Metaphase Anaphase Research Articles

PLEKHA5 Regulates Mitotic Progression by Promoting APC/C Localization to Microtubules

Progression through major cell cycle checkpoints involves spatiotemporal coordination of cytoskeletal dynamics with changes in protein activities, chromosomal dynamics, and signaling events. A major role of the ubiquitin–proteasome system is to orchestrate cell cycle progression through temporally controlled protein degradation programs. The anaphase‐promoting complex/cyclosome (APC/C) is the primary E3 ligase controlling all aspects of mitosis, including G2/M transition, early mitosis, the metaphase–anaphase transition, and mitotic exit, via polyubiquitination of important mitotic effector proteins. The temporal control of APC/C‐mediated ubiquitination in these processes is well established, including identities of ubiquitination substrates and accessory factors that modulate E3 ligase activity. Much less is known, however, about the spatial organization of APC/C function, including its potential interplay with the microtubule cytoskeleton, which serves as a signaling platform and whose dynamic behavior is central to advancement through mitosis. Here, we investigate and describe pleckstrin homology domain‐containing family A, member 5 (PLEKHA5) as a new regulator of APC/C function and the ubiquitination pathway in mitosis. We found that PLEKHA5 localized to the microtubule network and interacted with APC/C. PLEKHA5 knockdown antagonized mitotic entry and progression, causing a buildup of APC/C substrates implicated in both the G2/M and metaphase–anaphase transitions, in a manner dependent upon the PLEKHA5 interaction with APC/C. In vitro ubiquitination assays showed that APC/C isolated from mitotic cells lacking PLEKHA5 had lower catalytic activity and a decreased association with its key mitotic co‐activator CDC20. We investigated the dynamic localizations of the APC/C and the potential role of PLEKHA5 in its regulation by developing a TurboID proximity biotinylation tool to assess microtubule localization of endogenous proteins. Microtubule‐targeted TurboIDs revealed that PLEKHA5, APC/C subunits, and the co‐activator CDC20 all localized to microtubules both in interphase and in M phase. Importantly, upon PLEKHA5 knockdown, a pool of APC/C, but not CDC20, lost its microtubule localization, which could explain the decreased APC/C‐CDC20 association. We propose that PLEKHA5 functions as a novel APC/C adaptor to promote its subcellular localization to microtubules and enable APC/C to search for its co‐activator CDC20 more efficiently in one dimension along the microtubule network. In this model PLEKHA5 acts to coordinate the spatial regulation of APC/CCDC20 and facilitate its efficient polyubiquitination of key mitotic effector proteins to ensure proper progression of mitosis.

The Cell Biology of Heterochromatin.

A conserved feature of virtually all higher eukaryotes is that the centromeres are embedded in heterochromatin. Here we provide evidence that this tight association between pericentric heterochromatin and the centromere is essential for proper metaphase exit and progression into telophase. Analysis of chromosome rearrangements that separate pericentric heterochromatin and centromeres indicates that they must remain associated in order to balance Cohesin/DNA catenation-based binding forces and centromere-based pulling forces during the metaphase–anaphase transition. In addition, a centromere embedded in heterochromatin facilitates nuclear envelope assembly around the entire complement of segregating chromosomes. Because the nuclear envelope initially forms on pericentric heterochromatin, nuclear envelope formation proceeds from the pole, thus providing time for incorporation of lagging and trailing chromosome arms into the newly formed nucleus. Additional analysis of noncanonical mitoses provides further insights into the functional significance of the tight association between heterochromatin and centromeres.

Ubc1 turnover contributes to the spindle assembly checkpoint in Saccharomyces cerevisiae.

