Forebrain Eml1 depletion reveals early centrosomal dysfunction causing subcortical heterotopia.

Subcortical heterotopia is a cortical malformation associated with epilepsy, intellectual disability, and an excessive number of cortical neurons in the white matter. Echinoderm microtubule-associated protein like 1 (EML1) mutations lead to subcortical heterotopia, associated with abnormal radial glia positioning in the cortical wall, prior to malformation onset. This perturbed distribution of proliferative cells is likely to be a critical event for heterotopia formation; however, the underlying mechanisms remain unexplained. This study aimed to decipher the early cellular alterations leading to abnormal radial glia. In a forebrain conditional Eml1 mutant model and human patient cells, primary cilia and centrosomes are altered. Microtubule dynamics and cell cycle kinetics are also abnormal in mouse mutant radial glia. By rescuing microtubule formation in Eml1 mutant embryonic brains, abnormal radial glia delamination and heterotopia volume were significantly reduced. Thus, our new model of subcortical heterotopia reveals the causal link between Eml1's function in microtubule regulation and cell position, both critical for correct cortical development.

Dynamic Mitotic Localization of the Centrosomal Kinases CDK1, Plk, AurK, and Nek2 in Dictyostelium amoebae.

The centrosome of the amoebozoan model Dictyostelium discoideum provides the best-established model for an acentriolar centrosome outside the Opisthokonta. Dictyostelium exhibits an unusual centrosome cycle, in which duplication is initiated only at the G2/M transition and occurs entirely during the M phase. Little is known about the role of conserved centrosomal kinases in this process. Therefore, we have generated knock-in strains for Aurora (AurK), CDK1, cyclin B, Nek2, and Plk, replacing the endogenous genes with constructs expressing the respective green fluorescent Neon fusion proteins, driven by the endogenous promoters, and studied their behavior in living cells. Our results show that CDK1 and cyclin B arrive at the centrosome first, already during G2, followed by Plk, Nek2, and AurK. Furthermore, CDK1/cyclin B and AurK were dynamically localized at kinetochores, and AurK in addition at nucleoli. The putative roles of all four kinases in centrosome duplication, mitosis, cytokinesis, and nucleolar dynamics are discussed.

Centrosome amplification primes ovarian cancer cells for apoptosis and potentiates the response to chemotherapy.

Centrosome amplification is a feature of cancer cells associated with chromosome instability and invasiveness. Enhancing chromosome instability and subsequent cancer cell death via centrosome unclustering and multipolar divisions is an aimed-for therapeutic approach. Here, we show that centrosome amplification potentiates responses to conventional chemotherapy in addition to its effect on multipolar divisions and chromosome instability. We perform single-cell live imaging of chemotherapy responses in epithelial ovarian cancer cell lines and observe increased cell death when centrosome amplification is induced. By correlating cell fate with mitotic behaviors, we show that enhanced cell death can occur independently of chromosome instability. We identify that cells with centrosome amplification are primed for apoptosis. We show they are dependent on the apoptotic inhibitor BCL-XL and that this is not a consequence of mitotic stresses associated with centrosome amplification. Given the multiple mechanisms that promote chemotherapy responses in cells with centrosome amplification, we assess such a relationship in an epithelial ovarian cancer patient cohort. We show that high centrosome numbers associate with improved treatment responses and longer overall survival. Our work identifies apoptotic priming as a clinically relevant consequence of centrosome amplification, expanding our understanding of this pleiotropic cancer cell feature.

Aurora A Kinase Begins to Localize to the Centrosome in the S-phase of the Cell Cycle in the XL2 Cell Line.

BACKGROUND: The centrosome is one of the principal cell hubs, where numerous proteins important for intracellular regulatory processes are concentrated. One of them, serine-threonine kinase 6, alias Aurora A, is involved in centrosome duplication and mitotic spindle formation and maintenance. METHODS: Long-term vital observations of cells, immunofluorescence analysis of protein localization, synchronization of cells at different phases of the cell cycle, Western blot analysis of protein content were used in the work. RESULTS: In this study, we investigated the dynamics of Aurora A protein accumulation and degradation in the XL2 Xenopus cell line during its 28-hour cell cycle. Using Western blot and immunofluorescence analyses, we demonstrated that Aurora A disappeared from the centrosome within one hour following mitosis and was not redistributed to other cell compartments. Using double Aurora A/Bromodeoxyuridine immunofluorescence labeling of the cells with precisely determined cell cycle stages, we observed that Aurora A reappeared in the centrosome during the S-phase, which was earlier than reported for all other known proteins with mitosis-specific centrosomal localization. Moreover, Aurora A accumulation in the centrosomal region and centrosome separation were asynchronous in the sister cells. CONCLUSIONS: The reported data allowed us to hypothesize that Aurora A is one of the primary links in coordinating centrosome separation and constructing the mitotic spindle.

