Proximity labeling reveals novel interactomes in live Drosophila tissue

作者：Katelynn M. Mannix, Rebecca M. Starble, Ronit S. Kaufman, Lynn Cooley · 发表于：Development · 年份：2019 · DOI：10.1242/dev.176644 · 被引用次数：55 · 研究领域：Biotin and Related Studies、Cellular transport and secretion、Click Chemistry and Applications

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 high-confidence 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 enzyme-catalyzed proximity labeling for protein interactome analysis in live tissue and expand our understanding of RC biology.