Bilaterian Giant Ankyrins Have a Common Evolutionary Origin and Play a Conserved Role in Patterning the Axon Initial Segment

<div><p>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 <i>Drosophila</i> 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 <i>Drosophila</i> 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.</p></div

Model for ankyrin family evolution based on the phylogenetic distribution of short and giant ankyrins.

<p><b>Numbered arrowhead indicate key points in Ankyrin evolution as follows:</b> (1) Ankyrins likely first evolved in an ancestor of extant eumetazoans because they can be found in placozoans, cnidarians and bilaterians, but are absent from sponges (Porifera) and comb jellies (Ctenophora). The ancestral Ankyrin lacked the long exon and could not encode giant isoforms. (2) The long exon was most likely inserted after divergence of the eumetazoans in an ancestor of extant bilaterians, because giant ankyrins encoded by inclusion of a long exon are present in all major groups of bilaterians that we examined. The original long exon was presumably inserted downstream of the death domain (DD) because it is found in this position in both protostomes and early diverging deuterostome lineages. (3) The long exon switches position prior to the emergence of chordates, which carry the long exon upstream of the DD. The position in cephalochordates is uncertain as we were unable to identify a DD in <i>Branchiostoma</i>. The DD has low homology and is typically split over several exons and is thus difficult to identify if it is not present in automated gene prediction and transcriptome data is not available. The ancestral long exon has been lost multiple times in bilaterian evolution including (4) two separate lineages within the Ecdysozoa and (5) the vertebrate AnkR lineage.</p

<i>Ciona intestinalis</i> ankyrin has a long exon comparable to mouse, fly and worm that can encode giant isoform(s).

<p>(A) A map of the Ciona intestinalis ankyrin locus showing the position of exons (color coded by domain) comprising an ankyrin ortholog gene prediction relative to the position of a giant ORF (red) that could represent a long exon. (B) <i>In situ</i> hybridization of mid (left) and late (right) tail bud stage larvae with a probe corresponding to an EST cluster from within the potential long exon (black box in A) shows expression in the sensory vesicle (SV) and the visceral ganglion (VG), which together comprise the bulk of the larval nervous system. Pictures are publically available from the Aniseed database (<b>A</b>scidian <b>N</b>etwork for <b>I</b>n <b>S</b>itu <b>E</b>xpression and <b>E</b>mbryological <b>D</b>ata; <a href="http://www.aniseed.cnrs.fr/" target="_blank">http://www.aniseed.cnrs.fr/</a>) where additional pictures and methods can be found (gene prediction KH.C1.943). (C) Comparison of the Ciona ankyrin long exon size with that of the average size of all ORFs (defined as > 300 nucleotides without a stop codon) in the Ciona genome draft. (D) Length distribution for the largest 1,000 ORFs in the Ciona genome in bins of 250 bp, reveals that the Ciona ankyrin long exons sits in the 2^nd largest ORF in the genome. (E-G) Similar length distribution graphs for the largest 1,000 ORFs in mouse, <i>Drosophila melanogaster</i> and <i>C</i>. <i>elegans</i> with the rank of long ankyring exons indicated.</p

Diffusion in the plasma membrane is restricted at the base of the axon compared to the base of the dendrite.

<p>A. Example images of dendrite and axon FRAP experiments at two different larval ages are shown. mCD8-RFP was expressed in class I dendritic arborization neurons. Bleaching was performed in the ddaE neuron either at the base of the comb-like dendrite or the base of the axon. The red circle indicates the bleach area. B. The average recovery of fluorescence into the bleach areas as shown in A is plotted on the graph; averages were calculated from 14 cells for the dendrite, 17 for axon 2 day and 13 for axon 3 day. Each cell was in a different animal. The error bars show the standard error of the mean. C. Bars show the recovery plateau (mean ± SEM) quantitated by averaging recovery values between 110 and 120 ms. The asterisk indicates significant difference (P < 0.01, t-test).</p

RNAi targeting Ank2L reduces the axonal diffusion barrier.

<p>A. mCD8-GFP was expressed in class I neurons, and proximal axons of ddaE neurons were photobleached. Example images of a control neuron are shown in the top row, and Ank2 RNAi neurons below. B. Quantitation of FRAP experiments in different genetic backgrounds is compiled in the graph. The average recovery is shown, with standard error shown as error bars. Number of cells tested for each genotype were: control-24, Ank2-19, Ank-12, CRMP-9. C. The recovery plateau (mean ± SEM) was quantitated by averaging recovery values between 110 and 120 ms. The asterisk indicates significant difference (p < 0.02, t-test).</p

Ankyrins are highly conserved between bilaterians, cnidarians and placozoans.

<p>(A) schematic diagram of a canonical ankyrin protein showing the 24 ankyrin repeats, the ZU5-ZU5-UPA cassette and the death domain (DD). The position of the long exon included in giant isoforms is shown for vertebrates (red star) and protostome invertebrates (green star). (B) Pairwise amino acid identity shared between mouse AnkG (Mmus_AnkG; Bilateria, deuterostome), <i>Drosophila melanogaster</i> Ank2 (Dmel_Ank2; Bilateria, protostome), <i>Nematostella vectensis</i> Ank (Nvec_Ank; Cnidaria) and <i>Trichoplax adhaerens</i> Ank (Tadh_Ank; Placozoa) for the shown alignment. (C) Amino acid alignment of core conserved domains is shown for bilaterian, cnidarian and placozoan ankyrins. Gene name is given at the left margin and amino acid position at the right margin. Residues identical in at least 3/4 sequences are shaded according to the domain color codes shown in (A). Domains boundaries are indicated with underlines; the 24 ankyrin repeats are underlined with alternating black and gray lines for clarity. The spectrin binding motif of vertebrate ankyrins is highlighted with a bright green outline.</p

Amino acid composition of ankyrin long exon polypeptides.

<p>(A) Amino acid compositions for 11 long exon-encoded polypeptides from 10 bilaterian ankyrin genes. Gene names are given at the left margin, and frequencies are given as percent composition for each amino acid. Frequencies are color coded with a blue (low) to red (high) and amino acids are arranged left to right in ascending order of average frequency. (B) An identical amino acid composition analysis as shown in (A) is given for the core ankyrin motifs (24 ankyrin repeats + the ZU5-ZU5-UPA cassette). Note the very different amino acid enrichment profiles (red highlights). For instance, Ala and Leu replace Glu and Ser as the top two most frequent residues. <i>Nematostella</i> ankyrin was included in the core analysis to demonstrate that the composition bias of the core has not changed since the divergence of cnidarians and bilaterians, likely soon after emergence of the first ankyrins. (C) Fold enrichment for each amino acid in long exon vs. core polypeptides, color coded blue (depleted) to red (enriched). The top 6 long exon amino acids are all enriched relative to the core with a > 2-fold enrichment for Glu and Ser. Fold enrichment was calculated for sequence sets shown in A and B by dividing the average amino acid frequency encoded in long exons (A, asterisk) by the average amino acid frequency found in the core (B, asterisk).</p