RESUMEN
Cryptococcus neoformans is an opportunistic, human fungal pathogen which undergoes fascinating switches in cell cycle control and ploidy when it encounters stressful environments such as the human lung. Here we carry out a mechanistic analysis of the spindle checkpoint which regulates the metaphase to anaphase transition, focusing on Mps1 kinase and the downstream checkpoint components Mad1 and Mad2. We demonstrate that Cryptococcus mad1Δ or mad2Δ strains are unable to respond to microtubule perturbations, continuing to re-bud and divide, and die as a consequence. Fluorescent tagging of Chromosome 3, using a lacO array and mNeonGreen-lacI fusion protein, demonstrates that mad mutants are unable to maintain sister-chromatid cohesion in the absence of microtubule polymers. Thus, the classic checkpoint functions of the SAC are conserved in Cryptococcus. In interphase, GFP-Mad1 is enriched at the nuclear periphery, and it is recruited to unattached kinetochores in mitosis. Purification of GFP-Mad1 followed by mass spectrometric analysis of associated proteins show that it forms a complex with Mad2 and that it interacts with other checkpoint signalling components (Bub1) and effectors (Cdc20 and APC/C sub-units) in mitosis. We also demonstrate that overexpression of Mps1 kinase is sufficient to arrest Cryptococcus cells in mitosis, and show that this arrest is dependent on both Mad1 and Mad2. We find that a C-terminal fragment of Mad1 is an effective in vitro substrate for Mps1 kinase and map several Mad1 phosphorylation sites. Some sites are highly conserved within the C-terminal Mad1 structure and we demonstrate that mutation of threonine 667 (T667A) leads to loss of checkpoint signalling and abrogation of the GAL-MPS1 arrest. Thus Mps1-dependent phosphorylation of C-terminal Mad1 residues is a critical step in Cryptococcus spindle checkpoint signalling. We conclude that CnMps1 protein kinase, Mad1 and Mad2 proteins have all conserved their important, spindle checkpoint signalling roles helping ensure high fidelity chromosome segregation.
Asunto(s)
Proteínas de Ciclo Celular , Cryptococcus neoformans , Proteínas Mad2 , Huso Acromático , Cryptococcus neoformans/genética , Cryptococcus neoformans/metabolismo , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas Mad2/metabolismo , Proteínas Mad2/genética , Huso Acromático/metabolismo , Huso Acromático/genética , Transducción de Señal , Proteínas Fúngicas/metabolismo , Proteínas Fúngicas/genética , Humanos , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Serina-Treonina Quinasas/genética , Puntos de Control de la Fase M del Ciclo Celular/genética , Mitosis/genética , Cinetocoros/metabolismo , Segregación Cromosómica/genética , Microtúbulos/metabolismo , Microtúbulos/genética , Proteínas Nucleares/metabolismo , Proteínas Nucleares/genéticaRESUMEN
Citrus macroptera (family Rutaceae), commonly known as Sat Kara, is a pharmacologically diverse medicinal plant. Various parts of this plant, specifically fruit, have an immense range of medicinal uses in folk medicine directed for a number of ailments. A plethora of active phytochemical constituents of this plant have been revealed so far, namely, limonene, beta-caryophyllene, beta-pinene, geranial edulinine, ribalinine, isoplatydesmine, and so forth. Several studies demonstrated the exploration of pharmacological potential of various parts such as fruits, leaves, and stems of C. macroptera as antioxidant, cytotoxic, antimicrobial, thrombolytic, hypoglycemic, anxiolytic, antidepressant, cardioprotective, and hepatoprotective. Furthermore, inhibition of in vitro α-amylase, inhibition of paracetamol induced hepatotoxicity, and potentiation of brain antioxidant enzyme are also ascertained. In present review, comprehensive study focused on knowledge regarding several phytopharmacological activities of Citrus macroptera has been described.