Structural Basis for Mitotic Centrosome Assembly in Flies.

In flies, Centrosomin (Cnn) forms a phosphorylation-dependent scaffold that recruits proteins to the mitotic centrosome, but how Cnn assembles into a scaffold is unclear. We show that scaffold assembly requires conserved leucine zipper (LZ) and Cnn-motif 2 (CM2) domains that co-assemble into a 2:2 complex in vitro. We solve the crystal structure of the LZ:CM2 complex, revealing that both proteins form helical dimers that assemble into an unusual tetramer. A slightly longer version of the LZ can form micron-scale structures with CM2, whose assembly is stimulated by Plk1 phosphorylation in vitro. Mutating individual residues that perturb LZ:CM2 tetramer assembly perturbs the formation of these micron-scale assemblies in vitro and Cnn-scaffold assembly in vivo. Thus, Cnn molecules have an intrinsic ability to form large, LZ:CM2-interaction-dependent assemblies that are critical for mitotic centrosome assembly. These studies provide the first atomic insight into a molecular interaction required for mitotic centrosome assembly.

Proteome dynamics at broken replication forks reveal a distinct ATM-directed repair response suppressing DNA double-strand break ubiquitination.

Cells have evolved an elaborate DNA repair network to ensure complete and accurate DNA replication. Defects in these repair machineries can fuel genome instability and drive carcinogenesis while creating vulnerabilities that may be exploited in therapy. Here, we use nascent chromatin capture (NCC) proteomics to characterize the repair of replication-associated DNA double-strand breaks (DSBs) triggered by topoisomerase 1 (TOP1) inhibitors. We reveal profound changes in the fork proteome, including the chromatin environment and nuclear membrane interactions, and identify three classes of repair factors according to their enrichment at broken and/or stalled forks. ATM inhibition dramatically rewired the broken fork proteome, revealing that ataxia telangiectasia mutated (ATM) signalling stimulates DNA end resection, recruits PLK1, and concomitantly suppresses the canonical DSB ubiquitination response by preventing accumulation of RNF168 and BRCA1-A. This work and collection of replication fork proteomes provide a new framework to understand how cells orchestrate homologous recombination repair of replication-associated DSBs.

BUB1 and CENP-U, Primed by CDK1, Are the Main PLK1 Kinetochore Receptors in Mitosis.

Reflecting its pleiotropic functions, Polo-like kinase 1 (PLK1) localizes to various sub-cellular structures during mitosis. At kinetochores, PLK1 contributes to microtubule attachments and mitotic checkpoint signaling. Previous studies identified a wealth of potential PLK1 receptors at kinetochores, as well as requirements for various mitotic kinases, including BUB1, Aurora B, and PLK1 itself. Here, we combine ectopic localization, in vitro reconstitution, and kinetochore localization studies to demonstrate that most and likely all of the PLK1 is recruited through BUB1 in the outer kinetochore and centromeric protein U (CENP-U) in the inner kinetochore. BUB1 and CENP-U share a constellation of sequence motifs consisting of a putative PP2A-docking motif and two neighboring PLK1-docking sites, which, contingent on priming phosphorylation by cyclin-dependent kinase 1 and PLK1 itself, bind PLK1 and promote its dimerization. Our results rationalize previous observations and describe a unifying mechanism for recruitment of PLK1 to human kinetochores.

A bifunctional kinase-phosphatase module balances mitotic checkpoint strength and kinetochore-microtubule attachment stability.

Two major mechanisms safeguard genome stability during mitosis: the mitotic checkpoint delays mitosis until all chromosomes have attached to microtubules, and the kinetochore-microtubule error-correction pathway keeps this attachment process free from errors. We demonstrate here that the optimal strength and dynamics of these processes are set by a kinase-phosphatase pair (PLK1-PP2A) that engage in negative feedback from adjacent phospho-binding motifs on the BUB complex. Uncoupling this feedback to skew the balance towards PLK1 produces a strong checkpoint, hypostable microtubule attachments and mitotic delays. Conversely, skewing the balance towards PP2A causes a weak checkpoint, hyperstable microtubule attachments and chromosome segregation errors. These phenotypes are associated with altered BUB complex recruitment to KNL1-MELT motifs, implicating PLK1-PP2A in controlling auto-amplification of MELT phosphorylation. In support, KNL1-BUB disassembly becomes contingent on PLK1 inhibition when KNL1 is engineered to contain excess MELT motifs. This elevates BUB-PLK1/PP2A complex levels on metaphase kinetochores, stabilises kinetochore-microtubule attachments, induces chromosome segregation defects and prevents KNL1-BUB disassembly at anaphase. Together, these data demonstrate how a bifunctional PLK1/PP2A module has evolved together with the MELT motifs to optimise BUB complex dynamics and ensure accurate chromosome segregation.

