidy which frequently occur in cancer cells.

Multiple determinants and consequences of cohesion fatigue in mammalian cells.

Cells delayed in metaphase with intact mitotic spindles undergo cohesion fatigue, where sister chromatids separate asynchronously, while cells remain in mitosis. Cohesion fatigue requires release of sister chromatid cohesion. However, the pathways that breach sister chromatid cohesion during cohesion fatigue remain unknown. Using moderate-salt buffers to remove loosely bound chromatin cohesin, we show that "cohesive" cohesin is not released during chromatid separation during cohesion fatigue. Using a regulated protein heterodimerization system to lock different cohesin ring interfaces at specific times in mitosis, we show that the Wapl-mediated pathway of cohesin release is not required for cohesion fatigue. By manipulating microtubule stability and cohesin complex integrity in cell lines with varying sensitivity to cohesion fatigue, we show that rates of cohesion fatigue reflect a dynamic balance between spindle pulling forces and resistance to separation by interchromatid cohesion. Finally, while massive separation of chromatids in cohesion fatigue likely produces inviable cell progeny, we find that short metaphase delays, leading to partial chromatid separation, predispose cells to chromosome missegregation. Thus, complete separation of one or a few chromosomes and/or partial separation of sister chromatids may be an unrecognized but common source of chromosome instability that perpetuates the evolution of malignant cells in cancer.

Predictive bioinformatics identifies novel regulators of proliferation in a cancer stem cell model.

The cancer stem cell model postulates that tumors are hierarchically organized with a minor population, the cancer stem cells, exhibiting unlimited proliferative potential. These cells give rise to the bulk of tumor cells, which retain a limited ability to divide. Without successful targeting of cancer stem cells, tumor reemergence after therapy is likely. However, identifying target pathways essential for cancer stem cell proliferation has been challenging. Here, using a transcriptional network analysis termed GAMMA, we identified 50 genes whose correlation patterns suggested involvement in cancer stem cell division. Using RNAi depletion, we found that 21 of these target genes showed preferential growth inhibition in a breast cancer stem cell model. More detailed initial analysis of 6 of these genes revealed 4 with clear roles in the fidelity of chromosome segregation. This study reveals the strong predictive potential of transcriptional network analysis in increasing the efficiency of successful identification of novel proliferation dependencies for cancer stem cells.

GTSE1 regulates spindle microtubule dynamics to control Aurora B kinase and Kif4A chromokinesin on chromosome arms.

In mitosis, the dynamic assembly and disassembly of microtubules are critical for normal chromosome movement and segregation. Microtubule turnover varies among different mitotic spindle microtubules, dictated by their spatial distribution within the spindle. How turnover among the various classes of spindle microtubules is differentially regulated and the resulting significance of differential turnover for chromosome movement remains a mystery. As a new tactic, we used global microarray meta-analysis (GAMMA), a bioinformatic method, to identify novel regulators of mitosis, and in this study, we describe G2- and S phase-expressed protein 1 (GTSE1). GTSE1 is expressed exclusively in late G2 and M phase. From nuclear envelope breakdown until anaphase onset, GTSE1 binds preferentially to the most stable mitotic spindle microtubules and promotes their turnover. Cells depleted of GTSE1 show defects in chromosome alignment at the metaphase plate and in spindle pole integrity. These defects are coupled with an increase in the proportion of stable mitotic spindle microtubules. A consequence of this reduced microtubule turnover is diminished recruitment and activity of Aurora B kinase on chromosome arms. This decrease in Aurora B results in diminished binding of the chromokinesin Kif4A to chromosome arms.

Live-cell fluorescence imaging for phenotypic analysis of mitosis.

Live-cell fluorescence microscopy is a powerful tool for characterizing aberrant mitotic phenotypes resulting from exposure to chemical inhibitors or after depletion of protein targets by RNA interference or other methods. Live imaging of cultured cells during mitotic progression presents challenges in maintaining optimal health of cells while achieving the temporal and spatial resolution to accomplish the goals of the study. We describe herein strategies to monitor and analyze mammalian cell mitosis with standard, inverted, fluorescence microscopy systems that are widely available.

