Ralph Menzel et al. (2003) International Worm Meeting "Xenobiotically induced cytochrome P450 genes in Caenorhabditis elegans: GFP-fusions and embryonic lethality by RNAi."

Ralph Menzel, Ruth Boose, Sarah Forster

[International Worm Meeting, 2003]

Cytochromes P450 (CYP) are a super family of heme containing and NADPH dependent monooxygenases which catalyze the oxidative metabolism of many exogenous and endogenous compounds. While the investigation of CYP induction pathways in mammals has made considerable progress, very little is known about P450s from invertebrates, such as the nematode Caenorhabditis elegans. C. elegans possesses, however, a remarkably high number of 80 different cytochrome P450 genes. The functions of the encoded proteins are almost unknown. In a previous study it could be demonstrated that the five different CYP forms 35A1-A5 are strongly inducible by PAHs, PCBs and different drugs [1]. In order to study this xenobiotically induced gene expression different transgenic C. elegans lines expressing GFP under control of the CYP35A1-5 promoter regions were constructed. With this tool it was possible to follow the GFP production in vivo depending on the applied inducer. Induction with -naphthoflavone resulted in all cases in an expression of the green fluorescent protein exclusively in the worm intestine, identifying this compartment as the likely tissue of P450 35A localization. For the CYP35A2 and A5 forms promoter-deletion experiments were performed revealing a 400 bp and a 250 bp fragment, respectively, to be sufficient to induce GFP production. While the 35A2 promoter caused GFP production in all developmental stages, the 35A5 based gene expression was mainly detectable in eggs and in L1/L2 larvae. In particular in case of the 35A2 promoter region a striking correspondence to mammalian xenobiotic response elements (XRE) has been found. To obtain more information about the biological functions of the five different CYP35A forms dsRNA producing E. coli strains for RNAi by feeding experiments were produced. Surprisingly, embryonic lethality was observed as the loss of function phenotype for all CYP35A forms with exception of 35A2. This might indicate that at least one or more CYP35A forms play an essential role in the embryogenesis of C. elegans. Using semi-quantitative RT-PCRs the cross-reactivity level of the individual RNAi constructs was determined. Future efforts will be focused to characterize the blocked embryonic development more in detail.[1] Menzel R., Bogaert T. and Achazi A. (2001) A systematical gene expression screen of the Caenorhabditis elegans cytochrome P450 genes revealed CYP35 as strong xenobiotic inducible. Arch. Biochem. Biophys. 395, 158-168.

Iwasa H et al. (2013) Exp Cell Res "Characterization of RSF-1, the Caenorhabditis elegans homolog of the Ras-association domain family protein 1."

Hata Y, Ikeda M, Iwasa H, Kuroyanagi H, Nakagawa K, Maimaiti S

[Exp Cell Res, 2013]

Mammals have 10 RASSF proteins, which are characterized by the Ras-association (RA) domain. Among them, RASSF1 to RASSF6 have the Salvador/RASSF/Hippo (SARAH) domain and form the subclass C-terminal RASSF proteins. Drosophila genome has a single C-terminal RASSF, dRASSF. All these RASSF proteins are related to the tumor suppressive Hippo pathway, and are considered to function as tumor suppressors. Caenorhabditis elegans T24F1.3 encodes a protein with the RA and the SARAH domains. The amino acid sequences are 40% and 55% similar to those of RASSF1 in the RA and the SARAH domains, respectively. We have characterized T24F1.3 gene product and named it RSF-1 as RASSF1 homolog. RSF-1 is widely expressed in epithelial cells. About 14% rsf-1 mutants exhibit defects in embryonal morphogenesis and the actin disorganization. The combinatorial synthetic lethal analysis demonstrates that the lethality is enhanced to more than 80% in rsf-1 mutants with the WASP-family verprolin homologous protein complex-related gene depletions and corroborates the implication of RSF-1 in the regulation of actin cytoskeleton. In rsf-1 mutants, the structures of muscle actin are preserved, but the swimming ability is impaired. Although we could not detect the direct physical interaction of LET-60 with RSF-1, rsf-1 mutants suppress the multivulva phenotype of the active let-60 mutants, suggesting that rsf-1 genetically interacts with the Ras signaling.

Tsuyama T et al. (2013) Anal Chem "In vivo fluorescent adenosine 5'-triphosphate (ATP) imaging of Drosophila melanogaster and Caenorhabditis elegans by using a genetically encoded fluorescent ATP biosensor optimized for low temperatures."

Noji H, Kishikawa JI, Harada Y, Yokoyama K, Tsubouchi A, Tsuyama T, Uemura T, Imamura H, Kakizuka A, Han YW

[Anal Chem, 2013]

Adenosine 5'-triphosphate (ATP) is the major energy currency of all living organisms. Despite its important functions, the spatiotemporal dynamics of ATP levels inside living multicellular organisms is unclear. In this study, we modified the genetically encoded Forster resonance energy transfer (FRET)-based ATP biosensor ATeam to optimize its affinity at low temperatures. This new biosensor, AT1.03NL, detected ATP changes inside Drosophila S2 cells more sensitively than the original biosensor did, at 25 C. By expressing AT1.03NL in Drosophila melanogaster and Caenorhabditis elegans, we succeeded in imaging the in vivo ATP dynamics of these model animals at single-cell resolution.