RIPK1 and Caspase-8 Ensure Chromosome Stability Independently of Their Role in Cell Death and Inflammation.

Receptor-interacting protein kinase (RIPK) 1 functions as a key mediator of tissue homeostasis via formation of Caspase-8 activating ripoptosome complexes, positively and negatively regulating apoptosis, necroptosis, and inflammation. Here, we report an unanticipated cell-death- and inflammation-independent function of RIPK1 and Caspase-8, promoting faithful chromosome alignment in mitosis and thereby ensuring genome stability. We find that ripoptosome complexes progressively form as cells enter mitosis, peaking at metaphase and disassembling as cells exit mitosis. Genetic deletion and mitosis-specific inhibition of Ripk1 or Caspase-8 results in chromosome alignment defects independently of MLKL. We found that Polo-like kinase 1 (PLK1) is recruited into mitotic ripoptosomes, where PLK1's activity is controlled via RIPK1-dependent recruitment and Caspase-8-mediated cleavage. A fine balance of ripoptosome assembly is required as deregulated ripoptosome activity modulates PLK1-dependent phosphorylation of downstream effectors, such as BUBR1. Our data suggest that ripoptosome-mediated regulation of PLK1 contributes to faithful chromosome segregation during mitosis.

Centrioles generate a local pulse of Polo/PLK1 activity to initiate mitotic centrosome assembly.

Mitotic centrosomes are formed when centrioles start to recruit large amounts of pericentriolar material (PCM) around themselves in preparation for mitosis. This centrosome "maturation" requires the centrioles and also Polo/PLK1 protein kinase. The PCM comprises several hundred proteins and, in Drosophila, Polo cooperates with the conserved centrosome proteins Spd-2/CEP192 and Cnn/CDK5RAP2 to assemble a PCM scaffold around the mother centriole that then recruits other PCM client proteins. We show here that in Drosophila syncytial blastoderm embryos, centrosomal Polo levels rise and fall during the assembly process-peaking, and then starting to decline, even as levels of the PCM scaffold continue to rise and plateau. Experiments and mathematical modelling indicate that a centriolar pulse of Polo activity, potentially generated by the interaction between Polo and its centriole receptor Ana1 (CEP295 in humans), could explain these unexpected scaffold assembly dynamics. We propose that centrioles generate a local pulse of Polo activity prior to mitotic entry to initiate centrosome maturation, explaining why centrioles and Polo/PLK1 are normally essential for this process.

Polo-like kinase 1 regulates immune synapse assembly and cytotoxic T cell function by driving microtubule dynamics.

Elimination of virally infected or tumoral cells is mediated by cytotoxic T cells (CTL). Upon antigen recognition, CTLs assemble a specialized signaling and secretory domain at the interface with their target, the immune synapse (IS). During IS formation, CTLs acquire a transient polarity, marked by re-orientation of the centrosome and microtubule cytoskeleton toward the IS, thus directing the transport and delivery of the lytic granules to the target cell. Based on the implication that the kinase Aurora A has a role in CTL function, we hypothesized that its substrate, the mitotic regulator Polo-like kinase 1 (PLK1), might participate in CTL IS assembly. We demonstrate that PLK1 is phosphorylated upon TCR triggering and polarizes to the IS. PLK1 silencing or inhibition results in impaired IS assembly and function, as witnessed by defective synaptic accumulation of T cell receptors (TCRs), as well as compromised centrosome and lytic granule polarization to the IS, resulting in impaired target cell killing. This function is achieved by coupling early signaling to microtubule dynamics, a function pivotal for CTL-mediated cytotoxicity. These results identify PLK1 as a new player in CTL IS assembly and function.

DNA Replication Determines Timing of Mitosis by Restricting CDK1 and PLK1 Activation.

