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The mitotic spindle is the bipolar, microtubule-based structure that segregates chromosomes at each cell division. Aberrant spindles are frequently observed in cancer cells, but how oncogenic transformation affects spindle mechanics and function, particularly in the mechanical context of solid tumors, remains poorly understood. Here, we constitutively overexpress the oncogene cyclin D1 in human MCF10A cells to probe its effects on spindle architecture and response to compressive force. We find that cyclin D1 overexpression increases the incidence of spindles with extra poles, centrioles, and chromosomes. However, it also protects spindle poles from fracturing under compressive force, a deleterious outcome linked to multipolar cell divisions. Our findings suggest that cyclin D1 overexpression may adapt cells to increased compressive stress, possibly contributing to its prevalence in cancers such as breast cancer by allowing continued proliferation in mechanically challenging environments.

Aurora-A is an essential cell-cycle kinase with critical roles in mitotic entry and spindle dynamics. These functions require binding partners such as CEP192 and TPX2, which modulate both kinase activity and localisation of Aurora-A. Here we investigate the structure and role of the centrosomal Aurora-A:CEP192 complex in the wider molecular network. We find that CEP192 wraps around Aurora-A, occupies the binding sites for mitotic spindle-associated partners, and thus competes with them. Comparison of two different Aurora-A conformations reveals how CEP192 modifies kinase activity through the site used for TPX2-mediated activation. Deleting the Aurora-A-binding interface in CEP192 prevents centrosomal accumulation of Aurora-A, curtails its activation-loop phosphorylation, and reduces spindle-bound TPX2:Aurora-A complexes, resulting in error-prone mitosis. Thus, by supplying the pool of phosphorylated Aurora-A necessary for TPX2 binding, CEP192:Aurora-A complexes regulate spindle function. We propose an evolutionarily conserved spatial hierarchy, which protects genome integrity through fine-tuning and correctly localising Aurora-A activity. Synopsis: The mechanisms underlying the spatiotemporal control of Aurora-A kinase activity during mitosis are not yet determined. This study elucidates both the structure of the human Aurora-A:CEP192 complex and its critical role at the apex of Aurora-A activation in mitosis. CEP192 wraps around the N-lobe of the Aurora-A kinase domain occupying sites that are typically used by the competing microtubule-associated partners TACC3 and TPX2. CEP192 binds the F-pocket/C-helix region of the N-lobe in a way that inhibits Aurora-A, whereas TPX2 binding to the same region activates the kinase. The centrosomal CEP192-Aurora-A complex is the primary driver of Aurora-A auto-phosphorylation during mitosis. Deleting the Aurora-A binding interface of CEP192 causes mitotic spindle defects reminiscent of Aurora-A inhibition. CEP192 facilitates Aurora-A:TPX2 complex formation and activity on the mitotic spindle. The structure of the human Aurora-A:CEP192 complex reveals its importance in facilitating Aurora-A autophosphorylation and activity on the mitotic spindle. [ABSTRACT FROM AUTHOR]

The epidermis is a stratified epithelium that functions as the first line of defense against pathogenic invasion and acts as a barrier preventing water loss. In this study, we aimed to decipher the role of 14‐3‐3ε in the development of the epidermis. We report that loss of 14‐3‐3ε in the epidermis of juvenile and adult mice reduces cell division in the basal layer and increases the percentage of cells with multiple centrosomes, leading to a reduction in the thickness of the basal and stratified layers. We also demonstrate a decrease in the expression of differentiation markers, although no gross morphological defects in the skin or adverse effects on the survival of the mice were observed. These results suggest that loss of 14‐3‐3ε in the epidermis may lead to defects in proliferation and differentiation. [ABSTRACT FROM AUTHOR]

Centrosomes have critical roles in microtubule organization, ciliogenesis, and cell signaling. 1,2,3,4,5,6,7,8 Centrosomal alterations also contribute to diseases, including microcephaly, cancer, and ciliopathies. 9,10,11,12,13 To date, over 150 centrosomal proteins have been identified, including several kinases and phosphatases that control centrosome biogenesis, function, and maintenance. 2,3,4,5,14,15,16,17,18,19,20,21 However, the regulatory mechanisms that govern centrosome function are not fully defined, and thus how defects in centrosomal regulation contribute to disease is incompletely understood. Using a systems genetics approach, we find here that PPP2R3C, a poorly characterized PP2A phosphatase subunit, is a distal centriole protein and functional partner of centriolar proteins CEP350 and FOP. We further show that a key function of PPP2R3C is to counteract the kinase activity of MAP3K1. In support of this model, MAP3K1 knockout suppresses growth defects caused by PPP2R3C inactivation, and MAP3K1 and PPP2R3C have opposing effects on basal and microtubule stress-induced JNK signaling. Illustrating the importance of balanced MAP3K1 and PPP2R3C activities, acute overexpression of MAP3K1 severely inhibits centrosome function and triggers rapid centriole disintegration. Additionally, inactivating PPP2R3C mutations and activating MAP3K1 mutations both cause congenital syndromes characterized by gonadal dysgenesis. 22,23,24,25,26,27,28 As a syndromic PPP2R3C variant is defective in centriolar localization and binding to centriolar protein FOP, we propose that imbalanced activity of this centrosomal kinase-phosphatase pair is the shared cause of these disorders. Thus, our findings reveal a new centrosomal phospho-regulatory module, shed light on disorders of gonadal development, and illustrate the power of systems genetics to identify previously unrecognized gene functions. • PP2A phosphatase subunit PPP2R3C localizes to the distal end of centrioles • PPP2R3C acts with FOP and CEP350 to oppose centrosomal kinase MAP3K1 • Imbalance of PPP2R3C/MAP3K1 activity impairs centrosome function and cell fitness • Impaired phospho-regulation at centrioles may underlie gonadal dysgenesis disorders Ganga et al. show that the PP2A phosphatase subunit PPP2R3C and the kinase MAP3K1 are centriolar proteins that functionally oppose each other. Imbalance of their activities impairs centrosome function and cell fitness and is further revealed as a shared basis of gonadal dysgenesis disorders caused by mutations in MAP3K1 and PPP2R3C. [ABSTRACT FROM AUTHOR]

