A toolkit for GFP-mediated tissue-specific protein degradation in C. elegans - PubMed (original) (raw)

. 2017 Jul 15;144(14):2694-2701.

doi: 10.1242/dev.150094. Epub 2017 Jun 15.

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A toolkit for GFP-mediated tissue-specific protein degradation in C. elegans

Shaohe Wang et al. Development. 2017.

Abstract

Proteins that are essential for embryo production, cell division and early embryonic events are frequently reused later in embryogenesis, during organismal development or in the adult. Examining protein function across these different biological contexts requires tissue-specific perturbation. Here, we describe a method that uses expression of a fusion between a GFP-targeting nanobody and a SOCS-box containing ubiquitin ligase adaptor to target GFP-tagged proteins for degradation. When combined with endogenous locus GFP tagging by CRISPR-Cas9 or with rescue of a null mutant with a GFP fusion, this approach enables routine and efficient tissue-specific protein ablation. We show that this approach works in multiple tissues - the epidermis, intestine, body wall muscle, ciliated sensory neurons and touch receptor neurons - where it recapitulates expected loss-of-function mutant phenotypes. The transgene toolkit and the strain set described here will complement existing approaches to enable routine analysis of the tissue-specific roles of C. elegans proteins.

Keywords: C. elegans; GFP nanobody; Protein degradation; ZIF-1; vhhGFP4.

© 2017. Published by The Company of Biologists Ltd.

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Conflict of interest statement

Competing interestsThe authors declare no competing or financial interests.

Figures

Fig. 1.

Fig. 1.

epiDEG efficiently degrades GFP-tagged proteins that localize to different subcellular localizations. (A) Schematic illustrating the method. (B) Top: schematic showing imaged region. Bottom: fluorescence confocal images of L3 stage worms expressing GFP::β-tubulin and plots of GFP::β-tubulin fluorescence intensity. (C) Left: schematics and fluorescence confocal images of late L4 stage worms expressing DLG-1::GFP. Right: plots of DLG-1::GFP fluorescence intensity. (D) Left: schematics and fluorescence confocal images of L3 stage worms expressing GFP::MAD-1. Yellow arrows indicate the body wall muscle nuclei. Right: plots of GFP::MAD-1 fluorescence intensity. n is the number of worms analyzed. Data were analyzed using a two-tailed Student's _t_-test. P values are the probability of obtaining the observed results assuming the test group is the same as control. Data are shown as mean±s.d. Scale bars: 10 µm.

Fig. 2.

Fig. 2.

GFP-mediated protein degradation is efficient in multiple C. elegans tissues. (A) Transgene schematics. (B-D) Top: schematics showing imaged region. Middle: fluorescence confocal images (maximum intensity projections in B and C, single _z_-slice in D) of L3 stage worms expressing GFP::MAD-1. Yellow arrows indicate nuclei. Bottom: plots of GFP::MAD-1 fluorescence intensity. n is the number of worms analyzed. Each data point in B and C represents an average of five nuclei from the same worm. Data were analyzed using a two-tailed Student's _t_-test. P values are the probability of obtaining the observed results assuming the test group is the same as control. Data are shown as mean±s.d. Scale bars: 10 µm.

Fig. 3.

Fig. 3.

GFP-mediated protein degradation is rapid in C. elegans intestine. (A) Still images from time-lapse confocal imaging of embryos expressing GFP::MAD-1 and either the intControl or intDEG cassettes (also see

Movie 1

). (B) Graph quantifying total per embryo GFP fluorescence for GFP::PP1GSP-2 and GFP::MAD-1. (C) Still images from time-lapse confocal imaging of control and intDEG embryos expressing GFP::PP1GSP-2 (also see

Movie 2

). Dashed yellow outlines show the location of the intestinal cells in A and C. (D) Plot of normalized GFP::PP1GSP-2 or mCherry::histone intensity in control or intDEG embryos. n is the number of embryos analyzed. Data are shown as mean±s.d. Scale bars: 10 µm.

Fig. 4.

Fig. 4.

Degradation of GFP::DLK-1 in the touch receptor neurons blocks axon regeneration. (A) Transgene schematic. (B) Schematic of the axon regeneration assay. (C) Plots of normalized touch neuron (PLM) regrowth at 24 h post laser axotomy. Number in each bar is the number of worms assayed. (D,E) Inverted grayscale images of the touch neuron (PLM) axon. Data were analyzed using a one-way ANOVA with Bonferroni's post test. ***P<0.001. n.s., not significant. Results are compared with control unless specified by the line. Data are shown as mean±s.d. Scale bars: 10 µm.

Fig. 5.

Fig. 5.

Degradation of GIP-2::GFP or PLK-1::sGFP in the intestine causes cell division defects and impairs growth. (A) Maximum intensity projections of confocal images of 1.5-fold C. elegans embryos expressing GIP-2::GFP with the intControl or intDEG cassettes. (B) Confocal images of pre-comma C. elegans embryos expressing PLK-1::sGFP with the intControl or intDEG cassettes. (C) Plots of intestinal GFP fluorescence for GIP-2::GFP in 1.5- to 1.8-fold embryos (left) and PLK-1::sGFP in pre-comma embryos (right). n is the number of embryos analyzed. (D) Plot of the number of intestinal nuclei in 1.5- to 1.8-fold embryos. n is the number of embryos analyzed. (E) Left: plot of body length for worms of the indicated genotypes. Right: representative images of worms at 72 h post timed egg-lay. n is the number of worms analyzed at 24, 48 and 72 h. Data were analyzed using a two-tailed Student's _t_-test. P values are the probability of obtaining the observed results, assuming the test group is the same as control. Data are shown as mean±s.d. Scale bars: 10 µm (or as indicated).

References

    1. Armenti S. T., Lohmer L. L., Sherwood D. R. and Nance J. (2014). Repurposing an endogenous degradation system for rapid and targeted depletion of C. elegans proteins. Development 141, 4640-4647. 10.1242/dev.115048 -DOI -PMC -PubMed
    1. Caussinus E., Kanca O. and Affolter M. (2012). Fluorescent fusion protein knockout mediated by anti-GFP nanobody. Nat. Struct. Mol. Biol. 19, 117-121. 10.1038/nsmb.2180 -DOI -PubMed
    1. Chen L., Wang Z., Ghosh-Roy A., Hubert T., Yan D., O'Rourke S., Bowerman B., Wu Z., Jin Y. and Chisholm A. D. (2011). Axon regeneration pathways identified by systematic genetic screening in C. elegans. Neuron 71, 1043-1057. 10.1016/j.neuron.2011.07.009 -DOI -PMC -PubMed
    1. Collet J., Spike C. A., Lundquist E. A., Shaw J. E. and Herman R. K. (1998). Analysis of osm-6, a gene that affects sensory cilium structure and sensory neuron function in Caenorhabditis elegans. Genetics 148, 187-200. -PMC -PubMed
    1. Deppe U., Schierenberg E., Cole T., Krieg C., Schmitt D., Yoder B. and von Ehrenstein G. (1978). Cell lineages of the embryo of the nematode Caenorhabditis elegans. Proc. Natl. Acad. Sci. USA 75, 376-380. 10.1073/pnas.75.1.376 -DOI -PMC -PubMed

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