Microtubule minus ends are thought to be stable in cells. Surprisingly, in Drosophila and zebrafish neurons, we observed persistent minus end growth, with runs lasting over 10 min. In Drosophila, extended minus end growth depended on Patronin, and Patronin reduction disrupted dendritic minus-end-out polarity. In fly dendrites, microtubule nucleation sites localize at dendrite branch points. Therefore, we hypothesized minus end growth might be particularly important beyond branch points. Distal dendrites have mixed polarity, and reduction of Patronin lowered the number of minus-end-out microtubules. More strikingly, extra Patronin made terminal dendrites almost completely minus-end-out, indicating low Patronin normally limits minus-end-out microtubules. To determine whether minus end growth populated new dendrites with microtubules, we analyzed dendrite development and regeneration. Minus ends extended into growing dendrites in the presence of Patronin. In sum, our data suggest that Patronin facilitates sustained microtubule minus end growth, which is critical for populating dendrites with minus-end-out microtubules.

In vertebrate neurons, the axon initial segment (AIS) is specialized for action potential initiation. It is organized by a giant 480 Kd variant of ankyrin G (AnkG) that serves as an anchor for ion channels and is required for a plasma membrane diffusion barrier that excludes somatodendritic proteins from the axon. An unusually long exon required to encode this 480Kd variant is thought to have been inserted only recently during vertebrate evolution, so the giant ankyrin-based AIS scaffold has been viewed as a vertebrate adaptation for fast, precise signaling. We re-examined AIS evolution through phylogenomic analysis of ankyrins and by testing the role of ankyrins in proximal axon organization in a model multipolar Drosophila neuron (ddaE). We find giant isoforms of ankyrin in all major bilaterian phyla, and present evidence in favor of a single common origin for giant ankyrins and the corresponding long exon in a bilaterian ancestor. This finding raises the question of whether giant ankyrin isoforms play a conserved role in AIS organization throughout the Bilateria. We examined this possibility by looking for conserved ankyrin-dependent AIS features in Drosophila ddaE neurons via live imaging. We found that ddaE neurons have an axonal diffusion barrier proximal to the cell body that requires a giant isoform of the neuronal ankyrin Ank2. Furthermore, the potassium channel shal concentrates in the proximal axon in an Ank2-dependent manner. Our results indicate that the giant ankyrin-based cytoskeleton of the AIS may have evolved prior to the radiation of extant bilaterian lineages, much earlier than previously thought.