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Conjugation with the small ubiquitin-like modifier (SUMO) modulates protein interactions and localisation. The kinase Aurora B, a key regulator of mitosis, was previously identified as a SUMOylation target in vitro and in assays with overexpressed components. However, where and when this modification genuinely occurs in human cells was not ascertained. Here, we have developed intramolecular Proximity Ligation Assays (PLA) to visualise SUMO-conjugated Aurora B in human cells in situ. We visualised Aurora B-SUMO products at centromeres in prometaphase and metaphase, which declined from anaphase onwards and became virtually undetectable at cytokinesis. In the mitotic window in which Aurora B/SUMO products are abundant, Aurora B co-localised and interacted with NUP358/RANBP2, a nucleoporin with SUMO ligase and SUMO-stabilising activity. Indeed, in addition to the requirement for the previously identified PIAS3 SUMO ligase, we found that NUP358/RANBP2 is also implicated in Aurora B-SUMO PLA product formation and centromere localisation. In summary, SUMOylation marks a distinctive window of Aurora B functions at centromeres in prometaphase and metaphase while being dispensable for functions exerted in cytokinesis, and RANBP2 contributes to this control, adding a novel layer to modulation of Aurora B functions during mitosis.

Nowadays, morphology and molecular analyses at the single-cell level have a fundamental role in understanding biology better. These methods are utilized for cell phenotyping and in-depth studies of cellular processes, such as mitosis. Fluorescence microscopy and optical spectroscopy techniques, including Raman micro-spectroscopy, allow researchers to examine biological samples at the single-cell level in a non-destructive manner. Fluorescence microscopy can give detailed morphological information about the localization of stained molecules, while Raman microscopy can produce label-free images at the subcellular level; thus, it can reveal the spatial distribution of molecular fingerprints, even in live samples. Accordingly, the combination of correlative fluorescence and Raman microscopy (CFRM) offers a unique approach for studying cellular stages at the single-cell level. However, subcellular spectral maps are complex and challenging to interpret. Artificial intelligence (AI) may serve as a valuable solution to characterize the molecular backgrounds of phenotypes and biological processes by finding the characteristic patterns in spectral maps. The major contributions of the manuscript are: (I) it gives a comprehensive review of the literature focusing on AI techniques in Raman-based cellular phenotyping; (II) via the presentation of a case study, a new neural network-based approach is described, and the opportunities and limitations of AI, specifically deep learning, are discussed regarding the analysis of Raman spectroscopy data to classify mitotic cellular stages based on their spectral maps.

Photoswitchable reagents are powerful tools for high-precision studies in cell biology. When these reagents are globally administered yet locally photoactivated in two-dimensional (2D) cell cultures, they can exert micron- and millisecond-scale biological control. This gives them great potential for use in biologically more relevant three-dimensional (3D) models and in vivo, particularly for studying systems with inherent spatiotemporal complexity, such as the cytoskeleton. However, due to a combination of photoswitch isomerization under typical imaging conditions, metabolic liabilities, and insufficient water solubility at effective concentrations, the in vivo potential of photoswitchable reagents addressing cytosolic protein targets remains largely unrealized. Here, we optimized the potency and solubility of metabolically stable, druglike colchicinoid microtubule inhibitors based on the styrylbenzothiazole (SBT) scaffold that are nonresponsive to typical fluorescent protein imaging wavelengths and so enable multichannel imaging studies. We applied these reagents both to 3D organoids and tissue explants and to classic model organisms (zebrafish, clawed frog) in one- and two-protein imaging experiments, in which spatiotemporally localized illuminations allowed them to photocontrol microtubule dynamics, network architecture, and microtubule-dependent processes in vivo with cellular precision and second-level resolution. These nanomolar, in vivo capable photoswitchable reagents should open up new dimensions for high-precision cytoskeleton research in cargo transport, cell motility, cell division, and development. More broadly, their design can also inspire similarly capable optical reagents for a range of cytosolic protein targets, thus bringing in vivo photopharmacology one step closer to general realization.

Lysosomes, as primary degradative organelles, are the endpoint of different converging pathways, including macroautophagy. To date, lysosome degradative function has been mainly studied in interphase cells, while their role during mitosis remains controversial. Mitosis dictates the faithful transmission of genetic material among generations, and perturbations of mitotic division lead to chromosomal instability, a hallmark of cancer. Heretofore, correct mitotic progression relies on the orchestrated degradation of mitotic factors, which was mainly attributed to ubiquitin-triggered proteasome-dependent degradation. Here, we show that mitotic transition also relies on lysosome-dependent degradation, as impairment of lysosomes increases mitotic timing and leads to mitotic errors, thus promoting chromosomal instability. Furthermore, we identified several putative lysosomal targets in mitotic cells. Among them, WAPL, a cohesin regulatory protein, emerged as a novel SQSTM1-interacting protein for targeted lysosomal degradation. Finally, we characterized an atypical nuclear phenotype, the toroidal nucleus, as a novel biomarker for genotoxic screenings. Our results establish lysosome-dependent degradation as an essential event to prevent chromosomal instability. Abbreviations: 3D: three-dimensional; APC/C: anaphase-promoting complex; ARL8B: ADP ribosylation factor like GTPase 8B; ATG: autophagy-related; BORC: BLOC-one-related complex; CDK: cyclin-dependent kinase; CENPE: centromere protein E; CIN: chromosomal instability; ConcA: concanamycin A; CQ: chloroquine; DAPI: 4,6-diamidino-2-penylinole; FTI: farnesyltransferase inhibitors; GFP: green fluorescent protein; H2B: histone 2B; KIF: kinesin family member; LAMP2: lysosomal associated membrane protein 2; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MEF: mouse embryonic fibroblast; MTOR: mechanistic target of rapamycin kinase; PDS5B: PDS5 cohesin associated factor B; SAC: spindle assembly checkpoint; PLEKHM2: pleckstrin homology and RUN domain containing M2; SQSTM1: sequestosome 1; TEM: transmission electron microscopy; ULK1: unc-51 like autophagy activating kinase 1; UPS: ubiquitin-proteasome system; v-ATPase: vacuolar-type H+-translocating ATPase; WAPL: WAPL cohesion release factor.

Cell division cycle 20 homologue (CDC20) is a well-known regulator of cell cycle, as it controls the correct segregation of chromosomes during mitosis. Many studies have focused on the biological role of CDC20 in cancer development, as alterations of its functionality have been linked to genomic instability and evidence demonstrated that high CDC20 expression levels are associated with poor overall survival in solid cancers. More recently, novel CDC20 functions have been demonstrated or suggested, including the regulation of apoptosis and stemness properties and a correlation with immune cell infiltration. Here, we here summarize and discuss the role of CDC20 inside and outside mitosis, starting from its network of interacting proteins. In the last years, CDC20 has also attracted more interest in the blood cancer field, being overexpressed and showing an association with prognosis both in myeloid and lymphoid malignancies. Preclinical findings showed that selective CDC20 and APC/CCDC20/APC/CCDH1 inhibitors, namely Apcin and proTAME, are effective against lymphoma and multiple myeloma cells, resulting in mitotic arrest and apoptosis and synergizing with clinically-relevant drugs. The evidence and hypothesis presented in this review provide the input for further biological and chemical studies aiming to dissect novel potential CDC20 roles and targeting strategies in hematological malignancies.

Summary: During cell division, dramatic microtubular rearrangements driven by cyclin B-cdk1 (Cdk1) kinase activity mark the onset of mitosis leading to dismantling of the interphase microtubular cytoskeleton and assembly of the mitotic spindle. During interphase, Cdk1 accumulates in an inactive state, phosphorylated at inhibitory sites by Wee1/Myt1 kinases. At mitosis onset, Cdc25 phosphatase dephosphorylates and activates Cdk1. Once activated, Cdk1 clears cytoplasmic microtubules by inhibiting microtubule-stabilizing and growth-promoting microtubule-associated proteins (MAPs). Nevertheless, some of these MAPs are required for spindle microtubule growth and spindle assembly, creating quite a conundrum. We show here that a Cdk1 fraction bound to spindle structures escapes Cdc25 action and remains inhibited by phosphorylation (i-Cdk1) in mitotic human cells. Loss or restoration of i-Cdk1 inhibits or promotes spindle assembly, respectively. Furthermore, polymerizing spindle microtubules foster i-Cdk1 aggregating with Wee1 and excluding Cdc25. Our data reveal that spindle assembly relies on compartmentalized control of Cdk1 activity.

See Uzquiano and Francis (doi:10.1093/brain/awz048) for a scientific commentary on this article. Mutations in RTTN, which encodes Rotatin, give rise to various brain malformations. Vandervore et al. reveal mitotic failure, aneuploidy, apoptosis and defective ciliogenesis in patient cells. Rotatin binds to myosin subunits in the leading edge of human neurons, which may explain the proliferation and migration defects observed., Recessive mutations in RTTN, encoding the protein rotatin, were originally identified as cause of polymicrogyria, a cortical malformation. With time, a wide variety of other brain malformations has been ascribed to RTTN mutations, including primary microcephaly. Rotatin is a centrosomal protein possibly involved in centriolar elongation and ciliogenesis. However, the function of rotatin in brain development is largely unknown and the molecular disease mechanism underlying cortical malformations has not yet been elucidated. We performed both clinical and cell biological studies, aimed at clarifying rotatin function and pathogenesis. Review of the 23 published and five unpublished clinical cases and genomic mutations, including the effect of novel deep intronic pathogenic mutations on RTTN transcripts, allowed us to extrapolate the core phenotype, consisting of intellectual disability, short stature, microcephaly, lissencephaly, periventricular heterotopia, polymicrogyria and other malformations. We show that the severity of the phenotype is related to residual function of the protein, not only the level of mRNA expression. Skin fibroblasts from eight affected individuals were studied by high resolution immunomicroscopy and flow cytometry, in parallel with in vitro expression of RTTN in HEK293T cells. We demonstrate that rotatin regulates different phases of the cell cycle and is mislocalized in affected individuals. Mutant cells showed consistent and severe mitotic failure with centrosome amplification and multipolar spindle formation, leading to aneuploidy and apoptosis, which could relate to depletion of neuronal progenitors often observed in microcephaly. We confirmed the role of rotatin in functional and structural maintenance of primary cilia and determined that the protein localized not only to the basal body, but also to the axoneme, proving the functional interconnectivity between ciliogenesis and cell cycle progression. Proteomics analysis of both native and exogenous rotatin uncovered that rotatin interacts with the neuronal (non-muscle) myosin heavy chain subunits, motors of nucleokinesis during neuronal migration, and in human induced pluripotent stem cell-derived bipolar mature neurons rotatin localizes at the centrosome in the leading edge. This illustrates the role of rotatin in neuronal migration. These different functions of rotatin explain why RTTN mutations can lead to heterogeneous cerebral malformations, both related to proliferation and migration defects.

