4 Papers found (0.008 seconds)

Almeida MV et al. (2018) EMBO J "GTSF-1 is required for formation of a functional RNA-dependent RNA Polymerase complex in Caenorhabditis elegans."

Redl S, Hildebrandt A, Ulrich HD, Dietz S, Renz C, Almeida MV, Konig J, Ketting RF, Butter F, Karaulanov E

[

EMBO J,

2018]

Argonaute proteins and their associated small RNAs (sRNAs) are evolutionarily conserved regulators of gene expression. Gametocyte-specific factor 1 (Gtsf1) proteins, characterized by two tandem CHHC zinc fingers and an unstructured C-terminal tail, are conserved in animals and have been shown to interact with Piwi clade Argonautes, thereby assisting their activity. We identified the Caenorhabditis elegans Gtsf1 homolog, named it gtsf-1 and characterized it in the context of the sRNA pathways of C.elegans We report that GTSF-1 is not required for Piwi-mediated gene silencing. Instead, gtsf-1 mutants show a striking depletion of 26G-RNAs, a class of endogenous sRNAs, fully phenocopying rrf-3 mutants. We show, both invivo and invitro, that GTSF-1 interacts with RRF-3 via its CHHC zinc fingers. Furthermore, we demonstrate that GTSF-1 is required for the assembly of a larger RRF-3 and DCR-1-containing complex (ERIC), thereby allowing for 26G-RNA generation. We propose that GTSF-1 homologs may act to drive the assembly of larger complexes that act in sRNA production and/or in imposing sRNA-mediated silencing activities.

Goehring A et al. (2014) Nat Protoc "Screening and large-scale expression of membrane proteins in mammalian cells for structural studies."

Lee CH, Fischer S, Althoff T, Michel JC, Gouaux E, Garcia KC, Baconguis I, Claxton DP, Wang KH, Goehring A

[

Nat Protoc,

2014]

Structural, biochemical and biophysical studies of eukaryotic membrane proteins are often hampered by difficulties in overexpression of the candidate molecule. Baculovirus transduction of mammalian cells (BacMam), although a powerful method to heterologously express membrane proteins, can be cumbersome for screening and expression of multiple constructs. We therefore developed plasmid Eric Gouaux (pEG) BacMam, a vector optimized for use in screening assays, as well as for efficient production of baculovirus and robust expression of the target protein. In this protocol, we show how to use small-scale transient transfection and fluorescence-detection size-exclusion chromatography (FSEC) experiments using a GFP-His8-tagged candidate protein to screen for monodispersity and expression level. Once promising candidates are identified, we describe how to generate baculovirus, transduce HEK293S GnTI(-) (N-acetylglucosaminyltransferase I-negative) cells in suspension culture and overexpress the candidate protein. We have used these methods to prepare pure samples of chicken acid-sensing ion channel 1a (cASIC1) and Caenorhabditis elegans glutamate-gated chloride channel (GluCl) for X-ray crystallography, demonstrating how to rapidly and efficiently screen hundreds of constructs and accomplish large-scale expression in 4-6 weeks.

Escobar-Restrepo JM et al. (2014) Worm "An intimate look at LET-23 EGFR trafficking in the vulval cells of live C. elegans larvae."

Hajnal A, Escobar-Restrepo JM

[

Worm,

2014]

Precise cell fate specification is essential for organ formation. A simple view is that one or several signal sending cells emit a ligand to a group of signal receiving cells that express the corresponding receptor, which transduces the signal through intracellular enzyme pathways. All these events must be spatio-temporally regulated to achieve the proper strength, duration and output of the signaling pathways. In particular, the production and secretion of the ligand has to be coordinated with the expression and accessibility of the receptor in the signal receiving cells. Furthermore, removal of the ligand or receptor is key to achieve proper signal termination and prevent excess cell differentiation and proliferation. Improper regulation of any of these events may cause developmental defects and human disease. C. elegans is an excellent model to systematically identify genes that control the localization and activity of the Epidermal Growth Factor Receptor (EGFR) homolog LET-23. To identify regulators of LET-23 trafficking, Haag etal. observed LET-23 localization in the vulva precursor cells (VPCs) of RNAi treated larvae by live fluorescent microscopy. In this comment, we provide an overview of the newly identified regulators of LET-23 trafficking and discuss the role of the Ezrin/Radixin/Moesin homolog ERM-1 as a temporal regulator of EGFR signaling.

Dupont C et al. (2017) Front Microbiol "Highly Diversified Pandoraea pulmonicola Population during Chronic Colonization in Cystic Fibrosis."

Aujoulat F, Condom P, Jumas-Bilak E, Chiron R, Dupont C, Marchandin H

[

Front Microbiol,

2017]

Several environmental bacteria are considered as opportunistic pathogens in cystic fibrosis (CF) and are able to persistently colonize the CF respiratory tract (CFRT). Beside Pseudomonas aeruginosa and Burkholderia cepacia complex, Pandoraea spp. are defined as pathogenic. During chronic colonization, adaptive evolution and diversified population have been demonstrated, notably for P. aeruginosa. However, the persistence of Pandoraea in the CFRT remains largely unexplored. We studied genomic and phenotypic traits of Pandoraea pulmonicola isolates successively recovered from the airways of a single CF patient and relate the results to qualitative and quantitative evolution of other cultivable pathogens and to patient clinical status. A total of 31 isolates recovered from 18 sputum samples over a 7-year period in a single CF patient were studied. Genome dynamics was assessed by pulsed-field gel electrophoresis, ERIC-PCR fingerprinting and 16S rRNA gene PCR-temporal temperature gel electrophoresis. Phenotypic features included antimicrobial susceptibility, motility, biofilm production, and virulence in Caenorhabditis elegans model. Variability was observed for all the characteristics studied leading to highly diversified patterns (24 patterns) for the 31 clonally related isolates. Some of these modifications, mainly genomic events were concomitantly observed with CFRT microbiota composition shifts and with severe exacerbations. The diversity of P. pulmonicola population studied, observed for isolates recovered from successive samples but also within a sample suggested that existence of a diversified population may represent a patho-adaptive strategy for host persistence in the heterogeneous and fluctuating CFRT environment.