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Title: In vitro reconstitution of confined microtubule cytoskeleton self-organisation
Author: Baumann, H.
ISNI:       0000 0004 5363 770X
Awarding Body: University College London (University of London)
Current Institution: University College London (University of London)
Date of Award: 2014
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The microtubule cytoskeleton determines the internal architecture of cells, crucial for their proper functioning. In many vertebrate cells the interphase microtubule cytoskeleton has an astral organisation with stable microtubule ends focused near the cell centre and dynamic microtubule ends emanating outwards towards the plasma membrane. This arrangement is determined by a complex self-organisation process involving the cell boundary and many protein activities within and at the periphery of a cell. This project aimed at the elucidation of the minimal set of activities required for this process to understand the rules governing the self-organisation of microtubule arrays within a boundary. I reconstituted self-organising microtubule arrays inside lipid monolayer surrounded droplets in oil from purified components and hence established a well-controlled system to systematically study the interplay of organising activities by fluorescence microscopy. My studies showed that restriction in space as such has no influence on de novo polymerisation of microtubule polymerisation, however on their arrangement. In presence of a surrounding lipid monolayer microtubule nucleation and polymerisation could be achieved using a lipid composition close to that of a natural plasma membrane and microtubule nucleation enhancing buffer conditions. Without any further organising activity, the arrangement of microtubules depends on the droplet diameter and microtubule nucleation efficiency. The microtubule-crosslinking and organising kinesin 14 forms single microtubules asters in larger droplets, in smaller droplets it bundles microtubules indicating a microtubule length – droplet diameter interdependency influencing the aster formation inside a confined volume. Therefore I investigated whether microtubule shortening by a kinesin-13 or severing by Spastin, either homogenously distributed in the droplet or located to the droplet boundary, allows motor-driven aster formation in small droplets. In order to transfer that basic model system into artificial liposomes and investigate the influence of a pliable boundary, I designed a flow chamber to appropriately image vesicles.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available