ATAT1-enriched vesicles promote microtubule acetylation via axonal transport

Microtubules are polymerized dimers of α- and β-tubulin that underlie a broad range of cellular activities. Acetylation of α-tubulin by the acetyltransferase ATAT1 modulates microtubule dynamics and functions in neurons. However, it remains unclear how this enzyme acetylates microtubules over long distances in axons. Here, we show that loss of ATAT1 impairs axonal transport in neurons in vivo, and cell-free motility assays confirm a requirement of α-tubulin acetylation for proper bidirectional vesicular transport. Moreover, we demonstrate that the main cellular pool of ATAT1 is transported at the cytosolic side of neuronal vesicles that are moving along axons. Together, our data suggest that axonal transport of ATAT1-enriched vesicles is the predominant driver of α-tubulin acetylation in axons.

ATAT1-enriched vesicles promote microtubule acetylation via axonal transport

Microtubules are polymerized dimers of α- and β-tubulin that underlie a broad range of cellular activities. Acetylation of α-tubulin by the acetyl-transferase ATAT1 modulates microtubule dynamics and functions in neurons. However, it remains unclear how and why this enzyme acetylates microtubules over long distances in axons. Here, we show that loss of ATAT1 impairs axonal transport in neurons and cell free motility assays confirm a requirement of tubulin acetylation for proper bidirectional vesicular transport. Moreover, we demonstrate that the main cellular pool of ATAT1 is transported at the cytosolic side of neuronal vesicles that are moving along axons. Altogether, our data suggest that axonal transport of ATAT1-enriched vesicles is the predominant driver of α-tubulin acetylation in axons.

Dynactin Is Required for Coordinated Bidirectional Motility, but Not for Dynein Membrane Attachment

Transport of cellular and neuronal vesicles, organelles, and other particles along microtubules requires the molecular motor protein dynein ( Mallik and Gross, 2004 ). Critical to dynein function is dynactin, a multiprotein complex commonly thought to be required for dynein attachment to membrane compartments ( Karki and Holzbaur, 1999 ). Recent work also has found that mutations in dynactin can cause the human motor neuron disease amyotrophic lateral sclerosis ( Puls et al., 2003 ). Thus, it is essential to understand the in vivo function of dynactin. To test directly and rigorously the hypothesis that dynactin is required to attach dynein to membranes, we used both a Drosophila mutant and RNA interference to generate organisms and cells lacking the critical dynactin subunit, actin-related protein 1. Contrary to expectation, we found that apparently normal amounts of dynein associate with membrane compartments in the absence of a fully assembled dynactin complex. In addition, anterograde and retrograde organelle movement in dynactin deficient axons was completely disrupted, resulting in substantial changes in vesicle kinematic properties. Although effects on retrograde transport are predicted by the proposed function of dynactin as a regulator of dynein processivity, the additional effects we observed on anterograde transport also suggest potential roles for dynactin in mediating kinesin-driven transport and in coordinating the activity of opposing motors ( King and Schroer, 2000 ).

Functional morphology of the platyhelminth nervous system

SUMMARYAs the most primitive metazoan phylum, the Platyhelminthes occupies a unique position in nervous system evolution. Centrally, their nervous system consists of an archaic brain from which emanate one or more pairs of longitudinal nerve cords connected by commissures; peripherally, a diverse arrangement of nerve plexuses of varying complexity innervate the subsurface epithelial and muscle layers, and in the parasitic taxa they are most prominent in the musculature of the attachment organs and egg-forming apparatus. There is a range of neuronal-cell types, the majority being multi- and bipolar. The flatworm neuron is highly secretory and contains a heterogeneity of vesicular inclusions, dominated by densecored vesicles, whose contents may be released synaptically or by paracrine secretion for presumed delivery to target cells via the extracellular matrix. A wide range of sense organ types is present in flatworms, irrespective of life-styles. The repertoire of neuronal substances identified cytochemically includes all of the major candidate transmitters known in vertebrates. Two groups of native flatworm neuropeptides have been sequenced, neuropeptide F and FMRFamide-related peptides (FaRPs), and immunoreactivities for these have been localised in dense-cored neuronal vesicles in representatives of all major fiatworm groups. There is evidence of co-localisation of peptidergic and cholinergic elements; serotoninergic components generally occupy a separate set of neurons. The actions of neuronal substances in flatworms are largely undetermined, but FaRPs and 5-HT are known to be myoactive in all of the major groups, and there is immuno-cytochemical evidence that they have a role in the mechanism of egg assembly.

Heterotypic binding between neuronal membrane vesicles and glial cells is mediated by a specific cell adhesion molecule.

By means of a multistage quantitative assay, we have identified a new kind of cell adhesion molecule (CAM) on neuronal cells of the chick embryo that is involved in their adhesion to glial cells. The assay used to identify the binding component (which we name neuron-glia CAM or Ng-CAM) was designed to distinguish between homotypic binding (e.g., neuron to neuron) and heterotypic binding (e.g., neuron to glia). This distinction was essential because a single neuron might simultaneously carry different CAMs separately mediating each of these interactions. The adhesion of neuronal cells to glial cells in vitro was previously found to be inhibited by Fab' fragments prepared from antisera against neuronal membranes but not by Fab' fragments against N-CAM, the neural cell adhesion molecule. This suggested that neuron-glia adhesion is mediated by specific cell surface molecules different from previously isolated CAMs . To verify that this was the case, neuronal membrane vesicles were labeled internally with 6-carboxyfluorescein and externally with 125I-labeled antibodies to N-CAM to block their homotypic binding. Labeled vesicles bound to glial cells but not to fibroblasts during a 30-min incubation period. The specific binding of the neuronal vesicles to glial cells was measured by fluorescence microscopy and gamma spectroscopy of the 125I label. Binding increased with increasing concentrations of both glial cells and neuronal vesicles. Fab' fragments prepared from anti-neuronal membrane sera that inhibited binding between neurons and glial cells were also found to inhibit neuronal vesicle binding to glial cells. The inhibitory activity of the Fab' fragments was depleted by preincubation with neuronal cells but not with glial cells. Trypsin treatment of neuronal membrane vesicles released material that neutralized Fab' fragment inhibition; after chromatography, neutralizing activity was enriched 50-fold. This fraction was injected into mice to produce monoclonal antibodies; an antibody was obtained that interacted with neurons, inhibited binding of neuronal membrane vesicles to glial cells, and recognized an Mr = 135,000 band in immunoblots of embryonic chick brain membranes. These results suggest that this molecule is present on the surfaces of neurons and that it directly or indirectly mediates adhesion between neurons and glial cells. Because the monoclonal antibody as well as the original polyspecific antibodies that were active in the assay did not bind to glial cells, we infer that neuron-glial interaction is heterophilic, i.e., it occurs between Ng-CAM on neurons and an as yet unidentified CAM present on glial cells.