Twinfilin-2a Is Dispensable for Mouse Development

Peer reviewe

MyosinVIIa Interacts with Twinfilin-2 at the Tips of Mechanosensory Stereocilia in the Inner Ear

In vertebrates hearing is dependent upon the microvilli-like mechanosensory stereocilia and their length gradation. The staircase-like organization of the stereocilia bundle is dynamically maintained by variable actin turnover rates. Two unconventional myosins were previously implicated in stereocilia length regulation but the mechanisms of their action remain unknown. MyosinXVa is expressed in stereocilia tips at levels proportional to stereocilia length and its absence produces staircase-like bundles of very short stereocilia. MyosinVIIa localizes to the tips of the shorter stereocilia within bundles, and when absent, the stereocilia are abnormally long. We show here that myosinVIIa interacts with twinfilin-2, an actin binding protein, which inhibits actin polymerization at the barbed end of the filament, and that twinfilin localization in stereocilia overlaps with myosinVIIa. Exogenous expression of myosinVIIa in fibroblasts results in a reduced number of filopodia and promotes accumulation of twinfilin-2 at the filopodia tips. We hypothesize that the newly described interaction between myosinVIIa and twinfilin-2 is responsible for the establishment and maintenance of slower rates of actin turnover in shorter stereocilia, and that interplay between complexes of myosinVIIa/twinfilin-2 and myosinXVa/whirlin is responsible for stereocilia length gradation within the bundle staircase

Identification of a common risk haplotype for canine idiopathic epilepsy in the ADAM23 gene

Background: Idiopathic epilepsy is a common neurological disease in human and domestic dogs but relatively few risk genes have been identified to date. The seizure characteristics, including focal and generalised seizures, are similar between the two species, with gene discovery facilitated by the reduced genetic heterogeneity of purebred dogs. We have recently identified a risk locus for idiopathic epilepsy in the Belgian Shepherd breed on a 4.4 megabase region on CFA37. Results: We have expanded a previous study replicating the association with a combined analysis of 157 cases and 179 controls in three additional breeds: Schipperke, Finnish Spitz and Beagle (p(c) = 2.9e-07, p(GWAS) = 1.74E-02). A targeted resequencing of the 4.4 megabase region in twelve Belgian Shepherd cases and twelve controls with opposite haplotypes identified 37 case-specific variants within the ADAM23 gene. Twenty-seven variants were validated in 285 cases and 355 controls from four breeds, resulting in a strong replication of the ADAM23 locus (p(raw) = 2.76e-15) and the identification of a common 28 kb-risk haplotype in all four breeds. Risk haplotype was present in frequencies of 0.49-0.7 in the breeds, suggesting that ADAM23 is a low penetrance risk gene for canine epilepsy. Conclusions: These results implicate ADAM23 in common canine idiopathic epilepsy, although the causative variant remains yet to be identified. ADAM23 plays a role in synaptic transmission and interacts with known epilepsy genes, LGI1 and LGI2, and should be considered as a candidate gene for human epilepsies.Peer reviewe

Mammalian twinfilin sequesters ADP-G-actin and caps filament barbed ends: implications in motility

Twinfilins are conserved actin-binding proteins composed of two actin depolymerizing factor homology (ADF-H) domains. Twinfilins are involved in diverse morphological and motile processes, but their mechanism of action has not been elucidated. Here, we show that mammalian twinfilin both sequesters ADP-G-actin and caps filament barbed ends with preferential affinity for ADP-bound ends. Twinfilin replaces capping protein and promotes motility of N-WASP functionalized beads in a biomimetic motility assay, indicating that the capping activity supports twinfilin's function in motility. Consistently, in vivo twinfilin localizes to actin tails of propelling endosomes. The ADP-actin-sequestering activity cooperates with the filament capping activity of twinfilin to finely regulate motility due to processive filament assembly catalyzed by formin-functionalized beads. The isolated ADF-H domains do not cap barbed ends nor promote motility, but sequester ADP-actin, the C-terminal domain showing the highest affinity. A structural model for binding of twinfilin to barbed ends is proposed based on the similar foldings of twinfilin ADF-H domains and gelsolin segments

Identification of a common risk haplotype for canine idiopathic epilepsy in the ADAM23 gene.

BackgroundIdiopathic epilepsy is a common neurological disease in human and domestic dogs but relatively few risk genes have been identified to date. The seizure characteristics, including focal and generalised seizures, are similar between the two species, with gene discovery facilitated by the reduced genetic heterogeneity of purebred dogs. We have recently identified a risk locus for idiopathic epilepsy in the Belgian Shepherd breed on a 4.4 megabase region on CFA37.ResultsWe have expanded a previous study replicating the association with a combined analysis of 157 cases and 179 controls in three additional breeds: Schipperke, Finnish Spitz and Beagle (p(c) = 2.9e-07, p(GWAS) = 1.74E-02). A targeted resequencing of the 4.4 megabase region in twelve Belgian Shepherd cases and twelve controls with opposite haplotypes identified 37 case-specific variants within the ADAM23 gene. Twenty-seven variants were validated in 285 cases and 355 controls from four breeds, resulting in a strong replication of the ADAM23 locus (p(raw) = 2.76e-15) and the identification of a common 28 kb-risk haplotype in all four breeds. Risk haplotype was present in frequencies of 0.49-0.7 in the breeds, suggesting that ADAM23 is a low penetrance risk gene for canine epilepsy.ConclusionsThese results implicate ADAM23 in common canine idiopathic epilepsy, although the causative variant remains yet to be identified. ADAM23 plays a role in synaptic transmission and interacts with known epilepsy genes, LGI1 and LGI2, and should be considered as a candidate gene for human epilepsies

Northern blot, RT-PCR, and Western blot analysis of twinfilin-2a knockout mice.