To maintain genome stability, cells need to replicate their DNA before dividing. Upon completion of bulk DNA synthesis, the mitotic kinases CDK1 and PLK1 become active and drive entry into mitosis. Here, we have tested the hypothesis that DNA replication determines the timing of mitotic kinase activation. Using an optimized double-degron system, together with kinase inhibitors to enforce tight inhibition of key proteins, we find that human cells unable to initiate DNA replication prematurely enter mitosis. Preventing DNA replication licensing and/or firing causes prompt activation of CDK1 and PLK1 in S phase. In the presence of DNA replication, inhibition of CHK1 and p38 leads to premature activation of mitotic kinases, which induces severe replication stress. Our results demonstrate that, rather than merely a cell cycle output, DNA replication is an integral signaling component that restricts activation of mitotic kinases. DNA replication thus functions as a brake that determines cell cycle duration.

Balancing Plk1 activity levels: The secret of synchrony between the cell and the centrosome cycle.

The accuracy of cell division requires precise regulation of the cellular machinery governing DNA/genome duplication, ensuring its equal distribution among the daughter cells. The control of the centrosome cycle is crucial for the formation of a bipolar spindle, ensuring error-free segregation of the genome. The cell and centrosome cycles operate in close synchrony along similar principles. Both require a single duplication round in every cell cycle, and both are controlled by the activity of key protein kinases. Nevertheless, our comprehension of the precise cellular mechanisms and critical regulators synchronizing these two cycles remains poorly defined. Here, we present our hypothesis that the spatiotemporal regulation of a dynamic equilibrium of mitotic kinases activities forms a molecular clock that governs the synchronous progression of both the cell and the centrosome cycles.

O-GlcNAcylation stimulates the deubiquitination activity of USP16 and regulates cell cycle progression.

Histone 2A monoubiquitination (uH2A) underscores a key epigenetic regulation of gene expression. In this report, we show that the deubiquitinase for uH2A, ubiquitin-specific peptidase 16 (USP16), is modified by O-linked N-acetylglucosamine (O-GlcNAc). O-GlcNAcylation involves the installation of the O-GlcNAc moiety to Ser/Thr residues. It crosstalks with Ser/Thr phosphorylation, affects protein-protein interaction, alters enzyme activity or protein folding, and changes protein subcellular localization. In our study, we first confirmed that USP16 is glycosylated on Thr203 and Ser214, as reported in a previous chemoenzymatic screen. We then discovered that mutation of the O-GlcNAcylation site Thr203, which is adjacent to deubiquitination-required Cys204, reduces the deubiquitination activity toward H2AK119ub in vitro and in cells, while mutation on Ser214 had the opposite effects. Using USP16 Ser552 phosphorylation-specific antibodies, we demonstrated that O-GlcNAcylation antagonizes cyclin-dependent kinase 1-mediated phosphorylation and promotes USP16 nuclear export. O-GlcNAcylation of USP16 is also required for deubiquitination of Polo-like kinase 1, a mitotic master kinase, and the subsequent chromosome segregation and cytokinesis. In summary, our study revealed that O-GlcNAcylation of USP16 at Thr203 and Ser214 coordinates deubiquitination of uH2A and Polo-like kinase 1, thus ensuring proper cell cycle progression.

PPP4C facilitates homologous recombination DNA repair by dephosphorylating PLK1 during early embryo development.

Mammalian early embryo cells have complex DNA repair mechanisms to maintain genomic integrity, and homologous recombination (HR) plays the main role in response to double-strand DNA breaks (DSBs) in these cells. Polo-like kinase 1 (PLK1) participates in the HR process and its overexpression has been shown to occur in a variety of human cancers. Nevertheless, the regulatory mechanism of PLK1 remains poorly understood, especially during the S and G2 phase. Here, we show that protein phosphatase 4 catalytic subunit (PPP4C) deletion causes severe female subfertility due to accumulation of DNA damage in oocytes and early embryos. PPP4C dephosphorylated PLK1 at the S137 site, negatively regulating its activity in the DSB response in early embryonic cells. Depletion of PPP4C induced sustained activity of PLK1 when cells exhibited DNA lesions that inhibited CHK2 and upregulated the activation of CDK1, resulting in inefficient loading of the essential HR factor RAD51. On the other hand, when inhibiting PLK1 in the S phase, DNA end resection was restricted. These results demonstrate that PPP4C orchestrates the switch between high-PLK1 and low-PLK1 periods, which couple the checkpoint to HR.

Japanese encephalitis virus NS1 and NS1' proteins induce vimentin rearrangement via the CDK1-PLK1 axis to promote viral replication.

