Ana-Joao Rodrigues et al. (2007) International Worm Meeting "Knockdown of Isu1 ortholog causes larval arrest, decreased life span and brood size, and developmental abnormalities in C.elegans."

Alex Siegel, Giovanni Coppola, Sophie Levan, Ana-Joao Rodrigues, Jeremy Davis-Turak, Patricia Maciel, Daniel Geschwind

[International Worm Meeting, 2007]

Deletions in mitochondrial genes often cause alterations in the life span of the nematode C.elegans. Interestingly, while some of these genes have been shown to extend life span, increase response to stress, and reduce aging processes, others are known to shorten life span and decrease stress resistance in the same model organism. Biosynthesis of Fe/S proteins in eukaryotes is mediated by a number of highly conserved components that are located in mitochondria. A key protein of this Fe/S cluster-assembly machinery in yeast is the mitochondrial protein Isu1, which binds iron and functions as a scaffold during the assembly of iron-sulfur clusters. Isu1 interacts physically and functionally with yeast frataxin (Yfh1p), a protein that is involved in Friedreich''s Ataxia. The C.elegans Y45F10D.4 gene encodes an orthologous protein of the yeast Isu1. The two proteins share domain and sequence similarities, which suggest that they can play a similar role on cells. Yeast Isu1 knockouts are apparently normal, probably because of the redundancy with the Isu2 protein. However, Isu1/Isu2 double knockouts are unviable. We aimed to have a C.elegans model for Isu1 deficiency by knocking down the C.elegans Isu-1 ortholog using RNA interference (RNAi). The isu-1 RNAi causes a striking phenotype with early larval arrest, decreased life span and brood size, and obvious morphological abnormalities. The phenotype worsens when the animals are maintained for several generations on the isu-1 RNAi plates. We are currently studying these and other mutants in mitochondrial proteins involved in the biosynthesis of Fe/S proteins at a more detailed level, and also conducting stress resistance experiments to further characterize these models.

Sophie von Elsner et al. (2006) European Worm Meeting "Maintenance of Asymmetry during Germ Line Cell Cleavages"

Ralf Schnabel, Henning Schmidt, Sophie von Elsner

[European Worm Meeting, 2006]

Sophie von Elsner, Henning Schmidt and Ralf Schnabel. In most organisms axis formation is one of the first processes taking place in the developing embryo. The establishment of the antero-posterior axis in the C. elegans zygote is a prime example with the PAR-proteins polarising the cell cortex after the sperm entry. The asymmetric distribution of these proteins around the cortex initiates the reorganisation of the cytoplasm with all included proteins like the P granules or the transcription factor PIE-1 previous to a division.. During the first division also the unequal distribution of germ line and soma begins which is maintained by only a minimal number of similar asymmetric cleavages. In the cib mutants (changed identity of blastomeres) cib-1 to cib-4 the maintenance of germ line identity during these divisions is defect. Depending on the gene activity the germ line cells P1 to P3 pause up to a full cell cycle and divide symmetrically. The division of the zygote P0 is never affected.. We will report on the function of cib-3 which has no homology or described domain. In a Y2H screen we found that CIB-3 interacts with two proteins which were so far not known to be involved in embryogenesis. In cib-3 RNAi experiments and in the cib-3 ts mutant PAR-2::GFP is localised to the cortex like in WT embryos before a symmetric division but cytoplasmic factors like MEX-5 or nuclear like PIE-1 are aberrant.. Therefore we suggest that CIB-3 is involved in transmitting the cortical polarity to cytoplasmic factors and therefore maintaining the asymmetry.

Matthews LR et al. (1997) International C. elegans Meeting "ZYG-9 IS A COMPONENT OF THE MEIOTIC AND MITOTIC SPINDLE POLES"

Matthews LR, Thierry-Mieg D, Kemphues KJ

[International C. elegans Meeting, 1997]

zyg-9 is a maternally acting gene required for cytoplasmic organization and spindle formation in the one-cell embryo. Mutations in zyg-9 result in disorganized meiotic spindles, incomplete pronuclear migration and formation of a mitotic spindle that contains abnormally short astral microtubules. We have previously reported the cloning and initial molecular characterization of this gene. ZYG-9 protein localizes to both the meiotic and mitotic spindle poles. During meiosis I and II, ZYG-9 is present throughout the meiotic spindle but is enriched at the poles. At early prophase of the first mitosis, this protein localizes to the newly duplicated centrosomes where it persists throughout mitosis, diminishing to undetectable levels by interphase. Interestingly, ZYG-9 is also present at the kinetochore region during the metaphase/anaphase transition. This pattern of localization is observed in all mitotic cells in the developing embryo and in mitotic cells in the gonad. ZYG-9 is 40-48% identical to the human ch-TOG protein over 35% of the predicted ZYG-9 sequence. Although the function of ch-TOG is unknown, this protein appears to be a homolog of the Xenopus protein XMAP215 (David Gard, Sophie Charasse personal communication). In vitro, XMAP215 accelerates the assembly of microtubules at the plus end resulting in longer microtubules. In vivo, this protein accumulates in oocytes and localizes in a cell cycle dependent manner to the mitotic spindle and centrosome of Xenopus oogonia and blastula stage embryos. Levels of XMAP215 decrease 10 fold during embryogenesis. Due to the limited available XMAP215 protein sequence, the similarity between this protein and ZYG-9 is unknown. However, the early expression of XMAP215, its apparent role in microtubule elongation, and similarities between its localization and that of ZYG-9 at the spindle raise the interesting possibility that these proteins play similar roles in microtubule elongation in the early embryo.

