In vertebrate neurons, axons have a uniform arrangement of microtubules with plus ends distal to the cell body (plus-end-out), and dendrites have equal numbers of plus-and minus-end-out microtubules. To determine whether microtubule orientation is a conserved feature of axons and dendrites, we analyzed microtubule orientation in invertebrate neurons. Using microtubule plus end dynamics, we mapped microtubule orientation in Drosophila sensory neurons, interneurons, and motor neurons. As expected, all axonal microtubules have plus-end-out orientation. However, in proximal dendrites of all classes of neuron, ϳ90% of dendritic microtubules were oriented with minus ends distal to the cell body. This result suggests that minus-end-out, rather than mixed orientation, microtubules are the signature of the dendritic microtubule cytoskeleton. Surprisingly, our map of microtubule orientation predicts that there are no tracks for direct cargo transport between the cell body and dendrites in unipolar neurons. We confirm this prediction, and validate the completeness of our map, by imaging endosome movements in motor neurons. As predicted by our map, endosomes travel smoothly between the cell body and axon, but they cannot move directly between the cell body and dendrites. INTRODUCTIONMany differentiated cells have highly polarized arrays of microtubules that likely play a large role in establishing their specialized architecture and function. Neurons are strikingly polarized and initially seemed that they would be the clearest example of cells in which microtubule orientation formed the basis of directional transport and cell polarity (Black and Baas, 1989). Most neurons have a cell body in which the bulk of proteins are synthesized, dendrites that are specialized to receive signals, and axons that are specialized to send them. Where examined, microtubules in vertebrate dendrites have mixed orientation, and in axons they have uniform orientation with all plus ends distal to the cell body. Thus, the simplest model for selective transport from the cell body to dendrites is use of a minus end-directed motor. However, current models of transport into dendrites rely on plus enddirected motors (Setou et al., 2004;Hirokawa and Takemura, 2005;Kennedy and Ehlers, 2006;Levy and Holzbaur, 2006). These models raise the question: are minus-end-out microtubules important for directional transport or neuronal polarity?Axonal microtubule orientation has been examined in a variety of neurons, all with the same result: Ͼ95% of plus ends are oriented away from the cell body (plus-end-out). Original studies on axonal microtubule orientation relied on decoration of microtubules with exogenous tubulin, which forms curved hooks on the sides of existing microtubules, and analysis by electron microscopy. The direction of hook curvature indicates microtubule polarity. This method was used to determine axonal microtubule orientation in many different types of vertebrate neurons (Burton and Paige, 1981;Heidemann et al., 1981;Baas et al., 1987Baas et al...

The receptor Notch and its ligands of the Delta/Serrate/LAG2 (DSL) family are the central components in the Notch pathway, a fundamental cell signaling system that regulates pattern formation during animal development. Delta is directly ubiquitinated by Drosophila and Xenopus Neuralized,and by zebrafish Mind bomb, two unrelated RING-type E3 ubiquitin ligases with common abilities to promote Delta endocytosis and signaling activity. Although orthologs of both Neuralized and Mind bomb are found in most metazoan organisms, their relative contributions to Notch signaling in any single organism have not yet been assessed. We show here that a Drosophilaortholog of Mind bomb (D-mib) is a positive component of Notch signaling that is required for multiple Neuralized-independent, Notch-dependent developmental processes. Furthermore, we show that D-mib associates physically and functionally with both Serrate and Delta. We find that D-mib uses its ubiquitin ligase activity to promote DSL ligand activity, an activity that is correlated with its ability to induce the endocytosis and degradation of both Delta and Serrate (see also Le Borgne et al., 2005). We further demonstrate that D-mib can functionally replace Neuralized in multiple cell fate decisions that absolutely require endogenous Neuralized, a testament to the highly similar activities of these two unrelated ubiquitin ligases in regulating Notch signaling. We conclude that ubiquitination of Delta and Serrate by Neuralized and D-mib is an obligate feature of DSL ligand activation throughout Drosophiladevelopment.

Asymmetric division is a fundamental mechanism for generating cellular diversity. In the central nervous system of Drosophila, neural progenitor cells called neuroblasts undergo asymmetric division along the apical-basal cellular axis. Neuroblasts originate from neuroepithelial cells, which are polarized along the apical-basal axis and divide symmetrically along the planar axis. The asymmetry of neuroblasts might arise from neuroblast-specific expression of the proteins required for asymmetric division. Alternatively, both neuroblasts and neuroepithelial cells could be capable of dividing asymmetrically, but in neuroepithelial cells other polarity cues might prevent asymmetric division. Here we show that by disrupting adherens junctions we can convert the symmetric epithelial division into asymmetric division. We further confirm that the adenomatous polyposis coli (APC) tumour suppressor protein is recruited to adherens junctions, and demonstrate that both APC and microtubule-associated EB1 homologues are required for the symmetric epithelial division along the planar axis. Our results indicate that neuroepithelial cells have all the necessary components to execute asymmetric division, but that this pathway is normally overridden by the planar polarity cue provided by adherens junctions.