ENTH/ANTH proteins and clathrin-mediated membrane budding

The epsin N-terminal homology (ENTH) domain is an evolutionarily conserved protein module found primarily in proteins that participate in clathrin-mediated endocytosis. Structural analyses and ligand-binding studies have shown that a set of proteins previously designated as harboring an ENTH domain in fact contain a highly similar, yet unique module referred to as an AP180 N-terminal homology (ANTH) domain. ENTH and ANTH (E/ANTH) domains bind both inositol phospholipids and proteins and contribute to the nucleation and formation of clathrin coats on membranes. ENTH domains also function in the development of membrane curvature through lipid remodeling during the formation of clathrin-coated vesicles. E/ANTH-bearing proteins have recently been shown to function with adaptor protein-1 and GGA adaptors at the trans-Golgi network, which suggests that E/ANTH domains are universal components of the machinery for clathrin-mediated membrane budding

Endophilin regulates JNK activation through its interaction with the germinal center kinase-like kinase

The endophilin family of proteins function in clathrin-mediated endocytosis. Here, we have identified and cloned the rat germinal center kinase-like kinase (rGLK), a member of the GCK (germinal center kinase) family of e-Jun N-terminal kinase (JNK) activating enzymes, as a novel endophilin I-binding partner. The interaction occurs both in vitro and in cells and is mediated by the Src homology 3 domain of endophilin I and a region of rGLK containing the endophilin consensus-binding sequence PPRPPPPR. Overlay analysis of rat brain extracts demonstrates that endophilin I is a major Src homology 3 domain-binding partner for rGLK. Overexpression of full-length endophilin I activates rGLK-mediated JNK activation, whereas N- and C-terminal fragments of endophilin I block JNK activation. Thus, endophilin I appears to have a novel function in JNK activation

A small protein coded within the mitochondrial canonical gene nd4 regulates mitochondrial bioenergetics

BACKGROUND: Mitochondria have a central role in cellular functions, aging, and in certain diseases. They possess their own genome, a vestige of their bacterial ancestor. Over the course of evolution, most of the genes of the ancestor have been lost or transferred to the nucleus. In humans, the mtDNA is a very small circular molecule with a functional repertoire limited to only 37 genes. Its extremely compact nature with genes arranged one after the other and separated by short non-coding regions suggests that there is little room for evolutionary novelties. This is radically different from bacterial genomes, which are also circular but much larger, and in which we can find genes inside other genes. These sequences, different from the reference coding sequences, are called alternatives open reading frames or altORFs, and they are involved in key biological functions. However, whether altORFs exist in mitochondrial protein-coding genes or elsewhere in the human mitogenome has not been fully addressed. RESULTS: We found a downstream alternative ATG initiation codon in the + 3 reading frame of the human mitochondrial nd4 gene. This newly characterized altORF encodes a 99-amino-acid-long polypeptide, MTALTND4, which is conserved in primates. Our custom antibody, but not the pre-immune serum, was able to immunoprecipitate MTALTND4 from HeLa cell lysates, confirming the existence of an endogenous MTALTND4 peptide. The protein is localized in mitochondria and cytoplasm and is also found in the plasma, and it impacts cell and mitochondrial physiology. CONCLUSIONS: Many human mitochondrial translated ORFs might have so far gone unnoticed. By ignoring mtaltORFs, we have underestimated the coding potential of the mitogenome. Alternative mitochondrial peptides such as MTALTND4 may offer a new framework for the investigation of mitochondrial functions and diseases

The influence of mitochondria in epigenetics revealed through naturally occurring fish cybrids

