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bioRxiv · 10.1101/542795

Proximity labeling reveals novel interactomes in live Drosophila tissue

Mannix, K. M.·Starble, R. M.·Kaufman, R. S.·Cooley, L.
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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) have a dynamic actin cytoskeleton that drives their expansion to a diameter of 10m. While multiple proteins have been identified as components of ring canals (RCs), we lack a basic understanding of how RC proteins interact together to form and regulate the RC cytoskeleton. 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 preys were identified. The RC-APEX baits produced almost entirely distinct interactomes that included both known RC proteins as well as uncharacterized proteins. The proximity ligation assay was used to validate close-proximity interactions between the RC-APEX baits and their respective preys. Further, an RNAi 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.

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Mannix, K. M., Starble, R. M., Kaufman, R. S., Cooley, L.. 2019-02-06. Proximity labeling reveals novel interactomes in live Drosophila tissue. https://doi.org/10.1101/542795

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