RESUMO
Aurora kinases have recently taken centre stage in the regulation of key cell cycle processes. Aurora A is emerging as a critical regulator of centrosome and spindle function. Aurora B mediates chromosome segregation by ensuring proper biorientation of sister chromatids, possibly through the regulation of microtubule dynamics. This enzyme also functions in cytokinesis apparently by interacting with a critical GTPase and a kinesin-like protein. Recent work on both kinases has revealed functional links between Aurora kinase activity and the mechanics of cell division.
Assuntos
Mitose , Proteínas Quinases/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Animais , Aurora Quinase B , Aurora Quinases , Proteínas de Ciclo Celular , Divisão Celular , Cromátides/química , Cromatina/metabolismo , Drosophila , Sistema da Enzima Desramificadora do Glicogênio/química , Humanos , Cinética , Microscopia de Fluorescência , Microtúbulos , Modelos Biológicos , Xenopus , Proteínas de XenopusRESUMO
The propensity of human embryonic stem cells to die upon enzymatic disaggregation or low-density plating is an obstacle to their isolation and routine use in drug discovery and basic research. Equally, the very low rate of establishment of implanted cells hinders cell therapy. In the present study we have developed a high-content assay for human embryonic stem cell survival and used this to screen a range of libraries of 'lead-like' small molecules and known bioactives. From this we identified 18 confirmed hits with four structural classes being represented by multiple compounds: a series of 5-(acyl/alkyl-amino)indazoles, compounds with a 4-(acylamino)pyridine core, simple N6,N6-dialkyladenines and compounds with a 5-(acylamino)indolinone core. In vitro kinase profiling indicated that the ROCK (Rho-associated kinase)/PRK2 (protein kinase C-related kinase 2) protein kinases are of pivotal importance for cell survival and identified previously unreported compound classes that inhibited this important biological activity. An evaluation using an extensive panel of protein kinases showed that six of our hit compounds exhibited better selectivity for ROCK inhibition than the routinely used commercially available ROCK inhibitor Y-27632. In this screen we also identified the K(+)-ATP channel opener pinacidil and show that it probably promotes cell survival, by 'off-target' inhibition of ROCK/PRK2. We have therefore identified novel pro-survival compounds of greater specificity, equivalent potency and reduced toxicity relative to the routinely employed ROCK inhibitor Y-27632.
Assuntos
Proliferação de Células/efeitos dos fármacos , Células-Tronco Embrionárias/efeitos dos fármacos , Compostos Heterocíclicos/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Amidas/farmacologia , Técnicas de Cultura de Células , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Avaliação Pré-Clínica de Medicamentos/métodos , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/metabolismo , Compostos Heterocíclicos/química , Humanos , Indazóis/química , Indazóis/farmacologia , Estrutura Molecular , Proteína Quinase C/antagonistas & inibidores , Proteína Quinase C/metabolismo , Inibidores de Proteínas Quinases/química , Piridinas/química , Piridinas/farmacologia , Fatores de Tempo , Quinases Associadas a rho/antagonistas & inibidores , Quinases Associadas a rho/metabolismoRESUMO
There is an urgent need to develop new methods for male contraception, however a major barrier to drug discovery has been the lack of validated targets and the absence of an effective high-throughput phenotypic screening system. To address this deficit, we developed a fully-automated robotic screening platform that provided quantitative evaluation of compound activity against two key attributes of human sperm function: motility and acrosome reaction. In order to accelerate contraceptive development, we screened the comprehensive collection of 12,000 molecules that make up the ReFRAME repurposing library, comprising nearly all the small molecules that have been approved or have undergone clinical development, or have significant preclinical profiling. We identified several compounds that potently inhibit motility representing either novel drug candidates or routes to target identification. This platform will now allow for major drug discovery programmes that address the critical gap in the contraceptive portfolio as well as uncover novel human sperm biology.