The spindle assembly checkpoint protects the integrity of the genome by ensuring that chromosomes are properly attached to the mitotic spindle before they are segregated during anaphase. Activation of the spindle checkpoint results in inhibition of the Anaphase-Promoting Complex (APC), an E3 ubiquitin ligase that triggers the metaphase–anaphase transition. Here, we show that levels of Ubc1, an E2 enzyme that functions in complex with the APC, modulate the response to spindle checkpoint activation in Saccharomyces cerevisiae. Overexpression of Ubc1 increased resistance to microtubule poisons, whereas Ubc1 shut-off sensitized cells. We also found that Ubc1 levels are regulated by the spindle checkpoint. Checkpoint activation or direct APC inhibition led to a decrease in Ubc1 levels, charging, and half-life. Additionally, stabilization of Ubc1 prevented its down-regulation by the spindle checkpoint and increased resistance to checkpoint-activating drugs. These results suggest that down-regulation of Ubc1 in response to spindle checkpoint signaling is necessary for a robust cell cycle arrest.

Energy restriction causes metaphase delay and chromosome mis-segregation in cancer cells

ABSTRACT ATP metabolism during mitosis needs to be coordinated with numerous energy-demanding activities, especially in cancer cells whose metabolic pathways are reprogramed to sustain rapid proliferation in a nutrient-deficient environment. Although strategies targeting the energy metabolic pathways have shown therapeutic efficacy in preclinical cancer models, how normal cells and cancer cells differentially respond to energy shortage is unclear. In this study, using time-lapse microscopy, we found that cancer cells displayed unique mitotic phenotypes in a dose-dependent manner upon decreasing ATP (i.e. energy) supply. When reduction in ATP concentration was moderate, chromosome movements in mitosis were barely affected, while the metaphase–anaphase transition was significantly prolonged due to reduced tension between the sister-kinetochores, which delayed the satisfaction of the spindle assembly checkpoint. Further reduction in ATP concentration led to a decreased level of Aurora-B at the centromere, resulting in increased chromosome mis-segregation after metaphase delay. In contrast to cancer cells, ATP restriction in non-transformed cells induced cell cycle arrest in interphase, rather than causing mitotic defects. In addition, data mining of cancer patient database showed a correlation between signatures of energy production and chromosomal instability possibly resulted from mitotic defects. Together, these results reveal that energy restriction induces differential responses in normal and cancer cells, with chromosome mis-segregation only observed in cancer cells. This points to targeting energy metabolism as a potentially cancer-selective therapeutic strategy.

SUMOylation stabilizes sister kinetochore biorientation to allow timely anaphase.

During mitosis, sister chromatids attach to microtubules from opposite poles, called biorientation. Sister chromatid cohesion resists microtubule forces, generating tension, which provides the signal that biorientation has occurred. How tension silences the surveillance pathways that prevent cell cycle progression and correct erroneous kinetochore-microtubule attachments remains unclear. Here we show that SUMOylation dampens error correction to allow stable sister kinetochore biorientation and timely anaphase onset. The Siz1/Siz2 SUMO ligases modify the pericentromere-localized shugoshin (Sgo1) protein before its tension-dependent release from chromatin. Sgo1 SUMOylation reduces its binding to protein phosphatase 2A (PP2A), and weakening of this interaction is important for stable biorientation. Unstable biorientation in SUMO-deficient cells is associated with persistence of the chromosome passenger complex (CPC) at centromeres, and SUMOylation of CPC subunit Bir1 also contributes to timely anaphase onset. We propose that SUMOylation acts in a combinatorial manner to facilitate dismantling of the error correction machinery within pericentromeres and thereby sharpen the metaphase-anaphase transition.

PP1 promotes cyclin B destruction and the metaphase-anaphase transition by dephosphorylating CDC20.

