TECHNIQUES AND RESOURCES RESEARCH ARTICLE

Proximity labeling reveals novel interactomes in live DrosophilatissueKatelynn M. Mannix1, Rebecca M. Starble1, Ronit S. Kaufman1 and Lynn Cooley1,2,3,*

ABSTRACTGametogenesis is dependent on intercellular communicationfacilitated by stable intercellular bridges connecting developinggerm cells. During Drosophila oogenesis, intercellular bridges(referred to as ring canals; RCs) have a dynamic actin cytoskeletonthat drives their expansion to a diameter of 10 μm. Although multipleproteins have been identified as components of RCs, we lack a basicunderstanding of how RC proteins interact together to form andregulate the RC cytoskeleton. Thus, here, we optimized a procedurefor proximity-dependent biotinylation in live tissue using the APEXenzyme to interrogate the RC interactome. APEX was fused to fourdifferent RC components (RC-APEX baits) and 55 unique high-confidence prey were identified. The RC-APEX baits producedalmost entirely distinct interactomes that included both known RCproteins and uncharacterized proteins. A proximity ligation assay wasused to validate close-proximity interactions between the RC-APEXbaits and their respective prey. Furthermore, an RNA interferencescreen revealed functional roles for several high-confidence preygenes in RC biology. These findings highlight the utility of enzyme-catalyzed proximity labeling for protein interactome analysis in livetissue and expand our understanding of RC biology.

KEYWORDS:Drosophila oogenesis, Ring canal, Actin cytoskeleton,Proximity labeling, APEX, Protein-protein interaction, Massspectrometry

INTRODUCTIONRing canals (RCs) are intercellular bridges connecting developinggermline cells during both male and female gametogenesis.Unlike other characterized RCs, Drosophila female germline RCsaccumulate an extensive F-actin cytoskeleton that drives theircircumferential expansion during development from a ∼1 μm-diameter arrested cleavage furrow to an impressive 10 μm-diametertunnel. Genetic analyses have led to the identification of key proteinsthat are involved in the RC structure (for a recent review, seeYamashita, 2018). Interestingly, proper RC development requires theordered addition and/or activity of key proteins (Cooley, 1998;Haglund et al., 2011; Robinson et al., 1994), highlighting thedynamic and complex nature of the RC interactome. Various RCproteins are also regulated through post-translational modifications,with ubiquitination and phosphorylation being the most well-

characterized modifications at RCs to date (Hamada-Kawaguchiet al., 2015; Hudson et al., 2019; Kelso et al., 2002; Kline et al., 2018;Morawe et al., 2011; Yamamoto et al., 2013).

Although it is evident that many RC proteins are regulated inspatially restricted and temporally defined contexts, we still have anincomplete understanding of how RCs are formed and regulatedduring development. Given their insoluble nature and sizeheterogeneity, classical biochemical purification and fractionationapproaches that rely on abundant starting material to isolate andanalyze RC proteins in native conditions are not practical. However,proximity labeling approaches have recently been developed (forreviews, see Chen and Perrimon, 2017; Kim and Roux, 2016) inwhich an enzyme is fused to a protein or targeted to a subcellularcompartment of interest where it can generate reactive ‘handle’molecules or ‘tags’ (usually biotin based) that covalently interact withproximal proteins in live cells. The resulting biotin-tagged proteinscan then be purified by conventional methods and identified by massspectrometry (MS). Importantly, these new techniques allow for theidentification of proteomes and interactomes within the nativeenvironment of the cell. Additionally, the use of biotin tags makesthe interrogation of insoluble cellular compartments possible becausedenaturing conditions can be used to solubilize, capture and identifypreviously inaccessible proteins owing to the strength of biotin-streptavidin noncovalent binding (Kd≈10-15 M; Green, 1975).

The two most common classes of enzyme used for proximitylabeling approaches are a bacterial biotin ligase (BirA) andperoxidase-based enzymes [horseradish peroxidase (HRP) andascorbate peroxidase (APEX)]. BirA (Roux et al., 2012) and itsnewer, optimized variants (Branon et al., 2018; Kim et al., 2016) havebeen most commonly used for ‘BioID’ studies of protein-proteininteractions (PPIs), whereas HRP- and APEX-based applicationshave been used almost exclusively to identify subcellular organelleproteomes (for a more comprehensive review, see Kim and Roux,2016). BioID uses membrane-permeable biotin for the substrate, butthe relatively low enzymatic activity of BirA requires labelingreaction times of hours to days and an optimal temperature of 37°C,which is not practical for use in vivo in flies and worms. By contrast,APEX uses a less membrane-permeable substrate, biotin-phenol, butits high enzymatic activity allows for short labeling reaction times(seconds to minutes) at a wider range of temperatures. However, themembrane impermeability of biotin-phenol has posed a significanttechnical challenge for use of APEX in live tissue.

Here, we report the application of APEX-mediated proximitylabeling to the study of PPIs in the highly regulated but largelyinsoluble F-actin cytoskeleton ofDrosophila female RCs. We fusedAPEX to known RC proteins (‘RC-APEX baits’) to probe theirinteraction partners and identified high-confidence interactors(‘prey’) either unique to each RC-APEX bait or common tomultiple RC-APEX baits. We used a proximity ligation assay andgenetic analysis to validate the high-confidence prey. This workdemonstrates that APEX-mediated biotinylation can be used toReceived 6 February 2019; Accepted 23 May 2019

1Department of Genetics, Yale University School of Medicine, New Haven,CT 06520, USA. 2Department of Cell Biology, Yale University School of Medicine,New Haven, CT 06520, USA. 3Department of Molecular, Cellular andDevelopmental Biology, Yale University, New Haven, CT 06520, USA.

*Author for correspondence (lynn.cooley@yale.edu)

K.M.M., 0000-0003-4541-5050; R.M.S., 0000-0002-7724-0761; L.C., 0000-0003-4665-1258

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uncover specific PPIs within an intact cytoskeletal structure inliving cells.

RESULTSUse of APEX to identify ring canal protein interactomesin live tissueTo interrogate RC protein interactomes, we made constructs inwhich APEX was fused to the RC proteins Pavarotti (Pav), Hu li taishao (Hts)-RC and Kelch, and a substrate-trapping Kelch construct,KREP (Fig. 1A,B), and generated transgenic flies expressing thesefusion proteins (RC-APEX baits): Pav::APEX, HtsRC::APEX,APEX::Kelch and APEX::KREP (Fig. 1B).Pav is a kinesin-like protein that is a component of all characterized

germline RCs as well as Drosophila somatic cell RCs (Adams et al.,1998; Airoldi et al., 2011; Minestrini et al., 2002). Pav and its knownbinding partner Tumbleweed (Tum), a Rac GTPase-activating protein(RacGAP), constitute the centralspindlin complex as a heterodimerictetramer (Adams et al., 1998; Tao et al., 2016). HtsRC is a RC-specific protein required for recruiting the RC actin cytoskeleton(Hudson et al., 2019; Petrella et al., 2007). Kelch is the substrateadaptor component of a Cullin3-RING ubiquitin ligase (CRL3) thattargets HtsRC (Hudson and Cooley, 2010; Hudson et al., 2015,2019). Given that the KREP protein comprises only the substrate-targeting domain of Kelch, it cannot bind to Cullin3 and, hence,cannot ubiquitinate its substrate, HtsRC, which leads to a dominant-negative kelch-like RC phenotype (Hudson and Cooley, 2010;Fig. 1A).