The spindle and kinetochore-associated (Ska) complex enhances binding of the anaphase-promoting complex/cyclosome (APC/C) to chromosomes and promotes mitotic exit.

The spindle and kinetochore-associated (Ska) protein complex is a heterotrimeric complex required for timely anaphase onset. The major phenotypes seen after small interfering RNA-mediated depletion of Ska are transient alignment defects followed by metaphase arrest that ultimately results in cohesion fatigue. We find that cells depleted of Ska3 arrest at metaphase with only partial degradation of cyclin B1 and securin. In cells arrested with microtubule drugs, Ska3-depleted cells exhibit slower mitotic exit when the spindle checkpoint is silenced by inhibition of the checkpoint kinase, Mps1, or when cells are forced to exit mitosis downstream of checkpoint silencing by inactivation of Cdk1. These results suggest that in addition to a role in fostering kinetochore-microtubule attachment and chromosome alignment, the Ska complex has functions in promoting anaphase onset. We find that both Ska3 and microtubules promote chromosome association of the anaphase-promoting complex/cyclosome (APC/C). Chromosome-bound APC/C shows significantly stronger ubiquitylation activity than cytoplasmic APC/C. Forced localization of Ska complex to kinetochores, independent of microtubules, results in enhanced accumulation of APC/C on chromosomes and accelerated cyclin B1 degradation during induced mitotic exit. We propose that a Ska-microtubule-kinetochore association promotes APC/C localization to chromosomes, thereby enhancing anaphase onset and mitotic exit.

Haspin inhibitors reveal centromeric functions of Aurora B in chromosome segregation.

Haspin phosphorylates histone H3 at threonine-3 (H3T3ph), providing a docking site for the Aurora B complex at centromeres. Aurora B functions to correct improper kinetochore-microtubule attachments and alert the spindle checkpoint to the presence of misaligned chromosomes. We show that Haspin inhibitors decreased H3T3ph, resulting in loss of centromeric Aurora B and reduced phosphorylation of centromere and kinetochore Aurora B substrates. Consequently, metaphase chromosome alignment and spindle checkpoint signaling were compromised. These effects were phenocopied by microinjection of anti-H3T3ph antibodies. Retargeting Aurora B to centromeres partially restored checkpoint signaling and Aurora B-dependent phosphorylation at centromeres and kinetochores, bypassing the need for Haspin activity. Haspin inhibitors did not obviously affect phosphorylation of histone H3 at serine-10 (H3S10ph) by Aurora B on chromosome arms but, in Aurora B reactivation assays, recovery of H3S10ph was delayed. Haspin inhibitors did not block Aurora B localization to the spindle midzone in anaphase or Aurora B function in cytokinesis. Thus, Haspin inhibitors reveal centromeric roles of Aurora B in chromosome movement and spindle checkpoint signaling.

Cohesion fatigue induces chromatid separation in cells delayed at metaphase.

BACKGROUND: Chromosome instability is thought to be a major contributor to cancer malignancy and birth defects. For balanced chromosome segregation in mitosis, kinetochores on sister chromatids bind and pull on microtubules emanating from opposite spindle poles. This tension contributes to the correction of improper kinetochore attachments and is opposed by the cohesin complex that holds the sister chromatids together. Normally, within minutes of alignment at the metaphase plate, chromatid cohesion is released, allowing each cohort of chromatids to move synchronously to opposite poles in anaphase, an event closely coordinated with mitotic exit. RESULTS: Here we show that during experimentally induced metaphase delay, spindle pulling forces can cause asynchronous chromatid separation, a phenomenon we term "cohesion fatigue." Cohesion fatigue is not blocked by inhibition of Plk1, a kinase essential for the "prophase pathway" of cohesin release from chromosomes, or by depletion of separase, the protease that normally drives chromatid separation at anaphase. Cohesion fatigue is inhibited by drug-induced depolymerization of mitotic spindle microtubules and by experimentally increasing the levels of cohesin on mitotic chromosomes. In cells undergoing cohesion fatigue, cohesin proteins remain associated with the separated chromatids. CONCLUSION: In cells arrested at metaphase, pulling forces originating from kinetochore-microtubule interactions can, with time, rupture normal sister chromatid cohesion. This cohesion fatigue, resulting in unscheduled chromatid separation in cells delayed at metaphase, constitutes a previously overlooked source for chromosome instability in mitosis and meiosis.