Epigenetic processes are important mechanisms for phenotypic changes that occur in response to the environment. As such, it is expected that the alteration of cytoplasmic composition (the immediate environment of nuclei) results in the modification of the methylome and the expression of the nuclear genome. Cytoplasmic hybrids (or cybrids) are an ideal model to study the influence of mitochondria on gene expression. In this study, we take advantage of the natural co-occurrence of two biotypes that have a similar nuclear genome type (Chrosomus eos), but harbor mitochondria from different species (C. eos in wild type or C. neogaeus in cybrids) to assess the effects of mitochondria on DNA methylation profiles and protein expression of the nuclear genome. Comparison between these biotypes is particularly relevant given their recent divergence and their low level of genetic differentiation. Variations of DNA methylation assessed on tissues from different embryonic origins revealed the distinct profiles of cybrid and wild type populations. Differences are more pronounced between wild type and cybrids than between populations of a given biotype. The proteome is also more different between biotypes than within a given biotype. These results indicate a strong influence of mitochondria on the nuclear genome, which remains detectable in different genetic and environmental contexts. These changes in the methylome and proteome of cybrids are expected to reflect the adjustments imposed by the coexistence of nuclear and mitochondrial genomes from different species [Current Zoology 58 (1): 138–145, 2012]

Méthylation de l’ADN mitochondrial

La méthylation de l’ADN est un mécanisme épigénétique essentiel à la plupart des organismes, notamment pour la régulation de l’expression génique. Dans le génome nucléaire des mammifères, elle est généralement restreinte aux cytosines précédant une guanine, alors qu’elle opère dans un contexte nucléotidique plus varié chez les bactéries. Curieusement, l’existence même de méthylation dans les mitochondries demeure en débat. Cette controverse pourrait être due aux différences entre ces génomes, et à des méthodologies plutôt adaptées à l’étude des méthylations du génome nucléaire. Des études récentes suggèrent ainsi que la méthylation de l’ADN mitochondrial se ferait davantage en contexte nucléotidique varié, comme chez leurs ancêtres bactériens

Itch Is Required for Lateral Line Development in Zebrafish

<div><p>The zebrafish posterior lateral line is formed during early development by the deposition of neuromasts from a migrating primordium. The molecular mechanisms regulating the regional organization and migration of the primordium involve interactions between Fgf and Wnt/-catenin signaling and the establishment of specific <i>cxcr4b</i> and <i>cxcr7b</i> cytokine receptor expression domains. Itch has been identified as a regulator in several different signaling pathways, including Wnt and Cxcr4 signaling. We identified two homologous <i>itch</i> genes in zebrafish, <i>itcha</i> and <i>itchb</i>, with generalized expression patterns. By reducing <i>itchb</i> expression in particular upon morpholino knockdown, we demonstrated the importance of Itch in regulating lateral line development by perturbing the patterns of <i>cxcr4b</i> and <i>cxcr7b</i> expression. Itch knockdown results in a failure to down-regulate Wnt signaling and overexpression of <i>cxcr4b</i> in the primordium, slowing migration of the posterior lateral line primordium and resulting in abnormal development of the lateral line.</p></div

<i>itchb</i> is involved in pLL primordium migration and lateral line development.