Pav localizes to the outer rim of RCs (Minestrini et al., 2002; Ongand Tan, 2010), whereas HtsRC and Kelch localize to the RCF-actin-rich inner rim (Robinson et al., 1994). Knowing that theseRC proteins have discrete ‘sublocalizations’ within the RC as wellas unique functions, our goal was to identify the local interactomesof each RC-APEX bait protein within the RC using APEX-mediated proximity biotinylation as the basis for subsequentproteomic analysis (see Fig. 1C for general methods workflowand proteomic approach).

Localization and expression of RC-APEX bait proteinsWe matched the expression of the RC-APEX baits as closely aspossible to their endogenous levels to minimize mislocalization thatcould lead to a high background and false-positive results (Hunget al., 2016; Varnaite and MacNeill, 2016). For Pav::APEX, theAPEX-coding region was fused to the 3′-end of pav in the context ofa bacterial artificial chromosome (BAC) encompassing the pavgene; previous C-terminal protein fusions to Pav did not perturb itsfunction (Goshima and Vale, 2005). To generate HtsRC::APEX, theAPEX-coding region was fused to the 3′-end of the ovhts cDNAunder control of the otu promoter, which closely matches theexpression levels of the endogenous ovhts promoter (Petrella et al.,2007). APEX::kelch and APEX::KREP were put under UASpcontrol and expressed using mat-GAL4 driver, which has beenshown previously to be an appropriate GAL4 driver for kelch andKREP constructs (Hudson and Cooley, 2010; Hudson et al., 2015,2019). We also made a control construct expressing unlocalizedAPEX by fusing Green Fluorescence Protein (GFP)-BindingProtein (GBP) (Rothbauer et al., 2008) to APEX under UASpcontrol. Epitope tags [FLAG, hemagglutinin (HA), or V5] wereincluded in all five constructs.

Immunofluorescence microscopy revealed that the RC-APEX baitconstructs localized properly to RCs (Fig. 2A-D and Fig. S1A-D),and the GBP::APEX control was present throughout the cytoplasm ofnurse cells (Fig. 2E-E′ and Fig. S1E-E′). As expected, the APEX::KREP protein induced kelch-like RCs (Fig. 2D″, yellow-boxedinset). Of note, Pav::APEX, HtsRC::APEX and APEX::Kelch allrescued in their respective null mutant backgrounds (data not shown),demonstrating that the RC-APEX fusion constructs were functional.All experiments with APEX::Kelch were done in a kelch mutantbackground.

We directly compared expression of the RC-APEX constructs withtheir endogenous counterparts by western blot analysis (Fig. 2G-K).Pav::APEX (Fig. 2G) and HtsRC (Fig. 2H) were expressed atcomparable levels to endogenous Pav and HtsRC. APEX::Kelchprotein expression was slightly higher than that of endogenous Kelch(Fig. 2I). We also compared expression of different RC-APEXconstructs relative to each other using epitope tag antibodies. GBP::APEX expression driven by mat-GAL4 was notably higher comparedwith Pav::APEX (Fig. 2J). Detection of HtsRC::APEX, APEX::Kelchand APEX::KREP proteins showed that APEX::KREP was the mosthighly expressed of the three (Fig. 2K), which is consistent with thehigh expression previously observed with other KREP-derivedconstructs (Hudson and Cooley, 2010). Overall, expression of theRC-APEX baits was comparable to endogenous levels.

Biotinylation of ring canal proteins by RC-APEX baits andstreptavidin purificationMost previous studies using APEX have been in cultured cells thatare sufficiently permeable to the biotin-phenol APEX substrate aftera 30-min incubation period. The sole Drosophila APEX study(Chen et al., 2015) used dissected Drosophila imaginal discs,

Fig. 1. General workflow used to identify RC protein interactomes withAPEX. (A) Fluorescence images showing localization of indicated proteins atRCs. Pav was visualized byGFP fluorescence of a (BAC) pav::GFP transgene.HtsRC and Kelch were visualized with anti-HtsRC and anti-Kelch antibodies,respectively. KREP was expressed as UASp-APEX::V5::KREP driven bymat-GAL4 and visualized by anti-V5 immunofluorescence. F-actin was visualizedby TRITC-Phalloidin. (B) Scheme of transgenic APEX fusion constructsgenerated for this study. Cartoons were made based on known structuralmodels of Pav, Kelch and KREP, with APEX positioned according to where itwas fused. No structural data are available for HtsRC. (C) (Left) Exampleworkflow of optimized APEX labeling in live ovary tissue with HtsRC::APEXconstruct. (Right) Overview of streptavidin purification and MS methods usedto identify and validate high-confidence prey. Scale bar: 2 μm.

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Fig. 2. RC-APEX bait constructs localized to RCs and were expressed at appropriate levels. (A-F) Stage 9 or 10 egg chambers expressingvarious RC-APEX baits were stained with TRITC-Phalloidin (A″-F″) and epitope tags (A′-F′) to confirm proper RC-APEX bait construct localization to RCs.(G-K) Ovary lysates expressing indicated RC-APEX fusion baits were analyzed by western blotting to assess RC-APEX bait expression levels. (I) Levels ofAPEX::Kelch, expressed in a kelchmutant background, were comparable with endogenous Kelch. (J)mat-GAL4-drivenGBP::APEX had higher expression than(BAC) pav::APEX. (K) mat-GAL4-driven APEX::KREP was the most abundantly expressed RC-APEX bait construct. Scale bar: 50 μm.

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salivary glands and larval muscles, all of which were thin enough toallow biotin-phenol entry during the 30-min incubation. However,the egg chamber is within a muscle sheath and basement membrane,and the germline cells are surrounded by a layer of somatic folliclecells. Our initial experiments using the standard APEX labelingprotocol developed by the Ting lab (Hung et al., 2016; Rhee et al.,2013) for use in cultured cells were unsuccessful. Suspecting abiotin-phenol permeability issue, we pretreated live egg chamberswith a small amount of detergent, which resulted in robust biotinlabeling of RCs (Fig. S2).After optimizing the biotin-phenol labeling protocol (Fig. 1C, left),

we were able to achieve consistent and robust RC biotinylation after a3-min incubation in biotin-phenol, followed by 30 s with H2O2. Afterquenching the labeling reaction, we used streptavidin-conjugatedantibodies to visualize biotinylation by immunofluorescence. TheRC-APEX fusions specifically biotinylated RCs in all egg chambers,including stage 6 or 7 (Fig. 3) and stage 9 or 10 (Fig. S3). Asexpected, Pav::APEX also biotinylated follicle cell RCs (Fig. 3A′,yellow arrows), and GBP::APEX showed cytoplasmic biotin staining(Fig. 3E′).Although treatment with detergent did not affect the structure,

stability or apparent composition of RCs (Robinson and Cooley,1997b), detergent treatment could pose a problem for studyingcellular structures that are more labile. Given that APEX wasinitially developed for use after fixation (Martell et al., 2012), wetested whether APEX remained active in egg chambers afterformaldehyde fixation (Fig. S4). APEX retained activity even after a10-min fixation in 2% paraformaldehyde (Fig. S4B,B″), suggestingthat APEX-mediated biotinylation will be broadly useful in fixedtissues. Nonetheless, to avoid potential fixation-induced crosslinksthat could obscure peptide identification, we did not use fixative forthe following experiments.To capture biotinylated proteins using streptavidin beads, we

