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See detailPromoter analysis of three HMA4 gene copies in the zinc hyperaccumulator Arabidopsis halleri
Nouet, Cécile ULg; Cebula, Justyna; Motte, Patrick ULg et al

Poster (2012, September 20)

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See detailZinc hyperaccumulation: a model to examine metal homeostasis in plants
Hanikenne, Marc ULg; Nouet, Cécile ULg; Charlier, Jean-Benoit et al

Conference (2012, September 20)

The plant Arabidopsis halleri exhibits naturally selected metal hypertolerance and extraordinarily high levels of leaf metal accumulation. Metal hyperaccumulator species attract interest as they represent ... [more ▼]

The plant Arabidopsis halleri exhibits naturally selected metal hypertolerance and extraordinarily high levels of leaf metal accumulation. Metal hyperaccumulator species attract interest as they represent an extreme end of natural variation of the metal homeostasis network. This might be useful to reveal the global functioning of metal homeostasis networks and uncover key nodes whose alterations can drastically modify metal accumulation and tolerance. In addition, metal hyperaccumulation is a compelling model to study adaptation. In the last few years, major progress has been achieved in our understanding of the mechanisms underlying metal hyperaccumulation in A. halleri. High rates of metal uptake by roots, root-to-shoot translocation and efficient shoot vacuolar sequestration play central roles in determining hyperaccumulation and hypertolerance. Enhanced functions of several metal transporter-encoding genes result from gene copy number amplification and/or (cis)-regulatory changes. We will describe the function of several transporters in zinc and cadmium hyperaccumulation and hypertolerance, and in the adjustment of nutrient homeostasis in A. halleri. Recent work aiming to determine if selection acted during the evolutionary history of A. halleri on a loci required for metal tolerance and accumulation will be presented. [less ▲]

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See detailFunctional study of Arabidopsis thaliana ASF/SF2-like pre-mRNA SR splicing factors
Stankovic, Nancy ULg; Tillemans, Vinciane; Leponce, Isabelle et al

Poster (2012, July 04)

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See detailEvolution of Metal Hyperaccumulation in Arabidopsis halleri
Hanikenne, Marc ULg; Kroymann, Juergen; Bernal, Maria et al

Conference (2012, February 06)

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See detailA Single Ancient Origin for Prototypical Serine/Arginine-Rich Splicing Factors1[W][OA]
Califice, Sophie; Baurain, Denis ULg; Hanikenne, Marc ULg et al

in Plant Physiology (2012), 158(2), 546-560

Eukaryotic pre-mRNA splicing is a process involving a very complex RNA-protein edifice. Serine/arginine-rich (SR) proteins play essential roles in pre-mRNA constitutive and alternative splicing, and have ... [more ▼]

Eukaryotic pre-mRNA splicing is a process involving a very complex RNA-protein edifice. Serine/arginine-rich (SR) proteins play essential roles in pre-mRNA constitutive and alternative splicing, and have been suggested to be crucial in plant-specific forms of developmental regulation and environmental adaptation. Despite their functional importance, little is known about their origin and evolutionary history. SR splicing factors have a modular organization featuring at least one RRM domain and a C-terminal region enriched in Ser/Arg dipeptides. To investigate the evolution of SR proteins, we infer phylogenies for >12,000 RRM domains representing >200 broadly sampled organisms. Our analyses reveal that the RRM domain is not restricted to eukaryotes and that all prototypical SR proteins share a single ancient origin, including the plant-specific SR45 protein. Based on these findings, we propose a scenario for their diversification into four natural families, each corresponding to a main SR architecture, and a dozen subfamilies, of which we profile both sequence conservation and composition. Finally, using operational criteria for computational discovery and classification, we catalogue SR proteins in 20 model organisms, with a focus on green algae and land plants. Altogether, our study confirms the homogeneity and antiquity of SR splicing factors, while establishing robust phylogenetic relationships between animal and plant proteins, which should enable functional analyses of lesser characterized SR family members, especially in green plants. [less ▲]

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See detailReconstruction of a human mitochondrial complex I mutation in the unicellular green alga Chlamydomonas.
Larosa, Véronique ULg; Coosemans, Nadine ULg; Motte, Patrick ULg et al

in Plant Journal (The) (2012), 70

Defects in complex I (NADH:ubiquinone oxidoreductase) are the most frequent cause of human respiratory disorders. The pathogenicity of a given human mitochondrial mutation can be difficult to demonstrate ... [more ▼]

