mRNA spindle localization and mitotic translational regulation by CPEB1 and CPEB4

Transition through cell cycle phases requires temporal and spatial regulation of gene expression to ensure accurate chromosome duplication and segregation. This regulation involves dynamic reprogramming of gene expression at multiple transcriptional and posttranscriptional levels. In transcriptionally silent oocytes, the CPEB-family of RNA-binding proteins coordinates temporal and spatial translation regulation of stored maternal mRNAs to drive meiotic progression. CPEB1 mediates mRNA localization to the meiotic spindle, which is required to ensure proper chromosome segregation. Temporal translational regulation also takes place in mitosis, where a large repertoire of transcripts is activated or repressed in specific cell cycle phases. However, whether control of localized translation at the spindle is required for mitosis is unclear, as mitotic and acentriolar-meiotic spindles are functionally and structurally different. Furthermore, the large differences in scale-ratio between cell volume and spindle size in oocytes compared to somatic mitotic cells may generate distinct requirements for gene expression compartmentalization in meiosis and mitosis. Here we show that mitotic spindles contain CPE-localized mRNAs and translating ribosomes. Moreover, CPEB1 and CPEB4 localize in the spindles and they may function sequentially in promoting mitotic stage transitions and correct chromosome segregation. Thus, CPEB1 and CPEB4 bind to specific spindle-associated transcripts controlling the expression and/or localization of their encoded factors that, respectively, drive metaphase and anaphase/cytokinesis.Fil: Pascual, Rosa. Barcelona Institute Of Science And Technology.; EspañaFil: Segura Morales, Carolina. Barcelona Institute Of Science And Technology.; EspañaFil: Omerzu, Manja. University of Utrecht; Países BajosFil: Bellora, Nicolás. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Patagonia Norte. Instituto Andino Patagónico de Tecnologías Biológicas y Geoambientales. Universidad Nacional del Comahue. Instituto Andino Patagónico de Tecnologías Biológicas y Geoambientales; ArgentinaFil: Belloc, Eulàlia. Barcelona Institute Of Science And Technology.; EspañaFil: Castellazzi, Chiara Lara. Barcelona Institute Of Science And Technology.; EspañaFil: Reina, Oscar. Barcelona Institute Of Science And Technology.; EspañaFil: Eyras, Eduardo. Universitat Pompeu Fabra; España. Institució Catalana de Recerca i Estudis Avançats; EspañaFil: Maurice, Madelon M.. University of Utrecht; Países BajosFil: Millanes Romero, Alba. Barcelona Institute Of Science And Technology.; EspañaFil: Méndez, Raúl. Barcelona Institute Of Science And Technology.; Españ

Conditional targeting of MAD1 to kinetochores is sufficient to reactivate the spindle assembly checkpoint in metaphase

Fidelity of chromosome segregation is monitored by the spindle assembly checkpoint (SAC). Key components of the SAC include MAD1, MAD2, BUB1, BUB3, BUBR1, and MPS1. These proteins accumulate on kinetochores in early prometaphase but are displaced when chromosomes attach to microtubules and/or biorient on the mitotic spindle. As a result, stable attachment of the final chromosome satisfies the SAC, permitting activation of the anaphase promoting complex/cyclosome (APC/C) and subsequent anaphase onset. SAC satisfaction is reversible, however, as addition of taxol during metaphase stops cyclin B1 degradation by the APC/C. We now show that targeting MAD1 to kinetochores during metaphase is sufficient to reestablish SAC activity after initial silencing. Using rapamycin-induced heterodimerization of FKBP-MAD1 to FRB-MIS12 and live monitoring of cyclin B1 degradation, we show that timed relocalization of MAD1 during metaphase can stop cyclin B1 degradation without affecting chromosome-spindle attachments. APC/C inhibition represented true SAC reactivation, as FKBP-MAD1 required an intact MAD2-interaction motif and MPS1 activity to accomplish this. Our data show that MAD1 kinetochore localization dictates SAC activity and imply that SAC regulatory mechanisms downstream of MAD1 remain functional in metaphase. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s00412-014-0458-9) contains supplementary material, which is available to authorized users

