The cells were then transfected with CA-Kinesin-1-mCherry and soluble YFP, allowed to express the exogenous proteins under additional treatment for 45 h, and then fixed and imaged. neurons. In contrast, microtubule modifications were enriched in a subset of neurites in unpolarized stage 2 cells and the developing axon in polarized stage 3 cells. This enrichment correlated with the selective accumulation of constitutively active Kinesin-1 motors. Increasing tubulin acetylation, without altering the levels of other tubulin modifications, did not alter the selectivity of Kinesin-1 accumulation in polarized cells. However, globally enhancing tubulin acetylation, detyrosination, and polyglutamylation by Taxol treatment or inhibition of glycogen synthase kinase 3 decreased the selectivity of Kinesin-1 translocation and led to the formation of multiple axons. Although microtubule acetylation enhances the motility of Kinesin-1, the preferential translocation of Kinesin-1 on axonal microtubules in polarized neuronal cells is not determined by acetylation alone but is probably specified by a combination of tubulin modifications. == INTRODUCTION == Unidirectional signal transduction by neuronal cells is usually intimately linked to their highly polarized morphology. The biogenesis and maintenance of distinct axonal and dendritic compartments depends on the selective transport of specific vesicles and proteins along microtubules Cytarabine hydrochloride to these distinct cellular regions. Thus, one of the keys to understanding neuronal morphology and function involves discovering the molecular mechanisms responsible for the polarized transport of kinesin motors to axons or dendrites. Recent work has shown that constitutively active (CA) forms of Kinesin-1 (also known as KIF5 or conventional kinesin) selectively accumulate within axonal but not dendritic growth cones of hippocampal neurons in culture (Nakata and Hirokawa, 2003). This ability to Cytarabine hydrochloride distinguish among different neurites begins before morphological polarization, as Kinesin-1 accumulates in only one or a small subset of neurites in unpolarized hippocampal neurons (Jacobsonet al., 2006). The Kinesin-1 cargo protein c-Jun NH2-terminal kinase-interacting protein (JIP)1 follows a similar pattern as it also localizes to the axon in polarized cells and to a subset of neurites in nonpolarized cells (Verheyet al., 2001;Reedet al., 2006;Dajas-Bailadoret al., 2008). Strikingly, Kinesin-1 accumulation in unpolarized stage 2 cells can be very dynamic, with the accumulation of active Kinesin-1 motors alternating between neurites within a matter of minutes (Jacobsonet al., 2006). This indicates that this molecular signals that direct Rabbit Polyclonal to CtBP1 the transport of Kinesin-1 must also be dynamic. What then are the molecular signals that drive the selective transport of Kinesin-1 motors to axons? As polarized accumulation is intrinsic to the Kinesin-1 motor domain, it seems likely that this motormicrotubule interaction is critical for specifying transport selectivity. In cultured neurons, axonal and dendritic microtubules differ in their populations of microtubule-associated proteins (MAPs), but it is not obvious how the differential localization of MAPs could influence the selectivity of kinesin transport. Axonal and dendritic microtubules also differ in their stability, as determined by resistance to depolymerization by nocodazole, and the local stabilization of axonal microtubules could influence kinesin translocation (Baaset al., 1991;Witteet al., 2008). The ability of Kinesin-1 motors to bind to, and in some cases move along, microtubules is usually enhanced by the presence of acetylation, detyrosination, or glutamylation modifications of tubulin (Larcheret al., 1996;Liao and Gundersen, 1998;Reedet al., 2006;Dompierreet al., 2007;Ikegamiet al., 2007;Dunnet al., 2008;Caiet al., 2009;Konishiet al., 2009). Thus, we wondered whether axonal microtubules are preferentially subject to modifications of tubulin subunits and if so, whether selective posttranslational modifications (PTMs) of axonal microtubules are responsible for the preferential accumulation of truncated Kinesin-1 at the tip of the axon in polarized cells. We show that axons are enriched in tubulin PTMs and that manipulations that globally enhance tubulin PTMs alter the selectivity of Kinesin-1 translocation, allowing it to Cytarabine hydrochloride accumulate in all neurites. Increasing tubulin acetylation, without alterations in the levels of other tubulin PTMs, was not sufficient to alter the selectivity of Kinesin-1 accumulation. == MATERIALS AND METHODS == == Plasmids and Antibodies == Constitutively active (CA) versions of the KHC subunit of Kinesin-1 [KHC(1-509) or KHC(1-560)] were generated from the rat KIF5C gene by using polymerase chain reaction (PCR) or convenient restriction sites and subcloned into plasmids for in-frame fusion to various fluorescent proteins and protein expression driven by Cytarabine hydrochloride the -actin promoter. All constructs were verified by DNA sequencing. The following antibodies were used: total -tubulin (E7; Developmental Studies Hybridoma Bank, University of Iowa, Iowa City, IA), total -tubulin (monoclonal DM1 [Sigma-Aldrich, St. Louis, MO]; polyclonal ab18251 or monoclonal ab7750 [Abcam, Cambridge, MA]), acetylated -tubulin (monoclonal 6-11B-1 or a rabbit polyclonal antibody generated against an -tubulin peptide that contains an acetylated lysine-40 residue [Sigma-Aldrich]), detyrosinated -tubulin (AB3201 or a rabbit polyclonal antibody generated against an -tubulin C-terminal peptide; Millipore Bioscience Research Reagents, Temecula, CA), polyglutamylated tubulin (GT335; a gift from C. Janke, CNRS, Montpellier, France), Tau-1 (MAB375; Millipore, Billerica, MA), MAP2 (AB5622; Millipore), and JIP1 polyclonal.