Histone H3 Thr-3 phosphorylation by Haspin positions Aurora B at centromeres in mitosis.

Aurora B is a component of the chromosomal passenger complex (CPC) required for correct spindle-kinetochore attachments during chromosome segregation and for cytokinesis. The chromatin factors that recruit the CPC to centromeres are unknown, however. Here we show that phosphorylation of histone H3 threonine 3 (H3T3ph) by Haspin is necessary for CPC accumulation at centromeres and that the CPC subunit Survivin binds directly to H3T3ph. A nonbinding Survivin-D70A/D71A mutant does not support centromeric CPC concentration, and both Haspin depletion and Survivin-D70A/D71A mutation diminish centromere localization of the kinesin MCAK and the mitotic checkpoint response to taxol. Survivin-D70A/D71A mutation and microinjection of H3T3ph-specific antibody both compromise centromeric Aurora B functions but do not prevent cytokinesis. Therefore, H3T3ph generated by Haspin positions the CPC at centromeres to regulate selected targets of Aurora B during mitosis.

The dynamics of DNA topoisomerase IIalpha in living cells.

Here, we describe methods to prepare a mammalian expression plasmid encoding EGFP fused to the amino-terminus of human DNA topoisomerase IIalpha (Topo IIalpha) for use in studying the dynamics of Topo IIalpha in living cells. In previous studies, this plasmid was transfected into LLC-Pk cells, a porcine epithelial cell line that remains relatively flat during mitosis. After selection for stable integration, cells were cloned by serial dilution in microwells and used to grow a stable cell line expressing EGFP-Topo IIalpha; this cell line was termed LPk-GT2. Using photobleaching methods with conventional and patterned photobleaching, LPk-GT2 cells were used to demonstrate the rapid dynamics of Topo IIalpha exchange in both interphase nuclei and mitotic chromosomes. These rapid dynamics are dependent on enzyme activity since ICRF159, a catalytic inhibitor of Topo IIalpha, slows dynamics significantly. The methods utilized in these studies are described herein.

Ska3 is required for spindle checkpoint silencing and the maintenance of chromosome cohesion in mitosis.

The mitotic spindle checkpoint monitors proper bipolar attachment of chromosomes to the mitotic spindle. Previously, depletion of the novel kinetochore complex Ska1/Ska2 was found to induce a metaphase delay. By using bioinformatics, we identified C13orf3, predicted to associate with kinetochores. Recently, three laboratories independently indentified C13orf3 as an additional Ska complex component, and therefore we adopted the name Ska3. We found that cells depleted of Ska3 by RNAi achieve metaphase alignment but fail to silence the spindle checkpoint or enter anaphase. After hours of metaphase arrest, chromatids separate but retain robust kinetochore-microtubule attachments. Ska3-depleted cells accumulate high levels of the checkpoint protein Bub1 at kinetochores. Ska3 protein accumulation at kinetochores in prometaphase is dependent on Sgo1 protein. Sgo1, which accumulates at the centromeres earlier, in prophase, is not dependent on Ska3. Sgo1-depleted cells show a stronger premature chromatid separation phenotype than those depleted of Ska3. We hypothesize that Ska3 functions to coordinate checkpoint signaling from the microtubule binding sites within a kinetochore by laterally linking the individual binding sites. We suggest that this network plays a major role in silencing the spindle checkpoint when chromosomes are aligned at metaphase to allow timely anaphase onset and mitotic exit.

