Centrosome Maturation Research Articles

LAT1 supports mitotic progression through Golgi unlinking in an amino acid transport activity-independent manner

Amino acid transporters play a vital role in cellular homeostasis by maintaining protein synthesis. L-type amino acid transporter 1 (LAT1/SLC7A5/CD98lc) is a major transporter of large neutral amino acids in cancer cells because of its predominant expression. Although amino acid restriction with various amino acid analog treatments is known to induce mitotic defects, the involvement of amino acid transporters in cell division remains unclear. In this study, we identified that LAT1 is responsible for mitotic progression in a transport activity-independent manner. LAT1 knockdown activates the spindle assembly checkpoint, leading to a delay in metaphase. LAT1 maintains proper spindle orientation with confinement of the lateral cortex localization of the NuMA protein, which mediates the pulling force against the mitotic spindle toward the lateral cortex. Unexpectedly, JPH203, an inhibitor of LAT1 amino acid transport activity, does not affect mitotic progression. Moreover, the transport activity-deficient LAT1 mutant maintains the proper spindle orientation and mitotic progression. LAT1 forms a heterodimer with CD98 (SLC3A2/CD98hc) both in interphase and mitosis. Although CD98 knockdown decreases the plasma membrane localization of LAT1, it does not affect mitotic progression. LAT1 is localized to the Golgi and ER not only at the plasma membrane in interphase, and promotes Golgi unlinking during the mitotic entry, leading to centrosome maturation. These results suggest that LAT1 supports mitotic progression in an amino acid transport activity-independent manner and that Golgi-localized LAT1 is important for mitotic progression through the acceleration of Golgi unlinking and centrosome maturation. These findings reveal a novel LAT1 function in mitosis.

Ninein domains required for its localization, association with partners dynein and ensconsin, and microtubule organization.

Ninein (Nin) is a microtubule (MT) anchor at the subdistal appendages of mother centrioles and the pericentriolar material (PCM) of centrosomes that also functions to organize MTs at noncentrosomal MT-organizing centers (ncMTOCs). In humans, the NIN gene is mutated in Seckel syndrome, an inherited developmental disorder. Here, we dissect the protein domains involved in Nin's localization and interactions with dynein and ensconsin (ens/MAP7) and show that the association with ens cooperatively regulates MT assembly in Drosophila fat body cells. We define domains of Nin responsible for its localization to the ncMTOC on the fat body cell nuclear surface, localization within the nucleus, and association with Dynein light intermediate chain (Dlic) and ens, respectively. We show that Nin's association with ens synergistically regulates MT assembly. Together, these findings reveal novel features of Nin function and its regulation of a ncMTOC.

Contribution of AurkA/TPX2 Overexpression to Chromosomal Imbalances and Cancer.

The AurkA serine/threonine kinase is a key regulator of cell division controlling mitotic entry, centrosome maturation, and chromosome segregation. The microtubule-associated protein TPX2 controls spindle assembly and is the main AurkA regulator, contributing to AurkA activation, localisation, and stabilisation. Since their identification, AurkA and TPX2 have been described as being overexpressed in cancer, with a significant correlation with highly proliferative and aneuploid tumours. Despite the frequent occurrence of AurkA/TPX2 co-overexpression in cancer, the investigation of their involvement in tumorigenesis and cancer therapy resistance mostly arises from studies focusing only on one at the time. Here, we review the existing literature and discuss the mitotic phenotypes described under conditions of AurkA, TPX2, or AurkA/TPX2 overexpression, to build a picture that may help clarify their oncogenic potential through the induction of chromosome instability. We highlight the relevance of the AurkA/TPX2 complex as an oncogenic unit, based on which we discuss recent strategies under development that aim at disrupting the complex as a promising therapeutic perspective.