Assuntos
Anticoncepcionais/farmacologia , Descoberta de Drogas/métodos , Ensaios de Triagem em Larga Escala/métodos , Espermatozoides/efeitos dos fármacos , Espermatozoides/fisiologia , Acrossomo/efeitos dos fármacos , Humanos , Masculino , Fenótipo , Motilidade dos Espermatozoides/efeitos dos fármacosRESUMO
Chromosome orientation and alignment within the mitotic spindle requires the Aurora B protein kinase and the mitotic centromere-associated kinesin (MCAK). Here, we report the regulation of MCAK by Aurora B. Aurora B inhibited MCAK's microtubule depolymerizing activity in vitro, and phospho-mimic (S/E) mutants of MCAK inhibited depolymerization in vivo. Expression of either MCAK (S/E) or MCAK (S/A) mutants increased the frequency of syntelic microtubule-kinetochore attachments and mono-oriented chromosomes. MCAK phosphorylation also regulates MCAK localization: the MCAK (S/E) mutant frequently localized to the inner centromere while the (S/A) mutant concentrated at kinetochores. We also detected two different binding sites for MCAK using FRAP analysis of the different MCAK mutants. Moreover, disruption of Aurora B function by expression of a kinase-dead mutant or RNAi prevented centromeric targeting of MCAK. These results link Aurora B activity to MCAK function, with Aurora B regulating MCAK's activity and its localization at the centromere and kinetochore.
Assuntos
Ciclo Celular/fisiologia , Centrômero/metabolismo , Cinesinas/metabolismo , Mitose/fisiologia , Proteínas Serina-Treonina Quinases/metabolismo , Sequência de Aminoácidos , Animais , Antineoplásicos Fitogênicos/farmacologia , Aurora Quinase B , Aurora Quinases , Autorradiografia , Células CHO , Ciclo Celular/efeitos dos fármacos , Cromatografia Líquida de Alta Pressão , Cromossomos/metabolismo , Cricetinae , Eletroforese em Gel Bidimensional , Recuperação de Fluorescência Após Fotodegradação/métodos , Imunofluorescência/métodos , Proteínas de Fluorescência Verde , Células HeLa , Humanos , Técnicas In Vitro , Cinetocoros/metabolismo , Proteínas Luminescentes/metabolismo , Mitose/efeitos dos fármacos , Modelos Biológicos , Mutação , Nocodazol/farmacologia , Paclitaxel/farmacologia , Fosforilação , RNA Interferente Pequeno/farmacologia , Ratos , TransfecçãoRESUMO
An integral part of cell division is the separation of daughter cells via cytokinesis. There is now good evidence that the completion of cytokinesis requires coordinated membrane trafficking to deliver new membrane to the tip of the furrow and to complete the abscission. Here we have examined membrane traffic in cytokinesis and describe several novel observations. First, we show that Rab11- and FIP3-containing recycling endosomes accumulate near the cleavage furrow and are required for successful completion of cytokinesis. Second, we demonstrate that the Rab11-FIP3 protein complex is intimately involved in the delivery of endosomes to the cleavage furrow. Significantly, although FIP3 recruitment to endosomes is Rab11 dependent, we find that the targeting of FIP3 to the midbody is independent of Rab11. Third, we show that the Rab11-FIP3 complex is required for a late stage of cytokinesis, possibly abscission. Finally, we demonstrate that localization of FIP3 is subject to substantial spatial and temporal regulation. These data provide the first detailed analysis of recycling endosomes in cell division and provide a new model for membrane traffic to the furrow. We propose that the dynamic Rab11-FIP3 interaction controls the delivery, targeting, and fusion of recycling endosomes with furrow during late cytokinesis and abscission.
Assuntos
Membrana Celular/metabolismo , Citocinese , Proteínas de Ligação a DNA/metabolismo , Endossomos/metabolismo , Fatores de Transcrição/metabolismo , Proteínas rab de Ligação ao GTP/metabolismo , Animais , Proteínas de Transporte/metabolismo , Ciclo Celular , Citometria de Fluxo , Transferência Ressonante de Energia de Fluorescência , Células HeLa , Humanos , Quinase I-kappa B , Camundongos , Microscopia Confocal , Microscopia de Vídeo , Células NIH 3T3 , Proteínas Serina-Treonina Quinases/metabolismo , Transporte Proteico , Interferência de RNA , Fatores de Tempo , Fatores Estimuladores UpstreamRESUMO
The small GTPase Ran has multiple roles during the cell division cycle, including nuclear transport, mitotic spindle assembly, and nuclear envelope formation. However, regulation of Ran during cell division is poorly understood. Ran-GTP is generated by the guanine nucleotide exchange factor RCC1, the localization of which to chromosomes is necessary for the fidelity of mitosis in human cells. Using photobleaching techniques, we show that the chromosomal interaction of human RCC1 fused to green fluorescent protein (GFP) changes during progression through mitosis by being highly dynamic during metaphase and more stable toward the end of mitosis. The interaction of RCC1 with chromosomes involves the interface of RCC1 with Ran and requires an N-terminal region containing a nuclear localization signal. We show that this region contains sites phosphorylated by mitotic protein kinases. One site, serine 11, is targeted by CDK1/cyclin B and is phosphorylated in mitotic human cells. Phosphorylation of the N-terminal region of RCC1 inhibits its binding to importin alpha/beta and maintains the mobility of RCC1 during metaphase. This mechanism may be important for the localized generation of Ran-GTP on chromatin after nuclear envelope breakdown and may play a role in the coordination of progression through mitosis.