RAB12-LRRK2 complex suppresses primary ciliogenesis and regulates centrosome homeostasis in astrocytes.

The leucine-rich repeat kinase 2 (LRRK2) phosphorylates a subset of RAB GTPases, and their phosphorylation levels are elevated by Parkinson's disease (PD)-linked mutations of LRRK2. However, the precise function of the LRRK2-regulated RAB GTPase in the brain remains to be elucidated. Here, we identify RAB12 as a robust LRRK2 substrate in the mouse brain through phosphoproteomics profiling and solve the structure of RAB12-LRRK2 protein complex through Cryo-EM analysis. Mechanistically, RAB12 cooperates with LRRK2 to inhibit primary ciliogenesis and regulate centrosome homeostasis in astrocytes through enhancing the phosphorylation of RAB10 and recruiting RILPL1, while the functions of RAB12 require a direct interaction with LRRK2 and LRRK2 activity. Furthermore, the ciliary and centrosome defects caused by the PD-linked LRRK2-G2019S mutation are prevented by Rab12 deletion in astrocytes. Thus, our study reveals a physiological function of the RAB12-LRRK2 complex in regulating ciliogenesis and centrosome homeostasis. The RAB12-LRRK2 structure offers a guidance in the therapeutic development of PD by targeting the RAB12-LRRK2 interaction.

The doublecortin-family kinase ZYG-8DCLK1 regulates microtubule dynamics and motor-driven forces to promote the stability of C. elegans acentrosomal spindles.

Although centrosomes help organize spindles in most cell types, oocytes of most species lack these structures. During acentrosomal spindle assembly in C. elegans oocytes, microtubule minus ends are sorted outwards away from the chromosomes where they form poles, but then these outward forces must be balanced to form a stable bipolar structure. Simultaneously, microtubule dynamics must be precisely controlled to maintain spindle length and organization. How forces and dynamics are tuned to create a stable bipolar structure is poorly understood. Here, we have gained insight into this question through studies of ZYG-8, a conserved doublecortin-family kinase; the mammalian homolog of this microtubule-associated protein is upregulated in many cancers and has been implicated in cell division, but the mechanisms by which it functions are poorly understood. We found that ZYG-8 depletion from oocytes resulted in overelongated spindles with pole and midspindle defects. Importantly, experiments with monopolar spindles revealed that ZYG-8 depletion led to excess outward forces within the spindle and suggested a potential role for this protein in regulating the force-generating motor BMK-1/kinesin-5. Further, we found that ZYG-8 is also required for proper microtubule dynamics within the oocyte spindle and that kinase activity is required for its function during both meiosis and mitosis. Altogether, our findings reveal new roles for ZYG-8 in oocytes and provide insights into how acentrosomal spindles are stabilized to promote faithful meiosis.

MYCBPAP is a central apparatus protein required for centrosome-nuclear envelope docking and sperm tail biogenesis in mice.

The structure of the sperm flagellar axoneme is highly conserved across species and serves the essential function of generating motility to facilitate the meeting of spermatozoa with the egg. During spermiogenesis, the axoneme elongates from the centrosome, and subsequently the centrosome docks onto the nuclear envelope to continue tail biogenesis. Mycbpap is expressed predominantly in mouse and human testes and conserved in Chlamydomonas as FAP147. A previous cryo-electron microscopy analysis has revealed the localization of FAP147 to the central apparatus of the axoneme. Here, we generated Mycbpap-knockout mice and demonstrated the essential role of Mycbpap in male fertility. Deletion of Mycbpap led to disrupted centrosome-nuclear envelope docking and abnormal flagellar biogenesis. Furthermore, we generated transgenic mice with tagged MYCBPAP, which restored the fertility of Mycbpap-knockout males. Interactome analyses of MYCBPAP using Mycbpap transgenic mice unveiled binding partners of MYCBPAP including central apparatus proteins, such as CFAP65 and CFAP70, which constitute the C2a projection, and centrosome-associated proteins, such as CCP110. These findings provide insights into a MYCBPAP-dependent regulation of the centrosome-nuclear envelope docking and sperm tail biogenesis.

Arl2 GTPase associates with the centrosomal protein Cdk5rap2 to regulate cortical development via microtubule organization.

