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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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Project description:Toxoplasma gondii is a ubiquitous, intracellular parasite that envelops its parasitophorous vacuole with a protein-laden membrane (PVM). The PVM is critical for interactions with the infected host cell, such as nutrient transport and immune defense. Only a few parasite and host proteins have so far been identified on the host-cytosolic side of the Toxoplasma PVM. We report here the use of human foreskin fibroblasts expressing the proximity-labeling enzyme miniTurbo, fused to a domain that targets it to this face of the PVM, in combination with quantitative proteomics to specifically identify proteins present at this interface. Out of numerous human and parasite proteins with candidate PVM localization, we validate three parasite proteins (TGGT1_269950 [GRA61], TGGT1_215360 [GRA62], and TGGT1_217530 [GRA63]) and four new host proteins (PDCD6IP/ALIX, PDCD6, CC2D1A, and MOSPD2) as localized to the PVM in infected human cells through immunofluorescence microscopy. These results significantly expand our knowledge of proteins present at the Toxoplasma PVM and, given that three of the validated host proteins are components of the ESCRT (endosomal sorting complexes required for transport) machinery, they further suggest that novel biology is operating at this crucial host-pathogen interface. IMPORTANCE Toxoplasma is an intracellular pathogen which resides and replicates inside a membrane-bound vacuole in infected cells. This vacuole is modified by both parasite and host proteins which participate in a variety of host-parasite interactions at this interface, including nutrient exchange, effector transport, and immune modulation. Only a small number of parasite and host proteins present at the vacuolar membrane and exposed to the host cytosol have thus far been identified. Here, we report the identification of several novel parasite and host proteins present at the vacuolar membrane using enzyme-catalyzed proximity-labeling, significantly increasing our knowledge of the molecular players present and novel biology occurring at this crucial interface.

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Proximity labeling reveals novel interactomes in live Drosophila tissue.

Publications

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

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