Oxidative stress induces meiotic defects of oocytes in a mouse psoriasis model.

Psoriasis, an immune-mediated inflammatory disease, is associated with poor pregnancy outcomes. Emerging evidence indicates that these defects are likely attributed to compromised oocyte competence. Nevertheless, little is known about the underlying associated mechanisms between psoriasis and poor oocyte quality. In this study, we construct an imiquimod-induced chronic psoriasis-like mouse model to review the effects of psoriasis on oocyte quality. We discover that oocytes from psoriasis-like mice display spindle/chromosome disorganization, kinetochore-microtubule mis-attachment, and aneuploidy. Importantly, our results show that melatonin supplement in vitro and in vivo not only increases the rate of matured oocytes but also significantly attenuates oxidative stress and meiotic defects by restoring mitochondrial function in oocytes from psoriasis-like mice. Altogether, our data uncover the adverse effects of psoriasis symptoms on oocytes, and melatonin supplement ameliorates oxidative stress and meiotic defects of oocytes from psoriatic mice.

Kolaflavanone, a biflavonoid derived from medicinal plant Garcinia, is an inhibitor of mitotic kinesin Eg5.

Mitotic kinesin Eg5 remains a validated target in antimitotic therapy because of its essential role in the formation and maintenance of bipolar mitotic spindles. Although numerous Eg5 inhibitors of synthetic origin are known, only a few inhibitors derived from natural products have been reported. In our study, we focused on identifying novel Eg5 inhibitors from medicinal plants, particularly Garcinia species. Herein, we report the inhibitory effect of kolaflavanone (KLF), a Garcinia biflavonoid, on the ATPase and microtubule-gliding activities of mitotic kinesin Eg5. Additionally, we showed the interaction mechanism between Eg5 and KLF via in vitro and in silico analyses. The results revealed that KLF inhibited both the basal and microtubule-activated ATPase activities of Eg5. The inhibitory mechanism is allosteric, without a direct competition with adenosine-5'-diphosphate for the nucleotide-binding site. KLF also suppressed the microtubule gliding of Eg5 in vitro. The Eg5-KLF model obtained from molecular docking showed that the biflavonoid exists within the α2/α3/L5 (α2: Lys111-Glu116 and Ile135-Asp149, α3: Asn206-Thr226; L5: Gly117-Gly134) pocket, with a binding pose comparable to known Eg5 inhibitors. Overall, our data suggest that KLF is a novel allosteric inhibitor of mitotic kinesin Eg5.

Nicotinamide Mononucleotide Supplementation Reverses the Declining Quality of Maternally Aged Oocytes.

Advanced maternal age is highly associated with a decline in oocyte quality, but effective approaches to improve it have still not been fully determined. Here, we report that in vivo supplementation of nicotinamide mononucleotide (NMN) efficaciously improves the quality of oocytes from naturally aged mice by recovering nicotinamide adenine dinucleotide (NAD+) levels. NMN supplementation not only increases ovulation of aged oocytes but also enhances their meiotic competency and fertilization ability by maintaining the normal spindle/chromosome structure and the dynamics of the cortical granule component ovastacin. Moreover, single-cell transcriptome analysis shows that the beneficial effect of NMN on aged oocytes is mediated by restoration of mitochondrial function, eliminating the accumulated ROS to suppress apoptosis. Collectively, our data reveal that NMN supplementation is a feasible approach to protect oocytes from advanced maternal age-related deterioration, contributing to the improvement of reproductive outcome of aged women and assisted reproductive technology.

Treating Cancer by Spindle Assembly Checkpoint Abrogation: Discovery of Two Clinical Candidates, BAY 1161909 and BAY 1217389, Targeting MPS1 Kinase.

Inhibition of monopolar spindle 1 (MPS1) kinase represents a novel approach to cancer treatment: instead of arresting the cell cycle in tumor cells, cells are driven into mitosis irrespective of DNA damage and unattached/misattached chromosomes, resulting in aneuploidy and cell death. Starting points for our optimization efforts with the goal to identify MPS1 inhibitors were two HTS hits from the distinct chemical series "triazolopyridines" and "imidazopyrazines". The major initial issue of the triazolopyridine series was the moderate potency of the HTS hits. The imidazopyrazine series displayed more than 10-fold higher potencies; however, in the early project phase, this series suffered from poor metabolic stability. Here, we outline the evolution of the two hit series to clinical candidates BAY 1161909 and BAY 1217389 and reveal how both clinical candidates bind to the ATP site of MPS1 kinase, while addressing different pockets utilizing different binding interactions, along with their synthesis and preclinical characterization in selected in vivo efficacy models.

