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.

Fig 1. Probing the proximity interactome of centrosomal proteins by TurboID.

(A) Centrosomes and centrosome-related structures in C. elegans. Centrosome assembly and function has been primarily examined in the early embryo, although some limited work has also been performed on the acentriolar centrosome at the ciliary base of sensory neurons [10, 11] and the non-centrosomal microtubule organizing center in the intestine [12,13]. Blast cells are two pairs of cells in the L1 larva that will give rise to the somatic gonad and germline in the adult worm. At the L1 larval stage these cells are arrested in G1 and G2 phase of the cell cycle, respectively, with what appear to be canonical interphase centrosomes [60,61]. (B) Schematic of direct TurboID [19] which in our implementation also includes a GFP tag to visualize the TurboID fusion. Note that experimental and control TurboID fusions (lacking the protein of interest, POI) are expressed as separate transgenes, though under the same promoter and 3’ regulatory sequences. (C) TurboID applied to the PCM scaffolding protein SPD-5. Immunofluorescence micrograph of early embryo expressing GFP:TurboID:SPD-5 stained for GFP, biotin (streptavidin) and TAC-1 as a PCM countermarker. Biotinylation signal is observed at centrosomes without supplemental biotin addition. (D) Result of LC-MS/MS analysis for direct TurboID on SPD-5 from mixed-stage embryos. Volcano plot of -log10 p-values against log2 fold change (sample/control). Significantly enriched proteins (Log2 enrichment >1, p-value <0.05) are indicated in pink, with selected proteins highlighted. See also S1 Table and S2C Fig. (E) TurboID applied to the dynamically localized PCM regulator PLK-1. Immunofluorescence micrograph of early embryo expressing GFP:TurboID:PLK-1 stained for GFP, biotin (streptavidin) and TAC-1 as a PCM countermarker. Biotinylation signal is observed at centrioles coincident with GFP:PLK-1 signal. (D) Result of LC-MS/MS analysis for direct TurboID on PLK-1 from mixed-stage embryos. See also S1 Table and S2D Fig. Scale bars in C and E are 10μm.

Fig 2. GFP nanobody-directed TurboID as an improved method for proximity-dependent labeling.

(A) Schematic of indirect TurboID method, whereby the biotin ligase is targeted to an endogenous GFP fusion via a GFP nanobody [41]. Note that experimental and control strains utilize the same TurboID fusion, which may be expressed under a tissue or developmental stage-specific or inducible promoter, while the target protein is potentially expressed in a wide array of tissues and cell types. (B) GFP nanobody addition does not perturb PLK-1 mobility. Selected images and quantitation for fluorescence recovery after photobleaching (FRAP) analysis performed on PLK-1:GFP at centrosomes in prometaphase-stage embryos in the presence (n = 16 animals) or absence (n = 13) of the GFP nanobody:TurboID fusion. (C) Indirect TurboID applied to SPD-5. Immunofluorescence micrograph of early embryo from strain co-expressing a GFP nanobody:HA:TurboID fusion under the germline promoter pie-1 and endogenously GFP-tagged SPD-5 stained for GFP, biotin (streptavidin) and HA. Biotinylation signal is observed at centrosomes coincident with GFP:SPD-5 and the TurboID fusion. (D) Result of LC-MS/MS analysis for indirect TurboID on SPD-5 from mixed-stage embryos. Volcano plot of -log10 p-values against log2 fold change (sample/control). Significantly enriched proteins (Log2 enrichment >1, p-value <0.05) are indicated in pink, with selected proteins highlighted. Compare Fig 1D. See also S1 Table and S2E Fig. (E) Indirect TurboID applied to PLK-1. Immunofluorescence micrograph of early embryo from strain co-expressing a GFP nanobody:HA:TurboID fusion under the germline promoter pie-1 and endogenously GFP-tagged PLK-1 stained for GFP, biotin (streptavidin) and HA. Biotinylation signal is observed at centrosomes coincident with PLK-1:GFP and the TurboID fusion. (F) Result of LC-MS/MS analysis for indirect TurboID on PLK-1 from mixed-stage embryos. Compare Fig 1F. See also S1 Table and S2E Fig. Scale bars are 1μm (B), 10μm (C, E).

Fig 3. Tissue-specific labeling using GFP nanobody-directed TurboID.