needed to verify whether they were solubilized after lysis and presentin the soluble fraction after centrifugation and lysate clarification.RCs are known to remain intact after lysis with non-ionic detergents,such as TX-100 (Greenbaum et al., 2007; Robinson and Cooley,1997b). Therefore, we lysed egg chambers in harsh, denaturing lysisbuffer containing 8 M urea using a motorized spinning pestle, and theclarified lysate was incubated with magnetic streptavidin beads (seeMaterials and Methods for more specific details). The magneticstreptavidin beads were able to capture the biotinylated proteins(Fig. 3G), as shown by the depletion of the streptavidin-HRP signal inthe unbound fractions and the strong signal of biotinylated proteins inthe eluates. Interestingly, the no-APEX control w1118 sample alsoshowed a smear of biotinylated proteins by western blot (Fig. 3G),possibly because of the presence of endogenously biotinylatedproteins or proteins that were biotinylated by other endogenousperoxidases. In any event, the biotin signal was stronger in the eluatesfor the APEX samples compared with the no-APEX w1118 control.Before proceeding withMS analysis, we checked for the presence

or absence of known RC proteins in the eluates by western blotting(Fig. 3H-K). The results were encouraging. We observed that onlythe Pav::APEX sample showed enrichment over controls for a Pavprotein species (Fig. 3H, Pav::APEX band), which is consistentwith Pav being dimeric (Minestrini et al., 2002; Sommi et al., 2010;Tao et al., 2016). Similarly, only the APEX::Kelch sample showedenrichment for a Kelch protein species (Fig. 3I, APEX::Kelchband), which is also consistent with Kelch dimerization via its BTBdomain (Hudson and Cooley, 2010; Hudson et al., 2015; Robinsonand Cooley, 1997a). APEX::Kelch was expressed in a kelch mutantbackground, which is why endogenous Kelch was absent from that

lane. A faint band corresponding to endogenous Kelch wasdetectable in the other eluates, probably because of nonspecificbinding to the beads. We found that HtsRC was significantlyenriched in the APEX::KREP eluate (Fig. 3J), which is consistentwith it being the CRL3Kelch substrate (Hudson et al., 2019). We alsodetected HtsRC and HtsRC::APEX in the HtsRC::APEX eluate,which suggests that HtsRC is able to dimerize. Finally, we checkedthe eluate samples for the presence of Filamin, a key RC protein.Interestingly, Filamin was only detectable in the APEX::KREPsample (Fig. 3K), suggesting that Filamin is also in close proximityto the Kelch substrate-targeting KREP domain. These results gaveus proof of principle that the RC-APEX baits biotinylate uniquesubsets of proteins, which are probably their native interactionpartners at the RC.

Analysis of purified samples by mass spectrometryTo prepare samples for proteomic analysis, we subjected eggchambers from 100 flies (≈200 ovaries) of each genotype to ourbiotinylation labeling protocol. Two biological replicates were usedfor each of the six samples (Fig. 1B). Samples were subjected toliquid chromatography (LC)-MS/MS by MS Bioworks. Spectralcounts (SpC) were used as a semiquantitative measure of relativeprotein abundance. See Methods for more detailed information onthe MS workflow and proteomic data analysis.

As a quality control check, we assessed the correlation of spectralcounts of each identified protein across the biological replicates foreach RC-APEX bait (Fig. S5A). Linear regression analysis showed astrong correlation across replicates for each RC-APEX bait, asindicated by the large coefficient of determination (Fig. S5A). Toassess how each RC-APEX bait sample differed in total proteinidentification coverage, we generated density plots of the natural log(ln) of the normalized spectral abundance factor (NSAF) values forall proteins identified byMS (Fig. S5B). The density plots for all fourconstructs overlapped closely, indicating a similar extent of coverageof spectral counts between the samples, and eliminating any concernsof instrumentation- and sampling-based artifacts or significantdeviations in sample complexity or content.

Identification and characterization of RC-APEX high-confidence preyTo establish a list of high-confidence prey for each RC-APEX baitfrom our proteomic data, we used Significance Analysis ofINTeractome (SAINT) (Choi et al., 2011; Morris et al., 2014), acommon tool used to parse ‘true interactors’ from ‘false interactors’in affinity purification-MS data sets. We used a SAINT score cut-offof ≥0.8 (corresponding to a 5% false discovery rate), which resultedin 55 high-confidence prey (Fig. 4A, Table S1). Pav::APEX hadthree high-confidence prey: Pav, Tum and CG43333. HtsRC::APEX had five: Muscle-specific protein 300 kDa (Msp300),Filamin, HtsRC, CG43333 and Chorion protein c at 7F (Cp7Fc).APEX::Kelch and APEX::KREP had nine and 43 high-confidenceprey, respectively. Only a few prey (listed in the Venn diagram inFig. 4A) were shared between the RC-APEX baits.

We used Cytoscape to generate a network map of the RC-APEXbaits and prey (Fig. 4B). Known RC proteins were among the high-confidence prey (solid line outlining prey gene circle in Fig. 4B).Consistent with our western blot analysis of RC-APEX bait eluates(Fig. 3), Pav::APEX identified Pav; HtsRC::APEX and APEX::KREP identified HtsRC; APEX::Kelch identified Kelch; andAPEX::KREP identified Filamin. Interestingly, Filamin was alsoidentified by HtsRC::APEX, which shows the sensitivity achievedby MS analysis. We mined the Molecular Interaction Search Tool

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(MIST; Hu et al., 2018), and imported known interactions into theCytoscape network map (dotted lines connecting prey genes inFig. 4B). In addition, we searched publicly available expression dataat FlyBase (Thurmond et al., 2018) and found that 51 of the 55

identified proteins are expressed in the Drosophila ovary. Takentogether, these data indicate that each RC-APEX bait identifiedunique interactomes that include previously established interactors,new candidate interactors and known RC proteins.

Fig. 3. See next page for legend.

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Of the 55 RC-APEX prey genes identified, five were previouslyknown to be involved in RC biology (prey genes with solid outlinesin Fig. 4B): pav, tum, hts, cheerio (cher) and kel. That Pav::APEX

identified Pav and Tum was reassuring, given that Pav dimerizes(Minestrini et al., 2002; Sommi et al., 2010) and also interacts withTum (Adams et al., 1998; Tao et al., 2016). Kelchwas the top prey forAPEX::Kelch, consistent with Kelch dimerizing via its BTB domain(Hudson and Cooley, 2010; Hudson et al., 2015; Robinson andCooley, 1997a). HtsRC was identified by the CRL3Kelch substrate-trapping APEX::KREP construct, supporting the conclusion that theCRL3Kelch substrate is HtsRC (Hudson et al., 2019). HtsRC::APEXidentified HtsRC and Filamin as prey. Although HtsRC and Filaminare known RC proteins that genetically interact (Robinson et al.,1997; Sokol and Cooley, 1999), this is the first evidence of a physicalinteraction between them. Additionally, the finding that Filamin wasa top prey for APEX::KREP suggested that Filamin, HtsRC andKREP are all in close proximity at kelch-like RCs.

For a more global and quantitative visualization of the preyidentified by each RC-APEX bait, we generated dot plots using theProHits-viz tool (Knight et al., 2017) (Fig. 5A-D). The color of eachprey ‘dot’ is dependent on the NSAF log2(FoldChange) for each baitcompared with the control samples (see key in Fig. 5). The size ofeach dot is dependent on its relative abundance between baitsamples, meaning that the maximum-sized dot for each prey is

Fig. 3. RC-APEX baits biotinylate distinct proteins in live egg chambers.(A-F) Ovaries expressing indicated RC-APEX baits were permeabilized andbiotinylated, then egg chambers were fixed and stained with TRITC-Phalloidin(A″-F″) and streptavidin-AF488 (A′-F′) to visualize F-actin and biotin.Representative images are shown of stage 6 or 7 egg chambers. Note thespecific biotin signal at RCs for the RC-APEX fusions (A′-D′), the unlocalizedcytoplasmic biotin signal for GBP::APEX (E′) and the absence of signal in the no-APEXcontrol (F′). (A′) Yellowarrows denote biotinylated follicle cell RCs in (BAC)pav::GFP. (G-K) APEX-labeled ovaries were lysed in denaturing conditions,biotinylated proteins were captured by magnetic streptavidin beads, and eluateswere analyzed by western blotting. (G) Inputs, unbound fractions and eluateswere analyzed by western blotting with streptavidin-HRP to confirm thatbiotinylated proteins were present. (H-K)Western blot analysiswas performed onthe APEX eluates (labeled on top of lanes) to check for known RC proteins.(H) Pav::APEX protein was abundant in the Pav::APEX eluate. (I) APEX::Kelchwas enriched in the APEX::Kelch eluate, whereas a background endogenousKelch protein band could be detected in all other samples. (J) HtsRC::APEX andendogenous HtsRC were present in the HtsRC::APEX sample, whereasendogenousHtsRCwas highly enriched in the APEX::KREPsample. (K) Filaminwas observed only in the APEX::KREP sample. Scale bar: 50 μm.