In Silico Discovery of Novel Phytoconstituents of Amyris pinnata as a Mitotic Spindle Kinase Inhibitor.

BACKGROUND: Despite many successes in the discovery of numerous cancer chemotherapeutic agents from natural sources, some of the moieties were dropped because of its inefficiency or serious toxicity. Mitosis is an ordered series of fundamentally mechanical events in which identical copies of the genome are moved to two discrete locations within the dividing cell. The crucial role of the mitotic spindle in cell division has identified, which is an important target in cancer chemotherapy. In the present study, we are reporting molecular docking studies and in silico pharmacokinetic profiles of selected phytoconstituents obtained from Amyris pinnata. METHODS: Molecular docking studies of selected phytoconstituents were performed using iGEMDOCK. The crystal structure of the protein was exported from the protein data bank (PDB id: 4C4H). In silico pharmacokinetic profile of selected phytoconstituents was performed using the SWISSADME server. RESULTS: Compound AMNP6 showed higher binding affinity as compared to the standard ligand. All the selected phytoconstituents have passed the Lipinski rule of five and shown no violations. CONCLUSION: Good binding affinity and drug likeliness of the AMNP6 suggest that it can be further investigated and explored as mitotic spindle kinase inhibitor in cancer disease.

Nondisjunction and unequal spindle organization accompany the drive of Aegilops speltoides B chromosomes.

B chromosomes (Bs) are supernumerary chromosomes, which are often preferentially inherited. When transmission rates of chromosomes are higher than 0.5, not obeying the Mendelian law of equal segregation, the resulting transmission advantage is collectively referred to as 'chromosome drive'. Here we analysed the drive mechanism of Aegilops speltoides Bs. The repeat AesTR-183 of A. speltoides Bs, which also can be detected on the Bs of Aegilops mutica and rye, was used to track Bs during pollen development. Nondisjunction of CENH3-positive, tubulin interacting B sister chromatids and an asymmetric spindle during first pollen grain mitosis are key for the accumulation process. A quantitative flow cytometric approach revealed that, independent of the number of Bs present in the mother plant, Bs accumulate in the generative nuclei to > 93%. Nine out of 11 tested (peri)centromeric repeats were shared by A and B chromosomes. Our findings provide new insights into the process of chromosome drive. Quantitative flow cytometry is a useful and reliable method to study the drive frequency of Bs. Nondisjunction and unequal spindle organization accompany during first pollen mitosis the drive of A. speltoides Bs. The prerequisites for the drive process seems to be common in Poaceae.

Indirubin, a bis-indole alkaloid binds to tubulin and exhibits antimitotic activity against HeLa cells in synergism with vinblastine.

Indirubin, a bis-indole alkaloid used in traditional Chinese medicine has shown remarkable anticancer activity against chronic myelocytic leukemia. The present work was aimed to decipher the underlying molecular mechanisms responsible for its anticancer attributes. Our findings suggest that indirubin inhibited the proliferation of HeLa cells with an IC50 of 40 µM and induced a mitotic block. At concentrations higher than its IC50, indirubin exerted a moderate depolymerizing effect on the interphase microtubular network and spindle microtubules in HeLa cells. Studies with goat brain tubulin indicated that indirubin bound to tubulin at a single site with a dissociation constant of 26 ±â€¯3 µM and inhibited the in vitro polymerization of tubulin into microtubules in the presence of glutamate as well as microtubule-associated proteins. Molecular docking analysis and molecular dynamics simulation studies indicate that indirubin stably binds to tubulin at the interface of the α-ß tubulin heterodimer. Further, indirubin stabilized the binding of colchicine on tubulin and promoted the cysteine residue modification by 5,5'-dithiobis-2-nitrobenzoic acid, indicating towards alteration of tubulin conformation upon binding. In addition, we found that indirubin synergistically enhanced the anti-mitotic and anti-proliferative activity of vinblastine, a known microtubule-targeted agent. Collectively our studies indicate that perturbation of microtubule polymerization dynamics could be one of the possible mechanisms behind the anti-cancer activities of indirubin.

