RESUMEN
During the cell cycle, DNA duplication in S phase must occur before a cell divides in mitosis. In the intervening G2 phase, mitotic inducers accumulate, which eventually leads to a switch-like rise in mitotic kinase activity that triggers mitotic entry. However, when and how activation of the signaling network that promotes the transition to mitosis occurs remains unclear. We have developed a system to reduce cell-cell variation and increase accuracy of fluorescence quantification in single cells. This allows us to use immunofluorescence of endogenous marker proteins to assess kinetics from fixed cells. We find that mitotic phosphorylations initially occur at the completion of S phase, showing that activation of the mitotic entry network does not depend on protein accumulation through G2. Our data show insights into how mitotic entry is linked to the completion of S phase and forms a quantitative resource for mathematical models of the human cell cycle.
Asunto(s)
Fase G2/genética , Mitosis/genética , Fase S/genética , Proteínas Bacterianas/química , Ciclo Celular , Línea Celular Tumoral , Centrosoma/metabolismo , Replicación del ADN , Fibronectinas/química , Marcadores Genéticos , Humanos , Procesamiento de Imagen Asistido por Computador , Cinética , Cinetocoros/química , Proteínas Luminiscentes/química , Microscopía Fluorescente , Modelos Teóricos , Fosforilación , ARN Interferente Pequeño/metabolismo , Factores de TiempoRESUMEN
Some oomycetes, for instance Saprolegnia parasitica, are severe fish pathogens that cause important economic losses worldwide. Cellulose biosynthesis is a vital process for this class of microorganisms, but the corresponding molecular mechanisms are poorly understood. Of all cellulose synthesizing enzymes known, only some oomycete cellulose synthases contain a pleckstrin homology (PH) domain. Some human PH domains bind specifically to phosphoinositides, but most PH domains bind phospholipids in a non-specific manner. In addition, some PH domains interact with various proteins. Here we have investigated the function of the PH domain of cellulose synthase 2 from the oomycete Saprolegnia monoica (SmCesA2), a species closely related to S. parasitica. The SmCesA2 PH domain is similar to the C-terminal PH domain of the human protein TAPP1. It binds in vitro to phosphoinositides, F-actin and microtubules, and co-localizes with F-actin in vivo. Our results suggest a role of the SmCesA2 PH domain in the regulation, trafficking and/or targeting of the cell wall synthesizing enzyme.
Asunto(s)
Proteínas Sanguíneas/química , Glucosiltransferasas/química , Péptidos y Proteínas de Señalización Intracelular/química , Proteínas de la Membrana/química , Fosfoproteínas/química , Saprolegnia/enzimología , Actinas/metabolismo , Secuencia de Aminoácidos , Proteínas Sanguíneas/metabolismo , Línea Celular Tumoral , Biología Computacional , Glucosiltransferasas/metabolismo , Humanos , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteínas de la Membrana/metabolismo , Microtúbulos/metabolismo , Datos de Secuencia Molecular , Fosfatidilinositoles/química , Fosfatidilinositoles/metabolismo , Fosfoproteínas/metabolismo , Estructura Terciaria de Proteína , Alineación de SecuenciaRESUMEN
Cdk1 and Plk1/Plx1 activation leads to their inactivation through negative feedback loops. Cdk1 deactivates itself by activating the APC/C, consequently generating embryonic cell cycle oscillations. APC/C inhibition by the mitotic checkpoint in somatic cells and the cytostatic factor (CSF) in oocytes sustain the mitotic state. Plk1/Plx1 targets its co-activator Bora for degradation, but it remains unclear how embryonic oscillations in Plx1 activity are generated, and how Plk1/Plx1 activity is sustained during mitosis. We show that Plx1-mediated degradation of Bora in interphase generates oscillations in Plx1 activity and is essential for development. In CSF extracts, phosphorylation of Bora on the Cdk consensus site T52 blocks Bora degradation. Upon fertilization, Calcineurin dephosphorylates T52, triggering Plx1 oscillations. Similarly, we find that GFP-Bora is degraded when Plk1 activity spreads to somatic cell cytoplasm before mitosis. Interestingly, GFP-Bora degradation stops upon mitotic entry when Cdk1 activity is high. We hypothesize that Cdk1 controls Bora through an incoherent feedforward loop synchronizing the activities of mitotic kinases.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Quinasas Ciclina-Dependientes/metabolismo , Mitosis/fisiología , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Animales , Proteína Quinasa CDC2 , Transferencia Resonante de Energía de Fluorescencia , Células HEK293 , Humanos , Immunoblotting , Inmunoprecipitación , Mutagénesis Sitio-Dirigida , Fosforilación , Estabilidad Proteica , Proteínas Proto-Oncogénicas c-mos/metabolismo , Xenopus laevis , Quinasa Tipo Polo 1RESUMEN