Senescence, marked by permanent cell cycle arrest may contribute to the decline in regenerative potential and neuronal function, thereby promoting neurodegenerative disorders. In this study, we employed whole exome sequencing to identify a previously unreported biallelic missense variant in SVBP (p.Leu49Pro) in six patients from three unrelated families. These affected individuals present with a complex hereditary spastic paraplegia (HSP), peripheral neuropathy, verbal apraxia, and intellectual disability, exhibiting a milder phenotype compared to patients with nonsense SVBP mutations described previously. Consistent with SVBP's primary role as a chaperone necessary for VASH‐mediated tubulin detyrosination, both patient fibroblasts with the p.Leu49Pro mutation, and HeLa cells harboring an SVBP knockdown exhibit microtubule dynamic instability and alterations in pericentriolar material (PCM) component trafficking and centrosome cohesion. In patient fibroblasts, structural abnormalities in the centrosome trigger mitotic errors and cellular senescence. Notably, premature senescence characterized by elevated levels of p16INK4, was also observed in patient peripheral blood mononuclear cells (PBMCs). Taken together, our findings underscore the critical role of SVBP in the development and maintenance of the central nervous system, providing novel insights associating cytokinesis failure with cortical motor neuron disease and intellectual disability. [ABSTRACT FROM AUTHOR]

Abstract Background Abnormalities of centrosomes, the major microtubular organizing centers of animal cells and regulators of cell cycle progression, usually accelerate tumor progression, but their prognostic value in lung adenocarcinoma (LUAD) remains insufficiently explored. Methods We collected centrosome genes from the literature and identified LUAD-specific centrosome-related genes (CRGs) using the single-sample gene set enrichment analysis (ssGSEA) algorithm and weighted gene co-expression network analysis (WGCNA). Univariate Cox was performed to screen prognostic CRGs. Consistent clustering was performed to classify LUAD patients into two subgroups, and centrosome-related risk score signatures were constructed by Lasso and multivariate Cox regression to predict overall survival (OS). We further explored the correlation between CRS and patient prognosis, clinical manifestations, mutation status, tumor microenvironment, and response to different treatments. Results We constructed centrosome-associated prognostic features and verified that CRS could effectively predict 1-, 3-, and 5-year survival in LUAD patients. In addition, patients in the high-risk group exhibited elevated tumor mutational loads and reduced levels of immune infiltration, particularly of T and B cells. Patients in the high-risk group were resistant to immunotherapy and sensitive to 5-fluoropyrimidine and gefitinib. The key gene spermine synthase (SRM) is highly expressed at the mRNA and protein levels in LUAD. Discussion Our work develops a novel centrosome-related prognostic signature that accurately predicts OS in LUAD and can assist in clinical diagnosis and treatment.

Cytoplasmic divisions are thought to rely on nuclear divisions and mitotic signals. We demonstrate in Drosophila embryos that cytoplasm can divide repeatedly without nuclei and mitotic CDK/cyclin complexes. Cdk1 normally slows an otherwise faster cytoplasmic division cycle, coupling it with nuclear divisions, and when uncoupled, cytoplasm starts dividing before mitosis. In developing embryos where CDK/cyclin activity can license mitotic microtubule (MT) organizers like the spindle, cytoplasmic divisions can occur without the centrosome, a principal organizer of interphase MTs. However, centrosomes become essential in the absence of CDK/cyclin activity, implying that the cytoplasm can employ either the centrosome-based interphase or CDK/cyclin-dependent mitotic MTs to facilitate its divisions. Finally, we present evidence that autonomous cytoplasmic divisions occur during unperturbed fly embryogenesis and that they may help extrude mitotically stalled nuclei during blastoderm formation. We postulate that cytoplasmic divisions occur in cycles governed by a yet-to-be-uncovered clock mechanism autonomous from CDK/cyclin complexes.

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. [ABSTRACT FROM AUTHOR]

Primary congenital glaucoma (PCG) occurs in children due to developmental abnormalities in the trabecular meshwork and anterior chamber angle. Previous studies have implicated rare variants in CYP1B1, LTBP2, and TEK and their interactions with MYOC, FOXC1, and PRSS56 in the genetic complexity and clinical heterogeneity of PCG. Given that some of the gene-encoded proteins are localized in the centrosomes (MYOC) and perform ciliary functions (TEK), we explored the involvement of a core centrosomal protein, CEP164, which is responsible for ocular development and regulation of intraocular pressure. Deep sequencing of CEP164 in a PCG cohort devoid of homozygous mutations in candidate genes (n = 298) and controls (n = 1757) revealed CEP164 rare pathogenic variants in 16 cases (5.36%). Co-occurrences of heterozygous alleles of CEP164 with other genes were seen in four cases (1.34%), and a physical interaction was noted for CEP164 and CYP1B1 in HEK293 cells. Cases of co-harboring alleles of the CEP164 and other genes had a poor prognosis compared with those with a single copy of the CEP164 allele. We also screened INPP5E, which synergistically interacts with CEP164, and observed a lower frequency of pathogenic variants (0.67%). Our data suggest the potential involvements of CEP164 and INPP5E and the yet unexplored cilia-centrosomal functions in PCG pathogenesis. [ABSTRACT FROM AUTHOR]

In cells, microtubules (MTs) assemble from α/β‐tubulin subunits at nucleation sites containing the γ‐tubulin ring complex (γ‐TuRC). Within the γ‐TuRC, exposed γ‐tubulin molecules act as templates for MT assembly by interacting with α/β‐tubulin. The vertebrate γ‐TuRC is scaffolded by γ‐tubulin‐interacting proteins GCP2‐6 arranged in a specific order. Interestingly, the γ‐tubulin molecules in the γ‐TuRC deviate from the cylindrical geometry of MTs, raising the question of how the γ‐TuRC structure changes during MT nucleation. Recent studies on the structure of the vertebrate γ‐TuRC attached to the end of MTs came to varying conclusions. In vitro assembly of MTs, facilitated by an α‐tubulin mutant, resulted in a closed, cylindrical γ‐TuRC showing canonical interactions between all γ‐tubulin molecules and α/β‐tubulin subunits. Conversely, native MTs formed in a frog extract were capped by a partially closed γ‐TuRC, with some γ‐tubulin molecules failing to align with α/β‐tubulin. This review discusses these outcomes, along with the broader implications. [ABSTRACT FROM AUTHOR]

