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
-
- 9,527
- views
-
- 1,085
- downloads
-
- 47
- 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
-
- Cell Biology
- Plant Biology
Autophagy-related gene 6 (ATG6) plays a crucial role in plant immunity. Nonexpressor of pathogenesis-related genes 1 (NPR1) acts as a signaling hub of plant immunity. However, the relationship between ATG6 and NPR1 is unclear. Here, we find that ATG6 directly interacts with NPR1. ATG6 overexpression significantly increased nuclear accumulation of NPR1. Furthermore, we demonstrate that ATG6 increases NPR1 protein levels and improves its stability. Interestingly, ATG6 promotes the formation of SINCs (SA-induced NPR1 condensates)-like condensates. Additionally, ATG6 and NPR1 synergistically promote the expression of pathogenesis-related genes. Further results showed that silencing ATG6 in NPR1-GFP exacerbates Pst DC3000/avrRps4 infection, while double overexpression of ATG6 and NPR1 synergistically inhibits Pst DC3000/avrRps4 infection. In summary, our findings unveil an interplay of NPR1 with ATG6 and elucidate important molecular mechanisms for enhancing plant immunity.
-
- Cell Biology
Prostaglandin E2 (PGE2) is an endogenous inhibitor of glucose-stimulated insulin secretion (GSIS) and plays an important role in pancreatic β-cell dysfunction in type 2 diabetes mellitus (T2DM). This study aimed to explore the underlying mechanism by which PGE2 inhibits GSIS. Our results showed that PGE2 inhibited Kv2.2 channels via increasing PKA activity in HEK293T cells overexpressed with Kv2.2 channels. Point mutation analysis demonstrated that S448 residue was responsible for the PKA-dependent modulation of Kv2.2. Furthermore, the inhibitory effect of PGE2 on Kv2.2 was blocked by EP2/4 receptor antagonists, while mimicked by EP2/4 receptor agonists. The immune fluorescence results showed that EP1–4 receptors are expressed in both mouse and human β-cells. In INS-1(832/13) β-cells, PGE2 inhibited voltage-gated potassium currents and electrical activity through EP2/4 receptors and Kv2.2 channels. Knockdown of Kcnb2 reduced the action potential firing frequency and alleviated the inhibition of PGE2 on GSIS in INS-1(832/13) β-cells. PGE2 impaired glucose tolerance in wild-type mice but did not alter glucose tolerance in Kcnb2 knockout mice. Knockout of Kcnb2 reduced electrical activity, GSIS and abrogated the inhibition of PGE2 on GSIS in mouse islets. In conclusion, we have demonstrated that PGE2 inhibits GSIS in pancreatic β-cells through the EP2/4-Kv2.2 signaling pathway. The findings highlight the significant role of Kv2.2 channels in the regulation of β-cell repetitive firing and insulin secretion, and contribute to the understanding of the molecular basis of β-cell dysfunction in diabetes.
