Microtubules function as molecular tracks along which motor proteins transport a variety of cargo to discrete destinations within the cell. The carboxyl termini of ␣-and ␤-tubulin can undergo different posttranslational modifications, including polyglutamylation, which is particularly abundant within the mammalian nervous system. Thus, this modification could serve as a molecular ''traffic sign'' for motor proteins in neuronal cells. To investigate whether polyglutamylated ␣-tubulin could perform this function, we analyzed ROSA22 mice that lack functional PGs1, a subunit of ␣-tubulin-selective polyglutamylase. In wild-type mice, polyglutamylated ␣-tubulin is abundant in both axonal and dendritic neurites. ROSA22 mutants display a striking loss of polyglutamylated ␣-tubulin within neurons, including their neurites, which is associated with decreased binding affinity of certain structural microtubule-associated proteins and motor proteins, including kinesins, to microtubules purified from ROSA22-mutant brain. Of the kinesins examined, KIF1A, a subfamily of kinesin-3, was less abundant in neurites from ROSA22 mutants in vitro and in vivo, whereas the distribution of KIF3A (kinesin-2) and KIF5 (kinesin-1) appeared unaltered. The density of synaptic vesicles, a cargo of KIF1A, was decreased in synaptic terminals in the CA1 region of hippocampus in ROSA22 mutants. Consistent with this finding, ROSA22 mutants displayed more rapid depletion of synaptic vesicles than wild-type littermates after high-frequency stimulation. These data provide evidence for a role of polyglutamylation of ␣-tubulin in vivo, as a molecular traffic sign for targeting of KIF1 kinesin required for continuous synaptic transmission.kinesin ͉ microtubules ͉ synaptic vesicles ͉ trafficking ͉ tyrosination

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Microtubules form a cytoskeletal framework that influences cell shape and provides structural support for the cell. Microtubules in the nervous system undergo a unique post-translational modification, polyglutamylation of the C termini of their tubulin subunits. The mammalian enzymes that perform ␤-tubulin polyglutamylation as well as their physiological functions in the neuronal tissue remain elusive. We report identification of a mammalian polyglutamylase with specificity for ␤-tubulin as well as its distribution and function in neurite growth. To identify putative tubulin polyglutamylases, we searched tubulin tyrosine ligase-like (TTLL) proteins for those predominantly expressed in the nervous system. Of 13 TTLL proteins, TTLL7 was transcribed at the highest level in the nervous system. Recombinant TTLL7 catalyzed tubulin polyglutamylation with high preference to ␤-tubulin in vitro. When expressed in HEK293T cells, TTLL7 demonstrated specificity for ␤-tubulin and not for ␣-tubulin or nucleosome assembly protein 1. Consistent with these findings, knockdown of TTLL7 in a primary culture of superior cervical ganglion neurons caused a loss of polyglutamylated ␤-tubulin. Following stimulation of PC12 cells with nerve growth factor to differentiate, the level of TTLL7 increased concomitantly with polyglutamylation of ␤-tubulin. Short interference RNA-mediated knockdown of TTLL7 repressed nerve growth factor-stimulated MAP (microtubule-associated protein) 2-positive neurite growth in PC12 cells. Consistent with having a role in the growth of MAP2-positive neurites, TTLL7 accumulated within a MAP2-enriched somatodendritic portion of superior cervical ganglion, as did polyglutamylated ␤-tubulin. Anti-TTLL7 antibody revealed that TTLL7 was distributed in a somatodendritic compartment in the mouse brain. These findings indicate that TTLL7 is a ␤-tubulin polyglutamylase and is required for the growth of MAP2-positive neurites in PC12 cells.Microtubules have important functions in a variety of dynamic activities within the cell including intracellular transport, cell motility, and cell division. They provide structural support for the cell and are an important component of the cytoskeletal framework that generates cell morphology. In neurons microtubules are required for formation of specialized processes, i.e. dendrites and axons. Structural microtubule-associated proteins (MAPs), 2 such as MAP2 and tau, regulate the stability of microtubules in those processes.Microtubules are predominantly composed of tubes of polymerized dimers of ␣-and ␤-tubulin. A central question in cell biology is how functional heterogeneity is imparted to different types (e.g. dendritic, axonal, and centriolar) of microtubules. Two basic mechanisms have been proposed to explain this issue. The first involves duplication and divergence of genes encoding tubulin. The second mechanism involves the addition of a variety of post-translational modifications (PTM) to microtubules that further increases the tubulin heterogeneity (1). These PTMs are asso...

In Drosophila embryos, germ cell formation is induced by specialized cytoplasm at the posterior of the egg, the pole plasm. Pole plasm contains polar granules, organelles in which maternally produced molecules required for germ cell formation are assembled. An untranslatable RNA, called Polar granule component ( Pgc ), was identified and found to be localized in polar granules. Most pole cells in embryos produced by transgenic females expressing antisense Pgc RNA failed to complete migration and to populate the embryonic gonads, and females that developed from these embryos often had agametic ovaries. These results support an essential role for Pgc RNA in germline development.