Meiotic spindles are critical to ensure chromosome segregation during gamete formation. Oocytes lack centrosomes and use alternative microtubule-nucleation mechanisms for spindle building. How these mechanisms are regulated is still unknown. Aurora kinase A (AURKA) is essential for mouse oocyte meiosis because in pro-metaphase I it triggers microtubule organizing-center fragmentation and its expression compensates for the loss of the two other Aurora kinases (AURKB/AURKC). Although knockout mouse models were useful for foundational studies, AURK spatial and temporal functions are not yet resolved. We provide high-resolution analyses of AURKA/AURKC requirements during meiotic spindle-building and identify the subcellular populations that carry out these functions: 1) AURKA is required in early spindle assembly and later for spindle stability, whereas 2) AURKC is required in late pro-metaphase, and 3) Targeted AURKA constructs expressed in triple AURK knockout oocytes reveal that spindle pole-localized AURKA is the most important population controlling spindle building and stability mechanisms.

• Klíčová slova
• cell biology,
• Publikační typ
• časopisecké články MeSH

Homologous chromosome segregation during meiosis I (MI) in mammalian oocytes is carried out by the acentrosomal MI spindles. Whereas studies in human oocytes identified Ran GTPase as a crucial regulator of the MI spindle function, experiments in mouse oocytes questioned the generality of this notion. Here, we use live-cell imaging with fluorescent probes and Förster resonance energy transfer (FRET) biosensors to monitor the changes in Ran and importin β signaling induced by perturbations of Ran in mouse oocytes while examining the MI spindle dynamics. We show that unlike RanT24N employed in previous studies, a RanT24N, T42A double mutant inhibits RanGEF without perturbing cargo binding to importin β and disrupts MI spindle function in chromosome segregation. Roles of Ran and importin β in the coalescence of microtubule organizing centers (MTOCs) and MI spindle assembly are further supported by the use of the chemical inhibitor importazole, whose effects are partially rescued by the GTP hydrolysis-resistant RanQ69L mutant. These results indicate that RanGTP is essential for MI spindle assembly and function both in humans and mice.

• Klíčová slova
• RanGTP, importazole, importin β, meiosis I, oocyte,
• MeSH
• aparát dělícího vřeténka fyziologie   MeSH
• beta karyoferiny genetika   metabolismus   MeSH
• jaderné proteiny genetika   metabolismus   MeSH
• meióza fyziologie   MeSH
• mikrotubuly metabolismus   MeSH
• mutace MeSH
• myši MeSH
• oocyty cytologie   metabolismus   MeSH
• proteiny buněčného cyklu genetika   metabolismus   MeSH
• Ran protein vázající GTP genetika   metabolismus   MeSH
• segregace chromozomů MeSH
• výměnné faktory guaninnukleotidů genetika   metabolismus   MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH
• Názvy látek
• beta karyoferiny MeSH
• jaderné proteiny MeSH
• proteiny buněčného cyklu MeSH
• Ran protein vázající GTP MeSH
• Rcc1 protein, mouse MeSH Prohlížeč
• výměnné faktory guaninnukleotidů MeSH

Meiotic oocytes lack classic centrosomes and, therefore, bipolar spindle assembly depends on clustering of acentriolar microtubule-organizing centers (MTOCs) into two poles. However, the molecular mechanism regulating MTOC assembly into two poles is not fully understood. The kinase haspin (also known as GSG2) is required to regulate Aurora kinase C (AURKC) localization at chromosomes during meiosis I. Here, we show that inhibition of haspin perturbed MTOC clustering into two poles and the stability of the clustered MTOCs. Furthermore, we show that AURKC localizes to MTOCs in mouse oocytes. Inhibition of haspin perturbed the localization of AURKC at MTOCs, and overexpression of AURKC rescued the MTOC-clustering defects in haspin-inhibited oocytes. Taken together, our data uncover a role for haspin as a regulator of bipolar spindle assembly by regulating AURKC function at acentriolar MTOCs in oocytes.

• Klíčová slova
• Aurora kinase, Haspin, MTOC, Oocyte, Spindle,
• MeSH
• aparát dělícího vřeténka metabolismus   MeSH
• aurora kinasa C metabolismus   MeSH
• intracelulární signální peptidy a proteiny antagonisté a inhibitory   metabolismus   MeSH
• metafáze MeSH
• myši MeSH
• oocyty metabolismus   MeSH
• organizační centrum mikrotubulů metabolismus   MeSH
• protein-serin-threoninkinasy antagonisté a inhibitory   metabolismus   MeSH
• transport proteinů MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• aurora kinasa C MeSH
• Haspin protein, mouse MeSH Prohlížeč
• intracelulární signální peptidy a proteiny MeSH
• protein-serin-threoninkinasy MeSH

