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Role of the Kinases NEK6, NEK7 and NEK9 in the Regulation of the Centrosome CycleSdelci, Sara 13 December 2012 (has links)
This thesis project is focused on the study of the signaling module formed by the NIMA-related protein Nek6, Nek7, and Nek9 and their function during early mitosis, with particular interest in centrosome separation and maturation.
Nek9/Nercc1 was identified by Dr. Joan Roig. Nek9 is expressed in all cell lines and tissues studied is inactive during interphase while during mitosis is activated through phosphorylation by Plk1 which is in fact able to bind Nek9 and subsequently phosphorylates Nek9 on its activation loop.
During mitosis Nek6 and Nek7 bind the C-terminal of Nek9. Once active, Nek9 can phosphorylate Nek6 and Nek7, thus activating them. Active Nek9 localizes at centrosome, suggesting that Nek9/Nek6-7 has important functions in the organization of microtubules during cell division. Confirming this idea, it has been shown that the microinjection of anti-Nek9 module induces arrest in prometaphase with disorganized spindle structures and misaligned chromosomes, or leads to abnormal mitosis resulting in aneuploidy. In the same direction, interference with the function of Nek7 or Nek6 leads to abnormal mitotic progression and spindle formation.
We described how the Nek9/Nek6-7 module could provide a link connecting Plk1 and Eg5 in the context of centrosome separation. we analyzed the effects of Plk1, Eg5, Nek9, Nek6 or Nek7 down-regulation by RNAi on the extent of separation of duplicated centrosomes in prophase cells and we observed how this downregulation was affecting centrosome separation.
We determine whether the activation of Nek9 or Nek6 could induce centrosome separation trasfecting cells with the active form of these two kinases; a considerable amount of cells that were in interphase shown separate centrosome demonstrating that Nek9/Nek6 are sufficient to induce centrosome separation. To test whether active Nek9 and Nek6 exerted their effect through the regulation of Eg5 we simultaneously transfected the cells with Eg5 siRNAs and we completely lost the centrosome separation described above.
We demonstrated by immunofluorescence that the key event during centrosome separation was the recruitment of Eg5 at centrosomes and that the down-regulation of Plk1, Nek6, Nek7 or Nek9 resulted in prophase cells with unseparated centrosomes because Eg5 was not properly recruited.
To prove whether the phosphorylation on Ser-1033 controls the accumulation of Eg5 to centrosomes and centrosome separation during early mitosis we transfected cells with wild type Eg5 or Eg5 S1033A; the wild type form of the kinesin was able to localize at centrosome and rescue the normal phenotype while Eg5 S1033A was not able to localize and resulted in cells delayed in mitosis.
Plk1, the Nek9 activator, is involved in the regulation of centrosome maturation during early mitosis. Centrosome maturation refers to the process through which centrosomes increase size and microtubule nucleation activity and requires the accumulation of γ-TuRC complexes at centrosome. This recruitment depends on Nedd1 that acts as γ-Tubulin targeting factor. Plk1 depletion prevents accumulation of Nedd1 at centrosome.
Our experiments show the importance of Nek9 in the regulation of centrosome maturation downstream of Plk1. Depletion of Nek9 by siRNA determined a decrease of γ-Tubulin and Nedd1 at centrosome. Further we investigated the upstream role of Plk1 depleting Plk1 and trasfecting active Nek9 and it was able to rescue the normal phenotype.
Nek9 can interact with Nedd1 during mitosis and phosphorylates it provoking its accumulation at centrosome. The no-phosphorylable form of Nedd1 was not able to accumulate at centrosome and support the accumulation of γ-Tubulin there, determining a delay of the cells in prometaphase. Our results show that Nek9 is the link between Plk1 activity and the recruitment of Nedd1 to the centrosome and that the pathway formed by Plk1/Nek9/Nedd1 can be a key element in the control of mitotic centrosome maturation.
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Aurora A kinase function during anaphaseLioutas, Antonio, 1980- 09 November 2012 (has links)
Aurora A (AurA) is an important mitotic kinase mainly studied for its
involvement in cell cycle progression, centrosome maturation,
mitotic spindle pole organization and bipolar spindle formation. It
localizes to duplicated centrosomes and spindle microtubules (MTs)
during mitosis where it regulates various factors participating in
metaphase spindle formation. AurA is degraded late in mitosis
suggesting that it might also have a function in anaphase. In this
study we focused in understanding AurA function during anaphase
in two different experimental systems.
First, we kept AurA active in cycled Xenopus egg extracts and found
that MTs maintained their mitotic organization longer throughout
mitotic exit. We also observed chromosome segregation defects and
problematic nuclear envelope formation. These observations
indicate that AurA activity needs to be down-regulated for the
transition from metaphase back to interphase.
