In vivoproteomic mapping through GFP-directed proximity-dependent biotin labelling in zebrafish

Abstract

Protein interaction networks are crucial for complex cellular processes. However, the elucidation of protein interactions occurring within highly specialised cells and tissues is challenging. Here we describe the development, and application, of a new method for proximity-dependent biotin labelling in whole zebrafish. Using a conditionally stabilised GFP-binding nanobody to target a biotin ligase to GFP-labelled proteins of interest, we show tissue-specific proteomic profiling using existing GFP-tagged transgenic zebrafish lines. We demonstrate the applicability of this approach, termed BLITZ (Biotin Labelling In Tagged Zebrafish), in diverse cell types such as neurons and vascular endothelial cells. We applied this methodology to identify interactors of caveolar coat protein, cavins, in skeletal muscle. Using this system, we defined specific interaction networks within in vivo muscle cells for the closely related but functionally distinct Cavin4 and Cavin1 proteins.

Identification of SSX2IP/Msd1 as a Wtip-binding partner by targeted proximity biotinylation

“…Importantly, we identified SSX2IP using both BirA-GBP/GFP-WtipN and WtipN-BirA, increasing our confidence in the result. Thus, our findings and a similar report using zebrafish embryos (Xiong et al, 2021) indicate that TPB represents a versatile approach for protein interaction studies of various GFP-tagged proteins.…”

A toolbox for efficient proximity-dependent biotinylation in zebrafish embryos

“…Xiong et al (32) used a conditionally stabilized GFP-nanobody fused to TurboID to target proximal labeling to GFP tagged proteins in muscle, while Pronobis et al (26) used BioID2 targeted cardiomyocytes to investigate proteome changes during cardiac regeneration in adult fish. Both these studies showed promise for investigating proximal interactors in vivo in the zebrafish, and the Xiong et al, manuscript is particularly interesting in that it permits use of existing green fluorescent protein fusion transgenics directly after a cross with a universal GFP-nanobody expressing line (32). Our manuscript differs from these two studies in a number of ways.…”

“…The generation of molecularly evolved variants of the E. coli BirA enzyme, called TurboID and miniTurbo, that display 15 to 30 times the labeling efficiency of BirA* has been a major breakthrough in enabling more efficient proximity-dependent biotinylation in different in vivo models, including C. elegans (9), D. melanogaster (9,27), A. thaliana (28), N. benthamiana (28,29), S. pombe (30), and M. musculus (31). Using transgenics in zebrafish, Xiong et al recently demonstrated the applicability TurboID through the fusion of a GFP nanobody to TurboID: crosses to transgenic lines expressing fusions of proteins of interest to the Clover green fluorescent protein enabled labeling and recovery of proximal interactors to cavin proteins (32). Yet, proximity-dependent biotinylation approaches are still in their infancy, and more thorough optimization and benchmarking of the approaches is needed; it has also not been clearly determined whether the more rapid and scalable mRNA injection systems lead to high-quality proximal proteomes.…”

A Toolbox for Efficient Proximity-Dependent Biotinylation in Zebrafish Embryos

“…Two recent publications have already demonstrated the use of proximity-dependent labeling in zebrafish. Xiong et al (32) used a conditionally stabilized GFP-nanobody fused to TurboID to target proximal labeling to GFP tagged proteins in muscle, while Pronobis et al (26) used BioID2 targeted cardiomyocytes to investigate proteome changes during cardiac regeneration in adult fish. Both these studies showed promise for investigating proximal interactors in vivo in the zebrafish, and the Xiong et al, manuscript is particularly interesting in that it permits use of existing green fluorescent protein fusion transgenics directly after a cross with a universal GFP-nanobody expressing line (32).…”

“…The generation of molecularly evolved variants of the E. coli BirA enzyme, called TurboID and miniTurbo, that display 15 to 30 times the labeling efficiency of BirA* has been a major breakthrough in enabling more efficient proximity-dependent biotinylation in different in vivo models, including C. elegans (9), D. melanogaster (9, 27), A. thaliana (28), N. benthamiana (28,29), S. pombe (30), and M. musculus (31). Using transgenics in zebrafish, Xiong et al recently demonstrated the applicability TurboID through the fusion of a GFP nanobody to TurboID: crosses to transgenic lines expressing fusions of proteins of interest to the Clover green fluorescent protein enabled labeling and recovery of proximal interactors to cavin proteins (32). Yet, proximity-dependent biotinylation approaches are still in their infancy, and more thorough optimization and benchmarking of the approaches is needed; it has also not been clearly determined whether the more rapid and scalable mRNA injection systems lead to high-quality proximal proteomes.…”