<p>Total RNAs and proteins were isolated from 3-month-old wild-type and twinfilin-2a knockout littermates. (A) Northern blot analysis of the total RNA of wild type (+/+) and knockout (−/−) mice hybridized with a twinfilin-2 cDNA probe. Twinfilin-2 RNA is absent from all mutant tissues, except in heart and skeletal muscles. The remaining twinfilin-2 mRNA encodes for the twinfilin-2b isoform. Equal loading between the wild-type and knockout RNAs is demonstrated in the lower panel. (B) Agarose gel electrophoresis of products from RT-PCR of total tissue RNA of wild type (+/+) and knockout (−/−) mice. Twinfilin-2a mRNA is absent from all mutant tissues, whereas twinfilin-2b expression remains in heart and skeletal muscles of twinfilin-2a knockout mice. Lower panel shows control reactions carried out by using GAPDH-specific primers. The slower mobility band detected in liver and brain of knockout mice was isolated and sequenced and turned out to be an unspecific product of an unrelated protein. (C) Western blot of tissue homogenates of wild type (+/+) and knockout (−/−) mice probed with twinfilin-2 (Twf-2) or twinfilin-1 (Twf-1) specific polyclonal antibodies. Twinfilin-2 protein was absent from all other mouse tissues tested except heart and skeletal muscles. Please note that the twinfilin-2 antibody used here does not distinguish between twinfilin-2a and twinfilin-2b isoforms. GAPDH and actin are shown as loading controls.</p

Generation of twinfilin-2a knockout mice.

<p>(A) Partial map of the wild-type <i>Twf2</i> allele, the targeting construct and the recombinant allele, and locations of the probes. The white boxes show the exons of the <i>twinfilin-2</i> gene and the shaded box the neomycin cassette, black box indicates the pWH9 vector. The expected fragment sizes of the wild type and the recombinant allele after digestion with EcoRI and subsequent hybridization with the indicated external probe are 8,6 and 3,5 kb, and with the indicated internal probe, 8,6 and 11 kb, respectively. (B) Southern blot analysis with the external probe of EcoRI digested mouse tail DNAs derived from progeny of heterozygous mating reveals the wild-type (8,6 kb) and the targeted <i>Twf2</i> allele (3,5 kb). [+/+ wild type, +/− heterozygote, −/− knockout].</p

qRT-PCR analysis of twinfilin-1 and twinfilin-2b expression in wild-type and twinfilin-2a knockout tissues.

<p><i>Gapdh</i> and <i>beta-actin</i> amplification was used as a control. <i>Twf2b</i> is the most abundant isoform in heart and skeletal muscles, whereas <i>Twf1</i> is widely expressed in non-muscle tissues. The expression levels of either <i>Twf-1</i> or <i>Twf-2b</i> are not significantly altered in the tissues of <i>Twf-2a</i> knockout mouse compared to the wild-type tissues.</p

Relative amounts of twinfilin-1 and twinfilin-2a proteins in the brain lysates.

<p>Mouse cell extracts and indicated amounts of purified recombinant mouse twinfilin-1 (A) or twinfilin-2a (B) were run on polyacrylamide gels, and the proteins were visualized by Western blotting using isoform-specific twinfilin-1 (A) or twinfilin-2 (B) antibodies. Twinfilin-1 is 5–10 fold more abundant than twinfilin-2 in the brain lysate. Twinfilin-2 is almost non-detectable in the twinfilin-2a knockout brain lysate, suggesting that twinfilin-2a is the predominant twinfilin-2 isoform in the brain.</p

Novel origins of copy number variation in the dog genome

BACKGROUND: Copy number variants (CNVs) account for substantial variation between genomes and are a major source of normal and pathogenic phenotypic differences. The dog is an ideal model to investigate mutational mechanisms that generate CNVs as its genome lacks a functional ortholog of the PRDM9 gene implicated in recombination and CNV formation in humans. Here we comprehensively assay CNVs using high-density array comparative genomic hybridization in 50 dogs from 17 dog breeds and 3 gray wolves. RESULTS: We use a stringent new method to identify a total of 430 high-confidence CNV loci, which range in size from 9 kb to 1.6 Mb and span 26.4 Mb, or 1.08%, of the assayed dog genome, overlapping 413 annotated genes. Of CNVs observed in each breed, 98% are also observed in multiple breeds. CNVs predicted to disrupt gene function are significantly less common than expected by chance. We identify a significant overrepresentation of peaks of GC content, previously shown to be enriched in dog recombination hotspots, in the vicinity of CNV breakpoints. CONCLUSIONS: A number of the CNVs identified by this study are candidates for generating breed-specific phenotypes. Purifying selection seems to be a major factor shaping structural variation in the dog genome, suggesting that many CNVs are deleterious. Localized peaks of GC content appear to be novel sites of CNV formation in the dog genome by non-allelic homologous recombination, potentially activated by the loss of PRDM9. These sequence features may have driven genome instability and chromosomal rearrangements throughout canid evolution.Additional author: The LUPA Consortium (www.eurolupa.org)</p