ADP ribosylation factor-like GTPase 2 (Arl2) is crucial for controlling mitochondrial fusion and microtubule assembly in various organisms. Arl2 regulates the asymmetric division of neural stem cells in Drosophila via microtubule growth. However, the function of mammalian Arl2 during cortical development was unknown. Here, we demonstrate that mouse Arl2 plays a new role in corticogenesis via regulating microtubule growth, but not mitochondria functions. Arl2 knockdown (KD) leads to impaired proliferation of neural progenitor cells (NPCs) and neuronal migration. Arl2 KD in mouse NPCs significantly diminishes centrosomal microtubule growth and delocalization of centrosomal proteins Cdk5rap2 and γ-tubulin. Moreover, Arl2 physically associates with Cdk5rap2 by in silico prediction using AlphaFold multimer, which was validated by co-immunoprecipitation and proximity ligation assay. Remarkably, Cdk5rap2 overexpression significantly rescues the neurogenesis defects caused by Arl2 KD. Therefore, Arl2 plays an important role in mouse cortical development through microtubule growth via the centrosomal protein Cdk5rap2.

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.

Spindle assembly checkpoint-dependent mitotic delay is required for cell division in absence of centrosomes.

The spindle assembly checkpoint (SAC) temporally regulates mitosis by preventing progression from metaphase to anaphase until all chromosomes are correctly attached to the mitotic spindle. Centrosomes refine the spatial organization of the mitotic spindle at the spindle poles. However, centrosome loss leads to elongated mitosis, suggesting that centrosomes also inform the temporal organization of mitosis in mammalian cells. Here, we find that the mitotic delay in acentrosomal cells is enforced by the SAC in a MPS1-dependent manner, and that a SAC-dependent mitotic delay is required for bipolar cell division to occur in acentrosomal cells. Although acentrosomal cells become polyploid, polyploidy is not sufficient to cause dependency on a SAC-mediated delay to complete cell division. Rather, the division failure in absence of MPS1 activity results from mitotic exit occurring before acentrosomal spindles can become bipolar. Furthermore, prevention of centrosome separation suffices to make cell division reliant on a SAC-dependent mitotic delay. Thus, centrosomes and their definition of two spindle poles early in mitosis provide a 'timely two-ness' that allows cell division to occur in absence of a SAC-dependent mitotic delay.

Heterotypic interaction promotes asymmetric division of human hematopoietic progenitors.

Hematopoietic stem and progenitor cells (HSPCs) give rise to all cell types of the hematopoietic system through various processes, including asymmetric divisions. However, the contribution of stromal cells of the hematopoietic niches in the control of HSPC asymmetric divisions remains unknown. Using polyacrylamide microwells as minimalist niches, we show that specific heterotypic interactions with osteoblast and endothelial cells promote asymmetric divisions of human HSPCs. Upon interaction, HSPCs polarize in interphase with the centrosome, the Golgi apparatus, and lysosomes positioned close to the site of contact. Subsequently, during mitosis, HSPCs orient their spindle perpendicular to the plane of contact. This division mode gives rise to siblings with unequal amounts of lysosomes and of the differentiation marker CD34. Such asymmetric inheritance generates heterogeneity in the progeny, which is likely to contribute to the plasticity of the early steps of hematopoiesis.

Kinase activity of histone chaperone APLF maintains steady state of centrosomes in mouse embryonic stem cells.

Our recent studies revealed the role of mouse Aprataxin PNK-like Factor (APLF) in development. Nevertheless, the comprehensive characterization of mouse APLF remains entirely unexplored. Based on domain deletion studies, here we report that mouse APLF's Acidic Domain and Fork Head Associated (FHA) domain can chaperone histones and repair DNA like the respective human orthologs. Immunofluorescence studies in mouse embryonic stem cells showed APLF co-localized with γ-tubulin within and around the centrosomes and govern the number and integrity of centrosomes via PLK4 phosphorylation. Enzymatic analysis established mouse APLF as a kinase. Docking studies identified three putative ATP binding sites within the FHA domain. Site-directed mutagenesis showed that R37 residue within the FHA domain is indispensable for the kinase activity of APLF thereby regulating the centrosome number. These findings might assist us comprehend APLF in different pathological and developmental conditions and reveal non-canonical kinase activity of proteins harbouring FHA domains that might impact multiple cellular processes.

Eg5 UFMylation promotes spindle organization during mitosis.