Neurogenesis impairment is a key determinant of intellectual disability in Down syndrome (DS), a genetic pathology due to triplication of chromosome 21. Since neurogenesis ceases after birth, apart in the hippocampus and olfactory bulb, the only means to tackle the problem of neurogenesis impairment in DS at its root is to intervene during gestation. A few studies in DS mouse models show that this is possible, although the drugs used may raise caveats in terms of safety. We previously found that neonatal treatment with 7,8-dihydroxyflavone (7,8-DHF), a flavonoid present in plants, restores hippocampal neurogenesis in the Ts65Dn model of DS. The goal of the current study was to establish whether prenatal treatment with 7,8-DHF improves/restores overall brain proliferation potency. Pregnant Ts65Dn females received 7,8-DHF from embryonic day 10 until delivery. On postnatal day 2 (P2) the pups were injected with BrdU and were killed after either 2 h or 52–60 days (P52–60). Evaluation of the number of proliferating (BrdU+) cells in various forebrain neurogenic niches of P2 mice showed that in treated Ts65Dn mice proliferation potency was improved or even restored in most of the examined regions, including the hippocampus. Quantification of the surviving BrdU+ cells in the dentate gyrus of P52–60 mice showed no difference between treated and untreated Ts65Dn mice. At P52–60, however, treated Ts65Dn mice exhibited a larger number of granule cells in comparison with their untreated counterparts, although their number did not reach that of euploid mice. Results show that 7,8-DHF has a widespread impact on prenatal proliferation potency in Ts65Dn mice and exerts mild long-term effects. It remains to be established whether treatment extending into the neonatal period can lead to an improvement in brain development that is retained in adulthood.

[eng] This work is focused on Barrier-to-Autointegration Factor (BAF) protein. BAF is a nuclear envelope (NE) component that binds chromatin and is required for NE reassembly (NER) at mitosis exit. Previous work in our group showed that, in Drosophila, a small fraction of BAF (cenBAF) associates with the centromere. BAF function is regulated by cycles of phosphorylation and dephosphorylation. At the entry of mitosis, BAF is phosphorylated by VRK1/NHK1 kinase and is released from chromatin and the NE. At mitosis exit, protein phosphatase 2A (PP2A) dephosphorylates BAF, which resumes binding to chromatin and promotes NER. Here, we find that cenBAF remains bound to the centromere during mitosis by the action of protein phosphatase 4 (PP4), which is recruited to the centromere by the constitutive centromere component CenpC. At the same time, BAF stabilizes CenpC and PP4 at the centromere forming a functional centromeric network essential for faithful chromosome segregation. Disrupting centromeric localization of PP4 destabilizes cenBAF at centromeres and induces ectopic PP2A-mediated dephosphorylation of free phosphoBAF (pBAF) in mitosis, which results in the accumulation of BAF in a perichromosomal layer surrounding the chromosomes. Concomitantly, NE disassembly/reassembly during mitosis is altered resulting in micronuclei formation and cells with altered NE morphology. This suggests that CenpC, PP4 and cenBAF form a centromeric network that signals pBAF dephosphorylation at mitosis exit by regulating PP2A activity. We also identify T4 and S5 as the main BAF phosphosites in Drosophila and study the actual contribution of PP4 and PP2A to pBAF dephosphorylation., [cat] El present treball se centra en l'estudi de la proteïna Barrier-to-Autointegration Factor (BAF). BAF és una proteïna de la membrana nuclear (MN) que s'uneix a la cromatina i té un paper essencial en el reassemblatge de la MN al final de la mitosi. BAF ha estat descrit per primer cop en el nostre grup com una nova proteïna centromèrica (cenBAF) a Drosophila. La localització de BAF durant el cicle cel·lular està regulada per cicles de fosforilació i defosforilació. A l'entrada de mitosi BAF és fosforilat per la kinasa VRK1/NHK1 perdent la seva afinitat per unit cromatina i la MN. Aquesta situació es manté fins que PP2A desfosforila BAF al final de la mitosi, retornant al seu estat inicial. En el present treball es mostra que BAF es manté al centrómer durant la mitosi degut a l'acció de PP4 que és reclutada al centrómer per CenpC. Al seu torn cenBAF estabilitza la localització de CenpC i PP4 al centrómer formant un entramat centromèric responsable de garantir la correcta segregació dels cromosomes. L'alteració d'aquest entramat centromèric desemboca en la desregulació de l'activitat de PP2A sobre phosphoBAF (pBAF) soluble, observant-se com a resultat una acumulació de BAF desfosforilat envoltant els cromosomes a metafase i com a conseqüència, provocant la formació de micronuclis i alteracions en la morfologia nuclear. També hem indentificat T4 i S5 com els principals residus implicats en la fosforegulació de BAF a Drosophila i hem estudiat la contribució de PP4 i PP2A sobre la desfosforilació de BAF.

Epithelioid sarcoma (ES) is a rare disease representing &lt, 1% of soft tissue sarcomas. Current therapies are based on anthracycline alone or in combination with ifosfamide or other cytotoxic drugs. ES is still characterized by a poor prognosis with high rates of recurrence. Indeed, for years, ES survival rates have remained stagnant, suggesting that conventional treatments should be revised and improved. New therapeutic approaches are focused to target the key regulators of signaling pathways, the causative markers of tumor pathophysiology. To this end, we selected, among the drugs to which an ES cell line is highly sensitive, those that target signaling pathways known to be dysregulated in ES. In particular, we found a key role for GSK-3β, which results in up-regulation in tumor versus normal tissue samples and associated to poor prognosis in sarcoma patients. Following this evidence, we evaluated CHIR99021, a GSK-3 inhibitor, as a potential drug for use in ES therapy. Our data highlight that, in ES cells, CHIR99021 induces cell cycle arrest, mitotic catastrophe (MC) and autophagic response, resulting in reduced cell proliferation. Our results support the potential efficacy of CHIR99021 in ES treatment and encourage further preclinical and clinical studies.

Although different anaphase proteins bind with characteristically different strength to the central spindle, the overall central spindle dynamicity slows down as mitosis proceeds., During mitosis, the spindle undergoes morphological and dynamic changes. It reorganizes at the onset of the anaphase when the antiparallel bundler PRC1 accumulates and recruits central spindle proteins to the midzone. Little is known about how the dynamic properties of the central spindle change during its morphological changes in human cells. Using gene editing, we generated human cells that express from their endogenous locus fluorescent PRC1 and EB1 to quantify their native spindle distribution and binding/unbinding turnover. EB1 plus end tracking revealed a general slowdown of microtubule growth, whereas PRC1, similar to its yeast orthologue Ase1, binds increasingly strongly to compacting antiparallel microtubule overlaps. KIF4A and CLASP1 bind more dynamically to the central spindle, but also show slowing down turnover. These results show that the central spindle gradually becomes more stable during mitosis, in agreement with a recent “bundling, sliding, and compaction” model of antiparallel midzone bundle formation in the central spindle during late mitosis.

Although centrosome abnormalities are frequent in cancer, the mechanisms responsible for their accumulation are poorly understood. Here we comment on our recent publication identifying a new type of selective autophagy, named doryphagy, which preserves centrosome organization through targeting Centriolar Satellites (CS). Thus, doryphagy prevents inaccurate mitosis and genomic instability.

10 páginas, 5 figuras, Adaptation to environmental changes is crucial for cell fitness. In Saccharomyces cerevisiae, variations in external osmolarity trigger the activation of the stress-activated protein kinase Hog1 (high-osmolarity glycerol 1), which regulates gene expression, metabolism, and cell-cycle progression. The activation of this kinase leads to the regulation of G1, S, and G2 phases of the cell cycle to prevent genome instability and promote cell survival. Here we show that Hog1 delays mitotic exit when cells are stressed during metaphase. Hog1 phosphorylates the nucleolar protein Net1, altering its affinity for the phosphatase Cdc14, whose activity is essential for mitotic exit and completion of the cell cycle. The untimely release of Cdc14 from the nucleolus upon activation of Hog1 is linked to a defect in ribosomal DNA (rDNA) and telomere segregation, and it ultimately delays cell division. A mutant of Net1 that cannot be phosphorylated by Hog1 displays reduced viability upon osmostress. Thus, Hog1 contributes to maximizing cell survival upon stress by regulating mitotic exit., S.T. was the recipient of a contract for Investigador Doctor Junior (PDJ 2014, Generalitat de Catalunya) and a Juan de la Cierva fellowship. The study was supported by grants from the Spanish Ministry of Economy and Competitiveness (PGC2018-094136-B-I00 to F.P.; BFU2017-85152-P and FEDER to E.d.N.; BFU2016-77975-R and FEDER to E.Q.), the Catalan Government (2017 SGR 799), and Fundación Botín, by Banco Santander through its Santander Universities Global Division (to F.P.). We gratefully acknowledge institutional funding from the Spanish Ministry of Economy, Industry and Competitiveness (MINECO) through the Centres of Excellence Severo Ochoa Award, and from the CERCA Programme of the Catalan Government and the Unidad de Excelencia María de Maeztu, funded by the AEI (CEX2018-000792-M). F.P. is the recipient of an ICREA Acadèmia Award (Generalitat de Catalunya).

