1 |
Characterisation of a novel spindle domain in mammalian meiosisSeres, Karmen Bianka January 2019 (has links)
The organisation of microtubule networks into a bipolar spindle is essential for reliable chromosome segregation during cell division. A pair of centrioles surrounded by pericentriolar material (PCM), define the canonical centrosome that acts as the main microtubule organising centre (MTOC) during mitosis. In mammalian meiosis, centrioles are eliminated early on during oogenesis. Despite the absence of centrosomes, a large number of centrosomal proteins are highly expressed in mouse oocytes. Here, I characterise the localisation and function of centrosomal proteins at a previously undescribed meiotic spindle pole domain (MSPD). An initial protein screen identified a group of pericentriolar satellite proteins that localised to a previously undescribed spindle pole domain throughout meiotic maturation in mouse oocytes, including Pericentriolar material 1 protein (PCM1). This domain was distinct from spindle microtubules and the acentrosomal microtubule organising centres (aMTOCs). Initial characterisation focused on PCM1, the main centriolar satellite scaffold protein in somatic cells. Depletion of PCM1 revealed interdependence with the essential aMTOC component, Pericentrin. In the absence of PCM1, aMTOCs could no longer assemble or maintain their structural integrity. PCM1 degradation and disassembly of aMTOCs disrupted spindle assembly and reduced the total amount of nucleated microtubules throughout meiosis. In the absence of the main microtubule nucleating aMTOCs, oocytes relied on the Ran GTPase activity to form a small bipolar spindle. A similar mechanism was previously reported in human oocytes that lack prominent MTOCs. The extended centrosomal protein screen identified additional components of the MSPD. TACC3, under the regulation of Aurora-A at aMTOCs, drive assembly of the MSPD. This domain was absent in MTOC free human oocytes but a second population of TACC3 (identified in mouse oocytes) localised to the meiotic spindle and K-fibres was essential for maintaining spindle pole integrity. Establishing the Lightsheet Z.1 system for live cell imaging of human oocytes enabled us to observe the dynamic distribution of TACC3 in these oocytes. In the absence of prominent MTOCs and the MSPD, human oocytes likely rely on other spindle assembly factors and motor proteins to organise their spindle. Future work to address if the absence of the MSPD could account (in part) for the observed spindle instability in human oocytes is an exciting outlook.
|
2 |
Therole of microtubule plus-end binding protein TACC3 during axon outgrowth and guidance:Erdogan, Burcu January 2019 (has links)
Thesis advisor: Laura Anne Lowery / Axon guidance is a critical process in forming the connections between a neuron and its target. Development of a properly functioning nervous system relies heavily on how accurately an axon is guided to the right target. Defects in the guidance machinery may result in neurological disorders. The growth cone that is formed at the tip of a growing axon is responsible for navigating axons to their final targets. The growth cone steers the growing axon towards the appropriate direction by integrating extracellular guidance cues received by membrane-associated receptors at the growth cone periphery. Upon receiving guidance cues, a number of intracellular signal transduction pathways are initiated downstream of the guidance receptors, that can promote or halt growth cone advance. The growth cone generates these responses by remodeling its cytoskeletal components, which are actin network in the periphery and microtubules in the growth cone center. In this thesis, we focus on understanding the role of microtubule dynamics regulation within the growth cone as it makes guidance decisions. Specifically, we examine the role of TACC3 as a microtubule plus-end binding protein during axon outgrowth and guidance. We show that TACC3 localizes at microtubule plus-ends in embryonic Xenopus laevis growth cones and regulates microtubule growth parameters. We also show that TACC3 is important for promoting axon outgrowth in cultured neural tube explants. Furthermore, our data suggests that TACC3 affects axon guidance in vivo and ex vivo. Examination of embryos depleted of TACC3 revealed guidance defects in the spinal cord neurons, while TACC3-overexpressing cultured spinal neurons showed increased resistance to Slit2-induced growth cone collapse. Finally, in an attempt to delineate the mechanism behind TACC3-mediated axon guidance under Slit2, we studied the importance of tyrosine phosphorylation induced by Abelson tyrosine kinase. We find that retaining phosphorylatable tyrosines within the TACC domain is important for its microtubule plus-end tracking behavior and its impact on microtubule dynamics regulation, axon outgrowth and guidance. Together, this thesis contributes new insights to the understanding of the role of TACC3 as a microtubule plus-end binding protein and identifies TACC3 as a potential regulator of axon outgrowth and guidance during Xenopus laevis embryonic development. / Thesis (PhD) — Boston College, 2019. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Biology.
|
3 |
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.
|
Page generated in 0.0206 seconds