Proper assembly of the spindle apparatus is crucially important for faithful chromosome segregation during anaphase. Thanks to the effort over the last decades, we have very detailed information about many events leading to spindle assembly and chromosome segregation, however we still do not understand certain aspects, including, for example, spindle length control. When tight regulation of spindle size is lost, chromosome segregation errors emerge. Currently, there are several hypotheses trying to explain the molecular mechanism of spindle length control. The number of kinetochores, activity of molecular rulers, intracellular gradients, cell size, limiting spindle components, and the balance of the spindle forces seem to contribute to spindle size regulation, however some of these mechanisms are likely specific to a particular cell type. In search for a general regulatory mechanism, in our study we focused on the role of cell size and nuclear to cytoplasmic ratio in this process. To this end, we used relatively large cells isolated from 2-cell mouse embryos. Our results showed that the spindle size upper limit is not reached in these cells and suggest that accurate control of spindle length requires balanced ratio between nuclear and cytoplasmic volumes.

• MeSH
• aparát dělícího vřeténka metabolismus   MeSH
• cytoplazma metabolismus   MeSH
• jaderné proteiny MeSH
• metafáze MeSH
• myši inbrední ICR MeSH
• myši MeSH
• partenogeneze MeSH
• proteiny asociované s mikrotubuly MeSH
• proteiny buněčného cyklu MeSH
• velikost buněčného jádra * MeSH
• velikost buňky MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• jaderné proteiny MeSH
• proteiny asociované s mikrotubuly MeSH
• proteiny buněčného cyklu MeSH
• TPX2 protein, mouse MeSH Prohlížeč

Aurora kinase A (AURKA), which is a centrosome-localized serine/threonine kinase crucial for cell cycle control, is critically involved in centrosome maturation and spindle assembly in somatic cells. Active T288 phosphorylated AURKA localizes to the centrosome in the late G(2) and also spreads to the minus ends of mitotic spindle microtubules. AURKA activates centrosomal CDC25B and recruits cyclin B1 to centrosomes. We report here functions for AURKA in meiotic maturation of mouse oocytes, which is a model system to study the G(2) to M transition. Whereas AURKA is present throughout the entire GV-stage oocyte with a clear accumulation on microtubule organizing centers (MTOC), active AURKA becomes entirely localized to MTOCs shortly before germinal vesicle breakdown. In contrast to somatic cells in which active AURKA is present at the centrosomes and minus ends of microtubules, active AURKA is mainly located on MTOCs at metaphase I (MI) in oocytes. Inhibitor studies using Roscovitine (CDK1 inhibitor), LY-294002 (PI3K inhibitor) and SH-6 (PKB inhibitor) reveal that activation of AURKA localized on MTOCs is independent on PI3K-PKB and CDK1 signaling pathways and MOTC amplification is observed in roscovitine- and SH-6-treated oocytes that fail to undergo nuclear envelope breakdown. Moreover, microinjection of Aurka mRNA into GV-stage oocytes cultured in 3-isobutyl-1-methyl xanthine (IBMX)-containing medium to prevent maturation also results in MOTC amplification in the absence of CDK1 activation. Overexpression of AURKA also leads to formation of an abnormal MI spindle, whereas RNAi-mediated reduction of AURKA interferes with resumption of meiosis and spindle assembly. Results of these experiments indicate that AURKA is a critical MTOC-associated component involved in resumption of meiosis, MTOC multiplication, proper spindle formation and the metaphase I-metaphase II transition.

• MeSH
• aparát dělícího vřeténka metabolismus   MeSH
• aurora kinasa A MeSH
• blastodisk metabolismus   MeSH
• buněčný cyklus genetika   fyziologie   MeSH
• buňky NIH 3T3 MeSH
• cyklin-dependentní kinasy metabolismus   fyziologie   MeSH
• fosfatidylinositol-3-kinasy metabolismus   fyziologie   MeSH
• HeLa buňky MeSH
• kinasy aurora MeSH
• lidé MeSH
• meióza genetika   fyziologie   MeSH
• myši inbrední BALB C MeSH
• myši inbrední C57BL MeSH
• myši MeSH
• oocyty enzymologie   fyziologie   MeSH
• organizační centrum mikrotubulů metabolismus   MeSH
• protein-serin-threoninkinasy genetika   metabolismus   fyziologie   MeSH
• protoonkogenní proteiny c-akt metabolismus   fyziologie   MeSH
• vazba proteinů MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• myši MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• AURKA protein, human MeSH Prohlížeč
• Aurka protein, mouse MeSH Prohlížeč
• aurora kinasa A MeSH
• cyklin-dependentní kinasy MeSH
• kinasy aurora MeSH
• protein-serin-threoninkinasy MeSH
• protoonkogenní proteiny c-akt MeSH