To get insights into the role of AurA during metaphase-anaphase
transition we initially asked whether its kinase activity is still
necessary for the maintenance of the metaphase spindle. We saw
that the inhibition of AurA kinase activity in metaphase resulted to a
collapse of the established metaphase spindle in HeLa cells.
Indicating that AurA activity is necessary for the metaphase spindle
maintenance.
Then, we looked whether AurA kinase activity is still necessary
during anaphase. We inhibited AurA at the onset of anaphase in
Hela cells and found that anaphase spindles were smaller. We also
observed that the MT structure responsible for anaphase spindle
elongation, the central spindle, was defectively assembled and
organized. Moreover, in cells where AurA was inhibited segregation
of chromosomes was defective. These results indicate that AurA
kinase activity is necessary for anaphase spindle elongation, central
spindle assembly and organization and chromosome segregation.
To understand further how AurA regulates anaphase spindle
formation we looked known AurA substrates. We depleted TACC3,
a known AurA substrate involved in MT formation earlier in mitosis
and observed that TACC3 depletion phenocopied AurA inhibition.
This indicates that TACC3 has a function in MT organization and
chromosome segregation during anaphase and this function could
possibly be regulated by AurA.
In this study we have demonstrated that AurA activity is essential for
metaphase spindle maintenance. We also found that during
anaphase when AurA is either maintained active or inhibited MT
organization is greatly affected and chromosome segregation is
defective. Suggesting that AurA activity needs to be tightly controlled
during anaphase for a correct completion of mitosis. / Aurora A (AurA) es una quinasa mitótica importante que se ha
estudiado principalmente en su papel durante la progresión del ciclo
celular, la maduración del centrosoma, la organización y la
formación del polo y del huso mitótico. Durante la mitosis, AurA se
localiza en los centrosomas duplicados y en los microtúbulos (MTs)
del huso y se ha observado que regula varios factores que
participan en la formación del huso mitótico. AurA se degrada al
final de la mitosis indicando que pueda tener una función durante la
anafase. En este estudio nos hemos centrado en la comprensión de
la función de AurA durante la anafase en dos sistemas
experimentales diferentes.
En primer lugar, utilizando extractos de huevos de Xenopus hemos
mantenido AurA activa durante la transición de metafase a anafase
y hemos visto que los MTs del huso mitótico mantienen su
organización durante más tiempo. También hemos observado que
cuando AurA se mantiene activa existen defectos en la segregación
cromosómica y la formación de la membrana nuclear. Esto indica
que la actividad de AurA tiene un papel regulador sobre los MTs y la
chromatina durante la transición de la metafase a la interfase.
Para entender cual es la función de AurA durante la transición de
metafase a anafase primero hemos estudiado si la actividad de la
quinasa es necesaria para el mantenimiento del huso mitótico.
Hemos visto que la inhibición de la actividad quinasa AurA resultó
en el colapso del huso durante la metafase en células HeLa. Esto
indica que la actividad de AurA es necesaria para el mantenimiento
del huso mitótico de metafase.
A continuación hemos analizamos si la actividad quinasa de AurA
sigue siendo necesaria para la anafase. Para ello hemos inhibido
AurA en células Hela al inicio de la anafase. En estas condiciones
los husos de la anafase son más pequeños y la estructura de los
MTs responsable del alargamiento del huso mitótico durante la
anafase, el huso central, se organiza defectuosamente. Además, se
encontraron errores durante la segregación de los cromosomas.
Estos resultados indican que la actividad quinasa de AurA es
necesaria para el alargamiento del huso durante la anafase y la
organización y segregación cromosómica.
Para entender el mecanismo de la función de AurA durante la
anafase hemos estudiado a sustratos de AurA. Al estudiar TACC3 ,
un sustrato conocido de AurA que participa en la formación de MTs
en las fase iniciales de la mitosis hemos encontrado que su
eliminación de células HeLa produce el mismo fenotipo que la
inhibición de AurA. Esto indica que TACC3 tiene una función en la
organización de MT y la segregación de cromosomas durante la
anafase y que esta función podría estar regulada por la quinasa
AurA.
En este estudio hemos demostrado que la actividad quinasa de
AurA es esencial para el mantenimiento del huso mitótico. También
hemos encontrado que durante la anafase cuando la quinasa AurA
se mantiene activa o se inhibe la organización de los MTs del huso
mitótico se ve muy afectada y los cromosomas se segregan
defectuosamente. Por tanto los resultados de este estudio indican
que la actividad quinasa de AurA está estrechamente controlada
durante la anafase para el correcto cumplimiento de la mitosis.
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