REMOVAL OF MAP4 FROM MICROTUBULES IN-VIVO PRODUCES NO OBSERVABLE PHENOTYPE AT THE CELLULAR-LEVEL

Microtubule-associated protein 4 (MAP4) promotes MT assembly in vitro and is localized along MTs in vivo. These results and the fact that MA P4 is the major MAP in nonneuronal cells suggest that MAP4's normal fu nctions may include the stabilization of MTs in situ. To understand MA P4 function in vivo, we produced a blocking antibody (Ab) to prevent M AP4 binding to MTs. The COOH-terminal MT binding domain of MAP4 was ex pressed in Escherichia coli as a glutathione transferase fusion protei n and was injected into rabbits to produce an antiserum that was then affinity purified and shown to be monospecific for MAP4. This Ab block ed >95% of MAP4 binding to MTs in an in vitro assay. Microinjection of the affinity purified Ab into human fibroblasts and monkey epithelial cells abolished MAP4 binding to MTs as assayed with a rat polyclonal antibody against the NH2-terminal projection domain of MAP4. The remov al of MAP4 from MTs was accompanied by its sequestration into visible MAP4-Ab immunocomplexes. However, the MT network appeared normal. Tubu lin photoactivation and nocodazole sensitivity assays indicated that M T dynamics were not altered detectably by the removal of MAP4 from the MTs. Cells progressed to mitosis with morphologically normal spindles in the absence of MAP4 binding to MTs. Depleting MAP4 from MTs also d id not affect the state of posttranslational modifications of tubulin subunits. Further, no perturbations of MT-dependent organelle distribu tion were detected. We conclude that the association of MAP4 with MTs is not essential for MT assembly or for the MT-based functions in cult ured cells that we could assay. A significant role for MAP4 is not exc luded by these results, however, as MAP4 may be a component of a funct ionally redundant system.