The genetic basis of vertebrate emergence during metazoan evolution has remained largely unknown. Understanding vertebrate‐specific genes, such as the tight junction protein Occludin (Ocln), may help answer this question. Here, it is shown that mammary glands lacking Ocln exhibit retarded epithelial branching, owing to reduced cell proliferation and surface expansion. Interestingly, Ocln regulates mitotic spindle orientation and function, and its loss leads to a range of defects, including prolonged prophase and failed nuclear and/or cytoplasmic division. Mechanistically, Ocln binds to the RabGTPase‐11 adaptor FIP5 and recruits recycling endosomes to the centrosome to participate in spindle assembly and function. FIP5 loss recapitulates Ocln null, leading to prolonged prophase, reduced cell proliferation, and retarded epithelial branching. These results identify a novel role in OCLN‐mediated endosomal trafficking and potentially highlight its involvement in mediating membranous vesicle trafficking and function, which is evolutionarily conserved and essential. [ABSTRACT FROM AUTHOR]

The primary cilium is a small organelle protruding from the cell surface that receives signals from the extracellular milieu. Although dozens of studies have reported that several genetic factors can impair the structure of primary cilia, evidence for environmental stimuli affecting primary cilia structures is limited. Here, we investigated an extracellular stress that affected primary cilia morphology and its underlying mechanisms. Hyperosmotic shock induced reversible shortening and disassembly of the primary cilia of murine intramedullary collecting duct cells. The shortening of primary cilia caused by hyperosmotic shock followed delocalization of the pericentriolar material (PCM). Excessive microtubule and F-actin formation in the cytoplasm coincided with the hyperosmotic shockinduced changes to primary cilia and the PCM. Treatment with a microtubule-disrupting agent, nocodazole, partially prevented the hyperosmotic shock-induced disassembly of primary cilia and almost completely prevented delocalization of the PCM. An actin polymerization inhibitor, latrunculin A, also partially prevented the hyperosmotic shock-induced shortening and disassembly of primary cilia and almost completely prevented delocalization of the PCM. We demonstrate that hyperosmotic shock induces reversible morphological changes in primary cilia and the PCM in a manner dependent on excessive formation of microtubule and F-actin. [ABSTRACT FROM AUTHOR]

Centrosomes are the canonical microtubule organizing centers (MTOCs) of most mammalian cells, including spermatocytes. Centrosomes comprise a centriole pair within a structurally ordered and dynamic pericentriolar matrix (PCM). Unlike in mitosis, where centrioles duplicate once per cycle, centrioles undergo two rounds of duplication during spermatogenesis. The first duplication is during early meiotic prophase I, and the second is during interkinesis. Using mouse mutants and chemical inhibition, we have blocked centriole duplication during spermatogenesis and determined that non-centrosomal MTOCs (ncMTOCs) can mediate chromosome segregation. This mechanism is different from the acentriolar MTOCs that form bipolar spindles in oocytes, which require PCM components, including gamma-tubulin and CEP192. From an in-depth analysis, we identified six microtubule-associated proteins, TPX2, KIF11, NuMA, and CAMSAP1-3, that localized to the non-centrosomal MTOC. These factors contribute to a mechanism that ensures bipolar MTOC formation and chromosome segregation during spermatogenesis when centriole duplication fails. However, despite the successful completion of meiosis and round spermatid formation, centriole inheritance and PLK4 function are required for normal spermiogenesis and flagella assembly, which are critical to ensure fertility. Synopsis: PLK4 is required for centriole duplication and maturation during mammalian spermatogenesis. However, when duplication and maturation fail, spermatocytes form non-centrosomal microtubule organizing centers to complete chromosome segregation during meiosis I and II. PLK4 is the master regulator of centriole duplication and maturation during mammalian spermatogenesis. Spermatocytes are essential for the formation of mature spermatozoa that harbor a flagella. Spermatocytes can rely on a non-centrosomal microtubule organizing center (ncMTOC) to mediate chromosome segregation during meiosis I and II. PLK4 is required for centriole duplication and maturation during mammalian spermatogenesis. However, when duplication and maturation fail, spermatocytes form non-centrosomal microtubule organizing centers to complete chromosome segregation during meiosis I and II. [ABSTRACT FROM AUTHOR]

Background/aim: Spindle pole bodies (SPB), the functional equivalent of centrosomes in yeast, duplicate through generation of a new SPB next to the old one. However, SPBs are dynamic structures that can grow and exchange, and mechanisms that regulate SPB size remain largely unknown. This study aims to elucidate the role of Bud14 in SPB size maintenance in Saccharomyces cerevisiae. Materials and methods: We employed quantitative fluorescence microscopy to assess the relative and absolute amounts of SPB structural proteins at SPBs of wildtype cells and in cells lacking BUD14 (bud14Δ). Quantifications were performed using asynchronous cell cultures, as well as cultures synchronously progressing through the cell cycle and upon different cell cycle arrests. We also utilized mutants that allow the separation of Bud14 functions. Results: Our results indicate that higher levels of SPB inner, outer, and central plaque proteins are present at the SPBs of bud14Δ cells compared to wildtype cells during anaphase, as well as during nocodazole-induced M-phase arrest. However, during α-factor mediated G1 arrest, inner and outer plaque proteins responded differently to the absence of BUD14. A Bud14 mutant that cannot interact with the Protein Phosphatase 1 (Glc7) phenocopied bud14Δ in terms of SPB-bound levels of the inner plaque protein Spc110, whereas disruption of Bud14-Kel1-Kel2 complex did not alter Spc110 levels at SPBs. In cells synchronously released from α-factor arrest, lack of Bud14-Glc7 caused increase of Spc110 at the SPBs at early stages of the cell cycle. Conclusion: We identified Bud14 as a critical protein for SPB size maintenance. The interaction of Bud14 with Glc7, but not with the Kelch proteins, is indispensable for restricting levels of Spc110 incorporated into the SPBs. [ABSTRACT FROM AUTHOR]

The γ-tubulin ring complex (γTuRC) is the principal nucleator of cellular microtubules, and the microtubule-nucleating activity of the complex is stimulated by binding to the γTuRC-mediated nucleation activator (γTuNA) motif. The γTuNA is part of the centrosomin motif 1 (CM1), which is widely found in γTuRC stimulators, including CDK5RAP2. Here, we show that a conserved segment within CM1 binds to the γTuNA and blocks its association with γTuRCs; therefore, we refer to this segment as the γTuNA inhibitor (γTuNA-In). Mutational disruption of the interaction between the γTuNA and the γTuNA-In results in a loss of autoinhibition, which consequently augments microtubule nucleation on centrosomes and the Golgi complex, the two major microtubule-organizing centers. This also causes centrosome repositioning, leads to defects in Golgi assembly and organization, and affects cell polarization. Remarkably, phosphorylation of the γTuNA-In, probably by Nek2, counteracts the autoinhibition by disrupting the γTuNA‒γTuNA-In interaction. Together, our data reveal an on-site mechanism for controlling γTuNA function.