Aurora A Phosphorylation of YY1 during Mitosis Inactivates its DNA Binding Activity.

Successful execution of mitotic cell division requires the tight synchronisation of numerous biochemical pathways. The underlying mechanisms that govern chromosome segregation have been thoroughly investigated. However, the mechanisms that regulate transcription factors in coordination with mitotic progression remain poorly understood. In this report, we identify the transcription factor YY1 as a novel mitotic substrate for the Aurora A kinase, a key regulator of critical mitotic events, like centrosome maturation and spindle formation. Using in vitro kinase assays, we show that Aurora A directly phosphorylates YY1 at serine 365 in the DNA-binding domain. Using a new phospho-specific antibody, we show that YY1 phosphorylation at serine 365 occurs during mitosis, and that this phosphorylation is significantly reduced upon inhibition of Aurora A. Furthermore, we show, using electrophoretic mobility shift and chromatin immunoprecipitation assays, that phosphorylation of YY1 at this site abolishes its DNA binding activity in vitro and in vivo. In conformity with this loss of binding activity, phosphorylated YY1 also loses its transctivation ability as demonstrated by a luciferase reporter assay. These results uncover a novel mechanism that implicates Aurora A in the mitotic inactivation of transcription factors.

Pseudolaric acid B induces mitotic arrest and apoptosis in both 5-fluorouracil-sensitive and -resistant colorectal cancer cells.

5-fluorouracil (5-FU)-based chemotherapy is the main chemotherapeutic approach for colorectal cancer (CRC) treatment. Because chemoresistance occurs frequently and significantly limits CRC therapies, a novel agent is needed. Pseudolaric acid B (PAB), a small molecule derived from the Chinese medicinal herb ''Tujinpi'', exhibits strong cytotoxic effects on a variety of cancers. However, the detailed mechanisms by which PAB inhibits CRC cell growth and its potential role in overcoming 5-FU resistance have not been well studied. In this study, we showed that PAB significantly inhibited the viability of various CRC cell lines but induced minor cytotoxicity in normal cells. Both the in vitro and in vivo results showed that PAB induced proliferation inhibition, mitotic arrest and subsequently caspase-dependent apoptosis in both 5-FU-sensitive and -resistant CRC cells. Moreover, PAB was shown to interfere with CRC cell mitotic spindle apparatus and activate the spindle assembly checkpoint. Finally, CDK1 activity was involved in PAB-induced mitotic arrest and apoptosis in CRC cells. Taken together, these data reveal that PAB induces CRC cell mitotic arrest followed by apoptosis and overcomes 5-FU resistance in vitro and in vivo, suggesting that PAB may be a potential agent for CRC treatment, particularly for 5-FU-resistant CRC.

Modeling Tumor Treating Fields (TTFields) application in single cells during metaphase and telophase.

Effects of electric fields on biological cells have been extensively studied but primarily in the low and high frequency regimes. Low frequency AC fields have been investigated for applications to nerve and muscle stimulation or to examine possible environmental effects of 60 Hz excitation. High frequency fields have been studied to understand tissue heating and tumor ablation. Biological effects at intermediate frequencies (in the 100-500 kHz regime) have only recently been discovered and are now being used clinically to disrupt cell division, primarily for the treatment of recurrent glioblastoma multiforme. In this study, we develop a computational framework to investigate the mechanisms of action of these Tumor Treating Fields (TTFields) and to understand in vitro findings observed in cell culture. Using Finite Element Method models of isolated cells we show that the intermediate frequency range is unique because it constitutes a transition region in which the intracellular electric field, shielded at low frequencies, increases significantly. We also show that the threshold at which this increase occurs depends on the dielectric properties of the cell membrane. Furthermore, our models of different stages of the cell cycle and of the morphological changes associated with cytokinesis show that peak dielectrophoretic forces develop within dividing cells exposed to TTFields. These findings are in agreement with in vitro observations, and enhance our understanding of how TTFields disrupt cellular function.

Effects of 5α-dihydrotestosterone and 17ß-estradiol on the mouse ovarian follicle development and oocyte maturation.