Aristotle as a misogynist

Aristotel je ženske velikokrat označil kot manjvredne ter podrejene moškim, ta vidik pa je še posebej izražen v njegovem delu Politika. V ospredju pa je predvsem sedem seksističnih trditev, ki jih je moč zaslediti na področju Aristotelove biologije: (1) ženska je nepopoln moški(2) ženska predstavlja odklon od oblike(3) najbolj idealen potomec je sin, podoben svojemu očetu(4) ženska se anatomsko razlikuje od moškega(5) moški so tisti, ki vladajo, ženske pa tiste, ki ubogajo(6) vloga žensk v razmnoževanju je podrejena ter (7) ženske so sposobne resničnega mnenja, le moški pa védenja. Njegova stališča so naletela na močan odziv s strani kritikov, ki Aristotela označujejo kot enega največjih seksistov, na drugi strani pa številni avtorji navajajo, da (predvsem njegova biologija) ne temelji na seksističnih predsodkih. V diplomskem delu smo z metodo komparativne analize prikazali splošen pregled omenjene tematike, pri čemer smo v prvem delu obravnavali področje biologije, ta pogled pa smo v drugem delu dopolnili še z Aristotelovimi trditvami na področju politike in metafizike. Za Aristotela je ženska nežnejši, manj pogumen spol, ki se podreja moškemu. Ker so ženske sposobne le resničnega mnenja, je filozofija rezervirana izključno za moške. Manjvredna pa je tudi vloga žensk v razmnoževanju, saj prispevajo le materialni vzrok. Pokazali smo, da kljub njegovim izjemnim dognanjem ne le na področju biologije, temveč v celotni filozofiji, Aristotel žensko smatra kot neenakovredno in podrejeno moškemu.Aristotle often described women as inferior and subordinate to men, this point of view in particular is evident in his work Politics. There are numerous sexist statements in the foreground which can be found in the field of Aristotle\u27s biology: (1) a female is a a disabled male(2) females represent a deviation from the kind(3) the ideal offspring is a male that perfectly resembles his father(4) male and female are anatomically different(5) the relation of male to female is ruler to ruled(6) women\u27s role in reproduction is inferior(7) women are capable of a true opinion while males have the knowledge. His views were met with strong responses from the critics which describe Aristotle as one of the greatest sexists, on the other hand, many authors state (his biology in particular) is not based on sexist prejudices. In the diploma we showed a general overview of the themes mentioned above, with the method of comparative analysis, where we discussed the field of biology in the first part of the diploma and we supplemented this view with Aristotle\u27s statements on politics and metaphysics. For Aristotle, a female is a gentle, less brave sex that subjects herself to a male\u27s rule. Because women are capable only of a true opinion, the philosophy is reserved exclusively for men. The role of women in reproduction is also inferior to men because they contribute only a material cause. We have shown that despite his exceptionality not only in the field of biology but in the entire philosophy, Aristotle considered women as unequal to men

Mechanisms of RNA processing in the Wnt signalling pathway

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Notes on the gregarines (Protozoa: Apicomplexa: Eugregarinorida) of insects in Slovenia

Gregarine so parazitske praživali, ki jih sistematsko uvrščamo v deblo Apicomplexa. Njihov razvoji krog poteka znotraj enega nevretenčarskega gostitelja, kjer se gregarine pritrdijo na epitelne celice črevesja, po stadiju razmnoževanja pa se v obliki oocist izločijo na prosto. V diplomskem delu smo predstavili vrste gregarin, ki zajedajo v žuželkah v Sloveniji. Preiskali smo 80 potencialnih vrst gostiteljev, ki jih uvrščamo med Odonata, Orthoptera, Dermaptera, Blattodea, Hemiptera, Coleoptera, Neuroptera, Hymenoptera, Lepidoptera in Diptera. Med navedenimi redovi žuželk so gregarine vsebovali naslednji: Orthoptera, Dermaptera, Blattodea, Coleoptera ter Neuroptera. Gregarine smo identificirali do vrst na osnovi morfometrije. Zabeležili smo 14 vrst gregarin, ki jih uvrščamo v rodove Gregarina, Hirmocysits, Gamocystis, Hyalospora, Leidyana in Actinocephalus. Pokazali smo, da je stopnja okuženosti med gregarnimi žuželkami dokaj visoka ter da tudi za slovenske gregarine velja rodovna specifičnost.The gregarines are parasitic Protozoa that are sistematically classified in the phylum Apicomplexa. Their development takes place within one of the invertebrate hosts, where gregarines attach themselves to the epithelial cells of the intestine and after the reproductive stadium they get extracted in the open in the form of the occyst. In the diploma we have described the gregarine species that live in the insects, in Slovenia. We have examined 80 species of the potential hosts which are classified among the Odonata, Orthoptera, Dermaptera, Blattodea, Hemiptera, Coleoptera, Neuroptera, Hymenoptera, Lepidoptera and Diptera. Among listed orders of insects, the following contained gregarines: Orthoptera, Dermaptera, Blattodea, Coleoptera and Neuroptera. The gregarines were identified to the species level with the help of the morphometry. We have recorded fourteen gregarine species which belong to the genera Gregarina, Hirmocysits, Gamocystis, Hyalospora, Leidyana and Actinocephalus.. We have shown that the rate of the infection among insects infested with gregarines is quite high and that also applies to the Slovenian generic gregarine specifity