Fig. 4. Proteomic analysis reveals RC-APEX baits have unique interactomes that include known RC proteins. (A) Venn diagram showing the extent ofoverlap of the 55 high-confidence prey identified by the RC-APEX baits. High-confidence prey had a SAINT score ≥0.8, using SAINTexpress analysis.(B) Network map generated in Cytoscape of RC-APEX baits, indicated by colored squares, and their respective prey. The size of the prey gene circle wasdependent on its abundance in terms of spectral counts compared with control samples [log2(FoldChange]). Known RC proteins are outlined in black. Dottedlines indicate previously established interactions mined from MIST (Hu et al., 2018).

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Fig. 5. Proteomic analysis reveals that RC-APEX baits identified unique prey proteins. (A-D) Dot plots of high-confidence prey identified by Pav::APEX (A),HtsRC::APEX (B), APEX::Kelch (C) and APEX::KREP (D). Dot plots were generated using ProHits-viz (Knight et al., 2017), and high-confidence prey(SAINT score ≥0.8) are outlined in yellow. Each RC-APEX bait identified mostly distinct high-confidence prey. For example, in the APEX::Kelch column (C),only CG9336was identified by another RC-APEX bait, APEX::KREP. (E-H) Volcano plots for each RC-APEX bait showing statistical significance [-log10(P-value)]and average relative abundance compared with controls [NSAF log2(FoldChange)] of identified proteins across replicate experiments. Notable high-confidenceprey are listed and plotted as gray dots. For APEX::Kelch (G) and APEX::KREP (H), the y-axis is broken to allow room for the top-most row of protein preythat had an undefined P-value (e.g. Ef-Ts and Cyp6d4 in APEX::Kelch).

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allocated to the largest SpC value within that bait and the other dotsare scaled proportionately. High-confidence prey within each baithave a yellow outline (see key in Fig. 5). This visualization revealedhow each RC-APEX bait identified unique sets of prey proteins. Forexample, APEX::Kelch identified nine high-confidence prey (thirdcolumn of ‘dots’ all outlined yellow in Fig. 5C) and only one ofthose prey (CG9336) was shared with another RC-APEX bait(APEX::KREP) (yellow outline around CG9336 dot in fourthcolumn for APEX::KREP in Fig. 5C).To visualize the quantitative proteomic data relative to statistical

significance, we generated volcano plots for each RC-APEX bait(Fig. 5E-H). For each protein identified by MS, a P-value wascalculated for its spectral counts within the biological replicates foreach RC-APEX bait and control samples. For all identified proteins,the -log10(P-value) was plotted against the NSAF log2(FoldChange)compared with controls for each RC-APEX bait sample. Proteinsthat showed a high fold-change compared with controls appear onthe right side of the plot, whereas proteins that are statisticallysignificant compared with controls (in terms of SpC acrossreplicates) appear towards the top of the plot. Unsurprisingly,proteins in the top right of the plot generally corresponded to ourhigh-confidence prey (gray dots in Fig. 5E-H). For example, the fivehigh-confidence prey identified by HtsRC::APEX were the soleoccupants of the upper-right quadrant (gray-labeled dots in Fig. 5F).

Validation of RC-APEX high-confidence prey by proximityligation assayGiven that APEX-mediated biotinylation is proximity dependent,the top prey are potential direct interactors or binding partners of thebaits. Confirming physical interactions between these protein pairsin their native environments by conventional methods, such as co-immunoprecipitation, was essentially impossible because of the lowsolubility of the RC cytoskeleton. Therefore, we turned to theDuolink proximity ligation assay (PLA) to test whether RC-APEXbaits were in close proximity (<40 nm) to their prey proteins in eggchambers. We were able to test five prey using available antibodiesand GFP-tagged stocks. Initial PLA experiments using fixed whole-egg chambers were unsuccessful. Suspecting a penetration issue, weperformed the PLA in 20-μm ovary cryosections and succeeded indetecting PLA signals (see Methods for more details).To test whether Pav dimerization could be detected using the

proximity ligation assay, we used egg chambers expressing the pav::APEX::FLAG BAC transgene. The presence of the FLAG epitope inthe fusion protein allowed us to use FLAG antibodies generated in twospecies (mouse and rabbit) to target the same FLAG antigen. The PLAassay confirmed that Pav interacts with itself, as indicated byfluorescent foci throughout egg chambers and at RCs (Fig. 6A, toppanel) compared with the negative control sample in which one of theantibodies was omitted (Fig. 6A, bottom panel). We detected aninteraction between Pav and Tum using egg chambers expressing aGFP::Tum protein trap and the pav::APEX::FLAG BAC transgene.Fluorescence microscopy showed a striking accumulation of PLA focithroughout the entire egg chamber and at the RCs (Fig. 6B, top panel),whereas the negative control was devoid of PLA signal (Fig. 6B,bottom panel). Quantification of PLA foci showed that there weresignificantly more foci per egg chamber unit area in the {Pav}→Pavand {Pav}→Tum samples compared with their respective negativecontrols (Fig. 6G, left graph). These results indicate that the proximityligation assay can detect true PPIs at their native environment in tissue,and validates Pav and Tum as Pav::APEX prey.We also tested the previously unknown potential interaction

between HtsRC and Filamin in egg chambers expressing a cher::

GFP protein trap using rabbit anti-GFP and mouse anti-HtsRCantibodies. Compared with the negative control, there were abundantPLA foci throughout the egg chamber as well as a strong signal atRCs (Fig. 6C). Similarly, we tested potential oligomerization ofHtsRC in egg chambers expressing the otu-ovhts::V5::APEXtransgene, which allowed us to use mouse and rabbit antibodiestargeting the same V5 antigen. We detected PLA foci throughout theegg chamber and at RCs (Fig. 6D). Quantification of PLA foci peregg chamber unit area showed that the {HtsRC}→Filamin and{HtsRC}→HtsRC samples had significantly more foci than theirrespective controls (Fig. 6G, right graph). These results validate ourproteomic results, and support the conclusion that HtsRC candimerize or oligomerize as well as interact directly with Filamin, bothin the cytosol and at RCs.

HtsRC was a high-confidence prey for the substrate-trappingAPEX::KREP construct, which was expected based on previouswork (Hudson et al., 2019).We validated this result in egg chambersexpressing APEX::V5::KREP using mouse anti-HtsRC antibodiesand rabbit anti-V5 antibodies. As expected, we observed strongPLA signal throughout the cytoplasm and at RCs (Fig. 6E) withessentially no foci in the negative control (Fig. 6E, bottom panel;quantified in Fig. 6G, right). These results provide proof of principlethat APEX fusion constructs can be used to identify substrates of E3ubiquitin ligases in live tissue.