We have previously reported that androstenedione induces abnormalities of follicle development and oocyte maturation in the mouse ovary. In granulosa cells of the ovarian follicle, androstenedione is aromatized to 17ß-estradiol (E2). To determine whether the androgen or estrogen acts directly on the follicle to induce the above-mentioned abnormalities, we compared the effects of a non-aromatizable androgen, 5α-dihydrotestosterone (DHT), with those of E2 on murine follicular development and oocyte maturation in a single follicle culture system. The high dose (10(-6) M) of DHT prompted normal follicular development, and there was no effect on oocyte meiotic maturation after stimulation with human chorionic gonadotropin (hCG) and epidermal growth factor (EGF). In contrast, culture with the high dose (10(-6) M) of E2 delayed follicular growth and also suppressed proliferation of granulosa cells and antrum formation. Furthermore, culture with E2 delayed or inhibited oocyte meiotic maturation, such as chromosome alignment on the metaphase plate and extrusion of the first polar body, after addition of hCG and EGF. In conclusion, these findings demonstrate that E2, but not DHT, induces abnormalities of follicular development, which leads to delay or inhibition of oocyte meiotic maturation.

Deformation of nuclei and abnormal spindles assembly in the second male meiosis of polyploid tobacco plants.

The bipolar spindle is a major cytoskeletal structure, which ensures an equal chromosome distribution between the daughter nuclei. The spindle formation in animal cells depends on centrosomes activity. In flowering plant cells the centrosomes have not been identified as definite structures. The absence of these structures suggests that plants assemble their spindle via novel mechanisms. Nonetheless, the cellular and molecular mechanisms controlling the cytoskeleton remodeling during the spindle development in plants are still insufficiently clear. This article describes the results of a comparative analysis of the microtubular cytoskeleton dynamics during assembly of the second division spindle in tobacco microsporocytes with the normal and deformed nuclei. According to our observations, the bipolar spindle fibres are formed from short arrays of the disintegrated perinuclear cytoskeleton system, the perinuclear microtubular band. The microsporocytes of polyploid tobacco plants with deformed nuclei entirely lack this cytoskeleton structure. In such type of cells the overall prometaphase events are blocked, and the assembly of second division spindles is completely arrested.

Growth arrest by the antitumor steroidal lactone withaferin A in human breast cancer cells is associated with down-regulation and covalent binding at cysteine 303 of ß-tubulin.

Withaferin A (WA), a C5,C6-epoxy steroidal lactone derived from a medicinal plant (Withania somnifera), inhibits growth of human breast cancer cells in vitro and in vivo and prevents mammary cancer development in a transgenic mouse model. However, the mechanisms underlying the anticancer effect of WA are not fully understood. Herein, we report that tubulin is a novel target of WA-mediated growth arrest in human breast cancer cells. The G2 and mitotic arrest resulting from WA exposure in MCF-7, SUM159, and SK-BR-3 cells was associated with a marked decrease in protein levels of ß-tubulin. These effects were not observed with the naturally occurring C6,C7-epoxy analogs of WA (withanone and withanolide A). A non-tumorigenic normal mammary epithelial cell line (MCF-10A) was markedly more resistant to mitotic arrest by WA compared with breast cancer cells. Vehicle-treated control cells exhibited a normal bipolar spindle with chromosomes aligned along the metaphase plate. In contrast, WA treatment led to a severe disruption of normal spindle morphology. NMR analyses revealed that the A-ring enone in WA, but not in withanone or withanolide A, was highly reactive with cysteamine and rapidly succumbed to irreversible nucleophilic addition. Mass spectrometry demonstrated direct covalent binding of WA to Cys(303) of ß-tubulin in MCF-7 cells. Molecular docking indicated that the WA-binding pocket is located on the surface of ß-tubulin and characterized by a hydrophobic floor, a hydrophobic wall, and a charge-balanced hydrophilic entrance. These results provide novel insights into the mechanism of growth arrest by WA in breast cancer cells.

Does copper stress lead to spindle misposition-dependent cell cycle arrest?

Because of its specific electrochemical properties, copper is an essential heavy metal for living organisms. As with other heavy metals, high levels can provoke damage. We examined gene expression under copper stress in wild-type fission yeast (Schizosaccharomyces pombe) through differential display. After the EC(50) concentration of CuSO(4) was determined as 50 µM, total RNA was isolated from cells treated or not with copper. The expression level of SPCC1682.13, ppk1, SPBC2F12.05c, and adg2 genes increased significantly under copper stress. Considering the functions of these genes are related to the cell cycle, cell division and chromosome dynamics, we hypothesize that retardation of the cell cycle under copper stress is relevant to the events that depend on the functions of these genes.