Mechanisms of RNA processing in the Wnt signalling pathway

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Mapping the synthetic dosage lethality network of CDK1/CDC28

Cdk1 (Cdc28 in yeast) is a cyclin-dependent kinase (CDK) essential for cell cycle progression and cell division in normal cells. However, CDK activity also underpins proliferation of tumor cells, making it a relevant study subject. While numerous targets and processes regulated by Cdc28 have been identified, the exact functions of Cdc28 are only partially understood. To further explore the functions of Cdc28, we systematically overexpressed ∼4800 genes in wild-type (WT) cells and in cells with artificially reduced Cdc28 activity. This screen identified 366 genes that, when overexpressed, specifically compromised cell viability under conditions of reduced Cdc28 activity. Consistent with the crucial functions of Cdc28 in cell cycle regulation and chromosome metabolism, most of these genes have functions in the cell cycle, DNA replication, and transcription. However, a substantial number of genes control processes not directly associated with the cell cycle, indicating that Cdc28 may also regulate these processes. Finally, because the dataset was enriched for direct Cdc28 targets, the results from this screen will aid in identifying novel targets and process regulated by Cdc28

Three-dimensional analysis of single molecule FISH in human colon organoids

The culturing of mini-organs (organoids) in three-dimensions (3D) presents a simple and powerful tool to investigate the principles underlying human organ development and tissue self-organization in both healthy and diseased states. Applications of single molecule analysis are highly informative for a comprehensive understanding of the complexity underlying tissue and organ physiology. To fully exploit the potential of single molecule technologies, the adjustment of protocols and tools to 3D tissue culture is required. Single molecule RNA fluorescence in situ hybridization (smFISH) is a robust technique for visualizing and quantifying individual transcripts. In addition, smFISH can be employed to study splice variants, fusion transcripts as well as transcripts of multiple genes at the same time. Here, we develop a 3-day protocol and validation method to perform smFISH in 3D in whole human organoids.We provide a number of applications to exemplify the diverse possibilities for the simultaneous detection of distinctmRNAtranscripts, evaluation of their spatial distribution and the identification of divergent cell lineages in 3D in organoids

Sequential multisite phospho-regulation of KNL1-BUB3 interfaces at mitotic kinetochores

Regulated recruitment of the kinase-adaptor complex BUB1/BUB3 to kinetochores is crucial for correcting faulty chromosome-spindle attachments and for spindle assembly checkpoint (SAC) signaling. BUB1/BUB3 localizes to kinetochores by binding phosphorylated MELT motifs (MELpT) in the kinetochore scaffold KNL1. Human KNL1 has 19 repeats that contain a MELT-like sequence. The repeats are, however, larger than MELT, and repeat sequences can vary significantly. Using systematic screening, we show that only a limited number of repeats is "active." Repeat activity correlates with the presence of a vertebrate-specific SHT motif C-terminal to the MELT sequence. SHT motifs are phosphorylated by MPS1 in a manner that requires prior phosphorylation of MELT. Phospho-SHT (SHpT) synergizes with MELpT in BUB3/BUB1 binding in vitro and in cells, and human BUB3 mutated in a predicted SHpT-binding surface cannot localize to kinetochores. Our data show sequential multisite regulation of the KNL1-BUB1/BUB3 interaction and provide mechanistic insight into evolution of the KNL1-BUB3 interface