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

Area of Science:

• Cell Biology
• Molecular Biology
• Biochemistry

Background:

• Proximity-dependent labeling methods like BioID are valuable for studying protein-protein interactions, especially in challenging contexts like centrosomes.
• Traditional biochemical methods are often difficult to apply to invertebrate models such as C. elegans.
• Previous proximity labeling methods required long labeling times, limiting their application in C. elegans.

Purpose of the Study:

• To adapt and validate TurboID, a faster proximity labeling enzyme, for studying centrosomal protein interactomes in C. elegans.
• To develop an indirect proximity labeling strategy using GFP nanobody-TurboID fusions for tissue-specific interactome analysis in whole C. elegans.
• To identify novel centrosomal components in C. elegans using this new methodology.

Main Methods:

• Utilized the TurboID enzyme for direct proximity labeling of centrosomal proteins (SPD-5 and PLK-1) in C. elegans.
• Developed and implemented an indirect proximity labeling method by fusing GFP nanobodies with TurboID.
• Employed endogenous GFP fusions to target specific proteins for labeling within the whole animal, avoiding transgenesis and overexpression.

Main Results:

• TurboID successfully identified interactors for both stable (SPD-5) and dynamic (PLK-1) centrosomal proteins.
• The indirect GFP nanobody-TurboID method enabled tissue-specific identification of protein interactors.
• Identified homologs of conserved centriolar components, Cep97 and BLD10/Cep135, in various somatic tissues of C. elegans.
• Found that Cep97 and BLD10/Cep135 are not expressed in C. elegans early embryos.

Conclusions:

• TurboID is effective for centrosome proximity labeling in C. elegans, overcoming limitations of previous methods.
• The indirect GFP nanobody-TurboID approach provides a powerful tool for tissue-specific interactome studies in vivo.
• The discovery of Cep97 and BLD10/Cep135 in somatic tissues, but not embryos, highlights tissue-specific variations in centrosome composition and explains their previous elusiveness.
• This work expands the toolkit for C. elegans research and facilitates future studies on centrosome diversity.