"Bouquet arrest", monopolar chromosomes segregation, and correction of the abnormal spindle.

According to our data, the arrest of univalents in bouquet arrangement is a widespread meiotic feature in cereal haploids and allohaploids (wide hybrids F(1)). We have analyzed 83 different genotypes of cereal haploids and allohaploids with visualization of the cytoskeleton and found a bouquet arrest in 45 of them (in 30% to 100% pollen mother cells (PMCs)). The meiotic plant cell division in 26 various genotypes with a zygotene bouquet arrest was analyzed in detail. In three of them in PMCs, a very specific monopolar conic-shaped figure at early prometaphase is formed. This monopolar figure consists of mono-oriented univalents and their kinetochore fibers converging in pointed pole. Such figures are never observed at wild-type prometaphase or in asynaptic meiosis in the variants without a bouquet arrest. Later at prometaphase, the bipolar central spindle fibers join in this monopolar figure, and a bipolar spindle with all univalents connected to one pole is formed. As a result of monopolar chromosome segregation at anaphase and normal cytokinesis at telophase, a dyad with one member carrying a restitution nucleus and the other enucleated is formed. However, such phenotype has only three genotypes among 26 analyzed with a bouquet arrest. In the remaining 23 haploids and allohaploids, the course of prometaphase was altered after the conic monopolar figure formation. In these variants, the completely formed conic monopolar figure was disintegrated into a chaotic network of spindle fibers and univalents acquired a random orientation. This arrangement looks like a mid-prometaphase in the wild-type meiosis. At late prometaphase, a bipolar spindle is formed with the univalents distributed more or less equally between two poles, similar to the phenotypes without a bouquet arrest. The product of cell division is a dyad with aneuploid members. Thus, the spindle abnormality-monopolar chromosome orientation-is corrected. In some cells the correction of the prometaphase monopolus occurs by means of its splitting into two half-spindles and their rotation along the future division axis.

Augmin plays a critical role in organizing the spindle and phragmoplast microtubule arrays in Arabidopsis.

In higher plant cells, microtubules (MTs) are nucleated and organized in a centrosome-independent manner. It is unclear whether augmin-dependent mechanisms underlie spindle MT organization in plant cells as they do in animal cells. When AUGMIN subunit3 (AUG3), which encodes a homolog of animal dim γ-tubulin 3/human augmin-like complex, subunit 3, was disrupted in Arabidopsis thaliana, gametogenesis frequently failed due to defects in cell division. Compared with the control microspores, which formed bipolar spindles at the cell periphery, the mutant cells often formed peripheral half spindles that only attached to condensed chromosomes or formed elongated spindles with unfocused interior poles. In addition, defective cells exhibited disorganized phragmoplast MT arrays, which caused aborted cytokinesis. The resulting pollen grains were either shrunken or contained two nuclei in an undivided cytoplasm. AUG3 was localized along MTs in the spindle and phragmoplast, and its signal was pronounced in anaphase spindle poles. An AUG3-green fluorescent protein fusion exhibited a dynamic distribution pattern, similar to that of the γ-tubulin complex protein2. When AUG3 was enriched from seedlings by affinity chromatography, AUG1 was detected by immunoblotting, suggesting an augmin-like complex was present in vivo. We conclude that augmin plays a critical role in MT organization during plant cell division.

The Arabidopsis mutant jason produces unreduced first division restitution male gametes through a parallel/fused spindle mechanism in meiosis II.

In plants, whole-genome doubling (polyploidization) is a widely occurring process largely contributing to plant evolution and diversification. The generation and fusion of diploid gametes is now considered the major route of plant polyploidization. The parallel arrangement or fusion of meiosis II MII spindles (ps) is one of the most frequently reported mechanisms generating triploid offspring. Through a forward genetics screen of an Arabidopsis (Arabidopsis thaliana) ethyl methanesulfonate population, we identified Arabidopsis thaliana Parallel Spindles1 (AtPS1), which was recently reported as a major gene implicated in the control of the ps meiotic defect. In addition, we describe the isolation and characterization of a novel allele of JASON, involved in male gametophytic ploidy regulation in plants. Similar to atps1 mutants, jason produces more than 25% 2n pollen grains and spontaneously forms triploid offspring. By combining both cytological and genetic approaches, we demonstrate that loss of JASON causes the formation of parallel arranged and fused spindles in male MII, resulting in the production of unreduced first division restitution 2n spores. Although JASON encodes a protein of unknown function, we additionally show that the meiotic ps defect in jason is caused by a reduction in AtPS1 transcript levels, indicating that JASON positively regulates AtPS1 expression, allowing the proper organization and orientation of metaphase II spindle plates in MII.

