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

Mol Biol Cell. 2002 Oct;13(10):3627-45. doi: 10.1091/mbc.e02-01-0061.

Abstract

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adenomatous Polyposis Coli Protein / genetics
  • Adenomatous Polyposis Coli Protein / metabolism
  • Animals
  • Antibodies, Monoclonal / metabolism
  • Antineoplastic Agents / metabolism
  • Cell Line
  • Centrosome / metabolism*
  • Cytoskeleton / metabolism
  • Dynactin Complex
  • Dyneins / metabolism
  • Epitope Mapping
  • Golgi Apparatus / metabolism
  • Humans
  • Microscopy, Fluorescence
  • Microtubule-Associated Proteins / genetics
  • Microtubule-Associated Proteins / metabolism*
  • Microtubules / metabolism*
  • Neoplasm Proteins
  • Nocodazole / metabolism
  • Protein Binding
  • Recombinant Fusion Proteins / genetics
  • Recombinant Fusion Proteins / metabolism*
  • Tubulin / metabolism

Substances

  • Adenomatous Polyposis Coli Protein
  • Antibodies, Monoclonal
  • Antineoplastic Agents
  • DCTN1 protein, human
  • Dynactin Complex
  • EB1 microtubule binding proteins
  • Microtubule-Associated Proteins
  • Neoplasm Proteins
  • Recombinant Fusion Proteins
  • Tubulin
  • cytoplasmic linker protein 170
  • Dyneins
  • Nocodazole