Mitosis is an attractive target for the development of new anticancer drugs. In a search for novel mitotic inhibitors, we virtually screened for low molecular weight compounds that would possess similar steric and electrostatic features, but different chemical structure than rigosertib (ON 01910.Na), a putative inhibitor of phosphoinositide 3-kinase (PI3K) and polo-like kinase 1 (Plk1) pathways. Highest scoring hit compounds were tested in cell-based assays for their ability to induce mitotic arrest. We identified a novel acridinyl-acetohydrazide, here named as Centmitor-1 (Cent-1), that possesses highly similar molecular interaction field as rigosertib. In cells, Cent-1 phenocopied the cellular effects of rigosertib and caused mitotic arrest characterized by chromosome alignment defects, multipolar spindles, centrosome fragmentation, and activated spindle assembly checkpoint. We compared the effects of Cent-1 and rigosertib on microtubules and found that both compounds modulated microtubule plus-ends and reduced microtubule dynamics. Also, mitotic spindle forces were affected by the compounds as tension across sister kinetochores was reduced in mitotic cells. Our results showed that both Cent-1 and rigosertib target processes that occur during mitosis as they had immediate antimitotic effects when added to cells during mitosis. Analysis of Plk1 activity in cells using a Förster resonance energy transfer (FRET)-based assay indicated that neither compound affected the activity of the kinase. Taken together, these findings suggest that Cent-1 and rigosertib elicit their antimitotic effects by targeting mitotic processes without impairment of Plk1 kinase activity.
Asunto(s)
Acridonas/farmacología , Antimitóticos/farmacología , Glicina/análogos & derivados , Hidrazinas/farmacología , Sulfonas/farmacología , Acridonas/química , Antimitóticos/química , Puntos de Control del Ciclo Celular/efectos de los fármacos , Proteínas de Ciclo Celular/antagonistas & inhibidores , Centrosoma/metabolismo , Ensayos de Selección de Medicamentos Antitumorales , Glicina/química , Glicina/farmacología , Células HeLa , Ensayos Analíticos de Alto Rendimiento , Humanos , Hidrazinas/química , Microtúbulos/metabolismo , Mitosis/efectos de los fármacos , Estructura Molecular , Peso Molecular , Inhibidores de las Quinasa Fosfoinosítidos-3 , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas Proto-Oncogénicas/antagonistas & inhibidores , Sulfonas/química , Quinasa Tipo Polo 1RESUMEN
Förster resonance energy transfer (FRET)-based reporters(1) allow the assessment of endogenous kinase and phosphatase activities in living cells. Such probes typically consist of variants of CFP and YFP, intervened by a phosphorylatable sequence and a phospho-binding domain. Upon phosphorylation, the probe changes conformation, which results in a change of the distance or orientation between CFP and YFP, leading to a change in FRET efficiency (Fig 1). Several probes have been published during the last decade, monitoring the activity balance of multiple kinases and phosphatases, including reporters of PKA(2), PKB(3), PKC(4), PKD(5), ERK(6), JNK(7), Cdk(18), Aurora B(9) and Plk1(9). Given the modular design, additional probes are likely to emerge in the near future(10). Progression through the cell cycle is affected by stress signaling pathways( 11). Notably, the cell cycle is regulated differently during unperturbed growth compared to when cells are recovering from stress(12).Time-lapse imaging of cells through the cell cycle therefore requires particular caution. This becomes a problem particularly when employing ratiometric imaging, since two images with a high signal to noise ratio are required to correctly interpret the results. Ratiometric FRET imaging of cell cycle dependent changes in kinase and phosphatase activities has predominately been restricted to sub-sections of the cell cycle(8,9,13,14). Here, we discuss a method to monitor FRET-based probes using ratiometric imaging throughout the human cell cycle. The method relies on equipment that is available to many researchers in life sciences and does not require expert knowledge of microscopy or image processing.