UFMylation is a highly conserved ubiquitin-like post-translational modification that catalyzes the covalent linkage of UFM1 to its target proteins. This modification plays a critical role in the maintenance of endoplasmic reticulum proteostasis, DNA damage response, autophagy, and transcriptional regulation. Mutations in UFM1, as well as in its specific E1 enzyme UBA5 and E2 enzyme UFC1, have been genetically linked to microcephaly. Our previous research unveiled the important role of UFMylation in regulating mitosis. However, the underlying mechanisms have remained unclear due to the limited identification of substrates. In this study, we identified Eg5, a motor protein crucial for mitotic spindle assembly and maintenance, as a novel substrate for UFMylation and identified Lys564 as the crucial UFMylation site. UFMylation did not alter its transcriptional level, phosphorylation level, or protein stability, but affected the mono-ubiquitination of Eg5. During mitosis, Eg5 and UFM1 co-localize at the centrosome and spindle apparatus, and defective UFMylation leads to diminished spindle localization of Eg5. Notably, the UFMylation-defective Eg5 mutant (K564R) exhibited shorter spindles, metaphase arrest, spindle checkpoint activation, and a failure of cell division in HeLa cells. Overall, Eg5 UFMylation is essential for proper spindle organization, mitotic progression, and cell proliferation.

Lateral expansion of the mammalian cerebral cortex is related to anchorage of centrosomes in apical neural progenitors.

The centrosome is the main microtubule organizing center in stem cells, and its mother centriole, anchored to the cell membrane, serves as the basal body of the primary cilium. Prolonged anchorage of centrosomes and primary cilia to the apical segment of the membrane of apical neural progenitor cells is considered vital for interkinetic nuclear translocation and repetitive cycling in the ventricular zone. In contrast, the basolateral anchorage of primary cilia has been regarded as the first step in delamination and conversion of apical to basal neural progenitor cells or neurons. Using electron microscopy analysis of serial sections, we show that centrosomes, in a fraction of cells, anchor to the basolateral cell membrane immediately after cell division and before development of cilia. In other cells, centrosomes situate freely in the cytoplasm, increasing their probability of subsequent apical anchorage. In mice, anchored centrosomes in the cells shortly after mitosis predominate during the entire cerebral neurogenesis, whereas in macaque monkeys, cytoplasmic centrosomes are more numerous. Species-specific differences in the ratio of anchored and free cytoplasmic centrosomes appear to be related to prolonged neurogenesis in the ventricular zone that is essential for lateral expansion of the cerebral cortex in primates.

The core spindle pole body scaffold Ppc89 links the pericentrin orthologue Pcp1 to the fission yeast spindle pole body via an evolutionarily conserved interface.

Centrosomes and spindle pole bodies (SPBs) are important for mitotic spindle formation and serve as cellular signaling platforms. Although centrosomes and SPBs differ in morphology, many mechanistic insights into centrosome function have been gleaned from SPB studies. In the fission yeast Schizosaccharomyces pombe, the α-helical protein Ppc89, identified based on its interaction with the septation initiation network scaffold Sid4, comprises the SPB core. High-resolution imaging has suggested that SPB proteins assemble on the Ppc89 core during SPB duplication, but such interactions are undefined. Here, we define a connection between Ppc89 and the essential pericentrin Pcp1. Specifically, we found that a predicted third helix within Ppc89 binds the Pcp1 pericentrin-AKAP450 centrosomal targeting (PACT) domain complexed with calmodulin. Ppc89 helix 3 contains similarity to present in the N-terminus of Cep57 (PINC) motifs found in the centrosomal proteins fly SAS-6 and human Cep57 and also to the S. cerevisiae SPB protein Spc42. These motifs bind pericentrin-calmodulin complexes and AlphaFold2 models suggest a homologous complex assembles in all four organisms. Mutational analysis of the S. pombe complex supports the importance of Ppc89-Pcp1 binding interface in vivo. Our studies provide insight into the core architecture of the S. pombe SPB and suggest an evolutionarily conserved mechanism of scaffolding pericentrin-calmodulin complexes for mitotic spindle formation.

Centrosome age breaks spindle size symmetry even in cells thought to divide symmetrically.

