In vivo proteomic mapping through GFP-directed proximity-dependent biotin labelling in zebrafish
- Institute for Molecular Bioscience, University of Queensland, Australia
- QIMR Berghofer Medical Research Institute, Australia
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.
Data availability
All data generated or analysed during this study are included in the manuscript and supplementary files. Source data files have been provided for Figures 1, 2, 4 and 5
Article and author information
Author details
Funding
National Health and Medical Research Council (569542)
- Robert G Parton
National Health and Medical Research Council (1045092)
- Robert G Parton
National Health and Medical Research Council (APP1044041)
- Robert G Parton
National Health and Medical Research Council (APP1099251)
- Robert G Parton
- Thomas E Hall
Australian Research Council (DP200102559)
- Robert G Parton
- Thomas E Hall
Australian Research Council Centre of Excellence in Convergent Bio-Nano Science and Technology (CE140100036)
- Robert G Parton
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
Ethics
Animal experimentation: This study was approved by Institutional Biosafety Committee, Office of the Gene Technology Regulator, Australian Government Department of Health, and Molecular Biosciences Animal Ethics Committees, the University of Queensland. The ethics approval numbers are IMB/271/19/BREED and IMB/326/17. IBC/OGTR approval number are IBC/1080/IMB/2017.
Copyright
© 2021, Xiong et al.
This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.
Metrics
-
- 9,672
- views
-
- 1,089
- downloads
-
- 50
- citations
Views, downloads and citations are aggregated across all versions of this paper published by eLife.
Download links
A two-part list of links to download the article, or parts of the article, in various formats.
Downloads (link to download the article as PDF)
Open citations (links to open the citations from this article in various online reference manager services)
Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)
In vivo proteomic mapping through GFP-directed proximity-dependent biotin labelling in zebrafish
eLife 10:e64631.
https://doi.org/10.7554/eLife.64631
Further reading
-
- Cancer Biology
- Cell Biology
DNA base lesions, such as incorporation of uracil into DNA or base mismatches, can be mutagenic and toxic to replicating cells. To discover factors in repair of genomic uracil, we performed a CRISPR knockout screen in the presence of floxuridine, a chemotherapeutic agent that incorporates uracil and fluorouracil into DNA. We identified known factors, such as uracil DNA N-glycosylase (UNG), and unknown factors, such as the N6-adenosine methyltransferase, METTL3, as required to overcome floxuridine-driven cytotoxicity. Visualized with immunofluorescence, the product of METTL3 activity, N6-methyladenosine, formed nuclear foci in cells treated with floxuridine. The observed N6-methyladenosine was embedded in DNA, called 6mA, and these results were confirmed using an orthogonal approach, liquid chromatography coupled to tandem mass spectrometry. METTL3 and 6mA were required for repair of lesions driven by additional base-damaging agents, including raltitrexed, gemcitabine, and hydroxyurea. Our results establish a role for METTL3 and 6mA in promoting genome stability in mammalian cells, especially in response to base damage.
-
- Cell Biology
Dynamic interactions between gut mucosal cells and the external environment are essential to maintain gut homeostasis. Enterochromaffin (EC) cells transduce both chemical and mechanical signals and produce 5-hydroxytryptamine to mediate disparate physiological responses. However, the molecular and cellular basis for functional diversity of ECs remains to be adequately defined. Here, we integrated single-cell transcriptomics with spatial image analysis to identify 14 EC clusters that are topographically organized along the gut. Subtypes predicted to be sensitive to the chemical environment and mechanical forces were identified that express distinct transcription factors and hormones. A Piezo2+ population in the distal colon was endowed with a distinctive neuronal signature. Using a combination of genetic, chemogenetic, and pharmacological approaches, we demonstrated Piezo2+ ECs are required for normal colon motility. Our study constructs a molecular map for ECs and offers a framework for deconvoluting EC cells with pleiotropic functions.