A high throughput, whole cell screen for small molecule inhibitors of the mitotic spindle checkpoint identifies OM137, a novel Aurora kinase inhibitor.

In mitosis, the kinetochores of chromosomes that lack full microtubule attachments and/or mechanical tension activate a signaling pathway called the mitotic spindle checkpoint that blocks progression into anaphase and prevents premature segregation of the chromatids until chromosomes become aligned at the metaphase plate. The spindle checkpoint is responsible for arresting cells in mitosis in response to chemotherapeutic spindle poisons such as paclitaxel or vinblastine. Some cancer cells show a weakened checkpoint signaling system that may contribute to chromosome instability in tumors. Because complete absence of the spindle checkpoint leads to catastrophic cell division, we reasoned that drugs targeting the checkpoint might provide a therapeutic window in which the checkpoint would be eliminated in cancer cells but sufficiently preserved in normal cells. We developed an assay to identify lead compounds that inhibit the spindle checkpoint. Most cells respond to microtubule drugs by activating the spindle checkpoint and arresting in mitosis with a rounded morphology. Our assay depended on the ability of checkpoint inhibitor compounds to drive mitotic exit and cause cells to flatten onto the substrate in the continuous presence of microtubule drugs. In this study, we characterize one of the compounds, OM137, as an inhibitor of Aurora kinases. We find that this compound is growth inhibitory to cultured cells when applied at high concentration and potentiates the growth inhibitory effects of subnanomolar concentrations of paclitaxel.

Fine tuning the cell cycle: activation of the Cdk1 inhibitory phosphorylation pathway during mitotic exit.

Inactivation of cyclin-dependent kinase (Cdk) 1 promotes exit from mitosis and establishes G1. Proteolysis of cyclin B is the major known mechanism that turns off Cdk1 during mitotic exit. Here, we show that mitotic exit also activates pathways that catalyze inhibitory phosphorylation of Cdk1, a mechanism previously known to repress Cdk1 only during S and G2 phases of the cell cycle. We present evidence that down-regulation of Cdk1 activates Wee1 and Myt1 kinases and inhibits Cdc25 phosphatase during the M to G1 transition. If cyclin B/Cdk1 complex is present in G1, the inhibitory sites on Cdk1 become phosphorylated. Exit from mitosis induced by chemical Cdk inhibition can be reversed if cyclin B is preserved. However, this reversibility decreases with time after mitotic exit despite the continued presence of the cyclin. We show that this G1 block is due to phosphorylation of Cdk1 on inhibitory residues T14 and Y15. Chemical inhibition of Wee1 and Myt1 or expression of Cdk1 phosphorylation site mutants allows reversal to M phase even from late G1. This late Cdk1 reactivation often results in caspase-dependent cell death. Thus, in G1, the Cdk inhibitory phosphorylation pathway is functional and can lock Cdk1 in the inactive state.

Shugoshin 1 plays a central role in kinetochore assembly and is required for kinetochore targeting of Plk1.

Physical connection between the sister chromatids is mediated by the cohesin protein complex. During prophase, cohesin is removed from the chromosome arms while the centromeres remain united. Shugoshin1 (Sgo1) is required for maintenance of centromeric cohesion from prophase to the metaphase-anaphase transition. Furthermore, Sgo1 has been proposed to regulate kinetochore microtubule stability and sense interkinetochore tension, two tasks which are tightly coupled with the function of the Chromosomal Passenger Complex (CPC) and Polo-like kinase 1 (Plk1). Here we show that depletion or chemical inhibition of Aurora B kinase (AurB), the catalytic subunit of the CPC, disrupts accumulation of Sgo1 on the kinetochores in HeLa cells and causes Sgo1 to localize on the chromosome arms. RNAi assays show that depletion of Sgo1 did not affect AurB localization but diminished Plk1 kinetochore binding. Furthermore, we demonstrate that vertebrate Sgo1 is phosphorylated by both AurB and Plk1 in vitro. The data presented here includes an extensive analysis of kinetochore targeting interdependencies of mitotic proteins that propose a novel branch in kinetochore assembly where Sgo1 and Plk1 have central roles. Furthermore our studies implicate Sgo1 in the tension sensing mechanism of the spindle checkpoint by regulating Plk1 kinetochore affinity.