Ubiquitin-dependent proteolysis of cyclin B and securin initiates sister chromatid segregation and anaphase. The anaphase-promoting complex/cyclosome and its coactivator CDC20 (APC/CCDC20) form the main ubiquitin E3 ligase for these two proteins. APC/CCDC20 is regulated by CDK1-cyclin B and counteracting PP1 and PP2A family phosphatases through modulation of both activating and inhibitory phosphorylation. Here, we report that PP1 promotes cyclin B destruction at the onset of anaphase by removing specific inhibitory phosphorylation in the N-terminus of CDC20. Depletion or chemical inhibition of PP1 stabilizes cyclin B and results in a pronounced delay at the metaphase-to-anaphase transition after chromosome alignment. This requirement for PP1 is lost in cells expressing CDK1 phosphorylation–defective CDC206A mutants. These CDC206A cells show a normal spindle checkpoint response and rapidly destroy cyclin B once all chromosomes have aligned and enter into anaphase in the absence of PP1 activity. PP1 therefore facilitates the metaphase-to-anaphase transition by promoting APC/CCDC20-dependent destruction of cyclin B in human cells.

Topoisomerase IIα prevents ultrafine anaphase bridges by two mechanisms.

Topoisomerase IIα (Topo IIα), a well-conserved double-stranded DNA (dsDNA)-specific decatenase, processes dsDNA catenanes resulting from DNA replication during mitosis. Topo IIα defects lead to an accumulation of ultrafine anaphase bridges (UFBs), a type of chromosome non-disjunction. Topo IIα has been reported to resolve DNA anaphase threads, possibly accounting for the increase in UFB frequency upon Topo IIα inhibition. We hypothesized that the excess UFBs might also result, at least in part, from an impairment of the prevention of UFB formation by Topo IIα. We found that Topo IIα inhibition promotes UFB formation without affecting the global disappearance of UFBs during mitosis, but leads to an aberrant UFB resolution generating DNA damage within the next G1. Moreover, we demonstrated that Topo IIα inhibition promotes the formation of two types of UFBs depending on cell cycle phase. Topo IIα inhibition during S-phase compromises complete DNA replication, leading to the formation of UFB-containing unreplicated DNA, whereas Topo IIα inhibition during mitosis impedes DNA decatenation at metaphase–anaphase transition, leading to the formation of UFB-containing DNA catenanes. Thus, Topo IIα activity is essential to prevent UFB formation in a cell-cycle-dependent manner and to promote DNA damage-free resolution of UFBs.

Tumor-infiltrating lymphocytes and CD8+ T cells predict survival of triple-negative breast cancer

PurposeTumor inflammatory response was evaluated as a prognostic feature in triple-negative breast cancer (TNBC) and compared with the clinical prognosticators of breast cancer and selected biomarkers of cancer cell proliferation.MethodsTNBC patients (n = 179) with complete clinical data and up to 18-year follow-up were obtained from Auria biobank, Turku University Hospital, Turku, Finland. Tumor-infiltrating lymphocytes (TILs) and several subtypes of inflammatory cells detected with immunohistochemistry were evaluated in different tumor compartments in full tissue sections and tissue microarrays.ResultsDeficiency of stromal TILs and low number of CD8+ T cells independently predicted mortality in TNBC (HR 2.4, p 0.02 and HR 2.1, p 0.02, respectively). Each 10% decrease in stromal TILs resulted in 20% increased risk of mortality. An average of 13.2-year survival difference was observed between the majority (> 75%) of patients with low (< 14% of TILs) vs high (≥ 14% of TILs) frequency of CD8+ T cells. The prognostic value of TILs and CD8+ T cells varied when evaluated in different tumor compartments. TILs and CD8+ T cells were significantly associated with Securin and Separase, essential regulators of metaphase–anaphase transition of the cell cycle.DiscussionTILs and CD8+ T cells provide additional prognostic value to the established clinical prognostic markers in TNBC. However, possible clinical applications would still benefit from systematic guidelines for evaluating tumor inflammatory response. Increasing understanding on the interactions between the regulation of cancer cell proliferation and inflammatory response may in future advance treatment of TNBC.

Mitotic motor CENP-E cooperates with PRC1 in temporal control of central spindle assembly.