Assuntos
Proteínas de Ciclo Celular , Cromossomos/metabolismo , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Mitose/fisiologia , Proteínas Nucleares/metabolismo , Proteína ran de Ligação ao GTP/metabolismo , Sequência de Aminoácidos , Animais , Autorradiografia , Cromossomos/fisiologia , Eletroforese em Gel de Poliacrilamida , Recuperação de Fluorescência Após Fotodegradação , Proteínas de Fluorescência Verde , Humanos , Immunoblotting , Carioferinas/metabolismo , Proteínas Luminescentes , Microesferas , Modelos Moleculares , Dados de Sequência Molecular , Fosforilação , Alinhamento de Sequência , Células Tumorais Cultivadas , Xenopus , Proteínas de XenopusRESUMO
Protein phosphatase 1 (PP1) is a ubiquitous serine/threonine phosphatase that regulates many cellular processes, including cell division. When transiently expressed as fluorescent protein (FP) fusions, the three PP1 isoforms, alpha, beta/delta, and gamma1, are active phosphatases with distinct localization patterns. We report here the establishment and characterization of HeLa cell lines stably expressing either FP-PP1gamma or FP alone. Time-lapse imaging reveals dynamic targeting of FP-PP1gamma to specific sites throughout the cell cycle, contrasting with the diffuse pattern observed for FP alone. FP-PP1gamma shows a nucleolar accumulation during interphase. On entry into mitosis, it localizes initially at kinetochores, where it exchanges rapidly with the diffuse cytoplasmic pool. A dramatic relocalization of PP1 to the chromosome-containing regions occurs at the transition from early to late anaphase, and by telophase FP-PP1gamma also accumulates at the cleavage furrow and midbody. The changing spatio-temporal distribution of PP1gamma revealed using the stable PP1 cell lines implicates it in multiple processes, including nucleolar function, the regulation of chromosome segregation and cytokinesis.
Assuntos
Ciclo Celular/fisiologia , Fosfoproteínas Fosfatases/metabolismo , Divisão Celular/fisiologia , Nucléolo Celular/metabolismo , Células HeLa , Humanos , Fosfoproteínas Fosfatases/genética , Proteína Fosfatase 1 , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismoRESUMO
The Aurora kinases have been implicated in tumorigenesis and are important regulators of diverse cell cycle events, ranging from the entry into mitosis, centrosome function, mitotic spindle formation, chromosome biorientation and segregation, and cytokinesis. The recent identification of novel binding partners and key downstream effectors, together with new small-molecule inhibitors that display efficacy against tumours, heralds an upsurge of interest in these critical kinases. This review details new developments in the field and analyses the potential of Aurora kinases as anticancer targets.
Assuntos
Antineoplásicos/uso terapêutico , Transformação Celular Neoplásica , Neoplasias/prevenção & controle , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Serina-Treonina Quinases/uso terapêutico , Animais , Aurora Quinases , Ciclo Celular , Centrossomo/ultraestrutura , Cromossomos/ultraestrutura , Humanos , MitoseRESUMO
Pluripotent stem cells promise to revolutionize drug discovery and offer new modes of therapy. The ease with which they can be grown in bulk and their differentiation controlled in vitro is of importance for their widespread adoption by industry and their clinical efficacy. Small molecules have already had a positive impact on several areas of stem cell biology, from maintenance of pluripotency, the promotion of single cell survival and steering differentiation to involvement in reprogramming somatic cells. High-throughput technology has played an important role in identifying novel compounds, however to date there are few published examples of medicinal chemistry input in this area. This review discusses the potential of pluripotent stem cells, the successful uses of small molecules and future prospects.
