Echinoderm microtubule-associated protein like protein 4, a member of the echinoderm microtubule-associated protein family, stabilizes microtubules

Abstract

Echinoderm microtubule-associated protein (EMAP) is the major microtubule binding protein in dividing sea urchin (Strongylocentrotus purpuratus) eggs. Echinoderm microtubule-associated protein like protein 4 (Eml4, restrictedly overexpressed proliferation-associated protein 120 kDa (Ropp120)) is one of the five mammalian EMAP homologues, the cellular function of which remains to be elucidated. In our first set of experiments we determined the spatio-temporal expression pattern of Eml4 in mouse brain. Our results demonstrate that Eml4 is a highly developmentally regulated gene with high expression levels in the developing nervous system of E11 embryos declining to low levels in adult. Spatially, Eml4 expression becomes restricted to the olfactory bulb, hippocampus and cerebellum. Transient transfection of a fusion construct of full-length mouse Eml4 with green fluorescent protein (GFP-Eml4) into Cos7 and HeLa cells resulted in colocalization of GFP-Eml4 with microtubules. This colocalization was observed both with microtubules of non-dividing cells and with the mitotic spindle of dividing cells. In addition, transient overexpression of GFP-Eml4 in Cos7 cells resulted in microtubules that were resistant to nocodazole treatment suggesting that Eml4 stabilizes microtubules. A consequence of microtubule stabilization is a net reduction in the amount of free tubulin. Microtubule stabilizing proteins therefore are expected to indirectly decrease the microtubule growth rate. Indeed, transient transfection of GFP-Eml4 resulted in a marked decrease in the microtubule growth rate, which is in line with our hypothesis that Eml4 functions as a microtubule stabilizing protein. In summary, our results suggest that Eml4 is a developmentally regulated protein that colocalizes with and stabilizes microtubules.