Error-free cell division depends on the accurate assembly of the spindle midzone from dynamic spindle microtubules to ensure chromatid segregation during metaphase–anaphase transition. However, the mechanism underlying the key transition from the mitotic spindle to central spindle before anaphase onset remains elusive. Given the prevalence of chromosome instability phenotype in gastric tumorigenesis, we developed a strategy to model context-dependent cell division using a combination of light sheet microscope and 3D gastric organoids. Light sheet microscopic image analyses of 3D organoids showed that CENP-E inhibited cells undergoing aberrant metaphase–anaphase transition and exhibiting chromosome segregation errors during mitosis. High-resolution real-time imaging analyses of 2D cell culture revealed that CENP-E inhibited cells undergoing central spindle splitting and chromosome instability phenotype. Using biotinylated syntelin as an affinity matrix, we found that CENP-E forms a complex with PRC1 in mitotic cells. Chemical inhibition of CENP-E in metaphase by syntelin prevented accurate central spindle assembly by perturbing temporal assembly of PRC1 to the midzone. Thus, CENP-E-mediated PRC1 assembly to the central spindle constitutes a temporal switch to organize dynamic kinetochore microtubules into stable midzone arrays. These findings reveal a previously uncharacterized role of CENP-E in temporal control of central spindle assembly. Since CENP-E is absent from yeast, we reasoned that metazoans evolved an elaborate central spindle organization machinery to ensure accurate sister chromatid segregation during anaphase and cytokinesis.

CDK1-CCNB1 creates a spindle checkpoint-permissive state by enabling MPS1 kinetochore localization.

Spindle checkpoint signaling is initiated by recruitment of the kinase MPS1 to unattached kinetochores during mitosis. We show that CDK1-CCNB1 and a counteracting phosphatase PP2A-B55 regulate the engagement of human MPS1 with unattached kinetochores by controlling the phosphorylation status of S281 in the kinetochore-binding domain. This regulation is essential for checkpoint signaling, since MPS1S281A is not recruited to unattached kinetochores and fails to support the recruitment of other checkpoint proteins. Directly tethering MPS1S281A to the kinetochore protein Mis12 bypasses this regulation and hence the requirement for S281 phosphorylation in checkpoint signaling. At the metaphase-anaphase transition, MPS1 S281 dephosphorylation is delayed because PP2A-B55 is negatively regulated by CDK1-CCNB1 and only becomes fully active once CCNB1 concentration falls below a characteristic threshold. This mechanism prolongs the checkpoint-responsive period when MPS1 can localize to kinetochores and enables a response to late-stage spindle defects. By acting together, CDK1-CCNB1 and PP2A-B55 thus create a spindle checkpoint-permissive state and ensure the fidelity of mitosis.

Novel Ca2+ increases in the maturing oocytes of starfish during the germinal vesicle breakdown

It has been known that the intracellular Ca2+ level transiently rises at the specific stages of mitosis such as the moment of nuclear envelope breakdown and at the metaphase–anaphase transition. Comparable intracellular Ca2+ increases may also take place during meiosis, as was intermittently reported in mouse, Xenopus, and starfish oocytes. In a majority of starfish species, the maturing oocytes display an intracellular Ca2+ increase within few minutes after the addition of the maturation hormone, 1-methyladenine (1-MA). Although starfish oocytes at meiosis also manifest a Ca2+ increase at the time of polar body extrusion, a similar Ca2+ increase has never been observed during the envelope breakdown of the nucleus (germinal vesicle, GV). Here, we report, for the first time, the existence of an additional Ca2+ response in the maturing oocytes of Asterina pectinifera at the time of GV breakdown. In contrast to the immediate early Ca2+ response to 1-MA, which is independent of external Ca2+ and takes a form of intracellular Ca2+ wave traveling three times as fast as that in the fertilized eggs, this late stage Ca2+ response comprised a train of numerous spikes representing Ca2+ influx. These Ca2+ spikes coinciding with GV breakdown were mostly eliminated when the GV was removed from the oocytes prior to the addition of 1-MA, suggesting that the Ca2+ spikes are rather a consequence of the GV breakdown. In support of the idea that these Ca2+ spikes play a physiological role, the oocytes matured in calcium-free seawater had a higher rate of cleavage failure 2h after the fertilization in natural seawater. Specific inhibitors of L-type Ca2+ channels, verapamil and diltiazem, severely suppressed the amplitude of the individual Ca2+ spikes, but not their frequencies. On the other hand, latrunculin-A (LAT-A), which promotes net depolymerization of the actin cytoskeleton, had a dual effect on this late Ca2+ response. When added immediately after the hormone-dependent period, LAT-A inhibited the occurrence (frequency) of the spikes in a dose-dependent manner, but the amplitude of the prevailing Ca2+ spikes itself was rather significantly increased. These results suggest that the cortical actin cytoskeleton and some nuclear factors may play a role in regulating ion channel activities during this stage of meiotic progression.

