Proximity labeling reveals novel interactomes in live Drosophila tissue

Abstract

Gametogenesis is dependent on intercellular communication facilitated by stable intercellular bridges connecting developing germ cells. During Drosophila oogenesis, intercellular bridges (referred to as ring canals; RCs) have a dynamic actin cytoskeleton that drives their expansion to a diameter of 10 μm. Although multiple proteins have been identified as components of RCs, we lack a basic understanding of how RC proteins interact together to form and regulate the RC cytoskeleton. Thus, here, we optimized a procedure for proximity-dependent biotinylation in live tissue using the APEX enzyme to interrogate the RC interactome. APEX was fused to four different RC components (RC-APEX baits) and 55 unique highconfidence prey were identified. The RC-APEX baits produced almost entirely distinct interactomes that included both known RC proteins and uncharacterized proteins. A proximity ligation assay was used to validate close-proximity interactions between the RC-APEX baits and their respective prey. Furthermore, an RNA interference screen revealed functional roles for several high-confidence prey genes in RC biology. These findings highlight the utility of enzymecatalyzed proximity labeling for protein interactome analysis in live tissue and expand our understanding of RC biology.

Proximity‐dependent labeling methods for proteomic profiling in living cells: An update

“…As biotin-phenoxyl radicals are not membrane-permeable, APEX is excellent for proteomic profiling of membrane-enclosed subcellular compartments, such as the mitochondria Hung et al, 2014;Rhee et al, 2013) and autophagosomes (Le Guerroue et al, 2017). Nevertheless, APEX is not limited to membrane-enclosed organelles, and has been used successfully to map proteins in the cilia (Kohli et al, 2017;Mick et al, 2015), SGs (Markmiller et al, 2018), mitochondria-ER contact points (Cho et al, 2017;Hung et al, 2017), Drosophila ring canals (Mannix, Starble, Kaufman, & Cooley, 2019), mitochondrial nucleoid (Han et al, 2017), bacterial-host inclusion membrane (Olson et al, 2019), lipid droplets (Bersuker et al, 2017), and lysosome-RNA granule contact points (Liao et al, 2019). APEX also provides a good tool for identification of protein-protein interactions.…”

Proximity Ligation Assay for Detecting Protein‐Protein Interactions and Protein Modifications in Cells and Tissues in Situ

“…Scale bars, 10 μm. Starble, Kaufman, & Cooley, 2019;Mendez & Banerjee, 2017;Ruffmann et al, 2018;Sartori et al, 2019;Taura, Fernández-Dueñas, & Ciruela, 2015). By combining antigen detection via antibody with PCR amplification, interactions that would otherwise be difficult to detect can be visualized and quantified while eliminating background signals.…”

“…5). The use of PLA in mouse, Drosophila, and hu-man/clinical tissue has been applied in multiple studies to investigate protein-protein interactions (for examples, see Bagchi, Fredriksson, & Wallén-Mackenzie, 2015;Doebele et al, 2015;Harmon et al, 2013;Mannix, Hegazy et al Figure 3 Use of PLA to detect specific post-translational protein modifications in situ. EGFR is stabilized at the plasma membrane in part by the process of neddylation, the addition of Nedd8 moieties to the C-terminus.…”

HtsRC-mediated accumulation of F-actin regulates ring canal size during Drosophila melanogaster oogenesis

“…We previously identified HtsRC in two independent mass spectrometry approaches designed to identify ring canal-associated proteins (Hudson et al 2019;Mannix et al 2019). Based on our previous work showing that HtsRC is cleaved from the Ovhts polyprotein (Petrella et al 2007), we searched our MS/MS data for peptides resulting from semi-tryptic cleavage, reasoning that an N-terminal peptide beginning at the Ovhts cleavage site could be identified in this way.…”

“…Mass spectrometry data from Hudson et al (2019) and Mannix et al (2019) were searched for semi-tryptic peptides with up to two missed cleavages allowed. Briefly, mass spectrometry data were searched at MS Bioworks using Mascot (Matrix Science); searches were conducted against the UniProt Drosophila melanogaster database with common laboratory contaminants added as well as custom protein sequences for HtsRC that included known SNPs.…”