The ribonucleoprotein complex factor Ybx1 stabilizes the maternal mRNA of the <i>ssx2ip</i> gene encoding the centrosome maturation protein in <i>Xenopus laevis</i> embryogenesis

Our study investigates the mechanisms that regulate early developmental gene expression in Xenopus laevis frog embryos. Our previous study demonstrated that maternal mRNAs of two developmentally significant genes, the nuclear retinoic acid receptor rxrg and the pluripotency factor pou5f3, form complexes with ribonucleoprotein complex Ybx1. Based on the results of the present study, we determined and demonstrated that the stability of the maternal mRNA ssx2ip, which encodes a conserved protein, also called Msd1 or ADIP, which is involved in centrosome maturation, is dependent on Ybx1. This research shows that Ybx1 forms a ribonucleoprotein complex with ssx2ip mRNA, which is mediated by its cold shock domain (CSD). This study confirms our hypothesis of Ybx1 selectively binding to maternal transcripts. It opens up new opportunities to study new mechanisms of gene expression regulation at the earliest stages of development by searching for possible cis-motifs for recognition by trans-regulators such as Ybx1.

Polo-like kinase 1 prevents excess microtubule polymerization in C. elegans oocytes to ensure faithful meiosis.

Sexual reproduction relies on meiosis, a specialized cell division program that produces haploid gametes. Oocytes of most organisms lack centrosomes, and therefore chromosome segregation is mediated by acentrosomal spindles. Here, we explore the role of Polo-like kinase 1 (PLK-1) in C. elegans oocytes, revealing mechanisms that ensure the fidelity of this unique form of cell division. Previously, PLK-1 was shown to be required for nuclear envelope breakdown and chromosome segregation in oocytes. We now find that PLK-1 is also required for establishing and maintaining acentrosomal spindle organization and for preventing excess microtubule polymerization in these cells. Additionally, our studies revealed an unexpected new role for this essential kinase. While PLK-1 is known to be required for centrosome maturation during mitosis, we found that removal of this kinase from oocytes caused premature recruitment of pericentriolar material to the sperm-provided centrioles following fertilization. Thus, PLK-1 suppresses centrosome maturation during oocyte meiosis, which is opposite to its role in mitosis. Taken together, our work reveals multiple new roles for PLK-1 in oocytes, identifying PLK-1 as a key player that promotes faithful acentrosomal meiosis.

A novel role of AURKA kinase in erythroblast enucleation.

Generation of mammalian red blood cells requires the expulsion of polarized nuclei late in terminal erythroid differentiation. However, the mechanisms by which spherical erythroblasts determine the direction of nuclear polarization and maintain asymmetry during nuclear expulsion are poorly understood. Given the analogy of erythroblast enucleation to asymmetric cell division and the key role of Aurora kinases in mitosis, we sought to investigate the function of Aurora kinases in erythroblast enucleation. We found that AURKA (Aurora kinase A) is abundantly expressed in orthochromatic erythroblasts. Intriguingly, high-resolution confocal microscopy analyses revealed that AURKA co-localized with the centrosome on the side of the nucleus opposite its membrane contact point during polarization and subsequently translocated to the anterior end of the protrusive nucleus upon nuclear exit. Mechanistically, AURKA regulated centrosome maturation and localization via interaction with i-tubulin to provide polarization orientation for the nucleus. Furthermore, we identified ECT2 (epithelial cell transforming 2), a guanine nucleotide exchange factor, as a new interacting protein and ubiquitination substrate of AURKA. After forming the nuclear protrusion, AURKA translocated to the anterior end of the protrusive nucleus to directly degrade ECT2, which is partly dependent on kinase activity of AURKA. Moreover, knockdown of ECT2 rescued impaired enucleation caused by AURKA inhibition. Our findings have uncovered a previously unrecognized role of Aurora kinases in the establishment of nuclear polarization and eventual nuclear extrusion and provide new mechanistic insights into erythroblast enucleation.