Assuntos
Bibliotecas de Moléculas Pequenas/farmacologia , Células-Tronco/efeitos dos fármacos , Diferenciação Celular , Sobrevivência Celular , Reprogramação Celular , Avaliação Pré-Clínica de Medicamentos , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/efeitos dos fármacos , Humanos , Células-Tronco Pluripotentes/citologia , Células-Tronco Pluripotentes/efeitos dos fármacos , Medicina Regenerativa , Bibliotecas de Moléculas Pequenas/química , Células-Tronco/citologiaRESUMO
Survivin operates in a complex with aurora B kinase and is phosphorylated by it on threonine 117 in vitro. Here we ask whether phosphorylation of survivin by aurora B kinase regulates its function during mitosis in vivo. Using a phospho-specific antibody we first establish that survivin is phosphorylated at T117 during mitosis and is present at the midbody during cytokinesis. Next we use two independent RNAi complementation approaches to investigate threonine 117 mutants in survivin depleted cells. Our data suggest that while non-phosphorylatable survivin, survivin(T117A), can substitute for the wild type protein, a phosphomimic, survivin(T117E) cannot restore viability, nor can it complement chromosome congression and spindle checkpoint defects that arise due to depletion of endogenous survivin. Fluorescence imaging and fluorescence recovery after photobleaching analysis suggest that the phosphomimic has reduced affinity for centromeres compared with the non-phosphorylatable form. We conclude that survivin is phosphorylated at T117 during mitosis, and once phosphorylated, dephosphorylation is crucial for chromosome congression and progression into anaphase.
Assuntos
Proteínas Associadas aos Microtúbulos/metabolismo , Mitose/fisiologia , Proteínas de Neoplasias/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Aurora Quinase B , Aurora Quinases , Linhagem Celular Tumoral , Centrômero/metabolismo , Recuperação de Fluorescência Após Fotodegradação , Humanos , Immunoblotting , Proteínas Inibidoras de Apoptose , Microscopia de Fluorescência , Proteínas Associadas aos Microtúbulos/genética , Mutação/genética , Proteínas de Neoplasias/genética , Fosforilação , Interferência de RNA , SurvivinaAssuntos
Células/metabolismo , Processamento de Imagem Assistida por Computador/métodos , Engenharia Tecidual , Animais , Biomarcadores/metabolismo , Técnicas de Cultura de Células , Sobrevivência Celular/fisiologia , Células/citologia , Células Cultivadas , Proteínas de Fluorescência Verde/metabolismo , Substâncias Luminescentes/metabolismoAssuntos
Fenômenos Fisiológicos Celulares , Células/citologia , Processamento de Imagem Assistida por Computador/métodos , Microscopia de Fluorescência/métodos , Animais , Sobrevivência Celular , Células/metabolismo , Células Cultivadas , Imageamento Tridimensional , Microscopia de Fluorescência/instrumentação , Engenharia TecidualRESUMO
Digital fluorescence microscopy is now a standard technology for assaying molecular localisation in cells and tissues. The choice of laser scanning (LSM) and wide-field microscopes (WFM) largely depends on the type of sample, with LSMs performing best on thick samples and WFMs performing best on thin ones. These systems are increasingly used to collect large multidimensional datasets. We propose a unified image structure that considers space, time, and fluorescence wavelength as integral parts of the image. Moreover, the application of fluorescence imaging to large-scale screening means that large datasets are now routinely acquired. We propose that analysis of these data requires querying tools based on relational databases and describe one such system.
Assuntos
Microscopia Confocal/métodos , Microscopia de Fluorescência/métodos , Animais , Biologia Computacional , Bases de Dados como Assunto , Microscopia Confocal/instrumentação , Microscopia de Fluorescência/instrumentação , Fótons , SoftwareRESUMO
The outer epidermal layer of the skin is an epithelium with remarkable protective barrier functions, which is subject to pronounced physical stress in its day-to-day function. A major candidate component for absorbing this stress is the K5/K14 keratin intermediate filament network. To investigate the part played by keratins in stress resilience, keratinocyte cell lines were subjected to mechanical stress. Repeated stretch and relaxation cycles over increasing time produced reproducible changes in the configuration of the keratin network. When wild-type cells were compared with cells carrying a keratin mutation associated with severe epidermolysis bullosa simplex-type skin fragility, the mutant keratin filaments were unable to withstand the mechanical stress and progressively fragmented yielding aggregates and novel ring structures. The cell junctions into which the keratin filaments are normally anchored also progressively disassembled, with all components tested of the cytoplasmic plaques becoming relocated away from the membrane and onto the keratin rings, while integral membrane receptors integrins and cadherins remained at the plasma membrane. The results suggest that maintenance of desmosomes and hemidesmosomes may require some tension, normally mediated by keratin attachments.