<p>(A–E) Posterior lateral line at 48 hpf <i>cldnb:gfp</i> in embryos injected with vehicle (CTRL), <i>in vitro</i>-transcribed human <i>ITCH</i> mRNA (mRNA), MOs against <i>itcha</i> (MoA), MOs against <i>itchb</i> (MoB) or MOs against <i>itchb</i> and <i>in vitro</i>-transcribed human <i>ITCH</i> mRNA (Rescue). There was a marked reduction in the number of neuromasts in the pLL after <i>itchb</i> knockdown (D), but not in <i>itcha</i> knockdown, although the primordium had not yet reached the end of the tail at this time point (C). The <i>itchb</i> knockdown effect was partially rescued by injection of human <i>ITCH</i> mRNA (E). (F–I) Time-lapse confocal microscopy on 26–30 hpf <i>cldnb:gfp</i> embryos showed that the pLL primordium migration was slowed in <i>itchb</i> knockdown embryos (MoB) compared to vehicle (CTRL), human <i>ITCH</i> mRNA-injected (mRNA) or rescued embryos (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0111799#pone.0111799.s006" target="_blank">Movies S1</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0111799#pone.0111799.s009" target="_blank">S4</a>). (N) The number of neuromasts in the posterior lateral-line was counted after staining with DiAsp in 72 hpf WT embryos injected with vehicle (CTRL), <i>in vitro</i>-transcribed human <i>ITCH</i> mRNA (mRNA), MOs against <i>itcha</i> (MoA), MOs against <i>itchb</i> (MoB) or MOs against <i>itchb</i> and <i>in vitro</i>-transcribed human <i>ITCH</i> mRNA (Rescue). Whereas the number of neuromasts in the pLL was markedly reduced after injection of MOs against <i>itchb</i>, injection of <i>in vitro</i> transcribed <i>ITCH</i> mRNA alone or of MOs against <i>itcha</i> had no effect. The MOs phenotype was rescued by co-injection of <i>ITCH</i> mRNA in <i>itchb</i> knockdown. The number of embryos for each group is indicated at the bottom of the graph. represents statistical significance at . n.s.  =  non-significant. (J–M) Morphology of the migrating primordium in 26 hpf embryos (CTRL), <i>itcha</i> knockdown embryos (MoA) and <i>itchb</i> knockdown embryos (MoB). Cells at the tip of the primordium of MoB embryos were slightly disorganized, but the typical rosette formation occurred normally. (O) Migration of the pLL primordium was quantified by measuring the distance between the primordium leading edge in the first frame () compared to the last frame () as shown in F–I (doted line). The number of embryos counted or imaged is indicated at the bottom of the graph. Each graph summarizes four different experiments. represents statistical significance at . n.s. =  non-significant. EGFP fluorescence is presented as reversed black and white to facilitate visualization in all micrographs. Scale bars: (A–E) , (F–I) , (L–M) .</p

In vitro proliferation of Mytilus edulis male germ cell progenitors.

Our understanding of basic cellular processes has mostly been provided by mammalian cell culture, and by some non-mammalian vertebrate and few invertebrate cell culture models. Developing reliable culture conditions for non-model organisms is essential to allow investigation of more unusual cellular processes. Here, we investigate how cells isolated from different tissues of the marine mussel Mytilus edulis thrive and survive in vitro in the hope of establishing a suitable laboratory model for the investigation of cellular mechanisms specific to these bivalve mollusks. We found that cells dissociated from mantle tissue attached to the culture vessels and proliferated well in vitro, whereas cells isolated from gills, although remaining viable, did not maintain divisions over three to four weeks in culture. We used antibodies against the germ-line marker DEAD-box helicase 4 (DDX4), also known as VASA, and the epithelial cell marker cytokeratin to distinguish different cell types in culture. DDX4-positive cells were predominant in 25-day-old cultures from male mantles. Cells from other tissues remained in low numbers and did not seem to change in composition over time. Overall, the culture conditions described here allow an efficient selection of male germ cells that could be used to study specific cellular mechanisms in vitro

Expression of <i>itch</i> genes in zebrafish embryos.

<p>(A) RT-PCR experiment showing early expression of both <i>itcha</i> and <i>itchb</i> genes. RNA was extracted from embryos at approximately 6, 24 and 48 hpf. cDNA was obtained from each stage using 1 g total RNA. <i>GAPDH</i> was used as an internal control for cDNA amplification. (B–D) Weak, general staining of embryos at 26 hpf revealed with DIG-labelled probes complementary to <i>itcha</i> (B) or <i>itchb</i> (C) or sense sequence (D) as a control. (E–G) The primordium of 26 hpf embryos revealed with DIG-labelled probes complementary to <i>itcha</i> (E) or <i>itchb</i> (F) or sense sequence (G) as a control. Position of the primordium is underlined with a dotted line. Expression of <i>itch</i> genes is general, and <i>itchb</i> is expressed throughout the primordium. Scale bar: (B–D) 250 , (E–G) 25 .</p