Cep57 regulates human centrosomes through multivalent interactions

Human Cep57 is a coiled-coil scaffold at the pericentriolar matrix (PCM), controlling centriole duplication and centrosome maturation for faithful cell division. Genetic truncation mutations of Cep57 are associated with the mosaic-variegated aneuploidy (MVA) syndrome. During interphase, Cep57 forms a complex with Cep63 and Cep152, serving as regulators for centrosome maturation. However, the molecular interplay of Cep57 with these essential scaffolding proteins remains unclear. Here, we demonstrate that Cep57 undergoes liquid-liquid phase separation (LLPS) driven by three critical domains (NTD, CTD, and polybasic LMN). In vitro Cep57 condensates catalyze microtubule nucleation via the LMN motif-mediated tubulin concentration. In cells, the LMN motif is required for centrosomal microtubule aster formation. Moreover, Cep63 restricts Cep57 assembly, expansion, and microtubule polymerization activity. Overexpression of competitive constructs for multivalent interactions, including an MVA mutation, leads to excessive centrosome duplication. In Cep57-depleted cells, self-assembly mutants failed to rescue centriole disengagement and PCM disorganization. Thus, Cep57's multivalent interactions are pivotal for maintaining the accurate structural and functional integrity of human centrosomes.

Abstract 371: Hypoxia-induced centrosome loss as a driver of chromosomal instability in prostate cancer

Abstract PURPOSE: This study aims to identify a causal mechanism of centrosome loss previously observed in primary prostate cancer. Prostate cancer progression is accompanied by bursts of chromosomal instability (CIN), including chromosomal translocations, oncogene amplifications, and chromothripsis. While high CIN correlates with most metrics of aggressive disease, we lack a mechanistic understanding of how CIN originates in prostate cancer. Recently, we discovered that cells in primary prostate tumors lack centrosomes, cytoplasmic organelles that ensure the fidelity of chromosome segregation by organizing the shape of the mitotic spindle. Experimental elimination of centrosomes in immortalized, non-tumorigenic prostate cell lines was sufficient to generate extensive CIN, resulting in oncogenic transformation of these lines when sub-cutaneously injected into NSG mice. Because we identified centrosome loss as a potential driver of CIN, we sought to understand mechanisms that can trigger centrosome loss in the prostate. Although centrosome loss naturally occurs during the development of some tissues, for example centrosome elimination during oogenesis, the mechanisms that trigger loss are not known. Therefore, we first investigated microenvironmental changes in early prostate cancer as a potential cause. Hypoxia, pathologically low oxygen concentration, is common in the aging prostate due to loss of vasculature and is associated with poor prostate cancer prognosis and high CIN. Therefore, we hypothesized that hypoxic exposure induces centrosome loss in prostate cells. Indeed, we found that exposure to 1% oxygen concentrations leads to progressive centrosome loss in non-tumorigenic, immortalized prostate epithelial cell lines. Using immunofluorescence of centrosomes in cultured cells, we found that hypoxia-induced centrosome loss is independent of HIF transcription but requires activation of the confluence-dependent Hippo signaling pathway. Mechanistically, we found that centrosome disassembly begins with the removal of the pericentriolar material (PCM), the outer shell of the centrosome that nucleates microtubules. We were able to block centrosome disassembly by over-expressing a constitutively-active mutant of Polo-like Kinase 1, which normally strengthens the PCM during mitosis to promote centrosome maturation. Furthermore, centrosome loss was prevented by treatment with the Protein Phosphatase 2A (PP2A) inhibitor Okadaic Acid. Using RNAseq, we have identified potential PP2A regulatory subunits that are up-regulated in cells undergoing hypoxia-induced centrosome loss. Our model is that hypoxia induces dephosphorylation of the PCM, mechanically weakening the centrosome and leaving it vulnerable to disassembly. Together with previous findings, we conclude that hypoxia-induced centrosome disassembly is a plausible driver of CIN in early prostate cancer. Citation Format: John M. Ryniawec, Gregory C. Rogers, Anne E. Cress. Hypoxia-induced centrosome loss as a driver of chromosomal instability in prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 371.

Building the centrosome: PLK-1 controls multimerization of SPD-5.

