Use this URL to cite or link to this record in EThOS: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.550859
Title: Functional characterisation of human EB protein family member EB2
Author: Ruane, Peter Thomas
Awarding Body: University of Leeds
Current Institution: University of Leeds
Date of Award: 2010
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Abstract:
Dynamic protein filaments in eukaryotic cells make up cytoskeletal arrays that perform essential functions. Microtubules form part of the cytoskeleton, and impart structure, integrity and organisation to cells. End binding (EB) proteins are an evolutionarily conserved family which associate with the dynamic ends of microtubules, where they act to control microtubule growth and recruit other proteins that localise to this site (+ TIPs). EB2 is one of three vertebrate EB proteins but its role is unclear because it exhibits weak EB protein activity. This thesis describes molecular and cell . biological experiments designed to functionally characterise human EB2. Fourteen human cell lines were all shown to express EB2 at lower levels than EB 1, while isolation of EB2 in HeLa cells by siRNA-mediated knock down of EB 1 and EB3 confirmed reports that EB2 is outcompeted at microtubule ends by EB 1 and EB3. Analysis of individual microtubules corroborated proposals that EB2 has lower affinity for microtubule tips than EB 1, and also suggested that EB proteins interact more strongly with the proximal region of microtubule tips than the distal region. Furthermore, the + TIP CLIP-170 localised to this distal region independently of EB proteins, implicating an activatory rather than direct-coupling role for EB proteins in the recruitment of CLIP-170. Additionally, the functional effects of structural divergences in EB2 were examined by mutation. An N-terminal extension, EB2 Nose, was shown to attenuate + TIP binding through a functional interaction with 322pQ323 in the C- terminal tail of EB2. A putative SxIP motif, 25TIIp28, was identified within EB2 Nose which contributed to this attenuation. It is proposed from these findings that EB2 is autoinhibited for + TIP binding by intramolecular interactions, between EB2 Nose and 322pQ323, and between 25Tllp28 and the C-terminal EB domain. These data portray EB2 as an unproductive EB protein, and a '+ TIP spacer' function is postulated.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.550859  DOI: Not available
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