Haploid larvae in non-mammalian vertebrates are lethal, with characteristic organ growth retardation collectively called ‘haploid syndrome’. In contrast to mammals, whose haploid intolerance is attributed to imprinting misregulation, the cellular principle of haploidy-linked defects in non-mammalian vertebrates remains unknown. Here, we investigated cellular defects that disrupt the ontogeny of gynogenetic haploid zebrafish larvae. Unlike diploid control larvae, haploid larvae manifested unscheduled cell death at the organogenesis stage, attributed to haploidy-linked p53 upregulation. Moreover, we found that haploid larvae specifically suffered the gradual aggravation of mitotic spindle monopolarization during 1–3 days post-fertilization, causing spindle assembly checkpoint-mediated mitotic arrest throughout the entire body. High-resolution imaging revealed that this mitotic defect accompanied the haploidy-linked centrosome loss occurring concomitantly with the gradual decrease in larval cell size. Either resolution of mitotic arrest or depletion of p53 partially improved organ growth in haploid larvae. Based on these results, we propose that haploidy-linked mitotic defects and cell death are parts of critical cellular causes shared among vertebrates that limit the larval growth in the haploid state, contributing to an evolutionary constraint on allowable ploidy status in the vertebrate life cycle.

Abstract This review aims to outline mitotic kinase inhibitors’ roles as potential therapeutic targets and assess their suitability as a stand-alone clinical therapy or in combination with standard treatments for advanced-stage solid tumors, including triple-negative breast cancer (TNBC). Breast cancer poses a significant global health risk, with TNBC standing out as the most aggressive subtype. Comprehending the role of mitosis is crucial for understanding how TNBC advances from a solid tumor to metastasis. Chemotherapy is the primary treatment used to treat TNBC. Some types of chemotherapeutic agents target cells in mitosis, thus highlighting the need to comprehend the molecular mechanisms governing mitosis in cancer. This understanding is essential for devising targeted therapies to disrupt these mitotic processes, prevent or treat metastasis, and improve patient outcomes. Mitotic kinases like Aurora kinase A, Aurora Kinase B, never in mitosis gene A-related kinase 2, Threonine-Tyrosine kinase, and Polo-kinase 1 significantly impact cell cycle progression by contributing to chromosome separation and centrosome homeostasis. When these kinases go awry, they can trigger chromosome instability, increase cell proliferation, and activate different molecular pathways that culminate in a transition from epithelial to mesenchymal cells. Ongoing clinical trials investigate various mitotic kinase inhibitors as potential biological treatments against advanced solid tumors. While clinical trials against mitotic kinases have shown some promise in the clinic, more investigation is necessary, since they induce severe adverse effects, particularly affecting the hematopoietic system.

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 FROM AUTHOR]

This review aims to outline mitotic kinase inhibitors' roles as potential therapeutic targets and assess their suitability as a stand-alone clinical therapy or in combination with standard treatments for advanced-stage solid tumors, including triple-negative breast cancer (TNBC). Breast cancer poses a significant global health risk, with TNBC standing out as the most aggressive subtype. Comprehending the role of mitosis is crucial for understanding how TNBC advances from a solid tumor to metastasis. Chemotherapy is the primary treatment used to treat TNBC. Some types of chemotherapeutic agents target cells in mitosis, thus highlighting the need to comprehend the molecular mechanisms governing mitosis in cancer. This understanding is essential for devising targeted therapies to disrupt these mitotic processes, prevent or treat metastasis, and improve patient outcomes. Mitotic kinases like Aurora kinase A, Aurora Kinase B, never in mitosis gene A-related kinase 2, Threonine-Tyrosine kinase, and Polo-kinase 1 significantly impact cell cycle progression by contributing to chromosome separation and centrosome homeostasis. When these kinases go awry, they can trigger chromosome instability, increase cell proliferation, and activate different molecular pathways that culminate in a transition from epithelial to mesenchymal cells. Ongoing clinical trials investigate various mitotic kinase inhibitors as potential biological treatments against advanced solid tumors. While clinical trials against mitotic kinases have shown some promise in the clinic, more investigation is necessary, since they induce severe adverse effects, particularly affecting the hematopoietic system. [ABSTRACT FROM AUTHOR]

Centriolar satellites are ubiquitous membrane‐less organelles that play critical roles in numerous cellular and organismal processes. They were initially discovered through electron microscopy as cytoplasmic granules surrounding centrosomes in vertebrate cells. These structures remained enigmatic until the identification of pericentriolar material 1 protein (PCM1) as their molecular marker, which has enabled their in‐depth characterization. Recently, centriolar satellites have come into the spotlight due to their links to developmental and neurodegenerative disorders. This review presents a comprehensive summary of the major advances in centriolar satellite biology, with a focus on studies that investigated their biology associated with the essential scaffolding protein PCM1. We begin by exploring the molecular, cellular, and biochemical properties of centriolar satellites, laying the groundwork for a deeper understanding of their functions and mechanisms at both cellular and organismal levels. We then examine the implications of their dysregulation in various diseases, particularly highlighting their emerging roles in neurodegenerative and developmental disorders, as revealed by organismal models of PCM1. We conclude by discussing the current state of knowledge and posing questions about the adaptable nature of these organelles, thereby setting the stage for future research. [ABSTRACT FROM AUTHOR]