Centrosome maturation relies on the assembly of an underlying molecular scaffold. In this issue of JCB, Rios et al. (https://doi.org/10.1083/jcb.202306142) use cross-linking mass spectrometry to reveal how PLK-1 phosphorylation promotes intermolecular SPD-5 self-association that is essential for scaffold formation.

Prolonged overexpression of PLK4 leads to formation of centriole rosette clusters that are connected via canonical centrosome linker proteins

Centrosome amplification is a hallmark of cancer and PLK4 is one of the responsible factors for cancer associated centrosome amplification. Increased PLK4 levels was also shown to contribute to generation of cells with centriole amplification in mammalian tissues as olfactory neuron progenitor cells. PLK4 overexpression generates centriole rosette (CR) structures which harbor more than two centrioles each. Long term PLK4 overexpression results with centrosome amplification, but the maturation of amplified centrioles in CRs and linking of PLK4 induced amplified centrosomes has not yet been investigated in detail. Here, we show evidence for generation of large clustered centrosomes which have more than 2 centriole rosettes and define these structures as centriole rosette clusters (CRCs) in cells that have high PLK4 levels for 2 consecutive cell cycles. In addition, we show that PLK4 induced CRs follow normal centrosomal maturation processes and generate CRC structures that are inter-connected with canonical centrosomal linker proteins as C-Nap1, Rootletin and Cep68 in the second cell cycle after PLK4 induction. Increased PLK4 levels in cells with C-Nap1 and Rootletin knock-out resulted with distanced CRs and CRCs in interphase, while Nek2 knock-out inhibited separation of CRCs in prometaphase, providing functional evidence for the binding of CRC structures with centrosomal linker proteins. Taken together, these results suggest a cell cycle dependent model for PLK4 induced centrosome amplification which occurs in 2 consecutive cell cycles: (i) CR state in the first cell cycle, and (ii) CRC state in the second cell cycle.

The Eyes Absent family members EYA4 and EYA1 promote PLK1 activation and successful mitosis through tyrosine dephosphorylation

The Eyes Absent proteins (EYA1-4) are a biochemically unique group of tyrosine phosphatases known to be tumour-promoting across a range of cancer types. To date, the targets of EYA phosphatase activity remain largely uncharacterised. Here, we identify Polo-like kinase 1 (PLK1) as an interactor and phosphatase substrate of EYA4 and EYA1, with pY445 on PLK1 being the primary target site. Dephosphorylation of pY445 in the G2 phase of the cell cycle is required for centrosome maturation, PLK1 localization to centrosomes, and polo-box domain (PBD) dependent interactions between PLK1 and PLK1-activation complexes. Molecular dynamics simulations support the rationale that pY445 confers a structural impairment to PBD-substrate interactions that is relieved by EYA-mediated dephosphorylation. Depletion of EYA4 or EYA1, or chemical inhibition of EYA phosphatase activity, dramatically reduces PLK1 activation, causing mitotic defects and cell death. Overall, we have characterized a phosphotyrosine signalling network governing PLK1 and mitosis.

Eph signal inhibition potentiates the growth-inhibitory effects of PLK1 inhibition toward cancer cells

Anti-mitotic drugs are clinically used as anti-cancer treatments. Polo-like kinase 1 (PLK1) is a promising target against cancer cell division due to its importance in the whole process of mitosis, and thus PLK1-targeting agents have been developed in the last few decades. Clinical trial studies show that several PLK1 inhibitors are generally well-tolerated. However, the response rates are limited; therefore, it is needed to improve the efficacy of those drugs. Here, we show that NVP-BHG712, an erythropoietin-producing human hepatocellular (Eph) signaling inhibitor, potentiates the growth-inhibitory effects of the PLK1 inhibitors BI2536 and BI6727 in cancer cells. This combination treatment strongly suppresses cancer spheroid formation. Moreover, the combination drastically arrests cells at mitosis by continuous activation of the spindle assembly checkpoint (SAC), thereby inducing apoptosis. SAC activation caused by the combination of NVP-BHG712 and BI2536 is due to the inhibition of centrosome maturation and separation. Although the inactivation level of the PLK1 kinase is comparable between BI2536 treatment alone and combination treatment, the combination treatment strongly inactivates MAPK signaling in mitosis. Since inhibition of MAPK signaling potentiates the efficacy of BI2536 treatment, inactivation of PLK1 kinase and MAPK signaling contributes to the strong inhibition of centrosome separation. These results suggest that Eph signal inhibition potentiates the effect of PLK1 inhibition, leading to strong mitotic arrest via SAC activation and the subsequent reduction of cancer cell survival. The combination of PLK1 inhibition and Eph signal inhibition will provide a new effective strategy for targeting cancer cell division.