Defects in complex I (NADH:ubiquinone oxidoreductase) are the most frequent cause of human respiratory disorders. The pathogenicity of a given human mitochondrial mutation can be difficult to demonstrate because the mitochondrial genome harbors large numbers of polymorphic base changes that have no pathogenic significance. In addition, mitochondrial mutations are usually found in the heteroplasmic state, which could hide the biochemical effect of the mutation. We propose that the unicellular green alga Chlamydomonas could be used to study such mutations because (1) respiratory-deficient mutants are viable and mitochondrial mutations are found in the homoplasmic state, (2) transformation of the mitochondrial genome is feasible, (3) Chlamydomonas complex I is close to that of humans. To illustrate that, we have introduced a Leu157Pro substitution in the Chlamydomonas ND4 subunit of complex I of two different recipient strains by biolistic transformation, demonstrating that site-directed mutagenesis of the Chlamydomonas mitochondrial genome is possible. This substitution did not lead to any respiratory enzyme defect when it is present in the heteroplasmic state in a patient presenting chronic progressive external ophthalmoplegia. When present in the homoplasmic state in the alga, the mutation does not prevent the assembly of the 950 kDa whole complex I which conserves nearly all the NADH dehydrogenase activity of the peripheral arm. However, the NADH:duroquinone oxidoreductase activity is strongly reduced, suggesting that the substitution could affect ubiquinone fixation to the membrane domain. The in vitro defects are correlated in vivo with a decrease in dark respiration and growth rate. [less ▲]

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See detailThe HMG-Box Transcription Factor Sox4b Is Required for Pituitary Expression of gata2a and Specification of Thyrotrope and Gonadotrope Cells in Zebrafish.
Quiroz O'Donova, Yobhana ULg; Lopez, Mauricio; Mavropoulos, A et al

in Molecular Endocrinology (2012), 26(6), 1014-1027

The pituitary is a complex gland comprising different cell types each secreting specific hormones. The extensive network of signaling molecules and transcription factors required for determination and ... [more ▼]

The pituitary is a complex gland comprising different cell types each secreting specific hormones. The extensive network of signaling molecules and transcription factors required for determination and terminal differentiation of specific cell types is still not fully understood. The SRY-like HMG-box (SOX) transcription factor Sox4 plays important roles in many developmental processes and has two homologs in zebrafish, Sox4a and Sox4b. We show that the sox4b gene is expressed in the pituitary anlagen starting at 24 h after fertilization (hpf) and later in the entire head region including the pituitary. At 48 hpf, sox4b mRNA colocalizes with that for TSH (tshbeta), glycoprotein subunit alpha (gsualpha), and the Zn finger transcription factor Gata2a. Loss of Sox4b function, using morpholino knockdown or expression of a dominant-negative Sox4 mutant, leads to a drastic decrease in tshbeta and gsualpha expression and reduced levels of gh, whereas other anterior pituitary gland markers including prl, slbeta, pomc, and lim3 are not affected. Sox4b is also required for expression of gata2a in the pituitary. Knockdown of gata2a leads to decreased tshbeta and gsualpha expression at 48 hpf, similar to sox4b morphants. Injection of gata2a mRNA into sox4b morphants rescued tshbeta and gsualpha expression in thyrotrope cells. Finally, sox4b or gata2a knockdown causes a significant decrease of gonadotropin expression (lhbeta and fshbeta) at 4 d after fertilization. In summary, our results indicate that Sox4b is expressed in zebrafish during pituitary development and plays a crucial role in the differentiation of thyrotrope and gonadotrope cells through induction of gata2a expression in the developing pituitary. [less ▲]

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See detailRUNX3, EGR1 AND SOX9B FORM A REGULATORY CASCADE REQUIRED TO MODULATE BMP-SIGNALING DURING CRANIAL CARTILAGE DEVELOPMENT IN ZEBRAFISH.
Dalcq, Julia ULg; Pasque, Vincent; Ghaye, Aurélie ULg et al

in PLoS ONE (2012), in press

The cartilaginous elements forming the pharyngeal arches of the zebrafish derive from cranial neural crest cells. Their proper differentiation and patterning are regulated by reciprocal interactions ... [more ▼]

The cartilaginous elements forming the pharyngeal arches of the zebrafish derive from cranial neural crest cells. Their proper differentiation and patterning are regulated by reciprocal interactions between neural crest cells and surrounding endodermal, ectodermal and mesodermal tissues. In this study, we show that the endodermal factors Runx3 and Sox9b form a regulatory cascade with Egr1 resulting in transcriptional repression of the fsta gene, encoding a BMP antagonist, in pharyngeal endoderm. Using a transgenic line expressing a dominant negative BMP receptor or a specific BMP inhibitor (dorsomorphin), we show that BMP signaling is indeed required around 30 hpf in the neural crest cells to allow cell differentiation and proper pharyngeal cartilage formation. Runx3, Egr1, Sox9b and BMP signaling are required for expression of runx2b, one of the key regulator of cranial cartilage maturation and bone formation. Finally, we show that egr1 depletion leads to increased expression of fsta and inhibition of BMP signaling in the pharyngeal region. In conclusion, we show that the successive induction of the transcription factors Runx3, Egr1 and Sox9b constitutes a regulatory cascade that controls expression of Follistatin A in pharyngeal endoderm, the latter modulating BMP signaling in developing cranial cartilage in zebrafish. [less ▲]