The reversibility of mitotic exit in vertebrate cells.

A guiding hypothesis for cell-cycle regulation asserts that regulated proteolysis constrains the directionality of certain cell-cycle transitions. Here we test this hypothesis for mitotic exit, which is regulated by degradation of the cyclin-dependent kinase 1 (Cdk1) activator, cyclin B. Application of chemical Cdk1 inhibitors to cells in mitosis induces cytokinesis and other normal aspects of mitotic exit, including cyclin B degradation. However, chromatid segregation fails, resulting in entrapment of chromatin in the midbody. If cyclin B degradation is blocked with a proteasome inhibitor or by expression of non-degradable cyclin B, Cdk inhibitors will nonetheless induce mitotic exit and cytokinesis. However, if after mitotic exit, the Cdk1 inhibitor is washed free from cells in which cyclin B degradation is blocked, the cells can revert back to M phase. This reversal is characterized by chromosome recondensation, nuclear envelope breakdown, assembly of microtubules into a mitotic spindle, and in most cases, dissolution of the midbody, reopening of the cleavage furrow, and realignment of chromosomes at the metaphase plate. These findings demonstrate that proteasome-dependent degradation of cyclin B provides directionality for the M phase to G1 transition.

Lysed cell models and isolated chromosomes for the study of kinetochore/centromere biochemistry in vitro.

The centromere or kinetochore functions in both chromosome movement and in regulation of progression through mitosis. It appears likely that the signaling pathways involved are keenly dependent on solid phase cytoskeletal and karyoskeletal scaffolds that may mediate important physical signals such as tension. Understanding these pathways will be greatly aided by reconstructing the signaling in lysed cell models. Here we present approaches to the in vitro study of signaling pathways in mitotic cells, particularly those involved in protein phosphorylation changes at kinetochores that may control cell cycle progression in M phase.

Polo-like kinase 1 creates the tension-sensing 3F3/2 phosphoepitope and modulates the association of spindle-checkpoint proteins at kinetochores.

BACKGROUND: In mitosis, a mechanochemical system recognizes tension that is generated by bipolar microtubule attachment to sister kinetochores. This is translated into multiple outputs including the stabilization of microtubule attachments, changes in kinetochore protein dynamics, and the silencing of the spindle checkpoint. How kinetochores sense tension and translate this into various signals represent critical unanswered questions. The kinetochores of chromosomes not under tension are specifically phosphorylated at an epitope recognized by the 3F3/2 monoclonal antibody. Determining the kinase that generates the 3F3/2 phosphoepitope at kinetochores should reveal an important component of this system that regulates mitotic progression. RESULTS: We demonstrate that Polo-like kinase 1 (Plk1) creates the 3F3/2 phosphoepitope on mitotic kinetochores. In a permeabilized in vitro cell system, the depletion of Xenopus Plk1 from M phase extract leads to the loss of 3F3/2 kinase activity. Purified recombinant Plk1 is sufficient to generate the 3F3/2 phosphoepitope in this system. Using siRNA, we show that the reduction of Plk1 protein levels significantly diminishes 3F3/2 phosphoepitope expression at kinetochores. The consensus phosphorylation sites of Plk1 show strong similarity to the 3F3/2 phosphoepitope sequence determined by phosphopeptide mapping. The inhibition of Plk1 by siRNA alters the normal kinetochore association of Mad2, Cenp-E, Hec1/Ndc80, Spc24, and Cdc20 and induces a spindle-checkpoint-mediated mitotic arrest. CONCLUSIONS: Plk1 generates the 3F3/2 phosphoepitope at kinetochores that are not under tension and contributes to the normal kinetochore association of several key proteins important in checkpoint signaling. Mechanical tension regulates Plk1 accumulation at kinetochores and possibly its kinase activity.