El CaP és la segona neoplàsia maligna invasiva diagnosticada amb major freqüència i la taxa de supervivència a 5 anys dels homes amb malaltia metastàtica cau per sota del 30%. Els andrògens, a través del receptor d'andrògens, són crucials per a l'inici i la progressió del CaP i, per tant, la ADT ha estat el principal tractament del CaP localment avançat, metastàtic i recurrent. Les teràpies de deprivació androgènica són capaces d'aconseguir inicialment una resposta bioquímica en la majoria dels pacients; no obstant això, les remissions són temporals i la malaltia acaba progressant a un estat independent d'andrògens, també denominat CRPC. Després de la progressió a CRPC, la supervivència mitjana d'aquests pacients és de menys de 2 anys i la malaltia acaba sent pràcticament intractable. Els mecanismes moleculars que causen aquesta transició continuen sent en gran part desconeguts. La creixent evidència en els últims anys suggereix que les cèl·lules de CaP insensibles als andrògens han estat reprogramades genèticament per a regular de manera selectiva l'expressió de gens de la fase M del cicle cel·lular. Els taxans, dirigits als microtúbuls, ja s'estan utilitzant en la pràctica clínica per a pacients amb CaP avançat, però la supervivència continua sent modesta i els pacients acaben desenvolupant resistència a la teràpia. Pel fet que la progressió mitòtica és un procés altament regulat, la nostra hipòtesi és que les proteïnes de fase M expressades de manera aberrant poden conferir a les cèl·lules CaP un avantatge per al creixement en condicions de depleció d'andrògens i, en conseqüència, representen possibles dianes terapèutiques per a la intervenció molecular de pacients amb CRPC. Encara que diversos inhibidors del cicle cel·lular no han aconseguit demostrar benefici en l'entorn clínic del CRPC, continua havent-hi un gran interès en aquest enfocament i encara hi ha desafiaments importants per a intentar tractar a aquests pacients amb teràpies dirigides que siguin eficaces. En aquest context, l'objectiu principal d'aquesta tesi és obtenir nous coneixements moleculars sobre la progressió del CaP, amb especial èmfasi en la participació dels reguladors mitòtics en l'adquisició de la independència a andrògens dels tumors de pròstata. El CaP es la segunda neoplasia maligna invasiva diagnosticada con mayor frecuencia y la tasa de supervivencia a 5 años de los hombres con enfermedad metastásica cae por debajo del 30%. Los andrógenos, a través del receptor de andrógenos, son cruciales para el inicio y la progresión del CaP y, por tanto, la ADT ha sido el principal tratamiento del CaP localmente avanzado, metastásico y recurrente. Las terapias de deprivación androgénica son capaces de conseguir inicialmente una respuesta bioquímica en la mayoría de los pacientes; sin embargo, las remisiones son temporales y la enfermedad acaba progresando a un estado independiente de andrógenos, también denominado CRPC. Tras la progresión a CRPC, la supervivencia media de estos pacientes es de menos de 2 años y la enfermedad acaba siendo prácticamente intratable. Los mecanismos moleculares que causan esta transición siguen siendo en gran parte desconocidos. La creciente evidencia en los últimos años sugiere que las células de CaP insensibles a andrógenos han sido reprogramadas genéticamente para regular de forma selectiva la expresión de genes de la fase M del ciclo celular. Los taxanos, dirigidos a los microtúbulos, ya se están utilizando en la práctica clínica para pacientes con CaP avanzado, pero la supervivencia sigue siendo modesta y los pacientes acaban desarrollando resistencia a la terapia. Debido a que la progresión mitótica es un proceso altamente regulado, nuestra hipótesis es que las proteínas de fase M expresadas de manera aberrante pueden conferir a las células CaP una ventaja para el crecimiento en condiciones de depleción de andrógenos y, en consecuencia, representan posibles dianas terapéuticas para la intervención molecular de pacientes con CRPC. Aunque varios inhibidores del ciclo celular no han logrado demostrar beneficio en el entorno clínico del CRPC, sigue habiendo un gran interés en este enfoque y todavía hay desafíos importantes para intentar tratar a estos pacientes con terapias dirigidas que sean eficaces. En este contexto, el objetivo principal de esta tesis es obtener nuevos conocimientos moleculares sobre la progresión del CaP, con especial énfasis en la participación de los reguladores mitóticos en la adquisición de la independencia a andrógenos de los tumores de próstata. PCa is the second most frequently diagnosed invasive malignancy and the 5-year survival rate of men with metastatic disease drops below 30%. Androgens, through the AR, are crucial for the initiation and progression of PCa and thus, ADT has been the mainstay of treatment for locally advanced, metastatic and recurring PCa. Androgen-ablation therapies can initially achieve a biochemical response in the majority of patients; however, remissions are temporary and the disease invariably progresses to an androgen-independent state, also termed CRPC. Upon progression to CRPC, the median survival for those patients is less than 2 years, and the disease is essentially untreatable. The molecular mechanisms that cause this transition remain largely unknown. Increasing evidence in recent years suggest that androgen insensitive PCa cells have undergone a genetic reprogramming to selectively upregulate the expression of M-phase cell cycle genes. Microtubule-targeting taxanes are already being used in the clinical practice for patients with advanced PCa, but survival remains modest and resistance inevitably develops. Because mitotic progression is a highly regulated process, we hypothesized that aberrantly expressed M-phase proteins may confer PCa cells an advantage to growth in androgen-depleted conditions and consequently represent potential therapeutic targets for the molecular intervention of CRPC patients. Although several small molecule inhibitors of the cell cycle have failed to demonstrate benefit in the clinical setting of CRPC, there remains a keen interest in this approach and significant challenges persist to match patients with effective targeted therapies. In this context, the main goal of this thesis is to gain novel molecular insights into the progression of PCa, with special emphasis on the involvement of mitotic regulators in the acquisition of prostate tumors androgen independence. Universitat Autònoma de Barcelona. Programa de Doctorat en Bioquímica, Biologia Molecular i Biomedicina

Before anaphase onset, budding yeast cells must align the mitotic spindle parallel to the mother-bud axis to ensure proper chromosome segregation. The protein kinase Snf1/AMPK is a highly conserved energy sensor, essential for adaptation to glucose limitation and in response to cellular stresses. However, recent findings indicate that it plays important functions also in non-limiting glucose conditions. Here we report a novel role of Snf1/AMPK in the progression through mitosis in glucose-repressing condition. We show that active Snf1 is localized to the bud neck from bud emergence to cytokinesis in a septin-dependent manner. In addition, loss of Snf1 induces a delay of the metaphase to anaphase transition that is due to a defect in the correct alignment of the mitotic spindle. In particular, genetic data indicate that Snf1 promotes spindle orientation acting in parallel with Dyn1 and in concert with Kar9. Altogether this study describes a new role for Snf1 in mitosis and connects cellular metabolism to mitosis progression.

SummaryHuman centromeres are defined by chromatin containing the histone H3 variant CENP-A assembled onto repetitive alphoid DNA sequences. By inducing rapid, complete degradation of endogenous CENP-A, we now demonstrate that once the first steps of centromere assembly have been completed in G1/S, continued CENP-A binding is not required for maintaining kinetochore attachment to centromeres or for centromere function in the next mitosis. Degradation of CENP-A prior to kinetochore assembly is found to block deposition of CENP-C and CENP-N, but not CENP-T, thereby producing defective kinetochores and failure of chromosome segregation. Without the continuing presence of CENP-A, CENP-B binding to alphoid DNA sequences becomes essential to preserve anchoring of CENP-C and the kinetochore to each centromere. Thus, there is a reciprocal interdependency of CENP-A chromatin and the underlying repetitive centromere DNA sequences bound by CENP-B in the maintenance of human chromosome segregation.

The faithful inheritance of chromosomes is essential for the propagation of organisms. In eukaryotes, central to this process is the mitotic spindle. Recently, we have identified TRIM8 as a gene aberrantly expressed in gliomas whose expression reduces the clonogenic potential in the patients' glioma cells. TRIM8 encodes an E3 ubiquitin ligase involved in various pathological processes, including hypertrophy, antiviral defense, encephalopathy, and cancer development. To gain insights into the TRIM8 functions, we characterized the TRIM8 interactome in primary mouse embryonic neural stem cells using proteomics. We found that TRIM8 interacts with KIFC1, and KIF11/Eg5, two master regulators of mitotic spindle assembly and cytoskeleton reorganization. By exploring the TRIM8 role in the mitotic spindle machinery, we showed that TRIM8 localizes at the mitotic spindle during mitosis and plays a role in centrosome separation at the beginning of mitosis with a subsequent delay of the mitotic progression and impact on chromosomal stability.

13 páginas, 3 figuras, Protein phosphorylation is a common mechanism for the regulation of cell cycle progression. The opposing functions of cell cycle kinases and phosphatases are crucial for accurate chromosome segregation and exit from mitosis. Protein phosphatases 2A are heterotrimeric complexes that play essential roles in cell growth, proliferation, and regulation of the cell cycle. Here, we review the function of the protein phosphatase 2A family as the counteracting force for the mitotic kinases. We focus on recent findings in the regulation of mitotic exit and cytokinesis by PP2A phosphatases in S. cerevisiae and other fungal species., We thank the CERCA Program/Generalitat de Catalunya for institutional support. Our laboratory is funded by the Spanish Ministry of Economy, Industry, and Competitiveness (MINECO), which is part of the State Agency, through projects BFU2013-43132-P and BFU2016-77975-R (co-funded by the European Regional Development Fund, ERDF, a way to build Europe).

Saccharomyces cerevisiae has been widely used as a model system for the study of basic biological processes which are usually evolutionarily conserved from yeasts to multicellular eukaryotes. These studies are very important because they shed light on mechanisms that are altered in human diseases and help the development of new biomarkers and therapies. The mitotic spindle is a conserved apparatus that governs chromosome segregation during mitosis. Given its crucial role for genome stability and, therefore, for cell viability, its structure and function are strictly regulated. Recent findings reveal new levels of regulation in mitotic spindle dynamics and link spindle pole diversification with cell fate determination, health, disease and aging.

