9 Papers found (0.01 seconds)

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.

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.

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 Caenorhabditis elegans 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 set-2, encoding the C. elegans SET1 homologue, or CFP-1, encoding the chromatin targeting subunit of COMPASS, enhances germline chromosome organization defects and sterility of condensin-II depleted animals. set-2 loss also aggravates germline defects resulting from conditional inactivation of topoisomerase II, another structural component of chromosomes. Expression profiling of set-2 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 C. elegans, 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.

Wang P et al. (2022) J Biol Chem "Whole-cell FRET monitoring of transcription factor activities enables functional annotation of signal transduction systems in living bacteria."

Chen Z, Liu X, Xu Z, Wang P, Zhang G, Wang C, Yan A, Zheng C, Zhang H, Wang J

[

J Biol Chem,

2022]

Bacteria adapt to their constantly changing environments largely by transcriptional regulation through the activities of various transcription factors (TFs). However, techniques that monitor TF-promoter interactions in situ in living bacteria are lacking. Herein, we developed a whole-cell TF-promoter binding assay based on the intermolecular Forster resonance energy transfer (FRET) between an unnatural amino acid, L-(7-hydroxycoumarin-4-yl) ethylglycine (CouA), which labels TFs with bright fluorescence through genetic encoding (donor fluorophore) and the live cell nucleic acid stain SYTO 9 (acceptor fluorophore). We show that this new FRET pair monitors the intricate TF-promoter interactions elicited by various types of signal transduction systems, including one-component (CueR) and two-component systems (BasSR and PhoPQ), in bacteria with high specificity and sensitivity. We demonstrate that robust CouA incorporation and FRET occurrence is achieved in all regulatory systems tested based on either the crystal structures of TFs or their simulated structures, if 3D structures of the TFs were unavailable. Furthermore, using CueR and PhoPQ systems as models, we demonstrate that the whole-cell FRET assay is applicable for the identification and validation of complex regulatory circuit and novel modulators of regulatory systems of interest. Lastly, we show that the FRET system is applicable for single-cell analysis and for monitoring TF activities in E. coli colonizing a C. elegans host. In conclusion, we established a tractable and sensitive TF-promoter binding assay which not only complements currently available approaches for DNA-protein interactions, but also provides novel opportunities for functional annotation of bacterial signal transduction systems and studies of the bacteria-host interface.

Kaminski Schierle GS et al. (2011) Chemphyschem "A FRET sensor for non-invasive imaging of amyloid formation in vivo."

Schwedler S, Kaminski Schierle GS, Nollen EA, Schlachter S, Kaminski CF, van der Goot AT, Dobson CM, Esposito A, Kumita JR, van Ham T, Chan FT, Skepper J, Bertoncini CW

[

Chemphyschem,

2011]

Misfolding and aggregation of amyloidogenic polypeptides lie at the root of many neurodegenerative diseases. Whilst protein aggregation can be readily studied in vitro by established biophysical techniques, direct observation of the nature and kinetics of aggregation processes taking place in vivo is much more challenging. We describe here, however, a Forster resonance energy transfer sensor that permits the aggregation kinetics of amyloidogenic proteins to be quantified in living systems by exploiting our observation that amyloid assemblies can act as energy acceptors for variants of fluorescent proteins. The observed lifetime reduction can be attributed to fluorescence energy transfer to intrinsic energy states associated with the growing amyloid species. Indeed, for a-synuclein, a protein whose aggregation is linked to Parkinson's disease, we have used this sensor to follow the kinetics of the self-association reactions taking place in vitro and in vivo and to reveal the nature of the ensuing aggregated species. Experiments were conducted in vitro, in cells in culture and in living Caenorhabditis elegans. For the latter the readout correlates directly with the appearance of a toxic phenotype. The ability to measure the appearance and development of pathogenic amyloid species in a living animal and the ability to relate such data to similar processes observed in vitro provides a powerful new tool in the study of the pathology of the family of misfolding disorders. Our study confirms the importance of the molecular environment in which aggregation reactions take place, highlighting similarities as well as differences between the processes occurring in vitro and in vivo, and their significance for defining the molecular physiology of the diseases with which they are associated.