Finally, we tested an unexpected interaction between SUMO(encoded by the smt3 gene) and Kelch using Drosophila SUMOrabbit antibodies (Abgent) and mouse anti-Kelch antibodies.Excitingly, we observed PLA foci throughout the cytoplasm ofegg chambers in the experimental sample but not the negativecontrol (Fig. 6F; quantification in Fig. 6G, right). The resultssuggest that Kelch is in close proximity to the SUMO protein in thecytoplasm of egg chambers, but not at RCs. Future experiments areneeded to test whether Kelch is covalently SUMOylated orinteracting with SUMO, perhaps through its predicted SUMOinteraction motif (Zhao et al., 2014).

As an additional control, we used the PLA to test interactionsbetween RC-APEX bait proteins and proteins that were not identifiedas prey. Specifically, we tested for interactions between Pav andKelch (Fig. S6A), Kelch and Filamin (Fig. S6B), Pav and HtsRC(Fig. S6C), and HtsRC and Kelch (Fig. S6D). All interactions testedwere negative, and quantification revealed no significant enrichmentof PLA foci in the experimental samples compared with theirrespective negative controls (Fig. S6E). These results were allconsistent with our RC-APEX bait and prey protein pairs and supportour conclusion that the PLA was faithfully recapitulating real PPIs.

Identification of novel ring canal regulators through an RNAiscreen of RC-APEX prey genesTo test genetically whether the RC-APEX preys were involved inRC biology, we performed an RNA interference (RNAi) screenusing stocks from the Transgenic RNAi Project (TRiP). Wescreened 33 prey gene small hairpin (sh)RNA lines using themat-GAL4 driver for germline-specific expression. Of the 33 genes,19 had a noticeable phenotype; eight phenotypes were RC specificand 11 had general oogenesis defects (Fig. 7A). Fourteen genesshowed no phenotype, which could be because: (1) the RNAi linedid not work; (2) the gene functions in early oogenesis before themat-GAL4 driver is expressed; or (3) knocking down the gene hasno deleterious effects on the egg chamber.

Six of the eight RC-specific phenotypes [barentsz (btz), combgap(cg), Protein on ecdysone puffs (Pep), polychaetoid ( pyd), Splicingfactor 2 (SF2) and smallminded (smid)] were significantly enlarged

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Fig. 6. In situ proximity ligation assay confirms close-proximity interactions betweenRC-APEX bait proteins and respective prey. (A-F) Proximity ligationassays were performed on cryosectioned ovary tissues of the indicated genotypes to test for close-proximity interactions between different protein pairs,listed in white text in the top left of each panel (with brackets around the RC-APEX bait protein and an arrow pointing to the prey protein). A positive PPI wasindicated by the presence of PLA signal (left panels), which was visualized by fluorescent probes that bind to the PLA DNA product. Antibodies used for eachreaction are indicated on the left, and the bottom panels served as negative controls because one antibody was omitted. Boxed insets contain RCs showingF-actin, GFP::Tum, or Filamin::GFP (FLN::GFP) patterns, as indicated in the panel labels. The protein pairs tested exhibited a distinct RC signal, with theexception of the {Kelch}→SUMO (F) interaction, which was mostly dispersed throughout the cytoplasm. (G) Positive PLA foci in each egg chamber imagedwere counted in FIJI using thresholding and particle analysis, and graphed as a function of egg chamber unit area. Student’s t-test was used to compare thenumber of PLA foci per unit area in experimental samples versus controls. *P≤0.1; **P≤0.05; ***P≤0.001; ****P≤0.0001. Scale bars: 20 μm (main panel) and5 μm (insets).

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RCs (Fig. 7B-H; RC diameter quantified in Fig. 7J). RNAi-mediated knockdown of Pep also led to abnormal F-actin structuresas well as deformed and collapsed RCs (represented by red, yellow,and blue arrows in Fig. 7E′, respectively). Knockdown of pyd alsoresulted in deformed RCs (yellow arrows in Fig. 7F′), whereas smidknockdown caused abnormal excess cortical F-actin surroundingRCs in stage 10 egg chambers (red arrows in Fig. 7H). Spt5knockdown led to egg chamber degeneration in mid-to-late-stagedegg chambers (Fig. 7I) and collapsed RCs (blue arrows in Fig. 7I′).Finally, RNAi-mediated knockdown of smt3 (the DrosophilaSUMO gene) led to egg chamber arrest as well as deformed andcollapsed RCs (Fig. 7K, yellow and blue arrows). To examine a

possible role for SUMO in RC biology, we stained egg chamberswith anti-SUMO antibodies and found occasional RC localization(Fig. 7L, boxed insets).

Interestingly, none of the eight genes with RC-specificphenotypes were known previously to have functions related toRCs. We were particularly surprised to see that five of these genes(btz, cg, Pep, SF2 and Spt5) had established functions involvingDNA and/or RNA binding, processing or regulation, suggestingpossible ‘new’ roles in RC biology. In summary, this preliminaryRNAi screen revealed that prey genes are involved in RC biology,which provides additional support that the RC-APEX baitsidentified physiologically relevant protein interactors.

Fig. 7. RNAi screen reveals that preygenes are involved in RC biology.(A) Summary of results from RNAiscreening of 33 high-confidence prey[with each prey color coded to indicateits respective RC-APEX bait(s)]. pav,hts, cher and kel are included for 37genes in total. Of the 33 prey genesscreened, 19 had a phenotype, eight ofwhich were RC specific. (B-I) Eggchambers of indicated genotypes werestained with TRITC-Phalloidin andHtsRC (B′-I′) to reveal RC size andmorphology. Red arrows indicateabnormal F-actin structures. Yellowand blue arrows indicate deformed andcollapsed RCs, respectively. (J) RCdiameters of the indicated genotypeswere measured in FIJI using HtsRCstaining as a RC marker, and violinplots were used to represent the data.Thick-dashed white lines show medianRC diameter, and thinner upper andlower white lines denote the upper andlower quartiles, respectively. One-wayANOVA was used to compare themean RC diameter between the RNAilines and w1118 control. ***P≤0.0001;****P≤0.00001. (K) RNAi-mediatedknockdown of smt3 caused collapsedand deformed RCs, marked by blueand yellow arrows, respectively.(L) Some RCs were positive for SUMOstaining (inset). Scale bars: 20 μm in L,25 μm in K and 50 μm in B-I.

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DISCUSSIONAPEX-mediated proximity labeling can be used to identifyprotein-protein interactions in live tissueSince its introduction in 2013, APEX-mediated localizedbiotinylation has been used successfully in several studies toidentify organellar or cellular proteomes, with most of the workdone in cultured cells. Three studies reported APEX-mediatedlabeling in live tissue. Two of these studies were performed inCaenorhabditis elegans in a mutant background that compromisedcuticle integrity, which allowed for successful delivery of the biotin-phenol substrate into the worm tissue (Reinke et al., 2017a,b). Thethird labeled the mitochondrial proteome in dissected Drosophilaimaginal discs, salivary glands and larval muscles (Chen et al., 2015).These tissues are relatively thin, which permits sufficient entry ofbiotin-phenol into the cells without notable intervention. Here, weadapted the use of APEX-mediated proximity labeling to thickertissue by using digitonin as a means to sufficiently permeabilize cellmembranes and allow biotin-phenol substrate entry. Our approachcould be adapted to other organisms and tissues, live or formaldehydefixed, to enable the more widespread use of APEX in vivo.Of note, the Ting lab recently engineered more catalytically active

mutants of the BirA enzyme used in BioID approaches, referred to asTurboID and miniTurbo (Branon et al., 2018). Essentially, thesemutants combine the catalytic efficiency of APEX (i.e. a 10-minlabeling reaction time) with the ease-of-use of BioID (i.e.straightforward biotin delivery to cells or tissues of interest).However, when TurboID was used in flies and worms, biotinsupplementation took hours or days, which limits its potential forcapturing more dynamic processes that require fine temporal controland resolution of the labeling reaction. In these cases, our APEXprotocol could be used with dissected tissue to achieve robustbiotinylation on a faster (seconds to minutes) timescale. Furtheradaptation and optimization of these proximity labeling approacheswill undoubtedly open the door to proteomic experiments in livingorganisms.