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See detailPromoter analysis of the three HMA4 copies in the zinc hyperaccumulator Arabidopsis halleri
Nouet, Cécile ULg; Cebula, Justyna; Motte, Patrick ULg et al

Poster (2011, August)

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See detailExpression of the metal homeostasis gene FRD3 in two Arabidopsis species
Charlier, Jean-Benoît ULg; Polese, Catherine ULg; Krämer, Ute et al

Poster (2011, August)

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See detailRSZ22, a dynamic nucleocytoplasmic shuttling SR splicing factor
Tillemans, Vinciane ULg; Rausin, Glwadys; Stankovic, Nancy ULg et al

Poster (2011, March)

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See detailChloroplastic and mitochondrial metal homeostasis.
Nouet, Cécile ULg; Motte, Patrick ULg; Hanikenne, Marc ULg

in Trends in Plant Science (2011), 16(7), 395-404

Transition metal deficiency has a strong impact on the growth and survival of an organism. Indeed, transition metals, such as iron, copper, manganese and zinc, constitute essential cofactors for many key ... [more ▼]

Transition metal deficiency has a strong impact on the growth and survival of an organism. Indeed, transition metals, such as iron, copper, manganese and zinc, constitute essential cofactors for many key cellular functions. Both photosynthesis and respiration rely on metal cofactor-mediated electron transport chains. Chloroplasts and mitochondria are, therefore, organelles with high metal ion demand and represent essential components of the metal homeostasis network in photosynthetic cells. In this review, we describe the metal requirements of chloroplasts and mitochondria, the acclimation of their functions to metal deficiency and recent advances in our understanding of their contributions to cellular metal homeostasis, the control of the cellular redox status and the synthesis of metal cofactors. [less ▲]

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See detailDynamic Nucleocytoplasmic Shuttling of an Arabidopsis SR Splicing Factor: Role of the RNA-Binding Domains
Rausin, Glwadys ULg; Tillemans, Vinciane ULg; Stankovic, Nancy ULg et al

in Plant Physiology (2010), 153

Serine/arginine-rich (SR) proteins are essential nuclear-localized splicing factors. We have investigated the dynamic subcellular distribution of the Arabidopsis (Arabidopsis thaliana) RSZp22 protein, a ... [more ▼]

Serine/arginine-rich (SR) proteins are essential nuclear-localized splicing factors. We have investigated the dynamic subcellular distribution of the Arabidopsis (Arabidopsis thaliana) RSZp22 protein, a homolog of the human 9G8 SR factor. Little is known about the determinants underlying the control of plant SR protein dynamics, and so far most studies relied on ectopic transient overexpression. Here, we provide a detailed analysis of the RSZp22 expression profile and describe its nucleocytoplasmic shuttling properties in specific cell types. Comparison of transient ectopic- and stable tissue-specific expression highlights the advantages of both approaches for nuclear protein dynamic studies. By site-directed mutagenesis of RSZp22 RNA-binding sequences, we show that functional RNA recognition motif RNP1 and zinc-knuckle are dispensable for the exclusive protein nuclear localization and speckle-like distribution. Fluorescence resonance energy transfer imaging also revealed that these motifs are implicated in RSZp22 molecular interactions. Furthermore, the RNA-binding motif mutants are defective for their export through the CRM1/XPO1/Exportin-1 receptor pathway but retain nucleocytoplasmic mobility. Moreover, our data suggest that CRM1 is a putative export receptor for mRNPs in plants. [less ▲]

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See detailNkx6.1 and nkx6.2 regulate alpha- and beta-cell formation in zebrafish by acting on pancreatic endocrine progenitor cells.
Binot, Anne-Catherine; Manfroid, Isabelle ULg; Flasse, Lydie ULg et al

in Developmental Biology (2010), 340(2), 397-407

In mice, the Nkx6 genes are crucial to alpha- and beta-cell differentiation, but the molecular mechanisms by which they regulate pancreatic subtype specification remain elusive. Here it is shown that in ... [more ▼]