Chk1 is required for the metaphase–anaphase transition via regulating the expression and localization of Cdc20 and Mad2

AimsThe checkpoint kinase 1 (Chk1) functions not only in genotoxic stresses but also in normal cell cycle progression, particularly in the initiation, progression and fidelity of unperturbed mitosis. In this study, we investigated the role of Chk1 in regulating the metaphase–anaphase transition in mammalian cells. Main methodsThe mitotic progression was monitored by flow cytometry analysis. The levels of cyclin B1, Cdc20 and Mad2 were measured by Western blotting. Metaphase chromosome alignment and the subcellular localization of Cdc20 and Mad2 were analyzed by immunofluorescence and confocal microscopy. Key findingsCyclin B1 degradation and the metaphase–anaphase transition were severely blocked by Chk1 siRNA. Depletion of Chk1 induced chromosome alignment defect in metaphase cells. The kinetochore localization of Cdc20, Mad2 was disrupted in Chk1 depleted cells. Chk1 abrogation also dramatically reduced the protein expression levels of Cdc20 and Mad2. SignificanceThese results strongly suggest that Chk1 is required for the metaphase–anaphase transition via regulating the subcellular localization and the expression of Cdc20 and Mad2.

Dephosphorylation of CaMKII at T253 controls the metaphase–anaphase transition

Calcium/calmodulin-stimulated protein kinase II (CaMKII) is a multi-functional serine/threonine protein kinase that controls a range of cellular functions, including proliferation. The biological properties of CaMKII are regulated by multi-site phosphorylation and targeting via interactions with specific proteins. To investigate the role specific CaMKII phosphorylation sites play in controlling cell proliferation and cell cycle progression, we examined phosphorylation of CaMKII at two sites (T253 and T286) at various stages of the cell cycle, and also examined the effects of overexpression of wild-type (WT), T286D phosphomimic, T253D phosphomimic and T253V phosphonull forms of CaMKIIα in MDA-MB-231 breast cancer and SHSY5Y neuroblastoma cells on cellular proliferation and cell cycle progression. We demonstrate herein that whilst there is no change in total CaMKII expression or T286 phosphorylation throughout the cell cycle, a marked dephosphorylation of CaMKII at T253 occurs during the G2 and/or M phases. Additionally, we show by molecular inhibition, as well as pharmacological activation, that protein phosphatase 2A (PP2A) is the phosphatase responsible for this dephosphorylation. Furthermore, we show that inducible overexpression of WT, T286D and T253V forms of CaMKIIα in MDA-MB-231 and SHSY5Y cells increases cellular proliferation, with no alteration in cell cycle profiles. By contrast, overexpression of a T253D phosphomimic form of CaMKIIα significantly decreases proliferation, and cells accumulate in mitosis, specifically in metaphase. Taken together, these results strongly suggest that the dephosphorylation of CaMKII at T253 is involved in controlling the cell cycle, specifically the metaphase–anaphase transition.