The host cytoskeleton plays crucial roles in various stages of virus infection, including viral entry, transport, replication, and release. However, the specific mechanisms by which intermediate filaments are involved in orthoflavivirus infection have not been well understood. In this study, we demonstrate that the Japanese encephalitis virus (JEV) remodels the vimentin network, resulting in the formation of cage-like structures that support viral replication. Mechanistically, JEV NS1 and NS1' proteins induce the translocation of CDK1 from the nucleus to the cytoplasm and interact with it, leading to the phosphorylation of vimentin at Ser56. This phosphorylation event recruits PLK1, which further phosphorylates vimentin at Ser83. Consequently, these phosphorylation modifications convert the typically filamentous vimentin into non-filamentous "particles" or "squiggles." These vimentin "particles" or "squiggles" are then transported retrogradely along microtubules to the endoplasmic reticulum, where they form cage-like structures. Notably, NS1' is more effective than NS1 in triggering the CDK1-PLK1 cascade response. Overall, our study provides new insights into how JEV NS1 and NS1' proteins manipulate the vimentin network to facilitate efficient viral replication. IMPORTANCE: Japanese encephalitis virus (JEV) is a mosquito-borne orthoflavivirus that causes severe encephalitis in humans, particularly in Asia. Despite the availability of a safe and effective vaccine, JEV infection remains a significant public health threat due to limited vaccination coverage. Understanding the interactions between JEV and host proteins is essential for developing more effective antiviral strategies. In this study, we investigated the role of vimentin, an intermediate filament protein, in JEV replication. Our findings reveal that JEV NS1 and NS1' proteins induce vimentin rearrangement, resulting in the formation of cage-like structures that envelop the viral replication factories (RFs), thus facilitating efficient viral replication. Our research highlights the importance of the interplay between the cytoskeleton and orthoflavivirus, suggesting that targeting vimentin could be a promising approach for the development of antiviral strategies to inhibit JEV propagation.

UBAP2L-dependent coupling of PLK1 localization and stability during mitosis.

PLK1 is an important regulator of mitosis whose protein levels and activity fluctuate during the cell cycle. PLK1 dynamically localizes to various mitotic structures to regulate chromosome segregation. However, the signaling pathways linking localized PLK1 activity to its protein stability remain elusive. Here, we identify the Ubiquitin-Binding Protein 2-Like (UBAP2L) that controls both the localization and the protein stability of PLK1. We demonstrate that UBAP2L is a spindle-associated protein whose depletion leads to severe mitotic defects. UBAP2L-depleted cells are characterized by increased PLK1 protein levels and abnormal PLK1 accumulation in several mitotic structures such as kinetochores, centrosomes and mitotic spindle. UBAP2L-deficient cells exit mitosis and enter the next interphase in the presence of aberrant PLK1 kinase activity. The C-terminal domain of UBAP2L mediates its function on PLK1 independently of its role in stress response signaling. Importantly, the mitotic defects of UBAP2L-depleted cells are largely rescued by chemical inhibition of PLK1. Overall, our data suggest that UBAP2L is required to fine-tune the ubiquitin-mediated PLK1 turnover during mitosis as a means to maintain genome fidelity.

PLK1 promotes the mitotic surveillance pathway by controlling cytosolic 53BP1 availability.

53BP1 acts at the crossroads between DNA repair and p53-mediated stress response. With its interactors p53 and USP28, it is part of the mitotic surveillance (or mitotic stopwatch) pathway (MSP), a sensor that monitors the duration of cell division, promoting p53-dependent cell cycle arrest when a critical time threshold is surpassed. Here, we show that Polo-like kinase 1 (PLK1) activity is essential for the time-dependent release of 53BP1 from kinetochores. PLK1 inhibition, which leads to 53BP1 persistence at kinetochores, prevents cytosolic 53BP1 association with p53 and results in a blunted MSP. Strikingly, the identification of CENP-F as the kinetochore docking partner of 53BP1 enabled us to show that measurement of mitotic timing by the MSP does not take place at kinetochores, as perturbing CENP-F-53BP1 binding had no measurable impact on the MSP. Taken together, we propose that PLK1 supports the MSP by generating a cytosolic pool of 53BP1 and that an unknown cytosolic mechanism enables the measurement of mitotic duration.

A miR-361-5p/ ORC6/ PLK1 axis regulates prostate cancer progression.