[eng] Lysosomes are the primary degradative organelles in mammalian cells. Indeed, lysosome enzymatic cocktail allows the degradation of a vast repertoire of cellular material through different converging pathways including macroautophagy. Lysosome function relies on both its acidification capacity, mediated by the vacuolar ATPase, and its cytosolic positioning, driven by the motor proteins kinesins and dyneins. Emerging evidences point out a relevant role of lysosomes in cancer progression denoting their important function in cellular homeostasis maintenance. Indeed, lysosomotropic drugs, such as chloroquine-derivatives, are already being used in clinical trials for cancer treatment. This PhD thesis aims to study lysosome functions in cancer cells to identify novel vulnerabilities related to this organelle. Two different questions are addressed herein: 1) The implication of lysosomes on glucose-mediated E2F1-driven mTORC1 activation and 2) The role of lysosomes in cell division. E2F1 is overexpressed in numerous human cancers, including lung, breast and hepatocellular carcinomas. Traditionally, the major role reported for E2F1 in cancer is the activation of cell cycle. Previously, our group reported that E2F1 regulates cell growth, through the activation of mTORC1, a major regulator of protein synthesis and autophagy and demonstrated that E2F1 induces the anterograde movement of lysosomes, which is associated with translocation of mTOR to lysosomes and v-ATPase activation. Here we demonstrate that E2F1-dependent mTORC1 activation relies on glucose availability. E2F1 transcriptionally regulates several glycolytic enzymes, thus increasing glycolytic flux. More specifically, we described that E2F1 up-regulates the PFKFB3 isoenzyme of the PFK-2, a potent activator of glycolysis, and that PFKFB3 activity determines mTORC1 activation. We hypothesize that E2F1 is able to activate glycolysis and therefore increase v-ATPase and mTORC1 activity. Besides, E2F1 induces lysosome-dependent exocytosis which correlates with a metastatic phenotype. These novel functions of E2F1 in v-ATPase regulation and lysosomal trafficking provide insight into regulatory mechanisms by which E2F1 drives malignancy and highlight the potential role of lysosomes as a metabolic hub in mammalian cells. Studies on lysosome function are mainly focused in cells in interphase. However, the implication of these organelles during cell division remains unclear. Mitosis is a key event during cell cycle, in which cells finally divide into two daughter cells. Mitotic progression comprises five active phases involving a dramatic rearrangement of cellular components in a short period of time. Until now, degradation of mitotic factors has been attributed only to ubiquitination and proteasome-dependent degradation. In the present study, we show that impairment of lysosomal trafficking and function delays mitotic progression and increases mitotic errors, phenomena accompanied by an increase in toroidal-shaped nuclei a reflection of impaired mitosis. Finally, we use a proteomic approach to discover novel lysosome protein substrates involved in mitotic progression. Interestingly, we identified regulatory proteins of the cohesin complex necessary for correct chromosomal segregation. By characterizing a novel function of lysosomes specifically in mitosis, our work establishes a novel model of regulation of cell division beyond the proteasome. In summary, this work provides new insights into the function of lysosomes down-stream of E2F1 oncogenic signaling, which modulates G1/S transition and cell proliferation but also into lysosomal function in the completion of cell cycle by regulating mitotic progression. Our work highlights the importance of targeting lysosomes for novel cancer therapies., [cat] Els lisosomes són els principals orgànuls degradatius de la cèl·lula eucariota, capaços de degradar un ampli repertori de material cel·lular. La funció lisosomal depèn tant de la seva acidificació, sota el control de la v-ATPasa, com de la seva distribució citosòlica, regulada per kinesines i dineïnes. La funció lisosomal és essencial per preservar la homeòstasi cel·lular i la seva alteració està lligada al desenvolupament de malalties de diferent índole, des de trastorns neurodegeneratius fins al càncer. De fet, fàrmacs lisosomotròpics, tals com els derivats de la cloroquina, s’estudien actualment en assajos clínics com a tractament per un ampli ventall de tumors. L’objectiu d’aquesta tesi doctoral és l’estudi de les funcions lisosomals en cèl·lules canceroses per tal de definir noves vulnerabilitats relacionades a aquest orgànul. S’adrecen així doncs, dues preguntes experimentals: 1) La implicació dels lisosomes en l’activació de mTORC1 degut a E2F1 a través de la glucosa i 2) El rol dels lisosomes en la divisió cel·lular. La funció oncogènica d’E2F1 s’ha atribuït tradicionalment al seu paper com a regulador del cicle cel·lular. Actualment, però, es coneix la seva implicació en diverses funcions relacionades amb la progressió tumoral, entre elles, el creixement cel·lular a través de l’activació de mTORC1. En aquest projecte, hem demostrat que l’activació d’mTORC1 és amplificada en presència de glucosa. E2F1 activa la transcripció d’enzims glucolítics, específicament l’isoenzim de la PFK-2 PFKFB3 i d’aquesta manera incrementa el flux glucolític. Hem descrit que l’activitat de PFKFB3 està directament relacionada amb l’activació d’mTORC1 suggerint la regulació del complex pel flux glucolític. En paral·lel, a través de la regulació del tràfic lisosomal, E2F1 incrementa la exocitosi lisosomal àmpliament correlacionada amb el fenotip metastàtic. Les funcions descrites aquí destaquen la importància del lisosoma en el mecanisme oncogènic d’E2F1 i recolzen la idea dels lisosomes com a plataformes d’integració del metabolisme cel·lular. L’estudi de la funció lisosomal s’ha limitat majoritàriament a cèl·lules en interfase i pocs estudis es centren en la seva implicació en la divisió cel·lular (mitosi). La mitosi és un procés crucial en el cicle cel·lular en què la cèl·lula finalment es divideix en dues cèl·lules filles genèticament idèntiques. La progressió mitòtica requereix un ampli canvi morfològic, estrictament coordinat en un període de temps extremadament curt. En aquest context, la degradació de factors mitòtics és un element limitant per garantir la correcta progressió mitòtica. Fins ara, la degradació de factors mitòtics s’ha atribuït exclusivament al proteasoma deixant la funció lisosomal fora de l’escenari. En aquest estudi, demostrem que la inhibició de la funció lisosomal tant a nivell de tràfic com d’acidificació retarda la progressió mitòtica i incrementa la freqüència d’errors mitòtics, fenòmens acompanyats de l’aparició del nucli toroïdal el qual reflexa una mitosi aberrant. Finalment, hem usat una aproximació proteòmica per descobrir noves dianes lisosomals implicades en la mitosi. Entre aquestes, hem identificat proteïnes reguladores del complex de coesines, essencial pel correcte desenvolupament de la segregació cromosòmica. La caracterització de la funció lisosomal específicament en mitosi desenvolupada en aquest estudi implica un nou model de regulació de la divisió cel·lular més enllà de la degradació proteasomal. Aquest estudi dóna llum a la funció lisosomal sota el senyal oncogènic d’E2F1, un regulador clau de la transició G1/S del cicle cel·lular així com demostra la implicació dels lisosomes per la culminació del cicle a través de la regulació de la mitosi. En conclusió, el nostre treball recolza la importància del lisosoma com a possible diana terapèutica en el càncer.

Introduction and Objectives: Nail apparatus melanoma (NAM) is an uncommon tumor, especially in Caucasians. The prognosis of patients affected by NAM was analyzed and correlated with the histopathological criteria and the surgical management of the tumors. Materials and Methods: We collected data regarding NAM referred to the Skin Cancer Unit of the Dermatology Department of the University of Bologna, from 1992 to January 2012. Results: Out of 1,327 melanoma cases diagnosed between 1992 and 2012, 42 patients were affected by NAM (2.93%). All the patients were Caucasian. Two deceased patients with insufficient medical records and 1 woman with a personal history of breast cancer were excluded. Thirty-nine cases entered this study: 24 were women (67%) and 15 men (33%). The mean age at diagnosis of NAM was 57.3 years (range 29-88 years). Statistical analyses showed that prognosis was significantly correlated with the Breslow thickness (≥/Conclusions: In our experience, the surgical management (disarticulation with respect to functional surgical excision) did not influence the prognosis of NAM patients. The latter was affected by the histopathological characteristics (Breslow thickness, regression and mitoses) and location (fingers vs. foot).

[EN] Mitosis is a process that ensure the correct distribution of the chromosomes between the two newly generated cells, is tightly regulated by two main processes, protein degradation controlled by the APC and protein phosphorylation by different mitotic kinases. CDK1 is the master regulator of mitosis but in the last decades proteins from the Aurora or Polo or the NIMA family have been shown to play key roles in mitosis. The objective of this thesis is to identify new roles during the cell cycle and more specifically the late phases of mitosis of Nek9, a NIMA-related kinase. We aim to characterize new substrates and functions of the kinase by using different cell lines and genetically modified mice and interfering with Nek9 expression. The centrosome acts as the major microtubule-organizing center (MTOC) of the cell to maintain cytoskeleton in interphase and to organize the bipolar spindle in mitosis, and its duplication cycle is coupled with the cell cycle. When the cell enters mitosis, the duplicated centrosomes separate to the spindle poles and assemble the bipolar mitotic spindle for accurate chromosome separation and to maintain genomic stability. However, centrosome aberrations occur frequently and often lead to abnormal mitotic spindle formation, which can result in abnormal chromosome segregation and as a consequence tumorigenesis, microcephaly or ciliopathies. Nek9 is inactive during interphase and activated at centrosomes and spindle poles during mitosis by a two-step mechanism mediated by Plk1 and CDK1. Once active, Nek9 is able to bind Nek6 and Nek7 and directly phosphorylate these kinases inducing in turn their activation. Our group has shown that Nek6/7 phosphorylates the kinesin Eg5 at Ser1033 in the C-terminal domain, modulating the accumulation of Eg5 at or around centrosomes and their separation during prophase. Nek9 also phosphorylates the adapter NEDD1/GCP-WD, independently of Nek6/7, contributing to its recruitment to the centrosome and in consequence, to the recruitment of the microtubule nucleating complex formed by y-tubulin to the same organelle. Thus, Nek9, Nek7 and Nek6 regulate different aspects of the centrosome machinery during the entry in mitosis and have a role in spindle organization and correct mitotic progression. Here we show that animals with a single Nek9 KO allele are healthy and fertile but intercrosses between them have not resulted in any homozygous null animals among born offspring indicating that the deletion of Nek9 is embryonic lethal. Also embryos obtained from these intercrosses had a higher frequency of mitotic abnormalities that result in death during the first days of development. As Nek9 is important for the proper development of mitosis we checked whether the expression in heterozygosity of Nek9 results in tumors affecting the viability of the animals. Some differences in tumor-free lifespan between heterozygous and wild type animals have been observed, with the appearance of tumors and aneuploidy. In addition, elimination of Nek9 expression lead to the apparition of abnormal mitosis, aneuploidy and multiple centrosomes both in genetically engineered MEFs and human cells, resulting in accumulation of centrobin, a protein mostly associated with the daughter centrioles, in the amplified centrioles. In the present thesis we describe possible new functions and substrates of Nek9 in the centrosome cycle, closely linked to the cell division cycle, after interfering with its expression using different strategies., [ES] La mitosis es un proceso que asegura la distribución correcta de los cromosomas entre dos células recién generadas, está regulada por dos procesos principales, la degradación y la fosforilación de proteínas por diferentes quinasas mitóticas. CDK1 es el principal regulador de la mitosis, pero en las últimas décadas se ha demostrado que las proteínas de la familia Aurora o Polo o NIMA desempeñan un papel clave en la mitosis. El objetivo de esta tesis es identificar nuevas funciones de Nek9, una quinasa de la familia NIMA, durante el ciclo celular y más específicamente durante las fases tardías de la mitosis. Nuestro objetivo es caracterizar nuevos sustratos y funciones de la quinasa mediante el uso de diferentes líneas celulares y ratones genéticamente modificados que nos permiten interferir con la expresión de Nek9. El centrosoma actúa como el principal centro organizador de microtúbulos de la célula para mantener el citoesqueleto en interfase y para organizar el huso bipolar en la mitosis, su ciclo de duplicación va en sintonía con el ciclo celular. Cuando la célula entra en mitosis, los centrosomas duplicados se separan ensamblando el huso mitótico para segregar los cromosomas y para mantener la estabilidad genómica. Sin embargo, diferentes aberraciones ocurren con frecuencia en el centrosoma y a menudo conducen a la formación anormal del huso mitótico, que puede dar como resultado una segregación cromosómica anormal y, como consecuencia, tumorogénesis, microcefalia o ciliopatias. Nek9 está inactiva en interfase y se activa en los centrosomas durante la mitosis mediante un mecanismo de dos pasos mediado por Plk1 y CDK1. Una vez activo, Nek9 se puede unir a Nek6 y Nek7 y fosforilarlas induciendo a su vez su activación. Nuestro grupo ha demostrado que Nek6/7 fosforilan la quinesina Eg5, modulando la acumulación de Eg5 en los centrosomas y su separación durante la profase. Nek9 también fosforila el adaptador NEDD1 / GCP-WD, independientemente de Nek6/7, lo que contribuye a su reclutamiento en el centrosoma y, en consecuencia, al reclutamiento del complejo de nucleación de microtúbulos formado por y-tubulina. Aquí mostramos que los animales con un único alelo Nek9 KO están sanos y son fértiles. Sin embargo, los cruces entre ellos no dan lugar a ningún animal KO homocigoto, lo que indica que la eliminación de Nek9 es letal durante el desarrollo embrionario. Además, los embriones procedentes de estos cruces tienen una mayor frecuencia de defectos mitóticos que provocan la muerte durante los primeros días de desarrollo. Como Nek9 es importante para el correcto desarrollo de la mitosis, queríamos ver si la expresión en heterocigosis daba como resultado tumores que afectan la viabilidad de los animales. Se han observado algunas diferencias en la esperanza de vida libre de tumores entre los heterocigotos con cierta incidencia de cáncer y aneuploidía. Por otro lado, la eliminación de la expresión de Nek9 en células conduce a la aparición de mitosis anormales, aneuploidía y múltiples centrosomas, tanto en fibroblastos embrionarios de ratón genéticamente modificados como en células humanas teniendo como consecuencia la acumulación de centrobina, una proteína presente en los procentriolos. En la presente tesis describimos posibles nuevas funciones y sustratos de Nek9 en el ciclo del centrosoma, íntimamente ligado al ciclo de división celular, tras interferir con su expresión de diferentes formas.