Neurotropic alphaherpesviruses, including herpes simplex virus type 1 (HSV-1), recruit microtubule motor proteins to invade cells. The incoming viral particle traffics to nuclei in a two-step process. First, the particle uses the dynein-dynactin motor to sustain transport to the centrosome. In neurons, this step is responsible for long-distance retrograde axonal transport and is an important component of the neuroinvasive property shared by these viruses. Second, a kinesin-dependent mechanism redirects the particle from the centrosome to the nucleus. We have reported that the kinesin motor used during the second step of invasion is assimilated into nascent virions during the previous round of infection. Here, we report that the HSV-1 pUL37 tegument protein suppresses the assimilated kinesin-1 motor during retrograde axonal transport. Region 2 (R2) of pUL37 was required for suppression and functioned independently of the autoinhibitory mechanism native to kinesin-1. Furthermore, the motor domain and proximal coiled coil of kinesin-1 were sufficient for HSV-1 assimilation, pUL37 suppression, and nuclear trafficking. pUL37 localized to the centrosome, the site of assimilated kinesin-1 activation during infection, when expressed in cells in the absence of other viral proteins; however, pUL37 did not suppress kinesin-1 in this context. These results indicate that the pUL37 tegument protein spatially and temporally regulates kinesin-1 via the amino-terminal motor region in the context of the incoming viral particle. [ABSTRACT FROM AUTHOR]

Primary cilia are essential eukaryotic organelles required for signalling and secretion. Dynein-2 is a microtubule-motor protein complex and is required for ciliogenesis via its role in facilitating retrograde intraflagellar transport (IFT) from the cilia tip to the cell body. Dynein-2 must be assembled and loaded onto IFT trains for entry into cilia for this process to occur, but how dynein-2 is assembled and how it is recycled back into a cilium remain poorly understood. Here, we identify centrosomal protein of 170 kDa (CEP170) as a dynein-2-interacting protein in mammalian cells. We show that loss of CEP170 perturbs intraflagellar transport and hedgehog signalling, and alters the stability of dynein-2 holoenzyme complex. Together, our data indicate a role for CEP170 in supporting cilia function and dynein-2 assembly. [ABSTRACT FROM AUTHOR]

CRNDE is considered an oncogene expressed as long non-coding RNA. Our previous paper is the only one reporting CRNDE as a micropeptide-coding gene. The amino acid sequence of this micropeptide (CRNDEP) has recently been confirmed by other researchers. This study aimed at providing a mass spectrometry (MS)-based validation of the CRNDEP sequence and an investigation of how the differential expression of CRNDE(P) influences the metabolism and chemoresistance of ovarian cancer (OvCa) cells. We also assessed cellular localization changes of CRNDEP, looked for its protein partners, and bioinformatically evaluated its RNA-binding capacities. Herein, we detected most of the CRNDEP sequence by MS. Moreover, our results corroborated the oncogenic role of CRNDE, portraying it as the gene impacting carcinogenesis at the stages of DNA transcription and replication, affecting the RNA metabolism, and stimulating the cell cycle progression and proliferation, with CRNDEP being detected in the centrosomes of dividing cells. We also showed that CRNDEP is located in nucleoli and revealed interactions of this micropeptide with p54, an RNA helicase. Additionally, we proved that high CRNDE(P) expression increases the resistance of OvCa cells to treatment with microtubule-targeted cytostatics. Furthermore, altered CRNDE(P) expression affected the activity of the microtubular cytoskeleton and the formation of focal adhesion plaques. Finally, according to our in silico analyses, CRNDEP is likely capable of RNA binding. All these results contribute to a better understanding of the CRNDE(P) role in OvCa biology, which may potentially improve the screening, diagnosis, and treatment of this disease. [ABSTRACT FROM AUTHOR]

Mesenchymal stem cells (MSC) attract an increasing amount of attention due to their unique therapeutic properties. Yet, MSC can undergo undesirable genetic and epigenetic changes during their propagation in vitro. In this study, we investigated whether polyploidy can compromise MSC oncological safety and therapeutic properties. For this purpose, we compared the impact of polyploidy on the transcriptome of cancer cells and MSC of various origins (bone marrow, placenta, and heart). First, we identified genes that are consistently ploidy-induced or ploidy-repressed through all comparisons. Then, we selected the master regulators using the protein interaction enrichment analysis (PIEA). The obtained ploidy-related gene signatures were verified using the data gained from polyploid and diploid populations of early cardiomyocytes (CARD) originating from iPSC. The multistep bioinformatic analysis applied to the cancer cells, MSC, and CARD indicated that polyploidy plays a pivotal role in driving the cell into hypertranscription. It was evident from the upregulation of gene modules implicated in housekeeping functions, stemness, unicellularity, DNA repair, and chromatin opening by means of histone acetylation operating via DNA damage associated with the NUA4/TIP60 complex. These features were complemented by the activation of the pathways implicated in centrosome maintenance and ciliogenesis and by the impairment of the pathways related to apoptosis, the circadian clock, and immunity. Overall, our findings suggest that, although polyploidy does not induce oncologic transformation of MSC, it might compromise their therapeutic properties because of global epigenetic changes and alterations in fundamental biological processes. The obtained results can contribute to the development and implementation of approaches enhancing the therapeutic properties of MSC by removing polyploid cells from the cell population. [ABSTRACT FROM AUTHOR]

Breast cancer is the most prevalent malignancy among women worldwide. Despite significant advances in treatment, it remains one of the leading causes of female mortality. The inability to effectively treat advanced and/or treatmentresistant breast cancer demonstrates the need to develop novel treatment strategies and targeted therapies. Centrosomes and their associated proteins have been shown to play key roles in the pathogenesis of breast cancer and thus represent promising targets for drug and biomarker development. Centrosomes are fundamental cellular structures in the mammalian cell that are responsible for error-free execution of cell division. Centrosome amplification and aberrant expression of its associated proteins such as Polo-like kinases (PLKs), Aurora kinases (AURKs) and Cyclin-dependent kinases (CDKs) have been observed in various cancers, including breast cancer. These aberrations in breast cancer are thought to cause improper chromosomal segregation during mitosis, leading to chromosomal instability and uncontrolled cell division, allowing cancer cells to acquire new genetic changes that result in evasion of cell death and the promotion of tumor formation. Various chemical compounds developed against PLKs and AURKs have shown meaningful antitumorigenic effects in breast cancer cells in vitro and in vivo. The mechanism of action of these inhibitors is likely related to exacerbation of numerical genomic instability, such as aneuploidy or polyploidy. Furthermore, growing evidence demonstrates enhanced antitumorigenic effects when inhibitors specific to centrosome-associated proteins are used in combination with either radiation or chemotherapy drugs in breast cancer. This review focuses on the current knowledge regarding the roles of centrosome and centrosome-associated proteins in breast cancer pathogenesis and their utility as novel targets for breast cancer treatment. [ABSTRACT FROM AUTHOR]