Prostate cancer (PCa) is the most prevalent malignant tumor of the genitourinary system, and metastatic disease has a significant impact on the prognosis of PCa patients. As a result, knowing the processes of PCa development can help patients achieve better outcomes. Here, we investigated the expression and function of ORC6 in PCa. Our findings indicated that ORC6 was elevated in advanced PCa tissues. Patients with PCa who exhibited high levels of ORC6 had a poor prognosis. Following that, we investigated the function of ORC6 in PCa progression using a variety of functional experiments both in vivo and in vitro, and discovered that ORC6 knockdown inhibited PCa cell proliferation, growth, and migration. Furthermore, RNA-seq was employed to examine the molecular mechanism of PCa progression. The results revealed that ORC6 might promote the expression of PLK1, a serine/threonine kinase in PCa cells. We also discovered that ORC6 as a novel miR-361-5p substrate using database analysis, and miR-361-5p was found to lower ORC6 expression. Additionally, RNA immunoprecipitation (RIP) and luciferase reporter tests revealed that the transcription factor E2F1 could regulate ORC6 expression in PCa cells. PLK1 overexpression or miR-361-5p inhibitor treatment effectively removed the inhibitory effects caused by ORC6 silencing. Notably, our data showed that therapeutically targeting the miR-361-5p/ORC6/PLK1 axis may be a viable therapy option for PCa.

USP18 promotes the proliferation, invasion, and migration of head and neck squamous cell carcinoma by deubiquitinating PLK1.

The ubiquitin specific peptidase 18 (USP18), a well-established deubiquitinase, has been extensively implicated in the malignant progression of various human tumors. However, its role in head and neck squamous cell carcinoma (HNSC) requires further investigation. Here, we revealed that USP18 was significantly upregulated in HNSC and knockdown of USP18 markedly suppressed tumor growth in vivo. Furthermore, silencing USP18 attenuated HNSC cell proliferation, invasion, and migration, while overexpression of USP18 exerted converse effects. Mechanistically, USP18 diminished K48-linked ubiquitination of polo-like kinase 1 (PLK1) to stabilize the protein through its deubiquitinase activity. Subsequently, we validated that USP18 modulated PLK1 to activate the mTORC1 pathway, thereby facilitating HNSC cell proliferation, invasion, and migration. In conclusion, our findings demonstrate that elevated expression of USP18 in HNSC cells promotes tumorigenesis by regulating the PLK1-mTORC1 pathway.

PLK1-ELAVL1/HuR-miR-122 signaling facilitates hepatitis C virus proliferation.

The liver-specific microRNA, miR-122, plays an essential role in the propagation of hepatitis C virus (HCV) by binding directly to the 5'-end of its genomic RNA. Despite its significance for HCV proliferation, the host factors responsible for regulating miR-122 remain largely unknown. In this study, we identified the cellular RNA-binding protein, ELAVL1/HuR (embryonic lethal-abnormal vision-like 1/human antigen R), as critically contributing to miR-122 biogenesis by strong binding to the 3'-end of miR-122. The availability of ELAVL1/HuR was highly correlated with HCV proliferation in replicon, infectious, and chronically infected patient conditions. Furthermore, by screening a kinase inhibitor library, we identified rigosertib, an anticancer agent under clinical trials, as having both miR-122-modulating and anti-HCV activities that were mediated by its ability to target polo-like kinase 1 (PLK1) and subsequently modulate ELAVL1/HuR-miR-122 signaling. The expression of PLK1 was also highly correlated with HCV proliferation and the HCV positivity of HCC patients. ELAVL1/HuR-miR-122 signaling and its mediation of PLK1-dependent HCV proliferation were demonstrated by performing various rescue experiments and utilizing an HCV mutant with low dependency on miR-122. In addition, the HCV-inhibitory effectiveness of rigosertib was validated in various HCV-relevant conditions, including replicons, infected cells, and replicon-harboring mice. Rigosertib was highly effective in inhibiting the proliferation of not only wild-type HCVs, but also sofosbuvir resistance-associated substitution-bearing HCVs. Our study identifies PLK1-ELAVL1/HuR-miR-122 signaling as a regulatory axis that is critical for HCV proliferation, and suggests that a therapeutic approach targeting this host cell signaling pathway could be useful for treating HCV and HCV-associated diseases.

Targeted PLK1 suppression through RNA interference mediated by high-fidelity Cas13d mitigates osteosarcoma progression via TGF-ß/Smad3 signalling.

