Fitton, Ben (2016) A study of the interaction of end-binding proteins with microtubules. PhD thesis, University of Warwick.

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Abstract

Dynamic microtubules control cell shape, cell locomotion and the proper segregation of chromosomes. End Binding (EB) proteins are the key components of the microtubule (MT) plus tip (+TIP) protein network. EBs bind to the MT plus end and regulate microtubule dynamics. EBs localise to the microtubule tip by recognising the nucleotide state of tubulin. Mammalian cells express three members of the EB family (EB1, EB2 and EB3) that localise to spatial distinct sites on the microtubule in cells. Perturbation experiments in cells and in vitro reconstitution experiments have shown that EB1 and EB3 accelerate MT assembly and increase catastrophe frequency. This is a paradoxical effect, as an increase in growth speed should increase the size of the GTP cap thus decreasing the probability of catastrophe. To study this paradoxical effect an image analysis routine was developed to gain insight into any structural re-arrangement at the microtubule tip. An algorithm was developed to extract fluorescence intensity data along the length of a microtubule from time-lapse images. Curve fitting to these data allowed determination of the MT end position with sub-pixel resolution, the measurement of taper (i.e. the length difference of protofilaments at the microtubule end) and the quantitative analysis of the comet-shaped distributions of EB proteins at microtubule ends. The method was verified using synthetic images of MTs and then applied to time lapse movies of dynamic MTs from in vitro experiments where either the tubulin concentration or the EB3 concentration was varied. It was discovered that EB3 may increase microtubule taper, thereby de-stabilising the microtubule tip structure.

Binding of the three EB proteins to spatial distinct sites at the MT tip was carefully re-investigated in-vitro by pair-wise comparison, and in relation to the MT tip. All three EB proteins were found to localise to distinct sites with EB3 found to bind closest to the MT tip and EB2 being the furthest from the MT tip. Based on structural data that became available during the course of the project, and additional evidence of different nucleotide preferences between EB1/EB3 and EB2, a dual nucleotide recognition model was conceived to explain these spatially distinct locations. The model assumes that an EB protein is sensitive to the nucleotide state at both E-sites close to its binding site at the interface of 4 tubulin dimers. All three EB proteins showed evidence of dual nucleotide recognition in mixed nucleotide lattice experiments designed to directly test the model.

EB proteins recognise spatial distinct sites by recognising the pairwise nucleotide state of tubulin. As EB3 binds closest to the MT tip, it is best placed to affect microtubule dynamics by increasing taper, promoting a quicker growth phase and destabilising the microtubule. Within cells this is a useful concept as it can be up regulated to increase the dynamicity of MTs ensuring more efficient re-organisation of the cytoskeleton during cell differentiation or neuronal elongation.

Item Type:	Thesis (PhD)
Subjects:	Q Science > QH Natural history > QH301 Biology
Library of Congress Subject Headings (LCSH):	Microtubules
Official Date:	March 2016
Dates:	Date Event March 2016 Submitted
Institution:	University of Warwick
Theses Department:	Molecular Organisation and Assembly in Cells
Thesis Type:	PhD
Publication Status:	Unpublished
Supervisor(s)/Advisor:	Straube, Anne ; Rajpoot, Nasir M. (Nasir Mahmood) ; Grosskinsky, Stefan
Extent:	xiii, 168 leaves : illustrations, charts
Language:	eng

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