Abstract C146: VIC-1911, a selective Aurora kinase A inhibitor, synergizes with sacituzumab govitecan in triple-negative breast cancer

Abstract BACKGROUND: Triple-negative breast cancer (TNBC) accounts for 15-20% of breast cancer cases and 30-40% of U.S. breast cancer deaths. Although current precision treatments improve TNBC patient outcomes, TNBC heterogeneity has led to therapeutic targeting challenges. Also, the resistance of TNBC to conventional treatment remains a significant clinical problem. Therefore, informed evaluation of new targets can improve the treatment of TNBC, representing an unmet need. Dysregulation of the cell cycle is a hallmark of cancer for unlimited proliferation. Abnormal mitosis and cell-cycle checkpoints are crucial in leading to aneuploidy and genetic instability. Aurora kinase A (AURKA), a serine/threonine kinase, regulates mitosis in the early stages (G2/M phase) by regulating centrosome maturation and disjunction, thereby establishing a bipolar mitotic spindle. VIC-1911 (previously known as TAS-119), a novel, orally active, highly selective inhibitor of AURKA with a low toxicity profile, suppresses the growth of various cancer cell lines in vitro and in vivo. As AURKA can modulate DNA damage response, we hypothesized that VIC-1911 combined with sacituzumab govitecan (SG), which has a topoisomerase 1 inhibitor as a payload, has a synergistic antitumor effect on TNBC. MATERIALS AND METHODS: To evaluate the short-term in vitro efficacy of VIC-1911 alone and combination with SG, a sulforhodamine B cell proliferation assay was performed using 10 TNBC cell lines. For long-term treatment conditions, a colony formation assay was conducted. Western blot and immunofluorescent analyses were conducted to confirm treatment-mediated DNA damage and apoptosis. Human TNBC cell line–derived mammary fat pad xenograft models were used to evaluate the antitumor effect of VIC-1911 and SG. RESULTS: In vitro proliferation, data demonstrated that single-agent VIC-1911 had a nanomolar to micromolar range of half-maximal inhibitory concentrations (2.69 nM to 10.6 mM) in the tested TNBC cell lines. The half-maximal inhibitory concentration was correlated with c-Myc expression (R2=0.4876, p=0.0247). Exposure to the combination of VIC-1911 and SG led to significant death of VIC-1911–sensitive TNBC cell lines (p<0.05). Western blot and immunofluorescent analysis revealed that VIC-1911 and SG combination caused TNBC cell death by inducing expression of phosphorylated histone H2AX (DNA damage marker) and cleaved poly (ADP-ribose) polymerase (apoptosis marker). Furthermore, the combination of VIC-1911 and SG had significantly greater antitumor efficacy than did either agent alone in SUM149 TNBC xenograft models (tumor growth inhibition rate (TGI): VIC-1911, 61.4%; SG, 68.3%; combination, 82.9%; p<0.01) and HCC1806 TNBC xenograft models (TGI: VIC-1911, 49.3%; SG, 66.3%; combination, 85.9%; p<0.01). CONCLUSION: The AURKA inhibitor VIC-1911 has a synergistic antitumor effect with SG by inducing DNA damage in TNBC cells. PDX and additional mechanistic studies are planned to further evaluate VIC-1911 and SG as novel combination therapy for TNBC. Citation Format: Jangsoon Lee, YoungJin Gi, Thomas Myers, Linda Paradiso, Debu Tripathy, Naoto T. Ueno. VIC-1911, a selective Aurora kinase A inhibitor, synergizes with sacituzumab govitecan in triple-negative breast cancer [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr C146.