Porcine oocytes are most vulnerable to the mycotoxin deoxynivalenol during formation of the meiotic spindle.

Deoxynivalenol (DON, vomitoxin) is a secondary metabolite and mycotoxin produced by Fusarium species that occurs with a high prevalence in cereals and grains intended for human and animal consumption. Pigs are considered to be the most sensitive animal species and exposure to DON results in reduced feed intake, reduced performance and cause alterations in the expression of markers of inflammation and cell cycle regulation. The objective of this study was to determine how DON possibly affects the oocyte developmental potential in vitro at concentrations which correspond to those observed in practice. To evaluate DON toxicity during specific stages of oocyte meiosis, cumulus-oocyte complexes were exposed to 0.02, 0.2, or 2 microM DON. Exposure to the highest DON concentration inhibited cumulus expansion and induced cumulus cell death. After exposure for 42 h, DON at all concentrations reduced Metaphase II formation and led to malformations of the meiotic spindle. Despite spindle malformations, exposure to different concentrations of DON did not lead to increased percentages of blastomeres with abnormal ploidy in embryos. Spindle malformation occurred by DON exposure during formation of meiotic spindles at Metaphase I and II, but embryo development was also reduced when oocytes were exposed to DON during Prophase I. Together, these results indicate that exposure to DON via contaminated food or feed can affect oocyte developmental competence by interfering directly with microtubule dynamics during meiosis, and by disturbing oocyte cytoplasmic maturation through other as yet undetermined mechanisms.

Dynamics of protein phosphorylation during meiotic maturation.

PURPOSE: To ask whether distinct kinase signaling pathways mediate cytoplasmic or nuclear maturation of mouse oocytes and if in vitro maturation influences the distribution and timing of these phosphorylation events. METHODS: Mouse cumulus oocyte complexes (COCs) were matured under conditions known to influence oocyte quality (basal or supplemented media) and assayed with epitope specific antibodies that would distinguish between Cdk1 or tyrosine kinase targets at 0, 2, 4, 8, and 16 hrs. Semi-quantitative image analysis was used to assess the topographical patterns of protein phosphorylation during in vitro maturation. In vitro fertilization and embryo culture were used to examine the effects of culture conditions on developmental potential. RESULTS: Protein tyrosine phosphorylation increased during meiotic progression from methaphase-I to metaphase-II. Levels were significantly higher in the oocyte cortex. Levels of cortical staining are enhanced in oocytes matured in supplemented media that displayed higher developmental competence. In contrast, bulk substrates for Cdk1 kinase localize to the meiotic spindle while cytoplasmic levels of kinase activity increase throughout meiotic progression; culture media had no measurable effect. Ablation of the tyrosine kinase Fyn significantly reduced cortical levels of tyrosine phosphorylation. CONCLUSIONS: The findings indicate that distinct signaling pathways mediate nuclear and cytoplasmic maturation during in vitro maturation in a fashion consistent with a role for tyrosine kinases in cortical maturation and oocyte quality.

The small organic compound HMN-176 delays satisfaction of the spindle assembly checkpoint by inhibiting centrosome-dependent microtubule nucleation.

HMN-176 is a potential new cancer therapeutic known to retard the proliferation of tumor cell lines. Here, we show that this compound inhibits meiotic spindle assembly in surf clam oocytes and delays satisfaction of the spindle assembly checkpoint in human somatic cells by inducing the formation of short and/or multipolar spindles. HMN-176 does not affect centrosome assembly, nuclear envelope breakdown, or other aspects of meiotic or mitotic progression, nor does it affect the kinetics of Spisula or mammalian microtubule (MT) assembly in vitro. Notably, HMN-176 inhibits the formation of centrosome-nucleated MTs (i.e., asters) in Spisula oocytes and oocyte extracts, as well as from isolated Spisula or mammalian centrosomes in vitro. Together, these results reveal that HMN-176 is a first-in-class anticentrosome drug that inhibits proliferation, at least in part, by disrupting centrosome-mediated MT assembly during mitosis.