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.
Highlights
The understanding of the biological functions of a protein requires detailed knowledge of the molecules with which it interacts
We have developed BLITZ (Biotin Labelling In Tagged Zebrafish): a modular system for in vivo proteomic mapping (Figure 6)
BLITZ does not require extensive molecular biology steps to produce numerous expression constructs, or laborious embryonic manipulation. It instead relies on simple crossing of a TurboID-dGBP line with an existing GFP-tagged fish line of choice; a plethora of such lines currently exist in stock centres globally and, with the advent of nuclease directed genome editing, this number is rapidly increasing
Summary
The understanding of the biological functions of a protein requires detailed knowledge of the molecules with which it interacts. Instead of directly fusing the biotin ligase to a POI, we developed a modular system for GFPdirected proteomic mapping by combining BioID with a GFP-binding nanobody (HamersCasterman et al, 1993; Rothbauer et al, 2008; Tang et al, 2015; Ariotti et al, 2015a) This system couples the power of the BioID system with the ability to use existing GFP-tagged transgenic zebrafish lines for proteomic mapping between different tissues and/or different proteins. We demonstrate the application of this system in screening for proteins associated with the caveolar cast proteins, Cavin and Cavin, in differentiated skeletal muscle which has, to date, been difficult to achieve in culture These analyses reveal proteins and pathways that are both overlapping and specific to Cavin and Cavin
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