Novel UNC‐44 AO13 ankyrin is required for axonal guidance in C. elegans, contains six highly repetitive STEP blocks separated by seven potential transmembrane domains, and is localized to neuronal processes and the periphery of neural cell bodies

Abstract

Conventional ankyrins are cortical cytoskeletal proteins that form an ankyrin-spectrin meshwork underlying the plasma membrane. We report here the unusual structure of a novel ankyrin (AO13 ankyrin, 775,369 Da, 6994 aa, pI = 4.45) that is required for proper axonal guidance in Caenorhabditis elegans. AO13 ankyrin contains the ANK repeat and spectrin-binding domains found in other ankyrins, but differs from all others in that the acidic carboxyl region contains six blocks of serine/threonine/glutamic acid/proline rich (STEP) repeats separated by seven hydrophobic domains. The STEP repeat blocks are composed primarily of sequences related to ETTTTTTVTREHFEPED(E/D)X(n)VVESEEYSASGSPVPSE (E/K)DVE(H/R)VI, and the hydrophobic domains contain sequences related to PESGEESDGEGFGSKVLGFAKK[AGMVAGGVVAAPVALAAVGA]KAAYDALKKDDDEE, which includes a potential transmembrane domain (in brackets). Recombinant protein fragments of AO13 ankyrin were used to prepare polyclonal antisera against the spectrin-binding domain (AO271 Ab), the conventional ankyrin regulatory domain (AO280 Ab), the AO13 ankyrin STEP domain (AO346 Ab), the AO13 ankyrin STEP + hydrophobic domain (AO289 Ab), and against two carboxyl terminal domain fragments (AO263 Ab and AO327 Ab). Western blot analysis with these Ab probes demonstrated multiple protein isoforms. By immunofluorescence microscopy, the antispectrin-binding and regulatory domain (AO271 and AO280) antibodies recognized many cell types, including neurons, and stained the junctions between cells. The AO13 ankyrin-specific (AO289 and AO346) antibodies showed a neurally restricted pattern, staining nerve processes and the periphery of neural cell bodies. These results are consistent with a role for AO13 ankyrin in neural development.

Tissue-specific regulation of alternative polyadenylation represses expression of a neuronal ankyrin isoform in C. elegans epidermal development

“…Neither insertion disrupted unc-44 function, as the knock-in animals superficially resembled the wild type in movement and growth rate. In adults, the pattern of UNC-44C::GFP( ju1413) fluorescence was generally consistent with that described for UNC-44 common isoforms by immunostaining (Otsuka et al, 2002); however, we also observed strong expression in muscles and in gonadal spermathecal/sheath cells (Fig. 1B, upper panel).…”

“…S1A). The N-terminal common domains of the major UNC-44 isoforms contain multiple ankyrin repeats and a spectrin-binding domain followed by a regulatory domain (Otsuka et al, 2002). Previous studies of UNC-44 expression used antibodies raised against the spectrin-binding domain, the conventional regulatory domain, or the extended C-terminal set of STEP domains specific to the large isoform.…”

“…Previous studies of UNC-44 expression used antibodies raised against the spectrin-binding domain, the conventional regulatory domain, or the extended C-terminal set of STEP domains specific to the large isoform. These studies showed that the common ankyrin proteins were widely expressed in multiple tissues, including oocytes, epidermis, the vulva and the nervous system, whereas the long isoform was restricted to the nervous system (Otsuka et al, 2002).…”

“…The C. elegans unc-44 gene generates multiple ankyrin isoforms, of which the large isoform (UNC-44L) is generally thought to be neuron specific (Otsuka et al, 2002). We recently identified one mechanism that promotes UNC44L expression in neurons via nuclear alternative polyadenylation.…”

Lateral Membrane Biogenesis in Human Bronchial Epithelial Cells Requires 190-kDa Ankyrin-G

“…Section 1734 solely to indicate this fact. (23,24). We have used small interfering RNA (siRNA) encoded by a plasmid (25) to knock down the transcript encoding 190-kDa ankyrin-G in a polarized human bronchial epithelial cell line.…”