Chromosome Segregation in Budding Yeast: Sister Chromatid Cohesion and Related Mechanisms

Studies on budding yeast have exposed the highly conserved mechanisms by which duplicated chromosomes are evenly distributed to daughter cells at the metaphase–anaphase transition. The establishment of proteinaceous bridges between sister chromatids, a function provided by a ring-shaped complex known as cohesin, is central to accurate segregation. It is the destruction of this cohesin that triggers the segregation of chromosomes following their proper attachment to microtubules. Since it is irreversible, this process must be tightly controlled and driven to completion. Furthermore, during meiosis, modifications must be put in place to allow the segregation of maternal and paternal chromosomes in the first division for gamete formation. Here, I review the pioneering work from budding yeast that has led to a molecular understanding of the establishment and destruction of cohesion.

Coordinated regulation of p31Comet and Mad2 expression is required for cellular proliferation

p31Comet is a well-known interacting partner of the spindle assembly checkpoint (SAC) effector molecule Mad2. At the molecular level it is well established that p31Comet promotes efficient mitotic exit, specifically the metaphase–anaphase transition, by antagonizing Mad2 function. However, there is little knowledge of how p31Comet is regulated or the physiological importance of controlling p31Comet. Here, we show that the Rb–E2F pathway regulates p31Comet expression. In multiple tumor types (including breast and lung) p31Comet expression is increased along with Mad2. Expression of this antagonist–target pair is coordinated in cells and correlated in cancer. Moreover, a narrow range of p31Comet:Mad2 ratios is compatible with cellular viability. Our data suggest that coordinate regulation is important for the outgrowth of oncogenic cell populations. Our findings suggest that altered p31Comet:Mad2 expression ratios may provide new insight into altered SAC function and the generation of chromosomal instability in tumors.

Mitotic phosphorylation of Exo84 disrupts exocyst assembly and arrests cell growth

The rate of eukaryotic cell growth is tightly controlled for proper progression through each cell cycle stage and is important for cell size homeostasis. It was previously shown that cell growth is inhibited during mitosis when cells are preparing for division. However, the mechanism for growth arrest at this stage is unknown. Here we demonstrate that exocytosis of a select group of cargoes was inhibited before the metaphase-anaphase transition in the budding yeast Saccharomyces cerevisiae. The cyclin-dependent kinase, Cdk1, when bound to the mitotic cyclin Clb2, directly phosphorylated Exo84, a component of the exocyst complex essential for exocytosis. Mitotic phosphorylation of Exo84 disrupted the assembly of the exocyst complex, thereby affecting exocytosis and cell surface expansion. Our study demonstrates the coordination between membrane trafficking and cell cycle progression and provides a molecular mechanism by which cell growth is controlled during the cell division cycle.

PI 3-kinase-dependent phosphorylation of Plk1–Ser99 promotes association with 14-3-3γ and is required for metaphase–anaphase transition

Polo-like kinase 1 (Plk1) controls multiple aspects of mitosis and is activated through its phosphorylation at Thr210. Here we identify Ser99 on Plk1 as a novel mitosis-specific phosphorylation site, which operates independently of Plk1–Thr210 phosphorylation. Plk1–Ser99 phosphorylation creates a docking site for 14-3-3γ, and this interaction stimulates the catalytic activity of Plk1. Knockdown of 14-3-3γ or replacement of wild-type (WT) Plk1 by a Ser99-phospho-blocking mutant leads to a prometaphase/metaphase-like arrest due to the activation of the spindle assembly checkpoint. Inhibition of phosphatidylinositol 3-kinase (PI3K) and Akt significantly reduces the level of Plk1–Ser99 phosphorylation and delays metaphase to anaphase transition. Plk1–Ser99 phosphorylation requires not only Akt activity but also protein(s) associated with Plk1 in a mitosis-specific manner. Therefore, mitotic Plk1 activity is regulated not only by Plk1–Thr210 phosphorylation, but also by Plk1 binding to 14-3-3γ following Plk1–Ser99 phosphorylation downstream of the PI3K–Akt signalling pathway. This novel Plk1 activation pathway controls proper progression from metaphase to anaphase.

