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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??m. 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.

SUBMITTER: Mannix KM

PROVIDER: S-EPMC6679357 | biostudies-literature | 2019 Jul

REPOSITORIES: biostudies-literature

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Proximity labeling reveals novel interactomes in live <i>Drosophila</i> tissue.

Mannix Katelynn M KM Starble Rebecca M RM Kaufman Ronit S RS Cooley Lynn L

Development (Cambridge, England) 20190718 14

Gametogenesis is dependent on intercellular communication facilitated by stable intercellular bridges connecting developing germ cells. During <i>Drosophila</i> 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, h ...[more]

PMID: 31208963

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Proximity labeling reveals novel interactomes in live Drosophila tissue.

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Proximity labeling reveals novel interactomes in live <i>Drosophila</i> tissue.

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