Family with sequence similarity 161 (Fam161) is an ancient family of microtubule-binding proteins located at the centriole and cilium transition zone (TZ) lumen that exhibit rapid evolution in mice. However, their adaptive role is unclear. Here, we used flies to gain insight into their cell type-specific adaptations. Fam161 is the sole orthologue of FAM161A and FAM161B found in flies. Mutating Fam161 results in reduced male reproduction and abnormal geotaxis behaviour. Fam161 localizes to sensory neuron centrioles and their specialized TZ (the connecting cilium) in a cell type-specific manner, sometimes labelling only the centrioles, sometimes labelling the centrioles and cilium TZ and sometimes labelling the TZ with varying lengths that are longer than other TZ proteins, defining a new ciliary compartment, the extra distal TZ. These findings suggest that Fam161 is an essential centriole and TZ protein with a unique cell type-specific localization in fruit flies that can produce cell type-specific adaptations.

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.

Abstract The genetic basis of vertebrate emergence during metazoan evolution has remained largely unknown. Understanding vertebrate‐specific genes, such as the tight junction protein Occludin (Ocln), may help answer this question. Here, it is shown that mammary glands lacking Ocln exhibit retarded epithelial branching, owing to reduced cell proliferation and surface expansion. Interestingly, Ocln regulates mitotic spindle orientation and function, and its loss leads to a range of defects, including prolonged prophase and failed nuclear and/or cytoplasmic division. Mechanistically, Ocln binds to the RabGTPase‐11 adaptor FIP5 and recruits recycling endosomes to the centrosome to participate in spindle assembly and function. FIP5 loss recapitulates Ocln null, leading to prolonged prophase, reduced cell proliferation, and retarded epithelial branching. These results identify a novel role in OCLN‐mediated endosomal trafficking and potentially highlight its involvement in mediating membranous vesicle trafficking and function, which is evolutionarily conserved and essential.

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.

Ovarian cancer (OC) is a common and lethal gynaecological malignancy. RNA-binding proteins (RBPs) play a crucial role in governing RNA metabolism and have been implicated in the development and progression of diverse cancer types. Slight alterations in RBPs' expression or activity can induce substantial modifications in the regulatory network. THUMPD2, as member of the RBP family, was found to have differential expression in ovarian cancer, with the mechanism has not been studied yet. In this study, THUMPD2 protein was found to be weakly expressed in the early (I + II) stages of OC (P = 0.013), with a low expression rate of 78.6 %, and highly expressed in late (III + IV) stages (P = 0.009), with a high expression rate of 84.8 %. The shRNA-mediated knockdown of THUMPD2 in OVCAR3 and SKOV3 cells resulted in increased cell proliferation but inhibited metastasis, whereas THUMPD2 overexpression had the opposite effect. THUMPD2 overexpression suppressed tumour growth in vivo. Conversely, low THUMPD2 expression promoted tumour growth. Furthermore, we identified the potential target genes and pathways of THUMPD2 using GO and KEGG analyses, which were related to the centrosome, microtubules, cell cycle, and extracellular matrix. We demonstrated that low expression of THUMPD2 in the early stage promoted tumour growth and high expression in the late stage promoted tumour metastasis. Our findings reveal the dual function of THUMPD2 in OC and suggest that THUMPD2 may serve as a therapeutic target for the treatment of OC.

Wnt signal transduction is controlled by the destruction complex (DC), a condensate comprising scaffold proteins and kinases that regulate β-catenin stability. Overexpressed DC scaffolds undergo liquid-liquid phase separation (LLPS), but DC mesoscale organization at endogenous expression levels and its role in β-catenin processing were previously unknown. Here, we find that DC LLPS is nucleated by the centrosome. Through a combination of CRISPR-engineered custom fluorescent tags, finite element simulations, and optogenetic tools that allow for manipulation of DC concentration and multivalency, we find that centrosomal nucleation drives processing of β-catenin by colocalizing DC components to a single reaction crucible. Enriching GSK3β partitioning on the centrosome controls β-catenin processing and prevents Wnt-driven embryonic stem cell differentiation to mesoderm. Our findings demonstrate the role of nucleators in controlling biomolecular condensates and suggest tight integration between Wnt signal transduction and the cell cycle.

Zika virus (ZIKV) is a flavivirus transmitted via mosquitoes and sex to cause congenital neurodevelopmental defects, including microcephaly. Inherited forms of microcephaly (MCPH) are associated with disrupted centrosome organization. Similarly, we found that ZIKV infection disrupted centrosome organization. ZIKV infection disrupted the organization of centrosomal proteins including CEP63, a MCPH-associated protein. The ZIKV nonstructural protein NS3 bound CEP63, and expression of NS3 was sufficient to alter centrosome architecture and CEP63 localization. Loss of CEP63 suppressed ZIKV-induced centrosome disorganization, indicating that ZIKV requires CEP63 to disrupt centrosome organization. ZIKV infection or CEP63 loss decreased the centrosomal localization and stability of TANK-binding kinase 1 (TBK1), a regulator of the innate immune response. ZIKV infection also increased the centrosomal accumulation of the CEP63 interactor DTX4, a ubiquitin ligase that degrades TBK1. Therefore, we propose that ZIKV disrupts CEP63 function to increase centrosomal DTX4 localization and destabilization of TBK1, thereby tempering the innate immune response.