APC/CCdh1-dependent degradation of Cdc20 requires a phosphorylation on CRY-box by Polo-like kinase-1 during somatic cell cycle

Cdc20 is an activator of the anaphase-promoting complex (APC/C), and APC/CCdc20 is essential for metaphase–anaphase transition. To allow progression beyond mitosis, Cdc20 is degraded through KEN-box-dependent APC/CCdh1 activity. Mammalian Cdc20 contains the CRY box, a second APC/CCdh1-dependent degron, but the molecular mechanism in degradation process remains undefined. Polo-like kinase-1 (Plk1) is an essential mitotic kinase regulating various targets in kinetochore, centrosome, and midbody for proper mitotic progression. Plk1 directly bound to Cdc20 and phosphorylates it on serine-170 located in CRY-box. Whereas wild-type Cdc20 was degraded according to progress cell cycle beyond mitosis, the phosphorylation-defective mutant, which serine-170 was changed into alanine, was not destroyed in early G1 phase. The phosphorylation on serine-170 by Plk1 was important for ubiquitination and Cdh1-dependent proteolysis. However, this modification by Plk1 on CRY box had no effect on the subcellular localization of Cdc20 and the formation of APC/C-inhibitory checkpoint complexes under spindle assembly checkpoint. This mechanism will be the first finding of inhibitory phosphorylation related to Cdc20 instability.

Partial depletion of gamma‐actin suppresses microtubule dynamics

Actin and microtubule interactions are important for many cellular events, however these interactions are poorly described. Alterations in γ-actin are associated with diseases such as hearing loss and cancer. Functional investigations demonstrated that partial depletion of γ-actin affects cell polarity and induces resistance to microtubule-targeted agents. To determine whether γ-actin alterations directly affect microtubule dynamics, microtubule dynamic instability was analyzed in living cells following partial siRNA depletion of γ-actin. Partial depletion of γ-actin suppresses interphase microtubule dynamics by 17.5% due to a decrease in microtubule shortening rates and an increase in microtubule attenuation. γ-Actin partial depletion also increased distance-based microtubule catastrophe and rescue frequencies. In addition, knockdown of γ-actin delayed mitotic progression, partially blocking metaphase–anaphase transition and inhibiting cell proliferation. Interestingly, in the presence of paclitaxel, interphase microtubule dynamics were further suppressed by 24.4% in the γ-actin knockdown cells, which is comparable to 28.8% suppression observed in the control siRNA treated cells. Paclitaxel blocked metaphase–anaphase transition in both the γ-actin knockdown cells and the control siRNA cells. However, the extent of mitotic arrest was much higher in the control cells (28.4%), compared to the γ-actin depleted cells (8.5%). Therefore, suppression of microtubule dynamics by partial depletion of γ-actin is associated with marked delays in metaphase-anaphase transition and not mitotic arrest. This is the first demonstration that γ-actin can modulate microtubule dynamics by reducing the microtubule shortening rate, promoting paused/attenuated microtubules, and increasing transition frequencies suggesting a mechanistic link between γ-actin and microtubules. © 2013 Wiley Periodicals, Inc

MYCN-mediated overexpression of mitotic spindle regulatory genes and loss of p53-p21 function jointly support the survival of tetraploid neuroblastoma cells

High-risk neuroblastomas often harbor structural chromosomal alterations, including amplified MYCN, and usually have a near-di/tetraploid DNA index, but the mechanisms creating tetraploidy remain unclear. Gene-expression analyses revealed that certain MYCN/MYC and p53/pRB-E2F target genes, especially regulating mitotic processes, are strongly expressed in near-di/tetraploid neuroblastomas. Using a functional RNAi screening approach and live-cell imaging, we identified a group of genes, including MAD2L1, which after knockdown induced mitotic-linked cell death in MYCN-amplified and TP53-mutated neuroblastoma cells. We found that MYCN/MYC-mediated overactivation of the metaphase–anaphase checkpoint synergizes with loss of p53-p21 function to prevent arrest or apoptosis of tetraploid neuroblastoma cells.