Vukajlovic M et al. (2011) Mol Biol Cell "How kinesin-2 forms a stalk."

Okten Z, Dietz H, Schliwa M, Vukajlovic M

[Mol Biol Cell, 2011]

The heterotrimeric structure of kinesin-2 makes it a unique member of the kinesin superfamily; however, molecular details of the oligomer formation are largely unknown. Here we demonstrate that heterodimerization of the two distinct motor domains KLP11 and KLP20 of Caenorhabditis elegans kinesin-2 requires a dimerization seed of merely two heptads at the C terminus of the stalk. This heterodimeric seed is sufficient to promote dimerization along the entire length of the stalk, as shown by circular dichroism spectroscopy, Forster resonance energy transfer analysis, and electron microscopy. In addition to explaining the formation of the kinesin-2 stalk, the seed sequence identified here bears great potential for generating specific heterodimerization in other protein biochemical applications.

Ali Khan L et al. (1996) Worm Breeder's Gazette "emb-8 mislocalizes Glp-1 and P-granules"

Siddiqui SS, Ali Khan L

[Worm Breeder's Gazette, 1996]

We have previously reported the transformation rescue of the emb-8(hc69) mutant with a cosmid containing the gene cej-1(C. elegans Meeting 1995, abstract 310). emb-8 is an embryonic lethal mutant at nonpermissive temperature (25oC). Cell division arrest between one cell to 50 cells stage. The egg shell is osmotically sensitive. Recently we have come to know from Sarah Crittenden( Judith Kimble Lab.) that emb-8 mislocalizes Glp-1 & P-granules specific antigens. We have earlier observed symmetric distribution of P-granules in a fraction of embryos using a monoclonal antibody specific to germ cells. These observation suggest a role of emb-8 in early cell divisions of the embryos.

Lleres D et al. (2017) Cell Rep "Quantitative FLIM-FRET Microscopy to Monitor Nanoscale Chromatin Compaction InVivo Reveals Structural Roles of Condensin Complexes."

Lleres D, Perrin A, Feil R, Bailly AP, Norman DG, Xirodimas DP

[Cell Rep, 2017]

How metazoan genomes are structured at the nanoscale in living cells and tissues remains unknown. Here, we adapted a quantitative FRET (Forster resonance energy transfer)-based fluorescence lifetime imaging microscopy (FLIM) approach to assay nanoscale chromatin compaction in living organisms. Caenorhabditis elegans was chosen as a model system. By measuring FRET between histone-tagged fluorescent proteins, we visualized distinct chromosomal regions and quantified the different levels of nanoscale compaction in meiotic cells. Using RNAi and repetitive extrachromosomal array approaches, we defined the heterochromatin state and showed that its architecture presents a nanoscale-compacted organization controlled by Heterochromatin Protein-1 (HP1) and SETDB1 H3-lysine-9 methyltransferase homologs invivo. Next, we functionally explored condensin complexes. We found that condensin I and condensin II are essential for heterochromatin compaction and that condensin I additionally controls lowly compacted regions. Our data show that, in living animals, nanoscale chromatin compaction is controlled not only by histone modifiers and readers but also by condensin complexes.

Ali Khan L et al. (1997) International C. elegans Meeting "MATERNAL GENE EMB-8 IS NECESSARY FOR ASYMMETRIC DISTRIBUTION OF DEVELOPMENTAL POTENTIALS IN EARLY C. ELEGANS EMBRYOS"

Siddiqui SS, Ali Khan L, Ali MY, Miwa J, Kikuno R, Kohara Y, Siddiqui ZK

[International C. elegans Meeting, 1997]

A number of observations suggest that emb-8 gene plays critical roles in C. elegans development. At non-permissive temperature, emb-8 embryos divides symmetrically; in 50% the first division produces two equal sized cells, and in the second cleavage both of these cells divide symmetrically again to produce four equal sized cells. In some embryos, these four cells fuse togather and produce two asymmetric cells. emb-8 variably mislocalizes P-granules, they are uniformly distributed in 1-cell embryos, are localized on both anterior AB, and posterior P1 cells of the 2-cell embryos, and are found in all four blastomeres of the 4-cell embryos. Crittenden et al., (1996) also observed mislocalization of the P-granules and Glp-1 in emb-8 embryos. emb-8 also appears to control the orientation of early mitotic spindles, similar to the par-3 muatnts (K. Kemphues and others). However complementation tests show that emb-8 and par-3 are different genes. We thank Sarah Crittenden, Judith Kimble and Ken Kemphues for sharing unpublished results.

