An Ancient Role for Giant Ankyrins in Axon Initial Segment Organization

Abstract

The axon initial segment (AIS) of multipolar vertebrate neurons contains a specialized set of ion channels that allows it to serve as the site for action potential initiation. The AIS is organized by a giant variant of ankyrin G (AnkG) that links ion channels to a specialized cytoskeleton and is required for a plasma membrane diffusion barrier that excludes somatodendritic proteins from the axon. The ankyrin-based AIS has been viewed as a vertebrate adaptation for fast, precise signaling because the giant isoform required for AIS organization is believed to have evolved recently in the vertebrate lineage. Here we re-examine AIS origins with a detailed analysis of giant ankyrin evolution and testing the role of a giant ankyrin in organization of an AIS-like domain in the proximal axon of a model multipolar Drosophila neuron (ddaE). We found giant ankyrins in all major bilaterian phyla, and phylogenetic analysis points to a single origin for the unusual long exon that encodes giant ankyrins in an ancestor of all extant bilaterians. We therefore hypothesized that giant ankyrins may play a conserved role in organization of the proximal axon throughout the Bilateria. We examined this possibility by looking for conserved ankyrin-dependent AIS features in Drosophila ddaE neurons via live imaging. Using a FRAP assay, we found that ddaE neurons have a plasma membrane diffusion barrier in the proximal axon that requires a giant isoform of the neuronal ankyrin ank2. Two voltage-gated K+ channels, elk and shal, concentrate in this proximal axon. We found that the accumulation of shal, like the diffusion barrier, depends on ank2. Our results suggest that the giant ankyrin-based cytoskeleton of the AIS may not be a recent vertebrate innovation, but instead evolved in an ancestral bilaterian soon after the divergence from cnidarians.