Assuntos
Epidermólise Bolhosa Simples/metabolismo , Queratinócitos/metabolismo , Queratinas/química , Linhagem Celular , Membrana Celular/metabolismo , Fragmentação do DNA , Desmossomos/metabolismo , Humanos , Imuno-Histoquímica , Queratinas/genética , Queratinas/metabolismo , Microscopia Eletrônica de Transmissão , Microscopia de Fluorescência , Modelos Biológicos , Mutação , Estrutura Terciária de Proteína , Estresse MecânicoRESUMO
Toxoplasma gondii is an intracellular parasite that proliferates within most nucleated cells, an important human pathogen, and a model for the study of human and veterinary parasitic infections. We used a stable yellow fluorescent protein-alpha-tubulin transgenic line to determine the structure of the microtubule cytoskeleton in T. gondii. Imaging of living yellow fluorescent protein-alpha-tubulin parasites by laser-scanning confocal microscopy (LSCM) failed to resolve the 22 subpellicular microtubules characteristic of the parasite cytoskeleton. To understand this result, we analyzed sources of noise in the LSCM and identified illumination fluctuations on time scales from microseconds to hours that introduce significant amounts of noise. We confirmed that weakly fluorescent structures could not be imaged in LSCM by using fluorescent bead standards. By contrast, wide-field microscopy (WFM) did visualize weak fluorescent standards and the individual microtubules of the parasite cytoskeleton. We therefore measured the fluorescence per unit length of microtubule by using WFM and used this information to estimate the tubulin content of the conoid (a structure important for T. gondii infection) and in the mitotic spindle pole. The conoid contains sufficient tubulin for approximately 10 microtubule segments of 0.5-microm length, indicating that tubulin forms the structural core of the organelle. We also show that the T. gondii mitotic spindle contains approximately 1 microtubule per chromosome. This analysis expands the understanding of structures used for invasion and intracellular proliferation by an important human pathogen and shows the advantage of WFM combined with image deconvolution over LSCM for quantitative studies of weakly fluorescent structures in moderately thin living cells.
Assuntos
Microscopia Confocal/métodos , Microscopia de Fluorescência/métodos , Toxoplasma/metabolismo , Tubulina (Proteína)/metabolismo , Tubulina (Proteína)/ultraestrutura , Algoritmos , Animais , Animais Geneticamente Modificados , Separação Celular , Citometria de Fluxo , Microtúbulos/ultraestrutura , Codorniz , Proteínas Recombinantes de Fusão/metabolismo , Fatores de TempoRESUMO
Sla1p is a protein required for cortical actin patch structure and organisation in budding yeast. Here we use a combination of immunofluorescence microscopy and biochemical approaches to demonstrate interactions of Sla1p both with proteins regulating actin dynamics and with proteins required for endocytosis. Using Sla1p-binding studies we reveal association of Sla1p with two proteins known to be important for activation of the Arp2/3 complex in yeast, Abp1p and the yeast WASP homologue Las17p/Bee1p. A recent report of Sla1p association with Pan1p puts Sla1p in the currently unique position of being the only yeast protein known to interact with all three known Arp2/3-activating proteins in yeast. Localisation of Sla1p at the cell cortex is, however, dependent on the EH-domain-containing protein End3p, which is part of the yeast endocytic machinery. Using spectral variants of GFP on Sla1p (YFP) and on Abp1p (CFP) we show for the first time that these proteins can exist in discrete complexes at the cell cortex. However, the detection of a significant FRET signal means that these proteins also come close together in a single complex, and it is in this larger complex that we propose that Sla1p binding to Abp1p and Las17p/Bee1p is able to link actin dynamics to the endocytic machinery. Finally, we demonstrate marked defects in both fluid-phase and receptor-mediated endocytosis in cells that do not express SLA1, indicating that Sla1p is central to the requirement in yeast to couple endocytosis with the actin cytoskeleton.