Use this URL to cite or link to this record in EThOS: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.529227
Title: Functional characterisation of a novel microtubule-actin interacting protein, GAS2-like 3
Author: Stroud, Matthew
Awarding Body: University of Manchester
Current Institution: University of Manchester
Date of Award: 2010
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Abstract:
The microtubule (MT) and actin cytoskeletons are fundamental to cell integrity, as they control a host of cellular activities. Until recently, studies have investigated these cytoskeletal components as separate entities, however, it has become increasingly clear that the MT and actin cytoskeletons function in an interdependent way. Elucidating how the two components interact will be key to our further understanding of fundamental cellular processes such as cell division, growth, polarisation and migration. This study focused on a newly identified and highly conserved protein, named GAS2-like 3, which contains a putative actin binding calponin homology (CH) domain and a putative MT binding GAS2-related (GAR) domain. GAS2-like 3 predominantly localised to both MT and actin cytoskeletons. By using a series of truncation mutants, the fundamental regions of GAS2-like 3 involved in mediating these interactions were dissected, both in cells and in vitro. Data from sedimentation assays revealed the CH domain binds directly to F-actin, and the C-terminus binds directly to MTs, whereas the GAR domain does not interact directly with MTs. In order to assess the dynamics of GAS2-like 3 and its mutants in cells, Fluorescence Recovery After Photobleaching experiments were performed to reveal that the GAR domain of GAS2-like 3 modulates the binding strength of GAS2-like 3 to MTs and actin. GAS2-like 3 localisation is dependent on both MT and actin cytoskeletons, although MTs have a greater influence on GAS2-like 3 localisation. Interestingly, tubulin acetylation enhanced GAS2-like 3 localisation to MTs. This study also uncovered two binding motifs for the MT plus-end binding protein 1 (EB1). EB1 is characterised as a new interaction partner of GAS2-like 3, which directly interacts with the C-terminus of GAS2-like 3 in vitro. Furthermore, a molecular mechanism in which GAS2-like 3 is recruited to MTs via EB1 in cells is revealed. Interestingly, the interaction between EB1 and GAS2-like 3 requires the complete C-terminus, rather than the specific EB1-binding motifs themselves.Taken together, this study provides insights into the new protein, GAS2-like 3, which has the capacity to crosslink MTs and actin and provides a mechanistic insight into how GAS2-like 3 localisation is regulated in cells.
Supervisor: Ballestrem, Christoph Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.529227  DOI: Not available
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