The mitotic spindle is a very dynamic structure that is built de novo and destroyed at each round of cell division. In order to perform its fundamental function during chromosome segregation, mitotic spindle dynamics must be tightly coordinated with other cell cycle events. These changes are driven by several protein kinases, phosphatases and microtubule-associated proteins. In budding yeast, the kinase Swe1 and the phosphatase Mih1 act in concert in controlling the phosphorylation state of Cdc28, the catalytic subunit of Cdk1, the major regulator of the cell cycle. In this study we show that Swe1 and Mih1 are also involved in the control of mitotic spindle dynamics. Our data indicate that Swe1 and the Polo-like kinase Cdc5 control the balance between phosphorylated and unphosphorylated forms of Mih1, which is, in turn, important for mitotic spindle elongation. Moreover, we show that the microtubule-associated protein Bik1 is a phosphoprotein, and that Swe1 and Mih1 are both involved in controlling phosphorylation of Bik1. These results uncover new players and provide insights into the complex regulation of mitotic spindle dynamics

18 páginas, 5 figuras, 1 tabla, Background: Protein phosphatase 2A (PP2A) is a family of conserved serine/threonine phosphatases involved in several essential aspects of cell growth and proliferation. PP2ACdc55 phosphatase has been extensively related to cell cycle events in budding yeast; however, few PP2ACdc55 substrates have been identified. Here, we performed a quantitative mass spectrometry approach to reveal new substrates of PP2ACdc55 phosphatase and new PP2A-related processes in mitotic arrested cells. Results: We identified 62 statistically significant PP2ACdc55 substrates involved mainly in actin-cytoskeleton organization. In addition, we validated new PP2ACdc55 substrates such as Slk19 and Lte1, involved in early and late anaphase pathways, and Zeo1, a component of the cell wall integrity pathway. Finally, we constructed docking models of Cdc55 and its substrate Mob1. We found that the predominant interface on Cdc55 is mediated by a protruding loop consisting of residues 84-90, thus highlighting the relevance of these aminoacids for substrate interaction. Conclusions: We used phosphoproteomics of Cdc55-deficient cells to uncover new PP2ACdc55 substrates and functions in mitosis. As expected, several hyperphosphorylated proteins corresponded to Cdk1-dependent substrates, although other kinases' consensus motifs were also enriched in our dataset, suggesting that PP2ACdc55 counteracts and regulates other kinases distinct from Cdk1. Indeed, Pkc1 emerged as a novel node of PP2ACdc55 regulation, highlighting a major role of PP2ACdc55 in actin cytoskeleton and cytokinesis, gene ontology terms significantly enriched in the PP2ACdc55-dependent phosphoproteome., Work in our laboratory is supported by the Spanish Ministry of Science and Innovation (BFU2011–27568), Spanish Ministry of Economy and Competitively (BFU2013–43132-P and BFU2016–77975-R AEI/FEDER, UE cofounded by FEDER funds/European Regional Development Fund- a way to build Europe). MRL was supported by the Lundbeck foundation (Junior Group Leader Fellowship). This work was supported by a generous grant from the VILLUM Foundation to the VILLUM Centre for Bioanalytical Sciences at the University of Southern Denmark. SBB is a recipient of ISCIII grant 13FIS037. IDIBELL Proteomics Unit belongs to ProteoRed, PRB2-ISCIII, and is supported by grant PT13/0001/0033.

[eng] Mitosis is a process that ensure the correct distribution of the chromosomes between the two newly generated cells, is tightly regulated by two main processes, protein degradation controlled by the APC and protein phosphorylation by different mitotic kinases. CDK1 is the master regulator of mitosis but in the last decades proteins from the Aurora or Polo or the NIMA family have been shown to play key roles in mitosis. The objective of this thesis is to identify new roles during the cell cycle and more specifically the late phases of mitosis of Nek9, a NIMA-related kinase. We aim to characterize new substrates and functions of the kinase by using different cell lines and genetically modified mice and interfering with Nek9 expression. The centrosome acts as the major microtubule-organizing center (MTOC) of the cell to maintain cytoskeleton in interphase and to organize the bipolar spindle in mitosis, and its duplication cycle is coupled with the cell cycle. When the cell enters mitosis, the duplicated centrosomes separate to the spindle poles and assemble the bipolar mitotic spindle for accurate chromosome separation and to maintain genomic stability. However, centrosome aberrations occur frequently and often lead to abnormal mitotic spindle formation, which can result in abnormal chromosome segregation and as a consequence tumorigenesis, microcephaly or ciliopathies. Nek9 is inactive during interphase and activated at centrosomes and spindle poles during mitosis by a two-step mechanism mediated by Plk1 and CDK1. Once active, Nek9 is able to bind Nek6 and Nek7 and directly phosphorylate these kinases inducing in turn their activation. Our group has shown that Nek6/7 phosphorylates the kinesin Eg5 at Ser1033 in the C-terminal domain, modulating the accumulation of Eg5 at or around centrosomes and their separation during prophase. Nek9 also phosphorylates the adapter NEDD1/GCP-WD, independently of Nek6/7, contributing to its recruitment to the centrosome and in consequence, to the recruitment of the microtubule nucleating complex formed by y-tubulin to the same organelle. Thus, Nek9, Nek7 and Nek6 regulate different aspects of the centrosome machinery during the entry in mitosis and have a role in spindle organization and correct mitotic progression. Here we show that animals with a single Nek9 KO allele are healthy and fertile but intercrosses between them have not resulted in any homozygous null animals among born offspring indicating that the deletion of Nek9 is embryonic lethal. Also embryos obtained from these intercrosses had a higher frequency of mitotic abnormalities that result in death during the first days of development. As Nek9 is important for the proper development of mitosis we checked whether the expression in heterozygosity of Nek9 results in tumors affecting the viability of the animals. Some differences in tumor-free lifespan between heterozygous and wild type animals have been observed, with the appearance of tumors and aneuploidy. In addition, elimination of Nek9 expression lead to the apparition of abnormal mitosis, aneuploidy and multiple centrosomes both in genetically engineered MEFs and human cells, resulting in accumulation of centrobin, a protein mostly associated with the daughter centrioles, in the amplified centrioles. In the present thesis we describe possible new functions and substrates of Nek9 in the centrosome cycle, closely linked to the cell division cycle, after interfering with its expression using different strategies., [spa] La mitosis es un proceso que asegura la distribución correcta de los cromosomas entre dos células recién generadas, está regulada por dos procesos principales, la degradación y la fosforilación de proteínas por diferentes quinasas mitóticas. CDK1 es el principal regulador de la mitosis, pero en las últimas décadas se ha demostrado que las proteínas de la familia Aurora o Polo o NIMA desempeñan un papel clave en la mitosis. El objetivo de esta tesis es identificar nuevas funciones de Nek9, una quinasa de la familia NIMA, durante el ciclo celular y más específicamente durante las fases tardías de la mitosis. Nuestro objetivo es caracterizar nuevos sustratos y funciones de la quinasa mediante el uso de diferentes líneas celulares y ratones genéticamente modificados que nos permiten interferir con la expresión de Nek9. El centrosoma actúa como el principal centro organizador de microtúbulos de la célula para mantener el citoesqueleto en interfase y para organizar el huso bipolar en la mitosis, su ciclo de duplicación va en sintonía con el ciclo celular. Cuando la célula entra en mitosis, los centrosomas duplicados se separan ensamblando el huso mitótico para segregar los cromosomas y para mantener la estabilidad genómica. Sin embargo, diferentes aberraciones ocurren con frecuencia en el centrosoma y a menudo conducen a la formación anormal del huso mitótico, que puede dar como resultado una segregación cromosómica anormal y, como consecuencia, tumorogénesis, microcefalia o ciliopatias. Nek9 está inactiva en interfase y se activa en los centrosomas durante la mitosis mediante un mecanismo de dos pasos mediado por Plk1 y CDK1. Una vez activo, Nek9 se puede unir a Nek6 y Nek7 y fosforilarlas induciendo a su vez su activación. Nuestro grupo ha demostrado que Nek6/7 fosforilan la quinesina Eg5, modulando la acumulación de Eg5 en los centrosomas y su separación durante la profase. Nek9 también fosforila el adaptador NEDD1 / GCP-WD, independientemente de Nek6/7, lo que contribuye a su reclutamiento en el centrosoma y, en consecuencia, al reclutamiento del complejo de nucleación de microtúbulos formado por y-tubulina. Aquí mostramos que los animales con un único alelo Nek9 KO están sanos y son fértiles. Sin embargo, los cruces entre ellos no dan lugar a ningún animal KO homocigoto, lo que indica que la eliminación de Nek9 es letal durante el desarrollo embrionario. Además, los embriones procedentes de estos cruces tienen una mayor frecuencia de defectos mitóticos que provocan la muerte durante los primeros días de desarrollo. Como Nek9 es importante para el correcto desarrollo de la mitosis, queríamos ver si la expresión en heterocigosis daba como resultado tumores que afectan la viabilidad de los animales. Se han observado algunas diferencias en la esperanza de vida libre de tumores entre los heterocigotos con cierta incidencia de cáncer y aneuploidía. Por otro lado, la eliminación de la expresión de Nek9 en células conduce a la aparición de mitosis anormales, aneuploidía y múltiples centrosomas, tanto en fibroblastos embrionarios de ratón genéticamente modificados como en células humanas teniendo como consecuencia la acumulación de centrobina, una proteína presente en los procentriolos. En la presente tesis describimos posibles nuevas funciones y sustratos de Nek9 en el ciclo del centrosoma, íntimamente ligado al ciclo de división celular, tras interferir con su expresión de diferentes formas.

