Mammalian microtubule plus-end tracking proteins (+TlPs) specifically associate with the ends of growing microtubules. +TlPs are involved in many cellular processes, including mitosis, cell migration and neurite extension. Navigators are mammalian homologues of the C. elegans unc-53 protein, an ATPase that has been linked to the migration and Outgrowth of muscles, axons and excretory canals. Here we show that all three mammalian Navigators are +TlPs, consistent with a previous on Navigator 1 (NAV1) (Martinez-Lopez et al., Mol Cell Neurosci 2005:28:599-612). Overexpression of GFP-tagged Navigators causes displacement of CAP_GLY-motif containing +TlPs, Such as CLIP-170, from microtubule ends, suggesting that the Navigator-binding, sites on microtubule ends overlap with those of the CAP_GLY-motif proteins. In interphase cells, mammalian Navigators also prominently localize to centrosomes, a localization that does not depend on an intact microtubule network. Fluorescence recovery after photobleaching (FRAP) experiments indicate that NAV1 associates with intracellular structures other than microtubules or centrosomes. Expression of GFP-tagged Navigators induces the formation of neurite-like extensions in non-neuronal cells, showing that Navigators can dominantly alter cytoskeletal behavior. For NAV1 this function depends on its ATPase activity; it is not achieved by a classical type of MT bundling and stabilization. Combined our data suggest that Navigators are +TlPs that call reorganize the cytoskeleton to guide cell shape changes. Our data are consistent with a role for Navigators in neurite outgrowth. Cell Motil. Cytoskeleton 66: 824-838, 2009. (C) 2009 Wiley-Liss. Inc.