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Title: A molecular analysis of the microtubule associated protein MAP65-1
Author: Hsin Yu, Chang
ISNI:       0000 0004 2714 9710
Awarding Body: Durham University
Current Institution: Durham University
Date of Award: 2006
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Microtubules (MTs) play important roles in various cellular processes including cell division, organelle movement and the determination of cellular morphology. The dynamics and organization of microtubules are regulated by microtubule associated proteins (MAPs).MAP65 bundles microtubules and forms crossbridges between microtubules in vitro. MAP65 belongs to a group of phylogenetically divergent proteins which includes yeast (s. cerevisiae) Asel, insect (D. melanogaster) FEO, mammalian {H. sapiens) PRC1, and worm (C. elegans) SPD-1. All members of this group concentrate in the spindle midzone during anaphase and telophase and are important for successful cytokinesis. A gene family encoding nine MAP65-like proteins has been identified in Arabidopsis thaliana. These proteins have a sequence identity from 28% to 79%, suggesting that each protein might play a different role in microtubule organization. This study focuses on the MAP65-1 sub group of MAP-65 proteins. Members of this sub-group can bind and bundle microtubules without affecting their dynamics in vitro and they co-localize with subsets of interphase microtubules, the preprophase band, the midzone of the anaphase spindle and the phragmoplast. Accumulation of MAP65-1 proteins in the anaphase spindle midzone suggests that it might crossbridge anti-parallel microtubules. However, the molecular mechanism of the MAP65-1 function is still unclear. To study the dynamics of the interaction between MAP65-1 and microtubules in vivo, GFP: MAP65-1 chimeras were constructed and expressed in tobacco BY2 and A. thaliana plants. The localization of GFP was then analysed through the cell cycle and in different plant tissues. FRAP (fluorescence recovery after photobleaching) was used to study the MAP65-1 turnover. The data revealed that MAP65 has a higher turnover than tubulin and that it can associate/dissociate randomly along microtubules. The expression of GFP: MAP65-1 in A. thaliana showed that MAP65-1 decorates microtubules in most of the tissues. These results suggest that the properties of MAP65-1 make it ideal for the maintenance of spatial organisation of dynamic microtubules via cross-bridging. The location of the microtubule binding domain of MAP65-1 was mapped using truncated fragments and mutants. The results of these experiments demonstrate that the microtubule-binding domain lies in the C-terminal region of AtMAP65-1, whereas the dimerisation domain lies in the N-terminal region. However, a single amino acid substitution within the AtMAP65-1 microtubule binding domain (A409D and A420V) can significantly decrease the microtubule binding ability of AtMAP65- 1 in vitro. The cell cycle-specific binding of MAP65-1 to microtubules suggests that a specific mechanism controls this binding. Two possibilities were examined: (i) control of the protein level regulated through the cell cycle by specific degradation utilising the Destruction box and (ii) cell cycle-specific phosphorylation. Mutation of the Destruction box motif did not affect cell division or microtubule organization during the cell cycle, nor did it affect plant development. This suggests that MAP65-1 is not regulated in this way. However, MAP65-1 is hyperphosphorylated during prophase/metaphase and several phosphorylation motifs are predicted in the AtMAP65-1 protein. in vivo and in vitro experiments demonstrated that AtMAP65-1 is regulated by phosphorylation/dephosphorylation during the cell cycle by several distinct pathways including CDK and МАРК. Interestingly, over-expression of the non-phosphorylatable form of MAP65-1 induced excessive bundling of microtubules during mitosis, increased the number of pole-to-pole microtubule bundles in the mitotic spindle and caused a delay in mitosis. Therefore, precise control of microtubule bundling by MAP65-1 is essential for normal cell division.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available