THE ADENOMATOUS POLYPOSIS COLI-BINDING PROTEIN EB1 IS ASSOCIATED WITHCYTOPLASMIC AND SPINDLE MICROTUBULES

The evolutionarily conserved protein EB1 originally was identified by its physical association with the carboxyl-terminal portion of the ade nomatous polyposis coli (APC) tumor suppressor protein, an APC domain commonly mutated in familial and sporadic forms of colorectal neoplasi a, The subcellular localization of EB1 in epithelial cells was studied by using immunofluorescence and biochemical techniques. EB1 colocaliz ed both to cytoplasmic microtubules in interphase cells and to spindle microtubules during mitosis, with pronounced centrosome staining. The cytoskeletal array detected by anti-EB1 antibody was abolished by inc ubation of the cells with nocodazole, an agent that disrupts microtubu les; upon drug removal, EB1 localized to the microtubule-organizing ce nter. Immunofluorescence analysis of SW480, a colon cancer cell line t hat expresses only carboxyl-terminal-deleted APC unable to interact wi th EB1, demonstrated that EB1 remained localized to the microtubule cy toskeleton, suggesting that this pattern of subcellular distribution i s not mediated by its interaction with APC, rn vitro cosedimentation w ith taxol-stabilized microtubules demonstrated that a significant frac tion of EB1 associated with microtubules, Recent studies of the yeast EB1 homologues Mal3 and Bim1p have demonstrated that both proteins loc alize to microtubules and are important in vivo for microtubule functi on. Our results demonstrate that EB1 is a novel component of the micro tubule cytoskeleton in mammalian cells. Associating with the mitotic a pparatus, EB1 may play a physiologic role connecting APC to cellular d ivision, coordinating the control of normal growth and differentiation processes in the colonic epithelium.