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

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 tissuespecific 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.

A Neuronal piRNA Pathway Inhibits Axon Regeneration in C. elegans

“…To test this, we took advantage of a tissue-specific protein knockdown method that relies on GFP-DEGRON (GFP nanobody-ZIF-1 fusion) to target and degrade GFP-tagged proteins (Armenti et al, 2014; Wang et al, 2017) (Figure 3D). We expressed the GFP-DEGRON in mechanosensory neurons (trnDEGRON), and confirmed that it eliminated GFP fluorescence (Figure S3), and also did not alter axon morphology.…”

Reflections on the use of protein binders to study protein function in developmental biology

“…Degradation of GFP-tagged proteins via the expression of a GFP nanobody fused to a F-box is a powerful tool to investigate protein function. While the deGradFP approach has originally been reported to be effective in Drosophila, similar methods have in the meantime been reported in C. elegans (Wang et al, 2017), zebrafish (Daniel et al, 2018;Shin et al, 2015;Yamaguchi et al, 2019) and in plants (Baudisch, Pfort, Sorge, & Conrad, 2018).…”

“…Upon binding to the GFP-tagged POI, a GFP nanobody fused to an F-box domain can target the POI for degradation (Caussinus, Kanca, & Affolter, 2011) (Figure 1c). Several similar approaches have been recently developed to achieve protein degradation in different model organisms (Daniel et al, 2018;Wang et al, 2017;Yamaguchi, Colak-Champollion, & Knaut, 2019).…”

Recombinant expression of nanobodies and nanobody-derived immunoreagents

“…The deGradFP system exploits an anti-GFP-Fbox fusion nanobody that promotes the ubiquitination of the bound antigen and its consequent degradation mediated by the proteasome machinery. It was initially optimized for flies [54] but the concept has been recently adapted and extended to other model organisms such as C. elegans and zebrafish and to perform ultrastructural localization of protein interactions [55][56][57][58]. It is expected that in the future protein-specific nanobodies will substitute the anti-tag binders because in such a way more flexible multi-dimensional analyses will be possible.…”