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Post-proteomic characterization of a novel plant microtubule-associated protein:MAP70

Reference	BB/C507953/1
Principal Investigator / Supervisor	Professor Clive Lloyd
Co-Investigators / Co-Supervisors
Institution	John Innes Centre
Department	Cell and Develop Biology
Funding type	Research
Value (£)	226,083
Status	Completed
Type	Research Grant
Start date	01/02/2005
End date	31/01/2008
Duration	36 months

Abstract

In a proteomics survey for plant microtubule-associated proteins, we have detected a 70kDA Arabidopsis protein that binds microtubules in vitro. Expressed as a GFP fusion protein it decorates the cortical microtubule array that is essential for maintaining the axial pattern of plant growth. It is a member of a small multigene family. In the post-proteomic phase we propose to characterise this protein family. Each will be made into a Gateway entry clone so that it can be expressed as a GFP fusion protein and so that protein can be bacterially-expressed in order to raise antibodies. These approaches will allow us to visualise the proteins and see if different family members decorate some or all of the four microtubule arrays around the cell cycle. Some degree of functional specialisation can be expected in a multigene family and the patterns of expression will be studied in planta using GUS fusions. To study the function of the protein we will first detect the proteins on substrate-attached membrane disks containing a monolayer of attached cortical MTs. This will allow us to see how MAP70 decorates MTs in cells (i.e., end-binding, cross-bridging?). Next, we will use bacterially-expressed MAP70 to test if it affects the dynamics of tubulin assembly and/or MT bundling. Truncated proteins will be expressed to determine the MT-binding domain of this novel MAP. It is possible that MAP70 occurs as part of a binding complex and we will use our Proteomics Facility to identify such proteins in immunoprecipitates. Each member of the MAP70 family has at least one hit in a T-DNA insertional library. After checking that these are in-frame functional insertions we will examine the phenotype. Since MAP70 decorates the cortical MT array we will look for changes in cell elongation and effects on the growth axis. For those genes where the insertion does not lead to reduction of loss of transcript we will use RNAi.

Summary

unavailable

Committee	Closed Committee - Biochemistry & Cell Biology (BCB)
Research Topics	X – not assigned to a current Research Topic
Research Priority	X – Research Priority information not available
Research Initiative	X - not in an Initiative
Funding Scheme	X – not Funded via a specific Funding Scheme

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