RC-APEX baits identified known binding partners as well aspotential new interactorsWe set out to test the feasibility of using APEX in vivo to identifyPPIs as opposed to whole-organelle or whole-cell proteomes.Although several RC proteins have been characterized, a fullunderstanding of F-actin regulation in RCs requires more completeinformation about PPIs in the structure. Given that biotinylation ofnearby proteins is dependent on their proximity to the APEXenzyme, we envisioned that the RC-APEX bait proteins couldidentify new binding partners. Our results were promising. We wereespecially excited that the two top prey of Pav::APEX were Pav andTum, because identifying the Centralspindlin complex (Tao et al.,2016) provided a powerful positive control and a compelling proofof principle that the RC-APEX baits were capturing real proteininteractors of the respective RC bait proteins. Our results alsoindicated that APEX fused to different domains within proteins canidentify domain-specific interactors. APEX fused to the Kelch Nterminus, which is the site of Kelch homodimerization throughits BTB domain (Canning et al., 2013; Errington et al., 2012; Jiand Privé, 2013), labeled distinct proteins compared with APEX::KREP, which contains just the substrate-binding domain of Kelch.Kelch itself was a top prey for APEX::Kelch, whereas HtsRC was atop prey for APEX::KREP. Surprisingly, APEX::Kelch did notidentify Cullin3, a known binding partner of the Kelch BTB domain(Canning et al., 2013; Errington et al., 2012; Ji and Privé, 2013; Xuet al., 2003), as a top prey. This might be because of the transient

nature of theKelch-Cullin3 interaction or the relatively low abundanceof Cullin3 present at RCs compared with its strong cytoplasmiclocalization (Hudson and Cooley, 2010; Hudson et al., 2015).

Identification of E3 ubiquitin ligase substrates is challengingbecause substrates are short-lived and their interactions with theligase are transient. Those challenges, coupled with the insolubilityof the RC actin cytoskeleton, made identifying the CRL3Kelch RCsubstrate particularly hard. We recently identified HtsRC as thesubstrate of CRL3Kelch with other methods (Hudson et al., 2019),and of the 40 prey identified by APEX::KREP in this study, onlyone, HtsRC, contains the known Kelch-binding motif (PEAEQ)(Schumacher et al., 2014). Thus, our work suggests that APEX is avaluable new tool for identifying E3 ubiquitin ligase substrates,particularly in previously inaccessible contexts like in live tissue, orwithin insoluble cellular compartments, such as the cytoskeleton. Infact, ours is the first study of its kind to use APEX in vivo to identifyan E3 ubiquitin ligase substrate. The advent of substrate-trappingconstructs that can be fused to APEX and expressed in modelorganisms could lead to future breakthroughs for substrateidentification in tissue-specific and developmental contexts,which will help move the E3 ubiquitin ligase field intophysiologically relevant models.

The APEX::KREP bait identified a surprisingly large number ofhigh-confidence prey proteins compared with the other three baits.This could be because of its abundance both in the cytoplasm andRCs, leading to more efficient biotinylation of proteins. Given thatexpression of KREP leads to a dominant-negative phenotype ofkelch-like RCs, it is also possible that protein complexes werecaught or trapped in the RC cytoskeleton, which could havefacilitated their biotinylation. Interestingly, many APEX::KREPprey genes are involved in RNA binding or processing as well asribonucleoprotein complexes. RNPs are transported alongmicrotubules, through RCs from the nurse cells into the oocyte(Mische et al., 2007); thus, they might indeed have been trapped.However, RNAi-mediated knockdown of genes for several of theseRNP-associated APEX::KREP prey genes led to RC-specificphenotypes, suggesting a role for mRNA regulation and RNPcomplexes in RC biology. In any event, the smaller number ofproteins identified with HtsRC, Pav and Kelch baits compared withKREP suggests that we succeeded in finding specific interactors forthese proteins within intact, morphologically normal RCs.

Proximity labeling revealed new insights into the ring canalsubstructureRCs are massive structures that undergo dramatic and dynamicstructural and possibly compositional changes during egg chamberdevelopment. Given the complex nature of the RC cytoskeleton (e.g.F-actin-dependent membrane and RC expansion, actin recruitment,F-actin crosslinking and bundling, and cytoskeleton disassembly inthe RC lumen), it wouldmake sense that different subcomplexes existin RCs to carry out these processes. Evidence already exists fordistinct domains within the RC. HtsRC, Kelch and F-actin localize tothe inner rim of RCs (Robinson et al., 1994), Pav localizes to the outerrim (Minestrini et al., 2002; Ong and Tan, 2010), and Filamin ispresent throughout the entire RC cytoskeleton. However, a morefinely resolved picture of RC protein complex localization isemerging from our localized biotinylation results.

Our data showed that different RC-APEX bait constructsidentified unique subsets of RC proteins suggesting that there aresubcomplexes within the RC that orchestrate mechanical functions,such as nucleating actin filament assembly at the membrane toexpand the RC diameter or controlling HtsRC levels to maintain an

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open RC lumen. Of particular interest is the finding that HtsRC::APEX identified Filamin and HtsRC as top prey. Given thatlocalization of HtsRC to RCs is genetically dependent on the chergene (Robinson et al., 1997; Sokol and Cooley, 1999), we can nowspeculate that Filamin directly recruits HtsRC to the RC, whereHtsRC functions to recruit the abundant F-actin cytoskeleton(Hudson et al., 2019). This also suggests that HtsRC has the abilityto dimerize or oligomerize, possibly through its C-terminal coiled-coil domain (predicted using Phyre2; Kelley et al., 2015). It will beinteresting to test whether the oligomerization status of HtsRCdiffers based on its localization in the cytoplasm or at RCs andwhether this affects its actin-regulating activity.It is also possible that many of the RC-APEX bait-prey interactions

we identified occurred in the cytoplasm rather than the RC. Theproximity ligation assay showed RC-specific signals for most of theinteractions tested, but there were also positive foci present in thecytoplasm. Cytoplasmic interactions could be important forassembling protein complexes destined for RCs; for example,HtsRC could bind to Filamin in the cytoplasm before localizing toRCs. We expect that additional analysis of the RC-APEX prey willlead to new insights into RC formation, maintenance and growthduring development.

MATERIALS AND METHODSDrosophila husbandryDrosophila were maintained at 25°C on standard fly food medium. Beforeovary dissections, females were fattened on wet yeast paste overnight at25°C. See Table S2 for a detailed list of the fly stocks used in this study.

