E-MAP-115 (ensconsin) associates dynamically with microtubules in vivo andis not a physiological modulator of microtubule dynamics

Microtubule-associated proteins (MAPs) have been hypothesized to regulate m icrotubule dynamics and/or functions. To test hypotheses concerning E-MAP-1 15 (ensconsin) function, we prepared stable cell lines expressing conjugate s in which the full-length MAP (Ensc) or its microtubule-binding domain (EM TB) was conjugated to one or more green fluorescent protein (GFP) molecules . Because both distribution and microtubule-binding properties of GFP-Ensc, GFP-EMTB, and 2x, 3x, or 4xGFP-EMTB chimeras all appeared to be identical to those of endogenous E-MAP-115 (ensconsin), we used the 2xGFP-EMTB molecu le as a reporter for the behavior and microtubule-binding function of endog enous MAP. Dual wavelength time-lapse fluorescence imaging of 2xGFP-EMTB in cells microinjected with labeled tubulin revealed that this GFP-MAP chimer a associated with the lattice of all microtubules immediately upon polymeri zation and dissociated concomitant with depolymerization, suggesting that d ynamics of MAP:microtubule interactions were at least as rapid as tubulin:m icrotubule dynamics in the polymerization reaction. Presence of both GFP-EM TB chimeras and endogenous E-MAP-115 (ensconsin) along apparently all cellu lar microtubules at all cell cycle stages suggested that the MAP might func tion in modulating stability or dynamics of microtubules, a capability show n previously in transiently transfected cells. Although cells with extremel y high expression levels of GFP-EMTB chimera exhibited stabilized microtubu les, cells expressing four to ten times the physiological level of endogeno us MAP exhibited microtubule dynamics indistinguishable from those of untra nsfected cells, This result shows that E-MAP-115 (ensconsin) is unlikely to function as a microtubule stabilizer in vivo. Instead, this MAP most likel y serves to modulate microtubule functions or interactions with other cytos keletal elements.