(A) TurboID applied to SPD-5 in ciliated neurons. Immunofluorescence micrograph of head of L1 larva from strain co-expressing a GFP nanobody:HA:TurboID fusion under the ciliated neuron-specific promoter osm-6 and endogenously GFP-tagged SPD-5 stained for GFP, biotin (streptavidin) and HA. Biotinylation signal is observed at the ciliary base coincident with GFP:SPD-5 and the TurboID fusion. (B) Result of LC-MS/MS analysis for indirect TurboID on SPD-5 in ciliated neurons. Volcano plot of -log10 p-values against log2 fold change (sample/control). Significantly enriched proteins (Log2 enrichment >1, p-value <0.05) are indicated in pink, with selected proteins highlighted. See also S1 Table. (C) Comparison of LC-MS/MS results for direct and indirect TurboID on SPD-5 in ciliated neurons. Indirect TurboID identifies several additional SPD-5 proximity interactors. Full results for direct TurboID presented in S2A and S2B Fig and S1 Table. (D) Volcano plot of average log2 LFQ intensity (sample) against log2 fold change (sample/control) for indirect TurboID on SPD-5 in ciliated neurons. Significantly enriched proteins (Log2 enrichment >1, p-value <0.05) are indicated in pink, with selected proximity interactors highlighted. Note that these interactors are present at much lower levels in the sample compared to endogenously biotinylated proteins, most notably the carboxylases PCCA-1, PYC-1, POD-2 and MCCC-1 [20], highlighting the importance of proper sample normalization. (E) TurboID applied to SPD-5 in germ cell precursors. Immunofluorescence micrograph of the area of the blast cells in an L1 larva co-expressing a GFP nanobody:HA:TurboID fusion under the germline-specific promoter nos-2 and endogenously GFP-tagged SPD-5 stained for GFP, biotin (streptavidin) and HA. Biotinylation signal is observed at centrosomes in two cells (the primordial germ cells Z2, Z3) coincident with GFP:SPD-5 and the TurboID fusion. (F) Result of LC-MS/MS analysis for indirect TurboID on SPD-5 in germ cell precursors. See also S1 Table and S2H Fig. Scale bars in A and E are 10μm.

Fig 4. CCEP-97 and CCEP-135 as novel tissue-specific centrosomal components in C. elegans.

(A) AlphaFold [73] predicts a similar 3D architecture for C. elegans R02F11.4/CCEP-97 and its putative Drosophila and human orthologs Cep97, including a prominent leucine rich repeat that is highly conserved across Cep97 orthologs followed by long coiled coils (see S3A and S3B Fig). Additionally, a Fibronectin type III (FN-3) domain is predicted for the C. elegans protein, which is conserved in other nematode homologs but not in insects or vertebrates. (B) An endogenous promoter GFP transgene shows CCEP-97 to be expressed in late-stage embryos, germ cell precursors and ciliated neurons, localizing to centrioles (marked by immunofluorescence co-staining with SPD-5) and the ciliary base (likewise), respectively. (C) AlphaFold structure prediction shows C. elegans H06I04.1/CCEP-135 and its putative Drosophila and human orthologs Cep135/BLD10 to display a similar 3D architecture, composed primarily of coiled coils. These align in a rope-like manner when predicting the proteins to form dimers as has been shown for human and Chlamydomonas Cep135/BLD10 [77]. For hsCep135 and dmCEP135 only the N-terminal regions (1–600, hsCEP135; 1–547, dmCEP135) were used for homodimer prediction to reduce computational costs. We note that in the homodimeric dmCEP135 model some regions are overlapping suggesting that the native structure deviates from the prediction. For CCEP-135 the first 131 residues, predicted with lower confidence, are not displayed. See also S4A and S4B Figs. (D) An endogenous promoter GFP transgene shows CCEP-135 to be expressed in ciliated sensory neurons of the head (amphids and cephalic/labial neurons), localizing to the ciliary base (marked by immunofluorescence co-staining with SPD-5). (E) Comparison of selected SPD-5 proximity interactors identified in embryos, germ cell precursors and ciliated neurons (for full list see S1 Table). Common to all three tissue contexts are PCMD-1 and RSA-2, while SPD-2 is absent from post-mitotic sensory neurons. The previously uncharacterized proteins CCEP-97 and CCEP-135 are amongst the tissue-specific SPD-5 interactors, identified in germ cell precursors and ciliated neurons, respectively. Scale bars in B and D are 10μm.