Molecular cloning and Drosophila transgenesis(BAC) pav::HA::APEX2::FLAGAPEX2 was recombineered into a BAC containing the pav gene at its Cterminus (BAC ID 322-102N3) to create pav::HA::APEX2::FLAG. ThisBAC contains the entire pav locus on a 21-kb genomic fragment(chr3L:4,229,286…4,250,505, FlyBase release 6). Briefly, we used a two-step BAC recombineering protocol to first insert a Kanamycin resistancecassette (Wang et al., 2006), which was subsequently replaced by HA::APEX2::FLAG through streptomycin selection. The HA::APEX2::FLAGsequence was made through PCR amplification of APEX2 from pcDNA3-APEX2-NES (Addgene Plasmid #49386) with primers designed to add anN-terminal HA epitope tag sequence and a C-terminal FLAG epitope tagsequence. The APEX2 sequence contained a C-terminal nuclear export signal(NES), which was incorporated in an attempt to keep Pav::APEX localizedexclusively to RCs. The final plasmid was injected into BL#24872 into theattP3B site on chr2L at Rainbow Transgenic Flies, Inc.

otu-ovhts::V5::APEX1The V5::APEX1 coding sequence was amplified from pcDNA3-mito-V5::APEX1 (Rhee et al., 2013; Addgene Plasmid #42607) using primersdesigned to create a V5::APEX1 fragment flanked by 3′ XhoI and 5′ NotIrestriction sites. pCOH-ovhts::GFP (Petrella et al., 2007) was digested withXhoI and NotI to excise GFP and the V5::APEX1 fragment was ligated intothe plasmid in-frame and in place of GFP. Transgenic flies were generatedvia P-element-mediated insertion at GenetiVision.

UASp-APEX2::V5::kelchThe APEX2 coding sequence was amplified from pcDNA3-APEX2-NES(Addgene Plasmid #49386) and the entire kelch coding sequence wasamplified with primers designed to contain an N-terminal V5 tag sequence.Through overlap-extension PCR, the APEX2::V5::kelch coding sequencewas assembled with flanking attB1 and attB2 sites and recombined first intopDONR201 and then into pPW-attB (Table S2) in BP Clonase II and LRClonase II Gateway recombination reactions, respectively. The pPW-APEX2::V5::kelch plasmid was injected into a strain carrying the attP2phiC31 landing site on chr3L at Rainbow Transgenic Flies.

UASp-APEX2::V5::KREPThe APEX2 coding sequence was amplified from pcDNA3-APEX2-NES(Addgene Plasmid #49386) and the KREP coding sequence of kelch wasamplified with primers designed to contain an N-terminal V5 tag sequence.Through overlap-extension PCR, the APEX2::V5::KREP coding sequencewas assembled with flanking attB1 and attB2 sites and recombined first intopDONR201 and then into pPW-attB (Table S2) in BP Clonase II and LRClonase II Gateway recombination reactions, respectively. The pPW-APEX2::V5::KREP plasmid was injected into a strain carrying the attP2phiC31 landing site on chr3L at Rainbow Transgenic Flies.

UASp-GBP::FLAG::APEX2The GBP (Rothbauer et al., 2008) coding sequence was amplified withprimers designed to contain 3′ and 5′ SfiI restriction sites. The FLAG::APEX2sequence was amplified from pcDNA3-APEX2-NES (Addgene Plasmid#49386) with primers designed to contain a 3′ SfiI site and a 5′ BamHI site.The fragments were digested with their respective restriction enzymes andligated into a pUASp-attB plasmid modified to contain a polylinker sequencedownstream of UASp site and P-element transposons. Transgenic flies weregenerated via P-element-mediated insertion at Rainbow Transgenic Flies.

Tissue fixation, fluorescence microscopy and imagingSee Table S2 for more information regarding the antibodies and reagents usedin this study. Ovaries were dissected in ionically matched Drosophila saline(IMADS) buffer (Singleton and Woodruff, 1994) and fixed for 10 min ineither 6% formaldehyde, 75 mM KCl, 25 mM NaCl, 3 mM MgCl2, and17 mM potassium phosphate, pH 6.8 (Verheyen and Cooley, 1994) or 4%formaldehyde in PBT [PBS with 0.3% Triton X-100 and 0.5% bovine serumalbumin (BSA)]. Fixed tissue was washed in PBT and incubated with theindicated primary antibodies in PBT for ∼1-2 h at room temperature orovernight at 4°C. Following washes with PBT, tissue was incubated withsecondary antibodies conjugated to Alexa Fluor and DAPI in PBT for ∼1-2 hat room temperature. F-actin was labeled with Phalloidin conjugated toTetramethylrhodamine (TRITC). Samples were washed in PBT and mountedon slides in ProLong Diamond (Thermo Fisher Scientific). Samples wereimaged with a Leica SP8 laser scanning confocal microscope using a 40×1.3NA oil-immersion objective lens or a Zeiss Axiovert 200 m equipped witheither a CARV II spinning disc confocal imager or a CrEST X-light spinningdisc system and a Photometrics CoolSNAP HQ2 camera using a 40×1.2 NAwater-immersion objective.

Western blot analysis of ovary lysatesOvary lysates were generated by homogenizing dissected ovaries inSDS-PAGE sample buffer. One ovary equivalent was loaded per lane andseparated by SDS-PAGE. Proteins were transferred to a nitrocellulosemembrane, stained with amido black to visualize total protein, blocked in5% milk in Tris-buffered saline with 0.1% Tween-20 (TBST) for 30 min atroom temperature, and probed with the indicated primary antibodies for∼2 h at room temperature in 5% milk/TBST. After washing the blots threetimes in TBST for 5 min, the blots were incubated with HRP-conjugatedsecondary antibodies for ∼1 h at room temperature followed by enhancedchemiluminescence (ECL) development for 5 min. ACCD camerawas usedfor imaging. See Table S2 for more information on the antibodies used inthis study.

Proteomic analysis of APEX samplesIn vivo biotinylation of proteins using RC-APEX baitsBefore dissection, female flies were fattened overnight on wet yeast paste at25°C in a vial with males. For small-scale pilot experiments or experimentsin which fluorescence microscopy was the final experimental readout,ovaries from 10-20 flies were used for biotin-phenol proximity labeling. Forlarge-scale proteomic experiments, three batches of labeling were performedwith ovaries from 30-35 flies in each batch, yielding a total of 100 flies usedper sample. Ovaries were dissected in 1× PBS, pH 8.0 and gently pipetted upand down ∼ten times to splay open the ovaries. The pH of all solutions waskept above 7.5 to prevent precipitation of the biotin-phenol. Egg chamberswere incubated in 500 μl biotin-phenol labeling solution (1 mM biotin-

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phenol, 0.5% digitonin, 1× PBS pH 8.0) for 3 min with rotation at roomtemperature. To initiate APEX-mediated biotinylation, H2O2 was added to afinal concentration of 0.05 mM and the solution was gently inverted to mixfor 30 s. After 30 s, 10 mM NaN3 was added to quench the reaction. Eggchambers were washed 3× in 500 μl quenching buffer (10 mM NaN3 in 1xPBS, pH 8.0). For every labeling reaction performed, egg chambers werealways fixed and analyzed by fluorescence microscopy to ensure that thelabeling reaction was successful (i.e. that RCs were biotinylated). Standardfixation and fluorescence protocols were used, as described earlier. Tovisualize biotin signal in egg chambers, fixed and permeabilized eggchambers were incubated with streptavidin-AF488 or streptavidin-AF568 ata 1:500 dilution in PBT for ∼1 h at room temperature. For large-scalelabeling reactions, a very small amount of egg chambers was removed afterthe last wash in quenching buffer and analyzed by fluorescence microscopy.Immediately after the last wash, the remaining quenching buffer wasremoved from the egg chambers, and the tissue was frozen at -80°C.