In mice, the Nkx6 genes are crucial to alpha- and beta-cell differentiation, but the molecular mechanisms by which they regulate pancreatic subtype specification remain elusive. Here it is shown that in zebrafish, nkx6.1 and nkx6.2 are co-expressed at early stages in the first pancreatic endocrine progenitors, but that their expression domains gradually segregate into different layers, nkx6.1 being expressed ventrally with respect to the forming islet while nkx6.2 is expressed mainly in beta-cells. Knockdown of nkx6.2 or nkx6.1 expression leads to nearly complete loss of alpha-cells but has no effect on beta-, delta-, or epsilon-cells. In contrast, nkx6.1/nkx6.2 double knockdown leads additionally to a drastic reduction of beta-cells. Synergy between the effects of nkx6.1 and nkx6.2 knockdown on both beta- and alpha-cell differentiation suggests that nkx6.1 and nkx6.2 have the same biological activity, the required total nkx6 threshold being higher for alpha-cell than for beta-cell differentiation. Finally, we demonstrate that the nkx6 act on the establishment of the pancreatic endocrine progenitor pool whose size is correlated with the total nkx6 expression level. On the basis of our data, we propose a model in which nkx6.1 and nkx6.2, by allowing the establishment of the endocrine progenitor pool, control alpha- and beta-cell differentiation. [less ▲]

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See detailExpression of the metal homeostasis gene FRD3 in two Arabidopsis species
Charlier, Jean-Benoit ULg; Polese, Catherine; Motte, Patrick ULg et al

Poster (2010, January 26)

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See detailKnock-down of the COX3 and COX17 gene expression of cytochrome c oxidase in the unicellular green alga Chlamydomonas reinhardtii.
Remacle, Claire ULg; Coosemans, Nadine ULg; Jans, Frédéric ULg et al

in Plant Molecular Biology (2010), 74(3), 223-2363

The COX3 gene encodes a core subunit of mitochondrial cytochrome c oxidase (complex IV) whereas the COX17 gene encodes a chaperone delivering copper to the enzyme. Mutants of these two genes were isolated ... [more ▼]

The COX3 gene encodes a core subunit of mitochondrial cytochrome c oxidase (complex IV) whereas the COX17 gene encodes a chaperone delivering copper to the enzyme. Mutants of these two genes were isolated by RNA interference in the microalga Chlamydomonas. The COX3 mRNA was completely lacking in the cox3-RNAi mutant and no activity and assembly of complex IV were detected. The cox17-RNAi mutant presented a reduced level of COX17 mRNA, a reduced activity of the cytochrome c oxidase but no modification of its amount. The cox3-RNAi mutant had only 40% of the wild-type rate of dark respiration which was cyanide-insensitive. The mutant presented a 60% decrease of H(2)O(2) production in the dark compared to wild type, which probably accounts for a reduced electron leakage by respiratory complexes III and IV. In contrast, the cox17-RNAi mutant showed no modification of respiration and of H(2)O(2) production in the dark but a two to threefold increase of H(2)O(2) in the light compared to wild type and the cox3-RNAi mutant. The cox17-RNAi mutant was more sensitive to cadmium than the wild-type and cox3-RNAi strains. This suggested that besides its role in complex IV assembly, Cox17 could have additional functions in the cell such as metal detoxification or Reactive Oxygen Species protection or signaling. Concerning Cox3, its role in Chlamydomonas complex IV is similar to that of other eukaryotes although this subunit is encoded in the nuclear genome in the alga contrary to the situation found in all other organisms. [less ▲]

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See detailADAMTS-2 functions as anti-angiogenic and anti-tumoral molecule independently of its catalytic activity.
Dubail, Johanne ULg; Kesteloot, F.; Deroanne, Christophe ULg et al

in Cellular & Molecular Life Sciences (2010)

ADAMTS-2 is a metalloproteinase that plays a key role in the processing of fibrillar procollagen precursors into mature collagen molecules by excising the amino-propeptide. We demonstrate that recombinant ... [more ▼]

ADAMTS-2 is a metalloproteinase that plays a key role in the processing of fibrillar procollagen precursors into mature collagen molecules by excising the amino-propeptide. We demonstrate that recombinant ADAMTS-2 is also able to reduce proliferation of endothelial cells, and to induce their retraction and detachment from the substrate resulting in apoptosis. Dephosphorylation of Erk1/2 and MLC largely precedes the ADAMTS-2 induced morphological alterations. In 3-D culture models, ADAMTS-2 strongly reduced branching of capillary-like structures formed by endothelial cells and their long-term maintenance and inhibited vessels formation in embryoid bodies (EB). Growth and vascularization of tumors formed in nude mice by HEK 293-EBNA cells expressing ADAMTS-2 were drastically reduced. A similar anti-tumoral activity was observed when using cells expressing recombinant deleted forms of ADAMTS-2, including catalytically inactive enzyme. Nucleolin, a nuclear protein also found to be associated with the cell membrane, was identified as a potential receptor mediating the antiangiogenic properties of ADAMTS-2. [less ▲]

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