The Parkinson's-disease-associated LRRK2 kinase phosphorylates multiple Rab GTPases including Rab8 and Rab10, which enhances their binding to RILPL1 and RILPL2. The nascent interaction between phospho-Rab10 and RILPL1 blocks ciliogenesis in vitro and in the intact brain, and interferes with the cohesion of duplicated centrosomes in dividing cells. We show here that regulators of the LRRK2 signaling pathway including vps35 and PPM1H converge upon causing centrosomal deficits. The cohesion alterations do not require the presence of other LRRK2 kinase substrates including Rab12, Rab35 and Rab43 or the presence of RILPL2. Rather, they depend on the RILPL1-mediated centrosomal accumulation of phosphorylated Rab10. RILPL1 localizes to the subdistal appendage of the mother centriole, followed by recruitment of the LRRK2-phosphorylated Rab proteins to cause the centrosomal defects. The centrosomal alterations impair cell polarization as monitored by scratch wound assays which is reverted by LRRK2 kinase inhibition. These data reveal a common molecular pathway by which enhanced LRRK2 kinase activity impacts upon centrosome-related events to alter the normal biology of a cell.

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. [ABSTRACT FROM AUTHOR]

The primary cilium is an antenna-like projection from the plasma membrane that serves as a sensor of the extracellular environment and a crucial signaling hub. Primary cilia are generated in most mammalian cells, and their physiological significance is highlighted by the large number of severe developmental disorders or ciliopathies that occur when primary ciliogenesis is impaired. Primary ciliogenesis is a tightly regulated process, and a central early regulatory step is the removal of a key mother centriole capping protein, CP110 (also known as CCP110). This uncapping allows vesicles docked on the distal appendages of the mother centriole to fuse to form a ciliary vesicle, which is bent into a ciliary sheath as the microtubule-based axoneme grows and extends from the mother centriole. When the mother centriole migrates toward the plasma membrane, the ciliary sheath fuses with the plasma membrane to form the primary cilium. In this Review, we outline key early steps of primary ciliogenesis, focusing on several novel mechanisms for removal of CP110. We also highlight examples of ciliopathies caused by genetic variants that encode key proteins involved in the early steps of ciliogenesis. [ABSTRACT FROM AUTHOR]

Aggregation of high-affinity IgE receptors (FceRIs) on granulated mast cells triggers signaling pathways leading to a calcium response and release of inflammatory mediators from secretory granules. While microtubules play a role in the degranulation process, the complex molecular mechanisms regulating microtubule remodeling in activated mast cells are only partially understood. Here, we demonstrate that the activation of bone marrow mast cells induced by FceRI aggregation increases centrosomal microtubule nucleation, with G protein-coupled receptor kinase-interacting protein 2 (GIT2) playing a vital role in this process. Both endogenous and exogenous GIT2 were associated with centrosomes and γ-tubulin complex proteins. Depletion of GIT2 enhanced centrosomal microtubule nucleation, and phenotypic rescue experiments revealed that GIT2, unlike GIT1, acts as a negative regulator of microtubule nucleation in mast cells. GIT2 also participated in the regulation of antigen-induced degranulation and chemotaxis. Further experiments showed that phosphorylation affected the centrosomal localization of GIT2 and that during antigen-induced activation, GIT2 was phosphorylated by conventional protein kinase C, which promoted microtubule nucleation. We propose that GIT2 is a novel regulator of microtubule organization in activated mast cells by modulating centrosomal microtubule nucleation. [ABSTRACT FROM AUTHOR]

Mitotic centrosomes assemble when centrioles recruit large amounts of pericentriolar material (PCM) around themselves. In early C. elegans embryos, mitotic centrosome size appears to be set by the limiting amount of a key component. In Drosophila syncytial embryos, thousands of mitotic centrosomes are assembled as the embryo proceeds through 13 rounds of rapid nuclear division, driven by a core cell cycle oscillator. These divisions slow during nuclear cycles 11–13, and we find that centrosomes respond by reciprocally decreasing their growth rate, but increasing their growth period—so that they grow to a relatively consistent size at each cycle. At the start of each cycle, moderate CCO activity initially promotes centrosome growth, in part by stimulating Polo/PLK1 recruitment to centrosomes. Later in each cycle, high CCO activity inhibits centrosome growth by suppressing the centrosomal recruitment and/or maintenance of centrosome proteins. Thus, in fly embryos, mitotic centrosome size appears to be regulated predominantly by the core cell cycle oscillator, rather than by the depletion of a limiting component. Synopsis: Centrosomes grow in preparation for mitosis and, in worm embryos, centrosome size appears to be set by the availability of a limiting component. In contrast, this study shows that, in fly embryos, centrosome size appears to be set predominantly by the cell-cycle oscillator (CCO). In Drosophila embryos, centrosomes reciprocally adjust their growth rate and growth period in response to changes in nuclear-cycle length. This ensures that centrosomes grow to a relatively consistent size at each nuclear cycle, irrespective of cycle length, or centrosome number. The CCO influences both centrosome growth rate and growth period. The CCO initiates centrosome growth at the start of each cycle by promoting the recruitment of Polo/PLK1 to centrosomes. The CCO suppresses centrosome growth towards the end of each cycle by inhibiting centrosome protein interactions. In contrast to worm embryos in which centrosome size depends on a availability of a limiting component, fly embryos reciprocally adjust centrosome growth rate and growth period to nuclear division cycles. [ABSTRACT FROM AUTHOR]

Ninein is a centrosome protein that has been implicated in microtubule anchorage and centrosome cohesion. Mutations in the human NINEIN gene have been linked to Seckel syndrome and to a rare form of skeletal dysplasia. However, the role of ninein in skeletal development remains unknown. Here, we describe a ninein knockout mouse with advanced endochondral ossification during embryonic development. Although the long bones maintain a regular size, the absence of ninein delays the formation of the bone marrow cavity in the prenatal tibia. Likewise, intramembranous ossification in the skull is more developed, leading to a premature closure of the interfrontal suture. We demonstrate that ninein is strongly expressed in osteoclasts of control mice, and that its absence reduces the fusion of precursor cells into syncytial osteoclasts, whereas the number of osteoblasts remains unaffected. As a consequence, ninein-deficient osteoclasts have a reduced capacity to resorb bone. At the cellular level, the absence of ninein interferes with centrosomal microtubule organization, reduces centrosome cohesion, and provokes the loss of centrosome clustering in multinucleated mature osteoclasts. We propose that centrosomal ninein is important for osteoclast fusion, to enable a functional balance between bone-forming osteoblasts and bone-resorbing osteoclasts during skeletal development.

