A modified TurboID approach identifies tissue-specific centriolar components in C. elegans

Abstract

Proximity-dependent labeling approaches such as BioID have been a great boon to studies of protein-protein interactions in the context of cytoskeletal structures such as centrosomes which are poorly amenable to traditional biochemical approaches like immunoprecipitation and tandem affinity purification. Yet, these methods have so far not been applied extensively to invertebrate experimental models such as C. elegans given the long labeling times required for the original promiscuous biotin ligase variant BirA*. Here, we show that the recently developed variant TurboID successfully probes the interactomes of both stably associated (SPD-5) and dynamically localized (PLK-1) centrosomal components. We further develop an indirect proximity labeling method employing a GFP nanobody-TurboID fusion, which allows the identification of protein interactors in a tissue-specific manner in the context of the whole animal. Critically, this approach utilizes available endogenous GFP fusions, avoiding the need to generate multiple additional strains for each target protein and the potential complications associated with overexpressing the protein from transgenes. Using this method, we identify homologs of two highly conserved centriolar components, Cep97 and BLD10/Cep135, which are present in various somatic tissues of the worm. Surprisingly, neither protein is expressed in early embryos, likely explaining why these proteins have escaped attention until now. Our work expands the experimental repertoire for C. elegans and opens the door for further studies of tissue-specific variation in centrosome architecture.

Chemical Acetylation of Ligands and Two-Step Digestion Protocol for Reducing Codigestion in Affinity Purification–Mass Spectrometry

“…It allows efficient bait elution, protects from contamination with ligand–peptides, uses nontoxic, mild chemical conditions, and is simple to carry out. We have adopted the protocol as a standard procedure in our core facility, and it has been successfully used in several projects. , In conclusion, the protocol presented in this study offers a practical and efficient solution for preventing ligand contamination in AP–MS samples with potential applications in a variety of research settings.…”

Molecular architecture of the C. elegans centriole

“…This is reminiscent of the region to which Cep135 and Cep97 localize in the fly centriole (34). It has been suggested that a divergent Cep135 protein localizes to centrioles of C. elegans during certain developmental stages (49), and it will be interesting to use U-Ex-STED to address whether it maps to this density. Alternatively, it is possible that a segment of the proteins tested here and localizing to such a density would not be apparent from tagging the N-or C-terminal part of the protein, or with some of the antibodies.…”

Depletion of endogenously biotinylated carboxylases enhances the sensitivity of TurboID-mediated proximity labeling in Caenorhabditis elegans

“…Overexpressing a patronin::TurboID fusion protein in the worm gut successfully highlighted the interactome of this microtubule-binding protein (18). Fusing TurboID to an anti-GFP nanobody allowed tissue-specific analysis of the interactomes of the GFP-tagged centrosomal proteins PLK-1 and SPD-5 (19). We identified interactors of the presynaptic active zone protein ELKS-1 by knocking in a TurboID::mNeongreen cassette at the elks-1 locus (10).…”