Centrosomes are the main microtubule-organizing centers in animal cells. Due to the semiconservative nature of centrosome duplication, the two centrosomes differ in age. In asymmetric stem cell divisions, centrosome age can induce an asymmetry in half-spindle lengths. However, whether centrosome age affects the symmetry of the two half-spindles in tissue culture cells thought to divide symmetrically is unknown. Here, we show that in human epithelial and fibroblastic cell lines centrosome age imposes a mild spindle asymmetry that leads to asymmetric cell daughter sizes. At the mechanistic level, we show that this asymmetry depends on a cenexin-bound pool of the mitotic kinase Plk1, which favors the preferential accumulation on old centrosomes of the microtubule nucleation-organizing proteins pericentrin, γ-tubulin, and Cdk5Rap2, and microtubule regulators TPX2 and ch-TOG. Consistently, we find that old centrosomes have a higher microtubule nucleation capacity. We postulate that centrosome age breaks spindle size symmetry via microtubule nucleation even in cells thought to divide symmetrically.

Disruption of Plasmodium falciparum kinetochore proteins destabilises the nexus between the centrosome equivalent and the mitotic apparatus.

Plasmodium falciparum is the causative agent of malaria and remains a pathogen of global importance. Asexual blood stage replication, via a process called schizogony, is an important target for the development of new antimalarials. Here we use ultrastructure-expansion microscopy to probe the organisation of the chromosome-capturing kinetochores in relation to the mitotic spindle, the centriolar plaque, the centromeres and the apical organelles during schizont development. Conditional disruption of the kinetochore components, PfNDC80 and PfNuf2, is associated with aberrant mitotic spindle organisation, disruption of the centromere marker, CENH3 and impaired karyokinesis. Surprisingly, kinetochore disruption also leads to disengagement of the centrosome equivalent from the nuclear envelope. Severing the connection between the nucleus and the apical complex leads to the formation of merozoites lacking nuclei. Here, we show that correct assembly of the kinetochore/spindle complex plays a previously unrecognised role in positioning the nascent apical complex in developing P. falciparum merozoites.

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.

Kinesins regulate the heterogeneity in centrosome clustering after whole-genome duplication.

After whole-genome duplication (WGD), tetraploid cells can undergo multipolar mitosis or pseudo-bipolar mitosis with clustered centrosomes. Kinesins play a crucial role in regulating spindle formation. However, the contribution of kinesin expression levels to the heterogeneity in centrosome clustering observed across different cell lines after WGD remains unclear. We identified two subsets of cell lines: "BP" cells efficiently cluster extra centrosomes for pseudo-bipolar mitosis, and "MP" cells primarily undergo multipolar mitosis after WGD. Diploid MP cells contained higher levels of KIF11 and KIF15 compared with BP cells and showed reduced sensitivity to centrosome clustering induced by KIF11 inhibitors. Moreover, partial inhibition of KIF11 or depletion of KIF15 converted MP cells from multipolar to bipolar mitosis after WGD. Multipolar spindle formation involved microtubules but was independent of kinetochore-microtubule attachment. Silencing KIFC1, but not KIFC3, promoted multipolar mitosis in BP cells, indicating the involvement of specific kinesin-14 family members in counteracting the forces from KIF11/KIF15 after WGD. These findings highlight the collective role of KIF11, KIF15, and KIFC1 in determining the polarity of the mitotic spindle after WGD.

Altered centriolar cohesion by CEP250 and appendages impact outcome of patients with pancreatic cancer.

BACKGROUND: Pancreatic ductal adenocarcinoma (PDAC) is one of the leading cause of cancer death worldwide. PDACs are characterized by centrosome aberrations, but whether centrosome-related genes influence patient outcomes has not been tested. METHODS: Publicly available RNA-sequencing data of patients diagnosed with PDAC were interrogated with unsupervised approaches to identify centrosome protein-encoding genes with prognostic relevance. Candidate genes were validated by immunohistochemistry and multiplex immunofluorescence in a set of clinical PDAC and normal pancreatic tissues. RESULTS: Results showed that two genes CEP250 and CEP170, involved in centrosome linker and centriolar subdistal appendages, were expressed at high levels in PDAC tissues and were correlated with prognosis of PDAC patients in independent databases. Large clustered γ-tubulin-labelled centrosomes were linked together by aberrant circular and planar-shaped CEP250 arrangements in CEP250-high expressing PDACs. Furthermore, PDACs displayed prominent centrosome separation and reduced CEP164-centrosomal labelling associated with acetylated-tubulin staining compared to normal pancreatic tissues. Interestingly, in a small validation cohort, CEP250-high expressing patients had shorter disease free- and overall-survival and almost none of those who received gemcitabine plus nab-paclitaxel first-line therapy achieved a clinical response. In contrast, weak CEP250 expression was associated with long-term survivors or responses to medical treatments. CONCLUSIONS: Alteration of the centriolar cohesion and appendages has effect on the survival of patients with PDAC.