Identification and expression analysis of splice variants of mouse enabled homologue during development and in adult tissues

<p>Abstract</p> <p>Background</p> <p>The Enabled/Vasodilator stimulated phosphoprotein (Ena/VASP) gene family comprises three genes in vertebrates: <it>Vasp</it>, Enabled homologue (<it>Enah</it>) and Ena-VASP like (<it>Evl</it>). <it>Enah </it>has the most complex gene structure. It has extra alternatively included exons compared to <it>Vasp </it>and <it>Evl</it>, and possibly one alternatively excluded intron S. The aim of this mapping study was to probe the occurrence of combinations of exon usage in <it>Enah </it>thereby identifying possible vertebrate ENAH splice variants. We investigated this via an <it>in silico </it>analysis and by performing a reverse transcription-polymerase chain reaction (RT-PCR) screen on mouse samples. We further probed the expression pattern of mouse Enah splice variants during development and in a selection of mouse adult tissues and mouse cell lines.</p> <p>Results</p> <p><it>In silico </it>analysis of the vertebrate Ena/VASP gene family reveals that birds do not have <it>Vasp</it>, while fish have two <it>Evl </it>genes. Analysis of expressed sequence tags of vertebrate <it>Enah </it>splice variants confirms that an Enah transcript without alternative exons is ubiquitously expressed, but yields only limited information about the existence of other possible alternatively spliced Enah transcripts. Via a RT-PCR screen, we provide evidence that during mouse development and in adult mice at least eight and maximally sixteen different Enah transcripts are expressed. We also show that tissues and cell lines display specific expression profiles of these different transcripts. Exons previously associated with neuronal expression of Enah splice variants are also present in other tissues, in particular in heart.</p> <p>Conclusions</p> <p>We propose a more uniform nomenclature for alternative exons in <it>Enah</it>. We provide an overview of distinct expression profiles of mouse Enah splice variants during mouse development, in adult mouse tissues and in a subset of mouse cell lines.</p

The US3 kinase of pseudorabies virus leads to activation of the actin regulator cofilin to induce actin cytoskeleton changes

The US3 kinase is conserved amongst all Alphaherpesvirinae. We and others have shown that this kinase induces dramatic rearrangements of the actin cytoskeleton, including disassembly of actin stress fibers (resulting in cell rounding) and the formation of cellular projections, which are associated with increased viral spread (Favoreel et al., 2005, PNAS). For the alphaherpesvirus pseudorabies virus (PRV), we have found that the US3-induced changes in the actin cytoskeleton are mediated through p21-activated kinases (PAKs), central regulators in RhoGTPase signaling (Van den Broeke et al., 2009, PNAS). Apart from the involvement of PAKs, relatively little is known on the cellular factors that contribute to US3-mediated actin rearrangements. Cofilin, a member of the ADF/cofilin family, is a central player in actin dynamics and is known to be inactivated through phosphorylation on serine residue 3 (S3) (Moriyama et al., 1996, Genes Cells). Our aim is to investigate whether the US3 protein of the alphaherpesvirus pseudorabies virus (PRV) affects cofilin phosphorylation, and, if so, whether this contributes to the US3-mediated effects on the actin cytoskeleton. We report that US3 leads to strong cofilin dephosphorylation, which is inhibited by a PAK inhibitor, and that overexpression of a phosphomimetic cofilin variant interferes with US3-mediated actin rearrangements

Phenotypes induced by NM causing α-skeletal muscle actin mutants in fibroblasts, Sol 8 myoblasts and myotubes

<p>Abstract</p> <p>Background</p> <p>Nemaline myopathy is a neuromuscular disorder characterized by the presence of nemaline bodies in patient muscles. 20% of the cases are associated with α-skeletal muscle actin mutations. We previously showed that actin mutations can cause four different biochemical phenotypes and that expression of NM associated actin mutants in fibroblasts, myoblasts and myotubes induces a range of cellular defects.</p> <p>Findings</p> <p>We conducted the same biochemical experiments for twelve new actin mutants associated with nemaline myopathy. We observed folding and polymerization defects. Immunostainings of these and eight other mutants in transfected cells revealed typical cellular defects such as nemaline rods or aggregates, decreased incorporation in F-actin structures, membrane blebbing, the formation of thickened actin fibres and cell membrane blebbing in myotubes.</p> <p>Conclusion</p> <p>Our results confirm that NM associated α-actin mutations induce a range of defects at the biochemical level as well as in cultured fibroblasts and muscle cells.</p

Structural plasticity of functional actin: Pictures of actin binding protein and polymer interfaces (vol 11, pg 1279, 2004)

AbstractActin is one of the most conserved and versatile proteins capable of forming homopolymers and interacting with numerous other proteins in the cell. We performed an alanine mutagenesis scan covering the entire β-actin molecule. Somewhat surprisingly, the majority of the mutants were capable of reaching a stable conformation. We tested the ability of these mutants to bind to various actin binding proteins, thereby mapping different interfaces with actin. Additionally, we tested their ability to copolymerize with α-actin in order to localize regions in actin that contact neighboring protomers in the filament. Hereby, we could discriminate between two existing models for filamentous actin and our data strongly support the right-handed double-stranded helix model. We present data corroborating this model in vivo. Mutants defective in copolymerization do not colocalize with the actin cytoskeleton and some impair its normal function, thereby disturbing cell shape

alpha-Skeletal Muscle Actin Mutants Causing Different Congenital Myopathies Induce Similar Cytoskeletal Defects in Cell Line Cultures

Central core disease (CCD), congenital fibre type disproportion (CFTD), and nemaline myopathy (NM) are earlyonset clinically heterogeneous congenital myopathies, characterized by generalized Muscle weakness and hypotonia. All three diseases are associated with (alpha-skeletal muscle actin mutations. We biochemically characterized the CCD and CFTD causing actin mutants and show that all Mutants fold correctly and are stable. Expression studies in fibroblasts, myoblasts, and myotubes show that these mutants incorporate in filamentous structures. However they do not intercalate between the nascent z-lines it) differentiating muscle cell Cultures. We also show that the distribution of mitochondria and of the ryanodine receptors, and calcium release properties from ryanodine receptors, are unchanged in myotubes expressing the CCD causing mutants. CFTD causing mutants induce partly similar phenotypes as NM associated ones, such as rods and thickened actin fibers in cell Culture. Our results, Suggest that molecular mechanisms behind CFTD and NM may be partly related. Cell Motil. Cytoskeleton 66: 179-192, 2009. (C) 2009 Wiley-Liss, Inc