Evidence that an interaction between EB1 and p150(Glued) is required for the formation and maintenance of a radial microtubule array anchored at the centrosome.
EB1 is a microtubule tip-associated protein that interacts with the APC tumor suppressor protein and components of the dynein/dynactin complex. We have found that the C-terminal 50 and 84 amino acids (aa) 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 p150(Glued) was identified. EB1-GFP deletion mutants demonstrated a role for the N-terminus in mediating the EB1-microtubule interaction, whereas C-terminal regions contributed to both its microtubule tip localization and a centrosomal localization. Cells expressing the last 84 aa of EB1 fused to GFP (EB1-C84-GFP) displayed profound defects in microtubule organization 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 gamma-tubulin and p150(Glued) to centrosomes was also inhibited. None of these effects were seen in cells expressing the last 50 aa 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 p150(Glued) is required for microtubule minus end anchoring at centrosomes during the assembly and maintenance of a radial microtubule array.
Askham
J M
JM
Molecular Medicine Unit, University of Leeds, Clinical Sciences Building, St. James's University Hospital, Leeds LS9 7TF, United Kingdom. rmrjma@leeds.ac.uk
Vaughan
K T
KT
Goodson
H V
HV
Morrison
E E
EE
D025601:Adenomatous Polyposis Coli Protein / Q000235:genetics / Q000378:metabolism; D000818:Animals; D000911:Antibodies, Monoclonal / Q000378:metabolism; D000970:Antineoplastic Agents / Q000378:metabolism; D002460:Cell Line; D018385:Centrosome / Q000378:metabolism*; D003599:Cytoskeleton / Q000378:metabolism; D000072159:Dynactin Complex; D004398:Dyneins / Q000378:metabolism; D018604:Epitope Mapping; D006056:Golgi Apparatus / Q000378:metabolism; D006801:Humans; D008856:Microscopy, Fluorescence; D008869:Microtubule-Associated Proteins / Q000235:genetics / Q000378:metabolism*; D008870:Microtubules / Q000378:metabolism*; D009363:Neoplasm Proteins; D015739:Nocodazole / Q000378:metabolism; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism*; D014404:Tubulin / Q000378:metabolism
D025601:Adenomatous Polyposis Coli Protein; D000911:Antibodies, Monoclonal; D000970:Antineoplastic Agents; C000606360:DCTN1 protein, human; D000072159:Dynactin Complex; C405804:EB1 microtubule binding proteins; D008869:Microtubule-Associated Proteins; D009363:Neoplasm Proteins; D011993:Recombinant Fusion Proteins; D014404:Tubulin; C076744:cytoplasmic linker protein 170; D004398:Dyneins; D015739:Nocodazole