AURKA Kinase Controls Erythroblast Enucleation Via Regulation of Centrosome Localization and ECT2 Degradation

Mature red blood cells (RBCs) in the peripheral circulation of mammals are nuclear-free. Erythroblast enucleation is proposed to be analogous to asymmetric cell division based on cellular progress. the molecular mechanisms of how spherical erythroblasts determine the direction of nuclear polarization and accomplish the final separation of the nucleus from the nascent reticulocytes remain largely unclear, until now. Aurora kinases are highly conserved serine/threonine kinases required for regulation of mitosis (AURKA and AURKB). However, up to now, the role of Aurora kinases in erythroid cells, especially in erythroblast enucleation has been fully undefined. Here, we found that AURKA and AURKB messenger RNA (mRNA) expression levels were similar early in terminal differentiation, but followed by a prominent increase in AURKA levels in polychromatic and orthochromatic erythroblasts, whereas AURKB levels were dramatically downregulated. The expression patterns of Auraro kinases were further confirmed by western blotting analysis. More notably, the expression of Aurora kinases is low or absent in most adult tissues or cells due to their lower proliferation rates. However, AURKA and AURKB are still highly expressed in orthochromatic erythroblasts compared to other precursor erythroblasts. The unconventional expression profile of Aurora kinases, especially AURKA, during terminal erythroid differentiation suggests an extraordinary role in erythroblast enucleation. To explore the role of Aurora kinases in erythroblast enucleation, we first tested whether pharmacological inhibition of these kinases blocked the enucleation of human erythroblasts. Treatment of erythroblasts with MLN8237 (Aliseritib, AURKA selective inhibitor), AZD1152(Barasertib, AURKB selective inhibitor), or AT9283 (AURKA and AURKB inhibitor) dramatically blocked enucleation in a dose-dependent manner. Furthermore, we found that knockdown of Aurora kinases by siRNA indeed resulted in impaired enucleation. We next examined whether Aurora kinases regulated nuclear polarization, which is considered the onset of erythroblast enucleation, by measuring the △centroid distribution (distance between centers of the cytoplasm and nucleus). We found that erythroblast nuclear polarization is AURKA dependent, but not AURKB dependent. Intriguingly, high-resolution confocal microscopy analyses revealed that AURKA colocalized with the centrosome on the side of the nucleus opposite its membrane point of contact during polarization and subsequently solely translocated to the anterior end of the protrusive nucleus upon nuclear exit, whereas AURKB consistently localized to the actin contraction ring during enucleation. Colocalization of AURKA and centrosome during nuclear polarization raised the question whether AURKA controls polarization by regulating centrosome organization and maturation. Co-immunoprecipitation (co-IP) verified a direct interaction between AURKA and γ-tubulin. We found that AURKA loss did not alter the expression levels of γ-tubulin in erythroblasts. However, immunofluorescence showed that MLN8237 treatment obviously impaired the proper organization of centrosome. These phenotypes of centrinone-B, an inhibitor of the master centriole biogenesis regulator PLK4 treated erythroblasts mimic the results of AUKRA-deficient erythroblasts, suggesting a close connection between AUKRA and centrosome in polarization during erythroblast enucleation. We intriguingly found that ECT2 (Epithelial Cell Transforming 2), was only constricted at the rear of the thanslocating nucleus in enucleating erythroblasts. Inhibition of AURKA obviously impaired this transformation, exhibiting persistent cytoplasmic localization. MLN8237 treatment resulted in a significant increase of ECT2 proteins in K562 cells.In contrast to control scramble siRNA, ECT2 siRNA significantly increased the enucleation of MLN8237-treated erythroblast. The translocation of AURKA, which is originally restricted to centrosome relied on γ-tubulin interaction during nuclear polarization, was to directly degrade ECT2 at the anterior end of the protrusive nucleus for final nuclear expulsion. Our findings reveal a previously unrecognized localization and role of Aurora kinases in terminal erythroid differentiation and provide new mechanistic insights into erythroblast enucleation.

