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. Author summary ‘You can tell a lot about a person by the company they keep.’ This is as true for proteins as it is for people. Unfortunately, the methods traditionally used to probe protein-protein interactions, which rely on isolating stable multimolecular complexes, fail where such complexes do not exist or cannot be isolated from the cell. Proximity-dependent labeling methods such as TurboID, where interacting proteins are marked in vivo before isolation and which therefore do not require complexes to be maintained during extract preparation, have consequently become invaluable tools in cell biology. Normally, this requires the protein of interest to be tagged with an enzymatic marker such as the biotin ligase BirA*. Here, we developed a variant of this method, whereby the enzyme is targeted to the protein of interest using a genetically-encoded GFP nanobody, making existing fluorescent strains immediately available for interaction biochemistry. We show that this method successfully probes protein-protein interactions in a tissue-specific manner, even where those interactions occur in only a few cells in the context of the entire animal. While this work was conducted in the nematode C. elegans, the same method should be applicable to other genetic experimental models, as well as potentially vertebrate cultured cells.