To investigate the specific target of PKA in the regulation of cell cycle progression and cell size we developed a new approach using the yeast strain GG104 bearing a deletion in adenylate cyclase gene and permeable to cAMP ( cyr1Δ, pde2Δ, msn2Δ, msn4Δ). In this strain the PKA activity is absent and can be activated by addition of cAMP in the medium, without any other change of the growth conditions. In the present work we show that the activation of PKA by exogenous cAMP in the GG104 strain exponentially growing in glucose medium caused a marked increase of cell size and perturbation of cell cycle with a transient arrest of cells in G1, followed by an accumulation of cells in G2/M phase with a minimal change in the growth rate. Deletion of CLN1 gene, but not of CLN2, abolished the transient G1 phase arrest. Consistently we found that PKA activation caused a transcriptional repression of CLN1 gene. Transcription of CLN1 is controlled by SBF and MBF dual-regulated promoter. We found that also the deletion of SWI4 gene abolished the transient G1 arrest suggesting that Swi4 is a target responsible for PKA modulation of G1/S phase transition. We generated a SWI4 allele mutated in the consensus site for PKA (Swi4(S159A)) and we found that expression of Swi4(S159A) protein in the GG104-Swi4Δ strain did not restore the transient G1 arrest induced by PKA activation, suggesting that Swi4 phosphorylation by PKA regulates CLN1 gene expression and G1/S phase transition.

Background The latest AJCC classification has included the number of mitoses as a factor for upstaging thin melanomas. Meanwhile, while dermoscopy has often been used to predict melanoma thickness, its value in predicting number of mitoses remains unknown. Objective Our aim is to evaluate the correlation between dermoscopic features and the presence of mitoses in a consecutive cohort of thin melanomas. Methods A case control study has been performed to identify specific dermoscopic parameters that could differentiate thin melanomas with 1 or more mitoses per mm2 from those without mitoses. Results Of 177 melanomas equal to or thinner than 1 mm, 131 (74%) lesions had no mitoses and 46 (36%) lesions had at least 1 mitosis × mm2. Dermoscopic features associated with the presence of 1 or more mitoses were the following: peripheral streaks (OR 4.11; 95% CI 1.94–8.71) and black colour (OR 4.70; 95% CI; 2.28–9.68). In contrast, atypical pigment network (OR (0.30; 95% CI 0.15–0.61)) and brown colour (OR 0.36; 95% CI 0.18–0.75) were associated to melanomas without mitoses. The same variables were also associated to the increasing number of mitoses at linear regression. Conclusion Black colour and peripheral streaks can predict the presence of mitoses in thin melanoma, while atypical pigment network and brown colour are associated to thin melanoma without mitoses.

Successful chromosome segregation is crucial for the survival of a cell and to avoid diseases such as cancer. Anaphase bridges are a type of segregation defect that can arises from structurally compromised chromosomes. Little is known about the mechanisms that normally prevent them. In this study I screened for genes that normally prevent anaphase bridges in order to learn more about their origin. I found anaphase bridges to arise in replication mutants and it is possible to trigger these bridges by exposing cells to replication stress. Thus, impaired replication is one cause for anaphase bridges. Further I identified a role for the mitotic exit network (MEN) in chromosome segregation. MEN mutants display anaphase bridges and I present evidence that these bridges arise from telomeric regions and may involve un-replicated DNA., La correcta segregació dels cromosomes és esencial per la supervivencia de la cèl·lula i per evitar l’aparició de certes malalties com el càncer. Els ponts anafàsics són un tipus d’error de segregació que pot ser originat per defectes estructurals dels cromosomes. Es coneix molt poc sobre els mecanismes que eviten la formació d’aquests ponts anafàsics. En aquest estudi he fet un análisis global dels diferents gens que normalment eviten la formació d’aquests ponts, per abançar en la comprensió del seu origen. He vist que el ponts anaphasics es formen en mutants que tenen afectat el proces de replicació i que és posible de provocar la formació d’aquests ponts exposant les cèl·lules a estrés replicatiu. Per tant, els problemes en la replicació són una de les causes dels ponts d’anafase. He identificat el rol de “mitotic exit network (MEN)” en la segregació cromosómica. Els mutants per MEN formen ponts anafàsics i mostren evidències que aquests ponts probenen de regions telomèriques i podrien incloure DNA no replicat.

Tubulin-targeting drugs, like taxanes and vinca alkaloids, are among the most effective anticancer therapeutics used in the clinic today. Specifically, anti-microtubule cancer drugs (AMCDs) have proven to be effective in the treatment of castration-resistant prostate cancer and triple-negative breast cancer. AMCDs, however, have limiting toxicities that include neutropenia and neurotoxicity, and, in addition, tumor cells can become resistant to the drugs after long-term use. Co-targeting mitotic progression/slippage with inhibition of the protein kinases WEE1 and MYT1 that regulate CDK1 kinase activity may improve AMCD efficacy, reducing the acquisition of resistance by the tumor and side effects from the drug and/or its vehicle. Other possible treatments that improve outcomes in the clinic for these two drug-resistant cancers, including new formulations of the AMCDs and pursuing different molecular targets, will be discussed.

Stable kinetochore-microtubule attachment is essential for cell division. It requires recruitment of outer kinetochore microtubule binders by centromere proteins C and T (CENP-C and CENP-T). To study the molecular requirements of kinetochore formation, we reconstituted the binding of the MIS12 and NDC80 outer kinetochore subcomplexes to CENP-C and CENP-T. Whereas CENP-C recruits a single MIS12:NDC80 complex, we show here that CENP-T binds one MIS12:NDC80 and two NDC80 complexes upon phosphorylation by the mitotic CDK1:Cyclin B complex at three distinct CENP-T sites. Visualization of reconstituted complexes by electron microscopy supports this model. Binding of CENP-C and CENP-T to MIS12 is competitive, and therefore CENP-C and CENP-T act in parallel to recruit two MIS12 and up to four NDC80 complexes. Our observations provide a molecular explanation for the stoichiometry of kinetochore components and its cell cycle regulation, and highlight how outer kinetochore modules bridge distances of well over 100 nm. DOI: http://dx.doi.org/10.7554/eLife.21007.001

During the development of the nervous system the regulation of cell cycle, differentiation, and survival is tightly interlinked. Newly generated neurons must keep cell cycle components under strict control, as cell cycle re-entry leads to neuronal degeneration and death. However, despite their relevance, the mechanisms controlling this process remain largely unexplored. Here we show that Scratch2 is involved in the control of the cell cycle in neurons in the developing spinal cord of the zebrafish embryo. scratch2 knockdown induces postmitotic neurons to re-enter mitosis. Scratch2 prevents cell cycle re-entry by maintaining high levels of the cycle inhibitor p57 through the downregulation of miR-25. Thus, Scratch2 appears to safeguard the homeostasis of postmitotic primary neurons by preventing cell cycle re-entry.

Cockayne syndrome (CS) is a genetic disease inherited in an autosomal recessive pattern. CS patients are characterized by photosensitivity, severe growth retardation, cachectic dwarfism, feature of premature aging and progressive neurological abnormalities of the central nervous system including microcephaly, cerebellar atrophy and demyelinating peripheral neuropathy. The average life span of children with this syndrome is about 12 years of age. CS patients have been assigned to two genetic complementation groups (CS-A and CS-B), whose corresponding genes (csa and csb) have been cloned and characterized (Henning et al 1995; Troelstra et al., 1992; Lehmann, 1982). CSA and CSB proteins have critical roles in a sub pathway of nucleotide excision repair known as transcription-coupled repair (TCR). Although a defect in TCR pathway could potentially account for the enhanced photosensitivity of CS patients, other pathological features including neurological dysfunctions may not solely explained by a DNA repair defect and requires additional explanations. Accordingly, in the last years it has been suggested that CSA and CSB proteins play multiple pleiotropic functions. More recently, we and others have demonstrated that CSB mediates the transcriptional programs following exposure to cellular stressors such as UV, oxidative damage, inflammation and hypoxia. Therefore, abnormalities in the regulation of RNA pol I and II mediated transcription might provide plausible explanations for many of the somatic features, including aspects of neurological symptoms associated with CS. Observation of neurological symptoms detected either at birth or during early childhood raises the possibility that CSB may have a crucial role in the transcriptional programs that govern the plasticity and the maintenance of the central nervous system during (perinatal and postnatal) pediatric life. Our recent studies showed that CSB suppression affects the neuronal differentiation capability of human neural progenitor cells. CSB also plays a critical role in cell robustness, negatively modulating p53 activity after cellular stress, including DNA damage and hypoxia and counteracting p53-independent apoptosis. Finally, CSB, as anti-apoptotic factor is over-expressed in a variety of cancer cells and tissues, so it represents a strategic target for anticancer therapy: the inhibition or down regulation of CSB in cancer cells makes these cells hypersensitive to a variety of commonly used cancer chemotherapeutic agents. Interesting, an unknown extranuclear role of CSA and CSB proteins is emerging. This study reveals an unexpected subcellular localization and biological function of CSA and CSB proteins in cytokinesis. We discovered, in several tumoral cells line and in Cockayne fibroblast, that CSA and CSB proteins have a different cytoplasmic localization during mitosis in cytoskeletal structures such as the centrosomes and the midbody (the intercellular microtubule bridge connecting two daughter cells at the end of cytokinesis). Immunofluorescence (IF) staining for CSA and CSB during mitosis shows that in the early stages the cytoplasmic localization seems to be consequent of chromosomes condensation. However, from the onset of cleavage furrow the proteins take progressively a specific position. In particular, while during metaphase CSA and CSB exit from the nucleus yet condensed, in anaphase, during cleavage furrow contraction, they are associated with the spindle midzone; then, in telophase, they accumulate at the midbody, where they have a characteristic ring-like arrangement embraced by Aurora B in the so called bulge-zone. These observations suggest a potential role of CSA and CSB protein in the organization and functioning of cytoskeleton-mediated cellular activities encompassing polarity, motility and cellular division. Depletion of CSB proteins in tumoral cells line and CSA and CSB mutations in Cockayne fibroblasts, results in cytokinesis failure. In particular, we show the presence of syncytium-like cells, the increase of cells number at the midbody stage and abscission defects associated with the increase of aberrant mitosis and with the accumulation of multi- binucleated cells. These signs are usually observed in cells with impaired abscission, that support the role of this proteins in last stage of cell division. To investigate the mechanism of CSA and CSB protein in the abscission, we analyzed the spatiotemporal localization of a series of structural and functional proteins sequentially recruited during cytokinesis to assure proper cell division. In all cells analyzed at the early stages of cytokinesis, we observed that the localization patterns for all proteins examined are not affected during midzone formation and cleavage furrow ingression in the CSA and CSB mutated cells, indicating that the proteins are not mainly involved in the early events of cytokinesis. In contrast, in the subsequent stage of midbody formation, PLK1 and PRC1 proteins can be detected at the midbody but their distribution pattern is altered, becoming dispersed along the midbody microtubules and the midbodies appear elongated. Furthermore, by analyzing the proteins involved in abscission, in CSA and CSB mutated cells the critical abscission factors as ALIX and Spastin present several defects of organization localizing itself along the midbody. PLK1 and PRC1 proteins are closely associated in the regulation of midzone and midbody formation; the ubiquitination of both proteins it is required for the cytokinesis completion. CSA protein is a component of a CRL4 E3 ubiquitin ligase complex while CSB protein, whit a Cterminal UBD, is a substrate of CSA. We speculate that in CSA and CSB mutated cells, PRC1 and PLK1 proteins are delocalized because of the lack of CSA protein that don’t permit the protein degradation. Overall, these results indicate that CSA and CSB protein play a role at the terminal stage of cytokinesis by controlling the morphology of the midbody and organizing the localization of a few critical regulators of the abscission. La sindrome di Cockayne (CS) è una malattia genetica autosomica recessiva. I pazienti CS sono caratterizzati da fotosensibilità cutanea, grave ritardo nella crescita, nanismo e cachessia, caratteristiche di invecchiamento precoce e da anomalie neurologiche del sistema nervoso centrale, tra cui microcefalia e atrofia cerebellare. La durata media della vita dei bambini affetti da questa sindrome è di circa 12 anni di età. I pazienti CS sono stati assegnati a due gruppi genetici di complementazione (CS-A e CS-B), i cui corrispondenti geni (csa e csb) sono stati clonati e caratterizzati. Le proteine CSA e CSB hanno un ruolo critico nel sub-pathway di riparazione per escissione nucleotidica (TCR). Anche se un difetto nel TCR potenzialmente spiega la maggiore fotosensibilità dei pazienti CS, altre caratteristiche patologiche, tra cui le disfunzioni neurologiche potrebbero non possono essere spiegate solo da un difetto di riparazione del DNA. Di conseguenza, negli ultimi anni è emerso che CSA e CSB svolgono funzioni pleiotropiche. Recentemente, abbiamo dimostrato che CSB media i programmi trascrizionali in seguito ad esposizione a fattori di stress cellulari come UV, danno ossidativo, infiammazione e ipossia. Inoltre abbiamo dimostrato che la soppressione CSB colpisce la capacità di differenziamento neuronale di cellule progenitrici neurali umane. CSB modula negativamente l'attività di p53 dopo stress cellulare e contrasta l'apoptosi in maniera p53-indipendente. Infine, CSB, come fattore anti-apoptotico è sovraespresso in una varietà di cellule tumorali e tessuti, quindi rappresenta un obiettivo strategico per la terapia antitumorale: l'inibizione o down regulazione di CSB nelle cellule tumorali rende queste cellule ipersensibili ad una varietà di agenti chemioterapici comunemente utilizzati. Infine, questo studio rivela una nuova localizzazione subcellulare delle proteine CSA eCSB e il loro coinvolgimento nella citodieresi. Infatti, tramite studi di IF abbiamo dimostrato che la soppressione di CSA e CSB in diverse linee tumorali e la mutazione delle stesse in fibroblasti Cockayne determina il fallimento della citodieresi e un aumento di cellule bi-multinucleate. Dottorato di ricerca in Genetica e biologia cellulare