BACKGROUND: Identification of novel molecular target(s) is important for designing newer mechanistically driven approaches for the treatment of prostate cancer (PCa), which is one of the main causes of morbidity and mortality in men. In this study, we determined the role of polo-like kinase 4 (PLK4), which regulates centriole duplication and centrosome amplification (CA), in PCa. MATERIALS AND METHODS: Employing human PCa tissue microarrays, we assessed the prevalence of CA, correlated with Gleason score, and estimated major causes of CA in PCa (cell doubling vs. centriole overduplication) by staining for mother/mature centrioles. We also assessed PLK4 expression and correlated it with CA in human PCa tissues and cell lines. Further, we determined the effects of PLK4 inhibition in human PCa cells. RESULTS: Compared to benign prostate, human PCa demonstrated significantly higher CA, which was also positively correlated with the Gleason score. Further, most cases of CA were found to arise by centriole overduplication rather than cell doubling events (e.g., cytokinesis failure) in PCa. In addition, PLK4 was overexpressed in human PCa cell lines and tumors. Moreover, PLK4 inhibitors CFI-400945 and centrinone-B inhibited cell growth, viability, and colony formation of both androgen-responsive and androgen-independent PCa cell lines. PLK4 inhibition also induced cell cycle arrest and senescence in human PCa cells. CONCLUSIONS: CA is prevalent in PCa and arises predominantly by centriole overduplication as opposed to cell doubling events. Loss of centrioles is cellular stress that can promote senescence and suggests that PLK4 inhibition may be a viable therapeutic strategy in PCa.

Abstract Objective The centrosome is universally recognized as the microtubule organizing center of animal cells, but emerging evidence suggests that it has other important functions including primary cilia formation, DNA damage checkpoints, and cell cycle progression. Despite this, the role of individual components of the centrosome remains unclear. Previous studies suggest that one component, centriolin, has an important function in cytokinesis and cell cycle progression, although its exact role in these processes is not known. To determine how centriolin influences the progression through the cell cycle, we sought to identify interacting partners that may be involved in regulating its function. Results This study provides evidence that the ubiquitin E3 ligase HectD1 binds to centriolin and that this association likely accounts for our observation that HectD1 co-localizes with centriolin at the centrosome during mitosis. In addition to its centrosomal localization, we also show that the expression of HectD1 fluctuates throughout the cell cycle, with the highest levels during mitosis, coinciding with a marked reduction in centriolin expression. We propose that the interaction between HectD1 and centriolin may be necessary for normal cell cycle progression and we speculate that this function may involve HectD1-mediated degradation of centriolin.

The Hedgehog (Hh) signaling is one of the essential signaling pathways during embryogenesis and in adults. Hh signal transduction relies on primary cilium, a specialized cell surface organelle viewed as the hub of cell signaling. Protein kinase A (PKA) has been recognized as a potent negative regulator of the Hh pathway, raising the question of how such a ubiquitous kinase specifically regulates one signaling pathway. We reviewed recent genetic, molecular and biochemical studies that have advanced our mechanistic understanding of PKA's role in Hh signaling in vertebrates, focusing on the compartmentalized PKA at the centrosome and in the primary cilium. We outlined the recently developed genetic and optical tools that can be harvested to study PKA activities during the course of Hh signal transduction.

Cyclins and cyclin-dependent kinases (CDKs) localize to the centrosome, but their significance in the cell cycle is unclear. Recently, Roberts et al. revealed that centrosomal cyclin B-CDK is required for mitotic entry and phosphorylation of substrates. This suggests that the centrosome acts as a signaling hub controlling the cell cycle. [ABSTRACT FROM AUTHOR]

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.

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.

Based on epidemiology studies, Chlamydia trachomatis has been proposed as a co-factor for human papillomavirus (HPV) in the development of cervical cancer. These two intracellular pathogens have been independently reported to induce the production of extra centrosomes, or centrosome amplification, which is a hallmark of cancer cells. We developed a cell culture model to systematically measure the individual and combined effects of Chlamydia and HPV on the centrosome in the same host cell. We found that C. trachomatis caused centrosome amplification in a greater proportion of cells than HPV and that the effects of the two pathogens on the centrosome were additive. Furthermore, centrosome amplification induced by Chlamydia, but not by HPV, strongly correlated with multinucleation and required progression through mitosis. Our results suggest that C. trachomatis and HPV induce centrosome amplification through different mechanisms, with the chlamydial effect being largely due to a failure in cytokinesis that also results in multinucleation. Our findings provide support for C. trachomatis as a co-factor for HPV in carcinogenesis and offer mechanistic insights into how two infectious agents may cooperate to promote cancer. TAKE AWAYS: • Chlamydia and HPV induce centrosome amplification in an additive manner. • Chlamydia-induced centrosome amplification is linked to host cell multinucleation. • Chlamydia-induced centrosome amplification requires cell cycle progression. • Chlamydia and HPV cause centrosome amplification through different mechanisms. • This study supports Chlamydia as a co-factor for HPV in carcinogenesis.

Skeletal muscle differentiation occurs as muscle precursor cells (myoblasts) elongate and fuse to form multinucleated syncytial myotubes in which the highly-organized actomyosin sarcomeres of muscle fibers assemble. Although less well characterized, the microtubule cytoskeleton also undergoes dramatic rearrangement during myogenesis. The centrosome-nucleated microtubule array found in myoblasts is lost as the nuclear membrane acquires microtubule nucleating activity and microtubules emerge from multiple sites in the cell, eventually rearranging into a grid-like pattern in myotubes. In order to characterize perinuclear microtubule organization using a biochemically tractable system, we isolated nuclei from mouse C2C12 skeletal muscle cells during the course of differentiation and incubated them in cytoplasmic extracts prepared from eggs of the frog Xenopus laevis. Whereas centrosomes associated with myoblast nuclei gave rise to radial microtubule arrays in extracts, myotube nuclei produced a sun-like pattern with microtubules transiently nucleating from the entire nuclear envelope. Perinuclear microtubule growth was suppressed by inhibition of Aurora A kinase or by degradation of RNA, treatments that also inhibited microtubule growth from sperm centrosomes. Myotube nuclei displayed microtubule motor-based movements leading to their separation, as occurs in myotubes. This in vitro assay therefore recapitulates key features of microtubule organization and nuclear movement observed during muscle cell differentiation.