Booth, Daniel (2011) An ultrastructural study of the role of clathrin as an inter-microtubule bridge in kinetochore fibres. Doctoral thesis, University of Liverpool.
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The mitotic spindle is required for the accurate segregation of an equal number of chromosomes, into two separate daughter cells. The movement of chromosomes is carried out by kinetochore fibres (K-fibres) of the spindle apparatus, these extend from the cell pole and terminate at attachment on the kinetochore. K-fibres are composed of bundles of individual kinetochore microtubules (kMTs). Microtubule (MT) bundling is coordinated by inter-MT bridges, which are thought to promote MT stability and organisation. The inter-MT bridges in K-fibres have never been fully characterised. Clathrin is a triskelion-structured protein with a well-characterised role in membrane trafficking, where it forms the ‘coat’ of clathrin-coated vesicles. More recently clathrin has been shown to have an unrelated function during mitosis: a period of the cell cycle when membrane trafficking is dormant. The depletion of clathrin by RNAi results in a number of mitotic defects, all of which are a consequence of destabilised K-fibres. It has been proposed that a TACC3/ch-TOG/clathrin complex physically cross-braces kMTs to stabilise K-fibres. This project set-up correlative light electron microscopy (CLEM) methods allowing mitotic, clathrin-depleted cells to be analysed from the level of light microscopy to electron microcopy. A dual-axis ultrastructural methodology was also developed allowing K-fibre bundles and inter-MT bridges to be measured. This project used CLEM to reveal that a clathrin complex functions as a population of short inter-MT bridges in K-fibres. The depletion of clathrin or TACC3 resulted in: 1) a loss of kMTs, 2) a decrease in kMT organisation and 3) a loss of a population of short inter-kMT bridges. Immunogold labelling confirmed that clathrin was a component of the inter-kMT bridge. The literature typically refer to inter-MT bridges as electron dense ‘struts’ that cross-link MTs, however, a few classic EM studies have described bridges as ‘filamentous’ with ‘multiple projections’. A highly resolved view of bridge structures was sought. Using high-pressure freezing with freeze-substitution, tomography and 3D rendering, this project revealed that a novel structure was present that cross-linked kMTs, we have termed this structure ‘inter-kMT mesh’. The mesh was found exclusively between kMTs and appeared to promote kMT organisation through multiple contacts with MTs. The mesh responded to manipulated levels of TACC3 expression, resulting in disorganised K-fibre bundles, misshapen kMTs and an enhanced electron-dense inter-kMT mesh. Collectively, these new data contribute to the existing knowledge of inter-MT cross-linkers in K-fibres by providing novel insights into their structure and how they confer kMT stability and organisation.
|Item Type:||Thesis (Doctoral)|
|Subjects:||Q Science > QH Natural history > QH301 Biology|
|Departments, Research Centres and Related Units:||Academic Faculties, Institutes and Research Centres > Faculty of Medicine > School of Biomedical Sciences|
|Deposited On:||06 Aug 2012 10:57|
|Last Modified:||01 Aug 2014 01:00|
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