GFP chimeras of E-MAP-115 (ensconsin) domains mimic behavior of the endogenous protein in vitro and in vivo

E-MAP-115 (ensconsin) is a microtubule-associated protein (MAP) abundant in carcinoma and other epithelia-derived cells. We expressed chimeras of gree n fluorescent protein (GFP) conjugated to ensconsin's N-terminal MT-binding domain (EMTB), to study distribution, dynamics, and function of the MAP in living cells. We tested the hypothesis that behavior of expressed GFP-EMTB accurately matched behavior of endogenous ensconsin. Like endogenous MAP, GFP-EMTB was associated with microtubules in living or fixed cells, and mic rotubule association of either molecule was impervious to extraction with n onionic detergents. In cell lysates both GFP-EMTB and endogenous ensconsin were dissociated from microtubules by identical salt extraction conditions, and both molecules remained bound to a calcium-stable subset of Taxol-stab ilized microtubules. These data show that microtubule association of enscon sin was affected neither by the absence of domains other than its microtubu le-binding domain, nor by the presence of appended GFP. We took advantage o f this finding to generate constructs in which additional GFP moieties were attached to EMTB, to obtain a more intensely fluorescent reporter of in vi vo MAP binding. We show here that expression of chimeric proteins consistin g of five GFP molecules attached to a single EMTB molecule produces brightl y labeled microtubules without compromising the behavior of the MAP or the microtubules to which it is attached. Thus, me have demonstrated the utilit y of chimeric proteins containing GFP multimers as authentic reporters of e nsconsin distribution and dynamics; expression of these GFP-EMTB chimeric m olecules also provides a non-perturbing label of the microtubule system in living cells.