Dysregulation of the endoplasmic reticulum blocks recruitment of centrosome-associated proteins resulting in mitotic failure.

The endoplasmic reticulum (ER) undergoes a remarkable transition in morphology during cell division to aid in the proper portioning of the ER. However, whether changes in ER behaviors modulate mitotic events is less clear. Like many animal embryos, the early Drosophila embryo undergoes rapid cleavage cycles in a lipid-rich environment. Here, we show that mitotic spindle formation, centrosomal maturation, and ER condensation occur with similar time frames in the early syncytium. In a screen for Rab family GTPases that display dynamic function at these stages, we identified Rab1. Rab1 disruption led to an enhanced buildup of ER at the spindle poles and produced an intriguing 'mini-spindle' phenotype. ER accumulation around the mitotic space negatively correlates with spindle length/intensity. Importantly, centrosomal maturation is defective in these embryos, as mitotic recruitment of key centrosomal proteins is weakened after Rab1 disruption. Finally, division failures and ER overaccumulation is rescued by Dynein inhibition, demonstrating that Dynein is essential for ER spindle recruitment. These results reveal that ER levels must be carefully tuned during mitotic processes to ensure proper assembly of the division machinery.

Loss of the mitochondrial protein SPD-3 elevates PLK-1 levels and dysregulates mitotic events.

In metazoans, Polo-like kinase (PLK1) controls several mitotic events including nuclear envelope breakdown, centrosome maturation, spindle assembly and progression through mitosis. Here we show that a mutation in the mitochondria-localized protein SPD-3 affects mitotic events by inducing elevated levels of PLK-1 in early Caenorhabditis elegans embryos. SPD-3 mutant embryos contain abnormally positioned mitotic chromosomes, show a delay in anaphase onset and asymmetrically disassemble the nuclear lamina. We found that more PLK-1 accumulated on centrosomes, nuclear envelope, nucleoplasm, and chromatin before NEBD, suggesting that PLK-1 overexpression is responsible for some of the observed mitotic phenotypes. In agreement with this, the chromosome positioning defects of the spd-3(oj35) mutant could be rescued by reducing PLK-1 levels. Our data suggests that the mitochondrial SPD-3 protein affects chromosome positioning and nuclear envelope integrity by up-regulating the endogenous levels of PLK-1 during early embryogenesis in C. elegans This finding suggests a novel link between mitochondria and nuclear envelope dynamics and chromosome positioning by increasing the amount of a key mitotic regulator, PLK-1, providing a novel link between mitochondria and mitosis.

Hornerin mediates phosphorylation of the polo-box domain in Plk1 by Chk1 to induce death in mitosis

The centrosome assembles a bipolar spindle for faithful chromosome segregation during mitosis. To prevent the inheritance of DNA damage, the DNA damage response (DDR) triggers programmed spindle multipolarity and concomitant death in mitosis through a poorly understood mechanism. We identified hornerin, which forms a complex with checkpoint kinase 1 (Chk1) and polo-like kinase 1 (Plk1) to mediate phosphorylation at the polo-box domain (PBD) of Plk1, as the link between the DDR and death in mitosis. We demonstrate that hornerin mediates DDR-induced precocious centriole disengagement through a dichotomous mechanism that includes sequestration of Sgo1 and Plk1 in the cytoplasm through phosphorylation of the PBD in Plk1 by Chk1. Phosphorylation of the PBD in Plk1 abolishes the interaction with Sgo1 and phosphorylation-dependent Sgo1 translocation to the centrosome, leading to precocious centriole disengagement and spindle multipolarity. Mechanistically, hornerin traps phosphorylated Plk1 in the cytoplasm. Furthermore, PBD phosphorylation inactivates Plk1 and disrupts Cep192::Aurora A::Plk1 complex translocation to the centrosome and concurrent centrosome maturation. Remarkably, hornerin depletion leads to chemoresistance against DNA damaging agents by attenuating DDR-induced death in mitosis. These results reveal how the DDR eradicates mitotic cells harboring DNA damage to ensure genome integrity during cell division.