Mitosis is a tightly regulated process that aims to ensure the correct distribution of the chromosomes between the two newly generated cells. Many protein kinases have been defined as essential for this process: cyclin- dependent kinases, Aurora family and Polo family kinases are some of the most relevant players. The objective of this thesis is to characterize one of the less studied kinase pathways involved in this process, which is constituted by the NIMA-related kinases Nek9, Nek6 and Nek7. Nek9 is activated at the onset of mitosis by a double step mechanism mediated by CDK1 and Plk1. Once Nek9 is activated it can bind to Nek6 and Nek7 and phosphorylate them, promoting their activation. Finally, Nek6 and Nek7 are responsible for the phosphorylation of the kinesin Eg5, promoting Eg5 accumulation at centrosome, and consequently, centrosome separation. The kinesin eg5 motor protein is considered as one of the major players for centrosome separation and formation of the bipolar spindle. The tetramer configuration allows Eg5 to bind antiparallel microtubules and slide them apart, exerting a force that promotes centrosome separation and the maintenance of the bipolar spindle. Centrosome separation, however, is a highly intricate process that involves several pathways, including Eg5 activity. Dynein presents a directed activity towards the minus ends of microtubules, which has a redundant role to Eg5 in centrosome separation. Dynein accumulation at the cell cortex and the nuclear membrane, through its adaptor BicD2, is also involved in centrosome tethering at the nuclear envelope, a necessary step prior to separation. Furthermore, dynein can control the position of Eg5 at the spindle via TPX2, an event that could also happen before nuclear envelope breakdown (NEB). Here we describe the conditions required for Eg5 accumulation at the centrosmes after Ser1033 phosphorylation. During the development of this project we have explored the essential circumstances for correct Eg5 localization in cells. By using protein-protein interaction techniques and shRNA depletion of protein candidates we have determined that another motor protein, dynein, together with the adaptor BicD2 and the protein TPX2 are responsible for Eg5 accumulation around centrosomes. Additionally, we proposed TPX2 as a novel Nek9 substrate and we have investigated the role of this phosphorylation, which affects TPX2 localization during prophase, before NEB. We present with this thesis a model for Eg5 accumulation at microtubule minus ends and centrosome separation during prophase summarized in the following points: 1) Dynein complex transports Eg5 towards the centrosome. Dynein interacts with Eg5 independently of the Ser1033 phosphorylation. The adaptor BicD2, which interacts directly with Eg5 tail domain, mediates the interaction. Dynein motility towards microtubule minus ends and the presence of BicD2 on the complex are required for Eg5 localization at centrosomes. Thus, the dynein complex is required for Eg5 transport to the centrosomes during G2-M transition. 2) TPX2 inhibits Eg5 motility in response to Ser1033 phosphorylation. TPX2 is necessary for the correct localization of Eg5 at centrosomes during prophase. TPX2 mislocalization at centrosomes without altering its overall levels leads to failed Eg5 localization, therefore the presence of TPX2 at centrosomes during prophase is required for Eg5 localization. TPX2 interacts with Eg5 during mitosis and the interaction is abolished when the Ser1033 can’t be phosphorylated. Thus, TPX2 is able to respond to Eg5 Ser1033 phosphorylation, which we propose is promoting the interaction between these two proteins, and consequently inhibiting Eg5 motility at centrosomal levels. 3) TPX2 phosphorylation by Nek9 promotes its centrosomal localization. Nek9 phosphorylation of TPX2 is responsible for TPX2 localization at the spindle poles during prophase. Nek9 phosphorylates TPX2 at residues that are proximal to a NLS, making TPX2 localization more cytoplasmic and promoting its accumulation to the area where Nek9 is more active, the centrosome., La mitosis es un proceso altamente regulado cuyo objetivo es asegurar la correcta distribución de los cromosomas entre las dos células nuevamente generadas. Diferentes proteínas quinasas han sido definidas como esenciales en este proceso pero el objetivo de esta tesis es caracterizar una de las rutas de señalización menos estudiada, la cual la componen las NIMA quinasas Nek9, Nek6 y Nek7. Nek9 es activada al inicio de mitosis por un doble mecanismo mediado por CDK1 y Plk1. Una vez activada, se puede unir a Nek6 y Nek7 y fosforilarlas, promoviendo su activación. Finalmente, Nek6 y Nek7 son responsables de la fosforilación de la quinesina Eg5, promoviendo la acumulación de Eg5 en los centrosomas, y en consecuencia, la separación de los mismos en profase. Aquí describimos las condiciones necesarias para la acumulación de Eg5 en los centrosomas después de la fosforilación en la Ser1033. Durante el desarrollo de este trabajo hemos explorado las circunstancias esenciales para una correcta localización de Eg5 en las células. Usando técnicas de interacción proteína-proteína y técnicas de silenciamiento proteico de candidatos con shRNA hemos determinado que otra proteína motora, dineína, junto con el adaptador BicD2 y la proteína TPX2, son responsables de la acumulación de Eg5 alrededor de los centrosomas. Además, hemos propuesto a TPX2 como un nuevo substrato regulado por Nek9 y hemos investigado el papel de esta fosforilación, la cual afecta la localización de TPX2 durante profase, antes de la rotura de la membrana nuclear. Con esta tesis presentamos un modelo para la acumulación de Eg5 y la separación de los centrosomas en profase que puede ser resumido en los siguientes puntos: - El complejo de dineína transporta Eg5 hacia el centrosoma independientemente de la fosforilación en la Ser1033. El adaptador BicD2 media esta interacción uniéndose directamente al dominio C terminal de Eg5. -TPX2 inhibe movilidad de Eg5 en respuesta a la fosforilación en la Ser1033. - La presencia de TPX2 en los centrosomas es necesaria para la localización de Eg5. La fosforilación de TPX2 por Nek9 promueve la localización de TPX2 en los centrosomas durante la profase.

Ethnopharmacological relevance Xanthium strumarium L. is a member of the Asteraceae family popularly used with multiple therapeutic purposes. Whole extracts of this plant have shown anti-mitotic activity in vitro suggesting that some components could induce mitotic arrest in proliferating cells. Aim of the sudy Aim of the present work was to characterize the anti-mitotic properties of the X. strumarium whole extract and to isolate and purify active molecule(s). Materials and methods The capacity of the whole extract to inhibit mitotic progression in mammalian cultured cells was investigated to identify its anti-mitotic activity. Isolation of active component(s) was performed using a bioassay-guided multistep separation procedure in which whole extract was submitted to a progressive process of fractionation and fractions were challenged for their anti-mitotic activity. Results Our results show for the first time that X. strumarium whole extract inhibits assembly of the mitotic spindle and spindle-pole separation, thereby heavily affecting mitosis, impairing the metaphase to anaphase transition and inducing apoptosis. The purification procedure led to a fraction with an anti-mitotic activity comparable to that of the whole extract. Chemical analysis of this fraction showed that its major component was xanthatin. Conclusions The present work shows a new activity of X. strumarium extract, i.e. the alteration of the mitotic apparatus in cultured cells that may be responsible for the anti-proliferative activity of the extract. Anti-mitotic activity is shown to be mainly exerted by xanthatin.