Jolanta Polanowska et al. (2006) European Worm Meeting "A Proteomic Approach to Decipher LIN-26 Function"

Sophie Bamps, Jolanta Polanowska, Jerome Reboul., Jean-Francois Dierick

[European Worm Meeting, 2006]

Jolanta Polanowska, Sophie Bamps, Jean-Franois Dierick1 and Jrme Reboul. The main aim of our studies is to map the molecular network implicated in the establishment of epithelial identity (polarity, adhesion..). We have chosen a set of over 100 proteins described as involved in the establishment of cell polarity or containing domains present in polarity related proteins and subjected them to a stringent yeast two hybrid screening of C. elegans cDNA and normalized AD-ORF libraries. The analysis of the obtained network revealed an interesting cluster around LIN-26 nod. It was previously shown that ectopic expression of lin-26 can reprogram the normal fate of blastomeres to induce the expression of three markers that play differents roles in maintaining epithelial cell polarity (jam-1,dlg-1 and che-14) (1). Several observations including the DNA sequence of the gene coding for a putative zinc-finger protein led to the proposition that one of the aspects of lin-26 function is to act as a transcriptional regulator. The yeast two-hybrid cluster brought the hints for possible lin-26 interactors. In order to further enhance the confidence of these findings and therefore understand how lin-26 is involved in epithelial differentiation we have directed ourselves towards a second proteomic approach implicating tandem affinity purification of protein complexes associated with epitope-tagged (HA-6xHis-Myc) LIN-26 from stable transgenic animals. Complexes purification has been carried on the protein extracts covering all developmental stages and all tissues of the animal. Extraction was performed in a way to distinguish the soluble portion of LIN-26 from the one linked to chromatin. Complexes obtained from both fractions were resolved by one and two dimension electrophoresis and analyzed by LC-MS/MS. Combination of yeast two hybrid with TAP approaches let us put to evidence novel interactors of LIN-26, Progress and preliminary results will be reported.. (1) den Boer et al. Development 125, 3213 (1998)

Sophie Bamps et al. (2006) European Worm Meeting "C. elegans Sumoylome"

Sophie Bamps, Jerome Reboul, Jean-Francois Dierick, Jolanta Polanowska

[European Worm Meeting, 2006]

Sophie Bamps, Jolanta Polanowska, Jean-Franois Dierick and Jrme Reboul Ubiquitin-like proteins are believed to act as post-translational modifiers in a manner analogous to ubiquitin. SUMO (small ubiquitin-related modifier) has a potential to alter the conformation of the target or to convey a new surface resulting in the ability to interact with a new partner. This kind of modification can affect the nuclear targeting, stability of the protein and modulation of transcription factor activities. The list of sumo targets remains to be elucidated although the number of known targets continues to grown substantially in recent years. The goal of the presented study is to address at least in part few of the key questions such as: what are the sumo targets, where sumoylation specificity comes from and what are the consequences of this modification; all still poorly defined. We aim to establish a SUMOylome, an exhaustive protein-protein interaction network of protein candidates implicated from close or far in sumoylation processes in C.elegans. In this effort we are performing a middle scale yeast two-hybrid screening using 25 ORFs as baits. Evident candidates are proteins already described to be involved in the enzymatic pathways of sumoylation such as SMO-1, UBC-9, AOS-1, UBA2, potential sumo ligases (GEI-17, NPP-9), and others. Until now, our yeast two hybrid screens allowed identifying new potential substrates and actors of the sumoylation pathways. In regard to our initial results we feel the need to enlarge the obtained network complementing the yeast two-hybrid screen with a second proteomic approach, that is a tandem immunoaffinity purification of chosen proteins. This will first show the relevance in vivo of the interactions found in vitro and second, as the complexes retain the biological activity, will allow us to test in vitro the sumoylation activity of the co-purified proteins. The first candidate is UBC-9, a sumo conjugase for which we are at present purifying the complex of associated proteins and analyzing the identity of interactors by MS/MS. Advancements of this work will be discussed