Codon adaptation–based control of protein expression in C. elegans

Redemann, Stefanie; Schloissnig, Siegfried; Ernst, Susanne; Pozniakowsky, Andrey; Ayloo, Swathi; Hyman, Anthony A.; Bringmann, Henrik

doi:10.1038/nmeth.1565

Cited by 182 publications

(171 citation statements)

References 24 publications

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“…1; see Movie 1 and Fig. S1D in the supplementary material), both of which were designed with C. elegans codon bias (Redemann et al, 2010). GPR-1 and GPR-2 are nearly identical proteins (Colombo et al, 2003); we use GPR-1/2 to refer to the endogenous protein pair and GPR-1 to refer to our transgenic versions.…”

Section: Resultsmentioning

confidence: 99%

Dynamic localization of C. elegans TPR-GoLoco proteins mediates mitotic spindle orientation by extrinsic signaling

2011

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SUMMARYCell divisions are sometimes oriented by extrinsic signals, by mechanisms that are poorly understood. Proteins containing TPR and GoLoco-domains (C. elegans GPR-1/2, Drosophila Pins, vertebrate LGN and AGS3) are candidates for mediating mitotic spindle orientation by extrinsic signals, but the mechanisms by which TPR-GoLoco proteins may localize in response to extrinsic cues are not well defined. The C. elegans TPR-GoLoco protein pair GPR-1/2 is enriched at a site of contact between two cells -the endomesodermal precursor EMS and the germline precursor P 2 -and both cells align their divisions toward this shared cell-cell contact. To determine whether GPR-1/2 is enriched at this site within both cells, we generated mosaic embryos with GPR-1/2 bearing a different fluorescent tag in different cells. We were surprised to find that GPR-1/2 distribution is symmetric in EMS, where GPR-1/2 had been proposed to function as an asymmetric cue for spindle orientation. Instead, GPR-1/2 is asymmetrically distributed only in P 2 . We demonstrate a role for normal GPR-1/2 localization in P 2 division orientation. We show that MES-1/Src signaling plays an instructive role in P 2 for asymmetric GPR-1/2 localization and normal spindle orientation. We ruled out a model in which signaling localizes GPR-1/2 by locally inhibiting LET-99, a GPR-1/2 antagonist. Instead, asymmetric GPR-1/2 distribution is established by destabilization at one cell contact, diffusion, and trapping at another cell contact. Once the mitotic spindle of P 2 is oriented normally, microtubule-dependent removal of GPR-1/2 prevented excess accumulation, in an apparent negative-feedback loop. These results highlight the role of dynamic TPR-GoLoco protein localization as a key mediator of mitotic spindle alignment in response to instructive, external cues.

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Section: Resultsmentioning

confidence: 99%

Dynamic localization of C. elegans TPR-GoLoco proteins mediates mitotic spindle orientation by extrinsic signaling

2011

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“…Repair templates were provided as plasmid (mEos2-HIM-3), long ssDNA [mMaple3-HIM-3 (21)], or ssODNs (synthesized as "Ultramers" by IDT) for epitope tagging and rec-8(ie41), respectively. All coding sequences for epitope tags and fluorescent proteins were codon-optimized for C. elegans, and the protein coding sequences also contained short introns (82).…”

Section: Methodsmentioning

confidence: 99%

Superresolution microscopy reveals the three-dimensional organization of meiotic chromosome axes in intact Caenorhabditis elegans tissue

Köhler

Wojcik

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

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When cells enter meiosis, their chromosomes reorganize as linear arrays of chromatin loops anchored to a central axis. Meiotic chromosome axes form a platform for the assembly of the synaptonemal complex (SC) and play central roles in other meiotic processes, including homologous pairing, recombination, and chromosome segregation. However, little is known about the 3D organization of components within the axes, which include cohesin complexes and additional meiosis-specific proteins. Here, we investigate the molecular organization of meiotic chromosome axes in Caenorhabditis elegans through STORM (stochastic optical reconstruction microscopy) and PALM (photo-activated localization microscopy) superresolution imaging of intact germ-line tissue. By tagging one axis protein (HIM-3) with a photoconvertible fluorescent protein, we established a spatial reference for other components, which were localized using antibodies against epitope tags inserted by CRISPR/Cas9 genome editing. Using 3D averaging, we determined the position of all known components within synapsed chromosome axes to high spatial precision in three dimensions. We find that meiosis-specific HORMA domain proteins span a gap between cohesin complexes and the central region of the SC, consistent with their essential roles in SC assembly. Our data further suggest that the two different meiotic cohesin complexes are distinctly arranged within the axes: Although cohesin complexes containing the kleisin REC-8 protrude above and below the plane defined by the SC, complexes containing COH-3 or -4 kleisins form a central core, which may physically separate sister chromatids. This organization may help to explain the role of the chromosome axes in promoting interhomolog repair of meiotic double-strand breaks by inhibiting intersister repair.

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“…To increase FLP expression levels we used the C. elegans codon adapter tool (REDEMANN et al 2011) with the parameters "Optimize for weak mRNA structure at ribosome binding site" and "Avoid splice sites in coding region" resulting in a codon adaptation index (CAI) of 0.9808. The sequence, which we termed FLP G5, was synthesized by Eurofins Genomics (Ebersberg, Germany).…”

Section: Plasmid Constructionmentioning

confidence: 99%

An Efficient FLP-Based Toolkit for Spatiotemporal Control of Gene Expression in Caenorhabditis elegans

Muñoz-Jiménez¹,

Ayuso²,

Dobrzynska³

et al. 2017

Genetics

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Codon adaptation–based control of protein expression in C. elegans

Cited by 182 publications

References 24 publications

Dynamic localization of C. elegans TPR-GoLoco proteins mediates mitotic spindle orientation by extrinsic signaling

Dynamic localization of C. elegans TPR-GoLoco proteins mediates mitotic spindle orientation by extrinsic signaling

Superresolution microscopy reveals the three-dimensional organization of meiotic chromosome axes in intact Caenorhabditis elegans tissue

An Efficient FLP-Based Toolkit for Spatiotemporal Control of Gene Expression in Caenorhabditis elegans

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