[eng] The depletion of MZT1 in human cells causes severe mitotic spindle defects. Depleted cells lack centrosomal γ-tubulin and arrest in mitosis with a monopolar spindle configuration. Similarly, double deletion mutants of the plant MZT1 orthologs GIP1a and GIP1b are embryonic lethal due to abnormal spindle microtubule distribution and chromosome mis-segregation. Moreover GIP1a and GIP1b were shown to localize to active nucleation sites in the interphase cortical microtubule array. MZT1 function is conserved in fission yeast, where it interacts with GCP3, and is an essential component for the recruitment of the γ-tubulin complex to the spindle pole body, the centrosome equivalent, but not for assembly of the γ-tubulin complex. However, the molecular mechanisms underlying these effects remain unknown. The main goal of this project is to study how MZT1 regulates γTuRC to control MT nucleation and organization. In this work I confirm that in human cells MZT1 is a subunit of the γTuRC and is required for the targeting of the γTuRC to centrosomes and for proper spindle formation. By sucrose gradient fractionation I found that in mammalian cells, MZT1 is not required to assemble the γTuRC. Interestingly, I found that MZT1 is necessary for the interaction of the γTuRC with the targeting factor NEDD1/GCPWD. While in plants and in fission yeast MZT1 interacts with the N-terminal region of GCP3, I found that in mammalian cells MZT1 interacts with a conserve motif at the N-terminal extension of GCP2, GCP3, GCP5 and GCP6. Furthermore, by immunoprecipitation of FLAG-tagged GCPs MZT1 binding motif mutants, I found that the mutants can be integrated into the γTuRC but lost the interaction with GCP-WD and fail to be targeted to the centrosomes To study the role of MZT1 in MT nucleation I performed a MT regrowth experiment with U2OS cells over expressing the CDK5RAP2 nucleation-activating fragment (CDK5RAP2 CM1) and depleted of MZT1. The MT nucleation induced by CDK5RAP2 was lost upon the depletion of MZT1, suggesting that MZT1 in required for the MT nucleation activation mediated by CDK5RAP2. In summary MZT1 is required for all γTuRC-dependent functions including centriole duplication. MZT1 binds to a conserved motif present in the extended N-termini of GCP2, GCP3, GCP5 and GCP6, allowing specific recognition of fully assembled γTuRC. Binding of MZT1 primes γTuRC for interaction with the adapter NEDD1/GCP-WD for targeting γTuRC to centrosomes. In addition, MZT1-dependent priming is required for the CDK5RAP2 CM1 domain to activate γTuRC nucleation activity. Thus, by enabling specific recognition of γTuRC by targeting and activation factors, MZT1 spatially controls microtubule nucleation., [spa] En las células humanas la depleción de MZT1 provoca graves defectos del huso mitótico. Las células deplecionadas carecen de γ-tubulina centrosomal y presentan una detención de la mitosis con una configuración monopolar del huso mitótico. Del mismo modo, mutantes de deleción dobles de planta MZT1 con sus ortólogos GIP1a y GIP1b son letales para los embriones debido a la anormal distribución de los microtúbulos del huso mitótico y la mala segregación del cromosoma. Además, GIP1a y GIP1b localizan en sitios de nucleación activos de los microtúbulos corticales. La función de MZT1 se conserva en la levadura de fisión, donde interactúa con GCP3, y es un componente esencial para el reclutamiento del complejo de γ-tubulina en el huso polar del cuerpo apical, el equivalente al centrosoma, pero no para el montaje de la γ-tubulina compleja. Sin embargo, los mecanismos moleculares que subyacen a estos efectos siguen siendo desconocidos. El objetivo principal de este proyecto es estudiar cómo MZT1 regula la actividad del γTuRC en la nucleación y organización de los microtúbulos. En este trabajo encontré que MZT1 es necesaria para todas las funciones γTuRC-dependientes, como la duplicación de centríolos. MZT1 se une a un motivo conservado presente en la N-terminales extendida de GCP2, GCP3, GCP5 y GCP6, lo que permite el reconocimiento específico de γTuRC totalmente ensamblado. La unión de MZT1 al γTuRC “prepara” al complejo para la interacción con el adaptador NEDD1/GCP-WD para la orientación γTuRC a los centrosomas. Además, se requieren esta “preparación” para activar la actividad nucleadora del γTuRC mediada por CDK5RAP2 CM1. Por lo tanto, al permitir el reconocimiento específico de γTuRC por los factores de reclutamiento y los factores de activación, se observa que la MZT1 controla espacialmente la nucleación de microtúbulos.

Bipolar spindle assembly in the vertebrate oocyte relies on a self-organization chromosome-dependent pathway. Upon fertilization, the male gamete provides a centrosome, and the first and subsequent embryonic divisions occur in the presence of duplicated centrosomes that act as dominant microtubule organizing centres (MTOCs). The transition from meiosis to embryonic mitosis involves a necessary adaptation to integrate the dominant chromosome-dependent pathway with the centrosomes to form the bipolar spindle. Here, we took advantage of the Xenopus laevis egg extract system to mimic in vitro the assembly of the first embryonic spindle and investigate the respective contributions of the centrosome and the chromosome-dependent pathway to the kinetics of the spindle bipolarization. We found that centrosomes control the transition from the meiotic to the mitotic spindle assembly mechanism. By defining the kinetics of spindle bipolarization, the centrosomes ensure their own positioning to each spindle pole and thereby their essential correct inheritance to the two first daughter cells of the embryo for the development of a healthy organism. T.C. was supported by a Formación de Personal Investigador (FPI) fellowship (Ministerio de Economıa y Competitividad) [grant number BES-2010-031355]. This work was supported by the Ministerio de Economıa y Competitividad [grant numbers ́ BFU2009-10202 and BFU2012-37163]. We acknowledge the support of the Spanish Ministerio de Economıa y Competitividad programme ́ ‘Centro de Excelencia Severo Ochoa 2013–2017’ [grant number SEV-2012-0208]

The NSL1 subunit structures interactions between the MIS12, NDC80, and KNL1 kinetochore complexes (see also a related paper by Maskell et al. in this issue)., Kinetochores are nucleoprotein assemblies responsible for the attachment of chromosomes to spindle microtubules during mitosis. The KMN network, a crucial constituent of the outer kinetochore, creates an interface that connects microtubules to centromeric chromatin. The NDC80, MIS12, and KNL1 complexes form the core of the KMN network. We recently reported the structural organization of the human NDC80 complex. In this study, we extend our analysis to the human MIS12 complex and show that it has an elongated structure with a long axis of ∼22 nm. Through biochemical analysis, cross-linking–based methods, and negative-stain electron microscopy, we investigated the reciprocal organization of the subunits of the MIS12 complex and their contacts with the rest of the KMN network. A highlight of our findings is the identification of the NSL1 subunit as a scaffold supporting interactions of the MIS12 complex with the NDC80 and KNL1 complexes. Our analysis has important implications for understanding kinetochore organization in different organisms.

Background: We have previously shown that the sustained expression of MARK4L transcripts in glioma and neural progenitors (NHNPs) declines after exposure to antisense MARK4L oligonucleotides in glioblastoma cell lines. Array-CGH confirmed the genomic duplication of MARK4L identified by FISH in a glioblastoma cell line. This background together with literature data on the exogenous association of MARK4 with interphase centrosome prompted us to investigate the sub-cellular localization of the endogenous MARK4L protein aiming at achieving insights on its possible role in the pathomechanisms of glioma.Methods: Immunodetection was carried out to validate the specificity of MARK4L antibody in gliomas and NHNPs. Mass spectrometry was applied for MARK4L protein identification in a representative glioblastoma cell line. Combined biochemical fractionation and immunodetection analyses were performed to confirm the sub-cellular localization of MARK4L achieved by immunofluorescence in glioma cell lines.Results: By assigning MARK4L protein within the band immunoprecipitated by the specific antibody we validated our anti-MARK4L antibody. We demonstrated that the endogenous MARK4L: (i) colocalizes with centrosomes at all mitotic stages and resides in centrosome-enriched fractions; (ii) associates with the nucleolus and the midbody and respective fractions, and (iii) co-stains the aberrant centrosome configurations observed in glioma cell lines.Conclusions: The overall data merge on the multiplex entry of MARK4L into the cell cycle and link it to the aberrant centrosomes in glioma cell lines suggesting a possible role of this kinase in the abnormal mitotic processes of human glioma.

Background The high density of tandem repeat sequences (satellites) in nematode genomes and the availability of genome sequences from several species in the group offer a unique opportunity to better understand the evolutionary dynamics and the functional role of these sequences. We take advantage of the previously developed SATFIND program to study the satellites in four Caenorhabditis species and investigate these questions. Methods The identification and comparison of satellites is carried out in three steps. First we find all the satellites present in each species with the SATFIND program. Each satellite is defined by its length, number of repeats, and repeat sequence. Only satellites with at least ten repeats are considered. In the second step we build satellite families with a newly developed alignment program. Satellite families are defined by a consensus sequence and the number of satellites in the family. Finally we compare the consensus sequence of satellite families in different species. Results We give a catalog of individual satellites in each species. We have also identified satellite families with a related sequence and compare them in different species. We analyze the turnover of satellites: they increased in size through duplications of fragments of 100-300 bases. It appears that in many cases they have undergone an explosive expansion. In C. elegans we have identified a subset of large satellites that have strong affinity for the centromere protein CENP-A. We have also compared our results with those obtained from other species, including one nematode and three mammals. Conclusions Most satellite families found in Caenorhabditis are species-specific; in particular those with long repeats. A subset of these satellites may facilitate the formation of kinetochores in mitosis. Other satellite families in C. elegans are either related to Helitron transposons or to meiotic pairing centers.

We designed 39 new 2-phenylindole derivatives as potential anticancer agents bearing the 3,4,5-trimethoxyphenyl moiety with a sulfur, ketone, or methylene bridging group at position 3 of the indole and with halogen or methoxy substituent(s) at positions 4-7. Compounds 33 and 44 strongly inhibited the growth of the P-glycoprotein-overexpressing multi-drug-resistant cell lines NCI/ADR-RES and Messa/Dx5. At 10 nM, 33 and 44 stimulated the cytotoxic activity of NK cells. At 20-50 nM, 33 and 44 arrested >80% of HeLa cells in the G2/M phase of the cell cycle, with stable arrest of mitotic progression. Cell cycle arrest was followed by cell death. Indoles 33, 44, and 81 showed strong inhibition of the SAG-induced Hedgehog signaling activation in NIH3T3 Shh-Light II cells with IC50 values of 19, 72, and 38 nM, respectively. Compounds of this class potently inhibited tubulin polymerization and cancer cell growth, including stimulation of natural killer cell cytotoxic activity and repression of Hedgehog-dependent cancer.

Pixantrone is a novel aza-anthracenedione active against aggressive lymphoma and is being evaluated for use against various hematologic and solid tumors. The drug is an analog of mitoxantrone, but displays less cardiotoxicity than mitoxantrone or the more commonly used doxorubicin. Although pixantrone is purported to inhibit topoisomerase II activity and intercalate with DNA, exact mechanisms of how it induces cell death remain obscure. Here we evaluated the effect of pixantrone on a panel of solid tumor cell lines to understand its mechanism of cell killing. Initial experiments with pixantrone showed an apparent discrepancy between its anti-proliferative effects in MTS assays (short-term) compared with clonogenic assays (long-term). Using live cell videomicroscopy to track the fates of cells, we found that cells treated with pixantrone underwent multiple rounds of aberrant cell division before eventually dying after approximately 5 d post-treatment. Cells underwent abnormal mitosis in which chromosome segregation was impaired, generating chromatin bridges between cells or within cells containing micronuclei. While pixantrone-treated cells did not display γH2AX foci, a marker of DNA damage, in the main nuclei, such foci were often detected in the micronuclei. Using DNA content analysis, we found that pixantrone concentrations that induced cell death in a clonogenic assay did not impede cell cycle progression, further supporting the lack of canonical DNA damage signaling. These findings suggest pixantrone induces a latent type of DNA damage that impairs the fidelity of mitosis, without triggering DNA damage response or mitotic checkpoint activation, but is lethal after successive rounds of aberrant division.