Abstract. Caenorhabditis elegans unc-44 mutations result in aberrant axon guidance and fasciculation with inappropriate partners. The unc-44 gene was cloned by transposon tagging, and verified by genetic and molecular analyses of six transposon-induced alleles and their revertants. Nucleotide sequence analyses demonstrated that unc-44 encodes a series of putative ankyrin-related proteins, including AO49 ankyrin (1815 aa, 198.8 kD), AO66 ankyrin (1867 aa, 204 kD), and AOI3 ankyrin (x<4700 aa, ~<517 kD). In addition to the major set of •6 kb alternatively spliced transcripts, minor transcripts were observed at -,3, 5, 7, and 14 kb. Evidence is provided that mutations in the *14-kb AO13 ankyrin transcript are responsible for the neuronal defects. These molecular studies provide the first evidence that ankyrin-related molecules are required for axonal guidance.TrIOUGH the molecular basis of neural development has been the object of intense study in recent years, the detailed mechanisms of axon guidance remain unknown (for general reviews see Dodd and Jessell, 1988;Jessell, 1988;Takeichi, 1988;Sanes, 1989;Takeichi, 1991;Rathjen et al., 1992;Gumbiner, 1993; for C. elegans reviews see Hedgecock et al., 1987;Wadsworth and Hedgecock, 1992).Mutations in the unc-44 gene affect the direction of axonal outgrowth for many axons (Hedgecock et al., 1985;Siddiqui, 1990;Siddiqui and Culotti, 1991;Mclntire et al., 1992), including the postdeirid (PDE) ~ axon, which normally extends from the postdeirid sensillum on the lateral surface of the nematode to the ventral nerve cord (White et al., 1986). In unc-44 mutants, the initial direction of PDE axon outgrowth along the basement membrane is apparently random, and the misdirected PDE axon fasciculates with inappropriate partners (Hedgecock et al., 1985 The discovery that the C. elegans unc-6 gene encodes a laminin B chain-related product provided evidence that directed axonal outgrowth and cell migration require interactions with the extracellular matrix (Hedgecock et al., 1990), and that these interactions use laminin or related proteins in both invertebrates and vertebrates (Jessell, 1988;Sanes, 1989;Hedgecock et al., 1990;Serafini et al., 1994). The product of the unc-5 gene, which affects dorsalward cell migrations and axon outgrowth, has been proposed to be a cell surface protein which may interact with the extracellular matrix (Leung-Hagesteijn et al., 1992). Thus, it was likely that other mutations affecting axonal outgrowth and guidance were defects in cytoskeletal or extracellular matrix structures. The actin/a-actinin framework of growth cone filopodia or the spectrin/ankyrin network underlying the cytoplasmic surface of the plasma membrane could be the targets for mutations affecting axon outgrowth and growth cone adhesion. In this study, we have discovered that the wild-type unc-44 gene, which is required for proper axonal guidance, encodes a series of putative ankyrin-related proteins.Ankyrin (or bands 2.1 and 2.2) has been most thoroughly studied in erythrocyte "ghost...
Caveolae are vesicular organelles that represent an appendage of the plasma membrane. Caveolin, a 21-24-kDa integral membrane protein, is a principal component of caveolae membranes in vivo. Caveolin has been proposed to function as a plasma membrane scaffolding protein to organize and concentrate signaling molecules within caveolae, including heterotrimeric G proteins (␣ and ␥ subunits). In this regard, caveolin interacts directly with G ␣ subunits and can functionally regulate their activity. To date, three cDNAs encoding four subtypes of caveolin have been described in vertebrates. However, evidence for the existence of caveolin proteins in less complex organisms has been lacking. Here, we report the identification, cDNA sequence and genomic organization of the first invertebrate caveolin gene, Cav ce (for caveolin from Caenorhabditis elegans). The Cav ce gene, located on chromosome IV, consists of two exons interrupted by a 125-nucleotide intron sequence. The region of Cav ce that is strictly homologous to mammalian caveolins is encoded by a single exon in Cav ce. This suggests that mammalian caveolins may have evolved from the second exon of Cav ce. Cav ce is roughly equally related to all three known mammalian caveolins and, thus, could represent a common ancestor. Remarkably, the invertebrate Cav ce protein behaves like mammalian caveolins: (i) Cav ce forms a high molecular mass oligomer, (ii) assumes a cytoplasmic membrane orientation, and (iii) interacts with G proteins. A 20-residue peptide encoding the predicted G protein binding region of Cav ce possesses "GDP dissociation inhibitor-like activity" with the same potency as described earlier for mammalian caveolin-1. Thus, caveolin appears to be structurally and functionally conserved from worms to man. In addition, we find that there are at least two caveolin-related genes expressed in C. elegans, defining an invertebrate caveolin gene family. These results establish the nematode C. elegans as an invertebrate model system to study caveolae and caveolin in vivo. Caveolae are vesicular organelles that represent a subdivision of the plasma membrane (1, 2). Although they are found in most cell types, caveolae are most numerous in adipocytes, endothelial cells, fibroblasts, and muscle cells (smooth, skeletal, and cardiac). In transmission electron micrographs they can be distinguished by their characteristic shape: ϳ50-100-nm vesicles located at or near the plasma membrane (3, 4). Functionally, caveolae have been implicated in a variety of signal transduction related events, including signaling from G protein-coupled receptors and growth factor receptors (5-7). Caveolin, a 21-24-kDa protein, is an integral membrane component of caveolae (8-12). Caveolin co-purifies with lipid modified signaling molecules, such as G proteins (␣ and ␥ subunits), Src-family tyrosine kinases, and H-Ras (see Refs. 6, 9, and 13-18 and references cited within). These molecules appear to be tightly associated as a discrete complex with caveolin as shown using a polyhistidine-tagg...
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.
In Caenorhabditis elegans, unc-44 mutations affect axonal outgrowth and guidance, leading to locomotory defects. The wild-type unc-44 gene encodes a family of ankyrin proteins, which, in addition to the conventional ankyrins, includes a novel ankyrin isoform with an extended C-terminal domain, referred to AO13 ankyrin. Six spontaneous unc-44 mutations and their reversions were analyzed in order to localize regions critical for gene function. The q331::Tc1 and rh1013::Tc1 mutations were mapped to the portion of the gene encoding the conventional ankyrins, mn339 had an uncharacterized 2-kb insertion in the serine/threonine/glutamic acid/proline-rich (STEP) repeat block 5, st200::Tc5(variant) and rh1042::Tc1 were localized near the C-terminus, and mn259 resulted from two Tc1 insertions, one in STEP block 6 and the other near the C-terminus. Tc1 excisions in several revertants resulted either in the restoration of the wild-type sequence, or were associated with small in-frame deletions or insertions. Reversion of mn339 resulted in the net excision of 2463 bp of genomic DNA, including the region encoding parts of STEP blocks 5 and 6 and the intervening hydrophobic region. Interestingly, additional Tc1 insertions at a 5' exon/intron boundary were found in revertants of st200 and rh1042. Reversion of the st200::Tc5 mutation resulted in excision of the Tc5 element, and the insertion of two copies of Tc1 at different sites. The wild-type unc-44 gene produces multiple transcripts - shorter RNAs determined to be approximately 1, 3.2, 5, 6, and 7 kb long, and two large transcripts estimated to be 22 and 26 kb in length. The largest transcripts were affected by all unc-44 mutations and are proposed to be essential for axonal outgrowth and guidance.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.