Osteosarcoma is the most common primary bone malignancy in children and adolescents. Overexpression of polo-like kinase 1 (PLK1) is frequent in osteosarcoma and drives disease progression and metastasis, making it a promising therapeutic target. In this study, we explored PLK1 knockdown in osteosarcoma cells using RNA interference mediated by high-fidelity Cas13d (hfCas13d). PLK1 was found to be significantly upregulated in osteosarcoma tumour tissues compared to normal bone. sgRNA-mediated PLK1 suppression via hfCas13d transfection inhibited osteosarcoma cell proliferation, induced G2/M cell cycle arrest, promoted apoptosis, reduced cell invasion and increased expression of the epithelial marker E-cadherin. Proximity labelling by TurboID coupled with co-immunoprecipitation identified novel PLK1 interactions with Smad3, a key intracellular transducer of TGF-ß signalling. PLK1 knockdown impaired Smad2/3 phosphorylation and modulated TGF-ß/Smad3 pathway inactivation. Finally, in vivo delivery of hfCas13d vectors targeting PLK1 substantially attenuated osteosarcoma xenograft growth in nude mice. Taken together, this study highlights PLK1 as a potential therapeutic target and driver of disease progression in osteosarcoma. It also demonstrates the utility of hfCas13d-mediated gene knockdown as a strategy for targeted therapy. Further optimization of PLK1 suppression approaches may ultimately improve clinical outcomes for osteosarcoma patients.

Polo-like kinase 1 (PLK1) O-GlcNAcylation is essential for dividing mammalian cells and inhibits uterine carcinoma.

The O-linked ß-N-acetylglucosamine (O-GlcNAc) transferase (OGT) mediates intracellular O-GlcNAcylation modification. O-GlcNAcylation occurs on Ser/Thr residues and is important for numerous physiological processes. OGT is essential for dividing mammalian cells and is involved in many human diseases; however, many of its fundamental substrates during cell division remain unknown. Here, we focus on the effect of OGT on polo-like kinase 1 (PLK1), a mitotic master kinase that governs DNA replication, mitotic entry, chromosome segregation, and mitotic exit. We show that PLK1 interacts with OGT and is O-GlcNAcylated. By utilizing stepped collisional energy/higher-energy collisional dissociation mass spectrometry, we found a peptide fragment of PLK1 that is modified by O-GlcNAc. Further mutation analysis of PLK1 shows that the T291A mutant decreases O-GlcNAcylation. Interestingly, T291N is a uterine carcinoma mutant in The Cancer Genome Atlas. Our biochemical assays demonstrate that T291A and T291N both increase PLK1 stability. Using stable H2B-GFP cells, we found that PLK1-T291A and PLK1-T291N mutants display chromosome segregation defects and result in misaligned and lagging chromosomes. In mouse xenograft models, we demonstrate that the O-GlcNAc-deficient PLK1-T291A and PLK1-T291N mutants enhance uterine carcinoma in animals. Hence, we propose that OGT partially exerts its mitotic function through O-GlcNAcylation of PLK1, which might be one mechanism by which elevated levels of O-GlcNAc promote tumorigenesis.

PCTAIRE1 promotes mitotic progression and resistance against antimitotic and apoptotic signals.

PCTAIRE1 (also known as CDK16) is a serine-threonine kinase implicated in physiological processes like neuronal development, vesicle trafficking, spermatogenesis and cell proliferation. However, its exact role in cell division remains unclear. In this study, using a library screening approach, we identified PCTAIRE1 among several candidates that resisted mitotic arrest and mitotic cell death induced by polyomavirus small T (PolST) expression in mammalian cells. Our study showed that PCTAIRE1 is a mitotic kinase that localizes at centrosomes during G2 and at spindle poles as the cells enter mitosis, and then at the midbody during cytokinesis. We also report that PCTAIRE1 protein levels fluctuate through the cell cycle and reach their peak at mitosis, during which there is an increase in PCTAIRE1 phosphorylation as well. Interestingly, knockdown of PCTAIRE1 resulted in aberrant mitosis by interfering with spindle assembly and chromosome segregation. Further, we found that PCTAIRE1 promotes resistance of cancer cells to antimitotic drugs, and this underscores the significance of PCTAIRE1 as a potential drug target for overcoming chemotherapeutic resistance. Taken together, these studies establish PCTAIRE1 as a critical mediator of mitotic progression and highlight its role in chemotherapeutic resistance. This article has an associated First Person interview with the first author of the paper.