Koyiloth M et al. (2021) Biochim Biophys Acta Biomembr "Cholesterol interaction attenuates scramblase activity of SCRM-1 in the artificial membrane."

Koyiloth M, Gummadi SN

[Biochim Biophys Acta Biomembr, 2021]

Phospholipid (PL) scramblases are single-pass transmembrane protein mediating bidirectional PL translocation. Previously in silico analysis of human PL scramblases, predicted the presence of an uncharacterized cholesterol-binding domain spanning partly in the transmembrane helix as well as in the adjacent extracellular coil. This domain was found to be universally conserved in diverse organisms like Caenorhabditis elegans. In this study, we investigated the saturable cholesterol-binding domain of SCRM-1 using fluorescence sterol binding assay, Stern-Volmer quenching, Forster resonance energy transfer, and CD spectroscopy. We observed high-affinity interaction between cholesterol and SCRM-1. Our results support a previous report, which showed that the cholesterol ordering effect reduced the scramblase activity of hPLSCR1. Considering the presence of a high-affinity binding sequence, we propose that the reduction in activity could partly be due to the cholesterol binding. To validate this, we generated a C-terminal helix (CTH) deletion construct (CTH SCRM-1) and a point mutation in the putative cholesterol-binding domain I273D SCRM-1. Deletion construct greatly reduced cholesterol affinity along with loss of scramblase activity. In contrast to this, I273D SCRM-1 retained scrambling activity in proteoliposomes containing ~30mol% cholesterol but lost sterol binding ability. These results suggest that C-terminal helix is crucial for membrane insertion and in the lipid bilayer the scrambling activity of SCRM-1 is modulated through its interaction with cholesterol.

Parrilla A et al. (2016) Cell Cycle "Mitotic entry: The interplay between Cdk1, Plk1 and Bora."

Gotta M, Cirillo L, Parrilla A, Santamaria A, Thomas Y, Pintard L

[Cell Cycle, 2016]

Polo-like kinase 1 (Plk1) is an important mitotic kinase that is crucial for entry into mitosis after recovery from DNA damage-induced cell cycle arrest. Plk1 activation is promoted by the conserved protein Bora (SPAT-1 in C. elegans), which stimulates the phosphorylation of a conserved residue in the activation loop by the Aurora A kinase. In a recent article published in Cell Reports, we show that the master mitotic kinase Cdk1 contributes to Plk1 activation through SPAT-1/Bora phosphorylation. We identified 3 conserved Sp/Tp residues that are located in the N-terminal, most conserved part, of SPAT-1/Bora. Phosphorylation of these sites by Cdk1 is essential for Plk1 function in mitotic entry in C. elegans embryos and during DNA damage checkpoint recovery in mammalian cells. Here, using an untargeted Forster Resonance Energy Transfer (FRET) biosensor to monitor Plk1 activation, we provide additional experimental evidence supporting the importance of these phosphorylation sites for Plk1 activation and subsequent mitotic entry after DNA damage. We also briefly discuss the mechanism of Plk1 activation and the potential role of Bora phosphorylation by Cdk1 in this process. As Plk1 is overexpressed in cancer cells and this correlates with poor prognosis, understanding how Bora contributes to Plk1 activation is paramount for the development of innovative therapeutical approaches.

Herbette M et al. (2020) Cells "A Role for Caenorhabditis elegans COMPASS in Germline Chromatin Organization."

BAilly A, Feil R, Robert V, Herbette M, Gely L, Palladino F, Lleres D

[Cells, 2020]

Deposition of histone H3 lysine 4 (H3K4) methylation at promoters is catalyzed by the SET1/COMPASS complex and is associated with context-dependent effects on gene expression and local changes in chromatin organization. The role of SET1/COMPASS in shaping chromosome architecture has not been investigated. Here we used <i>Caenorhabditis elegans</i> to address this question through a live imaging approach and genetic analysis. Using quantitative FRET (Forster resonance energy transfer)-based fluorescence lifetime imaging microscopy (FLIM) on germ cells expressing histones eGFP-H2B and mCherry-H2B, we find that SET1/COMPASS influences meiotic chromosome organization, with marked effects on the close proximity between nucleosomes. We further show that inactivation of <i>set-2</i>, encoding the <i>C. elegans</i> SET1 homologue, or CFP-1, encoding the chromatin targeting subunit of COMPASS, enhances germline chromosome organization defects and sterility of condensin-II depleted animals. <i>set-2</i> loss also aggravates germline defects resulting from conditional inactivation of topoisomerase II, another structural component of chromosomes. Expression profiling of <i>set-2</i> mutant germlines revealed only minor transcriptional changes, suggesting that the observed effects are at least partly independent of transcription. Altogether, our results are consistent with a role for SET1/COMPASS in shaping meiotic chromosomes in <i>C. elegans</i>, together with the non-histone proteins condensin-II and topoisomerase. Given the high degree of conservation, our findings expand the range of functions attributed to COMPASS and suggest a broader role in genome organization in different species.