Aurora A polyubiquitinates the BRCA1-interacting protein OLA1 to promote centrosome maturation

The BRCA1-interacting protein Obg-like ATPase 1 (OLA1) functions in centriole duplication. In this study, we show the role of the mitotic kinase Aurora A in the reduction of centrosomal OLA1. Aurora A binds to and polyubiquitinates OLA1, targeting it for proteasomal degradation. NIMA-related kinase 2 (NEK2) phosphorylates the T124 residue of OLA1, increases binding of OLA1 to Aurora A and OLA1 polyubiquitination by Aurora A, and reduces centrosomal OLA1 in G2 phase. The kinase activity of Aurora A suppresses OLA1 polyubiquitination. The decrease in centrosomal OLA1 caused by Aurora A-mediated polyubiquitination promotes the recruitment of pericentriolar material proteins in G2 phase. The E3 ligase activity of Aurora A is critical for centrosome amplification induced by its overexpression. The results suggest a dual function of Aurora A as an E3 ubiquitin ligase and a kinase in the regulation of centrosomal OLA1, which is essential for proper centrosome maturation in G2 phase.

A Role of PI3K/Akt Signaling in Oocyte Maturation and Early Embryo Development.

A serine/threonine-specific protein kinase B (PKB), also known as Akt, is a key factor in the phosphoinositide 3-kinase (PI3K)/Akt signaling pathway that regulates cell survival, metabolism and proliferation. Akt phosphorylates many downstream specific substrates, which subsequently control the nuclear envelope breakdown (NEBD), centrosome maturation, spindle assembly, chromosome segregation, and cytokinesis. In vertebrates, Akt is also an important player during oogenesis and preimplantation development. In the signaling pathways regulating mRNA translation, Akt is involved in the control of mammalian target of rapamycin complex 1 (mTORC1) and thereby regulates the activity of a translational repressor, the eukaryotic initiation factor 4E (eIF4E) binding protein 1 (4E-BP1). In this review, we summarize the functions of Akt in mitosis, meiosis and early embryonic development. Additionally, the role of Akt in the regulation of mRNA translation is addressed with respect to the significance of this process during early development.

Emerging roles of Aurora-A kinase in cancer therapy resistance.

Aurora kinase A (Aurora-A), a serine/threonine kinase, plays a pivotal role in various cellular processes, including mitotic entry, centrosome maturation and spindle formation. Overexpression or gene-amplification/mutation of Aurora-A kinase occurs in different types of cancer, including lung cancer, colorectal cancer, and breast cancer. Alteration of Aurora-A impacts multiple cancer hallmarks, especially, immortalization, energy metabolism, immune escape and cell death resistance which are involved in cancer progression and resistance. This review highlights the most recent advances in the oncogenic roles and related multiple cancer hallmarks of Aurora-A kinase-driving cancer therapy resistance, including chemoresistance (taxanes, cisplatin, cyclophosphamide), targeted therapy resistance (osimertinib, imatinib, sorafenib, etc.), endocrine therapy resistance (tamoxifen, fulvestrant) and radioresistance. Specifically, the mechanisms of Aurora-A kinase promote acquired resistance through modulating DNA damage repair, feedback activation bypass pathways, resistance to apoptosis, necroptosis and autophagy, metastasis, and stemness. Noticeably, our review also summarizes the promising synthetic lethality strategy for Aurora-A inhibitors in RB1, ARID1A and MYC gene mutation tumors, and potential synergistic strategy for mTOR, PAK1, MDM2, MEK inhibitors or PD-L1 antibodies combined with targeting Aurora-A kinase. In addition, we discuss the design and development of the novel class of Aurora-A inhibitors in precision medicine for cancer treatment.