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A more recent version of this article appeared on October 1, 2002
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Submitted on January 31, 2002
Revised on July 5, 2002
Accepted on July 18, 2002
1 Molecular Medicine Unit, University of Leeds, Clinical Sciences Building, St. James's University Hospital, Leeds LS9 7TF, UK
2 Department of Biological Sciences, University of Notre Dame, Notre Dame, Indiana, USA
3 Department of Chemistry and Biochemistry, University of Notre Dame, Notre Dame, Indiana, USA
4 Molecular Medicine Unit, University of Leeds, Clinical Sciences Building, St. James's University Hospital, Leeds LS9 7TF, UK (present address: CRUK Clinical Centre at Leeds, Division of Cancer Medicine Research, St James's University Hospital, Leeds LS9 7TF)
* Corresponding author. E-mail address: rmrjma{at}leeds.ac.uk.
EB1 is a microtubule tip-associated protein that interacts with the APC tumour suppressor protein and components of the dynein/dynactin complex. We have found that the C-terminal 50 and 84 amino acids of EB1 were sufficient to mediate the interactions with APC and dynactin respectively. EB1 formed mutually exclusive complexes with APC and dynactin, and a direct interaction between EB1 and p150Glued was identified. EB1-GFP deletion mutants demonstrated a role for the N-terminus in mediating the EB1-microtubule interaction, while C-terminal regions contributed to both its microtubule tip localisation and a centrosomal localisation. Cells expressing the last 84aa of EB1 fused to GFP (EB1-C84-GFP) displayed profound defects in microtubule organisation and centrosomal anchoring. EB1-C84-GFP expression severely inhibited microtubule regrowth, focusing and anchoring in transfected cells during recovery from nocodazole treatment. The recruitment of 
-tubulin and p150Glued to centrosomes was also inhibited. None of these effects were seen in cells expressing the last 50aa of EB1 fused to GFP. Furthermore, EB1-C84-GFP expression did not induce Golgi apparatus fragmentation. We propose that a functional interaction between EB1 and p150Glued is required for microtubule minus end anchoring at centrosomes during the assembly and maintenance of a radial microtubule array.
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