E-MAP-115 (ensconsin) associates dynamically with microtubules in vivo and is not a physiological modulator of microtubule dynamics.
Microtubule-associated proteins (MAPs) have been hypothesized to regulate microtubule dynamics and/or functions. To test hypotheses concerning E-MAP-115 (ensconsin) function, we prepared stable cell lines expressing conjugates in which the full-length MAP (Ensc) or its microtubule-binding domain (EMTB) 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 molecule as a reporter for the behavior and microtubule-binding function of endogenous MAP. Dual wavelength time-lapse fluorescence imaging of 2xGFP-EMTB in cells microinjected with labeled tubulin revealed that this GFP-MAP chimera associated with the lattice of all microtubules immediately upon polymerization and dissociated concomitant with depolymerization, suggesting that dynamics of MAP:microtubule interactions were at least as rapid as tubulin:microtubule dynamics in the polymerization reaction. Presence of both GFP-EMTB chimeras and endogenous E-MAP-115 (ensconsin) along apparently all cellular microtubules at all cell cycle stages suggested that the MAP might function in modulating stability or dynamics of microtubules, a capability shown previously in transiently transfected cells. Although cells with extremely high expression levels of GFP-EMTB chimera exhibited stabilized microtubules, cells expressing four to ten times the physiological level of endogenous MAP exhibited microtubule dynamics indistinguishable from those of untransfected cells. This result shows that E-MAP-115 (ensconsin) is unlikely to function as a microtubule stabilizer in vivo. Instead, this MAP most likely serves to modulate microtubule functions or interactions with other cytoskeletal elements.
Faire
K
K
Department of Anatomy, Columbia University, College of Physicians & Surgeons, Rm BB1213, New York, NY 10032-3702, USA.
Waterman-Storer
C M
CM
Gruber
D
D
Masson
D
D
Salmon
E D
ED
Bulinski
J C
JC
D005260:Female; D006801:Humans; D001483:Base Sequence; D001943:Breast Neoplasms; D002460:Cell Line; D017931:DNA Primers; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins / Q000032:analysis / Q000235:genetics; D008869:Microtubule-Associated Proteins / Q000032:analysis / Q000235:genetics / Q000502:physiology; D008870:Microtubules / Q000502:physiology / Q000648:ultrastructure; D008940:Mitotic Index; D008969:Molecular Sequence Data; D011993:Recombinant Fusion Proteins / Q000032:analysis / Q000096:biosynthesis; D008941:Spindle Apparatus / Q000502:physiology / Q000648:ultrastructure; D014162:Transfection; D014407:Tumor Cells, Cultured
D017931:DNA Primers; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins; D008869:Microtubule-Associated Proteins; D011993:Recombinant Fusion Proteins; C000721969:MAP7 protein, human