Purification of biotinylated proteins from ovary lysatesLabeled and quenched egg chambers were thawed on ice and the tissue fromthe three batches of labeling was combined into one tube for each sample.Tissue was ground in 100-200 μl urea lysis buffer (8 M urea, 10 mM TrispH 8.0, 50 mM NaPi, 300 mM NaCl, 5 μg/ml each of chymostatin,leupeptin, antipain and pepstatin, 10 mM NaN3, 1 μM bortezomib, 10 mMN-ethylmaleimide, 5 mM Trolox, 10 mM sodium ascorbate) with amechanical homogenizer. After grinding, additional lysis buffer wasadded to a final volume of 1 ml and the lysate was incubated for anadditional 30 min at room temperature to ensure sufficient lysis andsolubilization. The crude lysate was centrifuged at 13,200 rpm (16,100 g)for 10 min at room temperature, and the clarified lysate was transferred to anew tube with 500 μl pre-equilibrated magnetic streptavidin beads andincubated for 1 h at room temperature with rotation. The unbound fractionwas removed from the beads and the beads were washed three times in 1 mlurea wash buffer (2 M urea, 10 mM Tris pH 8.0, 50 mM NaPi, 300 mMNaCl, 5 μg/ml each of chymostatin, leupeptin, antipain, pepstatin,10 mM NaN3, 1 μM bortezomib, 10 mM N-ethylmaleimide, 5 mMTrolox, 10 mM sodium ascorbate). To analyze the purified fraction bywestern blotting, 100 μl bead/wash solution was removed during the lastwash (i.e. 10% of bead fraction). Beads were eluted with vigorous mixing at95°C in 50 μl elution buffer [SDS sample buffer (0.1 M Tris pH 8.0, 4%SDS, 0.01% Bromophenol Blue, 5% β-mercaptoethanol) with 10 mMbiotin]. Following the last wash, the remaining wash solution was removedfrom the rest of the beads and the beads were frozen immediately at -80°C oron dry ice. Beads were shipped on dry ice overnight toMS Bioworks for MSanalysis using their IP-works platform.

Sample preparation for mass spectrometryProteins were eluted from streptavidin beads by incubation with 1.5×lithium dodecyl sulfate (LDS) buffer for 15 min at 100°C. Eluted proteinswere separated from the beads by centrifugation and magnetic separation.Then, 50% of each eluatewas processed by SDS-PAGEwith a 10%Bis-TrisNuPAGE gel using the MES buffer system (Invitrogen). The gel lanes wereexcised, sliced into ten equal-sized fragments, and subjected to in-geltrypsin digestion using a robot (ProGest, DigiLab). For the in-gel trypsindigestion, gel slices were washed with 25 mM ammonium bicarbonatefollowed by acetonitrile, reduced with 10 mM dithiothreitol at 60°C,alkylated with 50 mM iodoacetamide at room temperature, digested withsequencing grade trypsin (Promega) at 37°C for 4 h, and quenched withformic acid. The resultant supernatant was analyzed by MS.

LC-MS/MS data acquisition and processingHalf of each digested samplewas analyzed by nanoLC-MS/MSwith aWatersNanoAcquity HPLC system interfaced to a Thermo Fisher Scientific QExactive. Peptides were loaded on a trapping column and eluted over a 75-μmanalytical column at a flow rate of 350 nl/min. Both columns were packedwith LunaC18 resin (Phenomenex). TheMS instrument was operated in data-dependent mode with the Orbitrap operating at 780,000 FWHM for MS and17,500 FWHM for MS/MS. The 15 most abundant ions were selected for

MS/MS. Data were searched using a local copy of Mascot (Version 2.6.0;Matrix Science) using the UniProt Drosophila melanogaster database withcommon laboratory contaminants added as well as custom protein sequencesfor HtsRC that included known single nucleotide polymorphisms (SNPs) andan N-terminal cleavage site at V693, and the APEX1 and APEX2 proteinsequences. Trypsin was selected as the enzyme and two missed cleavageswere allowed. Cysteine carbamidomethylation was selected as a fixedmodification. Methionine oxidation, N-terminal acetylation, N-terminalpyroglutamylation, asparagine or glutamine deamidation, and tyrosinebiotinylation by biotin-tyramide (chemical formula: C18H23N3O3S) wereselected as variable modifications. A peptide mass tolerance of 10 ppm and afragment mass tolerance of 0.02 Da were used. The resultant Mascot DATfiles were parsed using Scaffold (Version 4.8.4; Proteome Software) forvalidation, filtering, and to create a nonredundant protein list per sample.Experiment-wide grouping with binary peptide-protein weights was selectedas the Protein Grouping Strategy. Peptide and Protein Thresholds were set to95% and 99%, respectively, with a 1 peptide minimum. These settingsresulted in a 0.0% peptide false discovery rate (FDR) and a 0.4% protein FDRas measured byDecoy matches. The Scaffold Samples report was exported asan Excel spreadsheet. Contaminant proteins were removed from thespreadsheet data set. Spectral peptide matches for the Ovhts polyproteinencoded by the ovhts transcript were manually parsed into its twocorresponding protein products: HtsF (AA 1-658) and HtsRC (AA 659-1156). Thus, spectral counts were manually entered into the spreadsheet forthe HtsF and HtsRC proteins across each sample.

Data analysis using SAINTThe Automated Processing of SAINT Templated Layouts (APOSTL)(Kuenzi et al., 2016) tool was used to preprocess the MS data to generatecorresponding data files that were compatible with analysis by SAINTexpress(Teo et al., 2014). The resultant SAINTexpress data set was processed furtherusing APOSTL; namely, normalized spectral abundance factor (NSAF)values were calculated for each prey based on its respective SpC observed ineach bait as well as the log2 fold-change compared with control[log2(FoldChange)].

Interactome network visualization with CytoscapeCytoscape was used to display the ‘interactomes’ of the RC-APEX baits andtheir respective high-confidence prey. RC-APEX baits and their prey geneswere indicated by colored squares and circles, respectively. The size of the preygene circle was scaled based on its average abundance compared with controlsamples [log2(FoldChange) NSAF]. Known RC genes were manually denotedwith a solid outline. The Molecular Interaction Search Tool (MIST) (Hu et al.,2018) was used to search for previously established interactions between allhigh-confidence prey genes, and positive interactions were indicated with adotted line.

Generation of dot plots and heatmaps with ProHits-vizThe ProHits-viz tool (Knight et al., 2017) was used to generate dot plots tovisualize proteomics data for any high-confidence prey across all fourRC-APEX bait samples. The color of each prey ‘dot’ was dependent on theNSAF log2(FoldChange) for each bait compared with the control samples.The size of each dot was dependent on its relative abundance between baitsamples, meaning that the maximum-sized dot for each prey was allocated tothe largest SpC value within that bait and the other dots were scaledproportionately. High-confidence prey within each bait had a yellow outline,corresponding to a SAINT score ≥0.8, whereas prey with a SAINT score<0.8 for that bait had a faint outline.

Generation of volcano plotsVolcano plots were generated for each RC-APEX to visualize thequantitative proteomic data relative to their statistical significance. Foreach protein identified by MS, a P-value was calculated for its spectralcounts within the biological replicates for each RC-APEX bait and controlsamples. For all identified proteins, the -log10(P-value) was plotted againstthe NSAF log2(FoldChange) compared with controls for each RC-APEXbait sample.

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Validation of RC-APEX high-confidence prey using proximityligation assayOvaries from∼50 flies were dissected and fixed in 6% formaldehyde, 75 mMKCl, 25 mM NaCl, 3 mM MgCl2 and 17 mM potassium phosphate, pH 6.8with heptane for 8 min.After washing three times for 10 min in PBT, the tissuewas incubated with 500 μl 30% sucrose solution with gentle rotation for30 min, at which point the ovaries settled to the bottom of the tube. Tissuewasrinsed with fresh sucrose solution and transferred to an Eppendorf tube cap,where it was fully submerged in cold optimal cutting temperature (OCT)compound. After a 1-2 h incubation, the tissue was embedded in a cryoblockmold in OCT by incubating on dry ice for 10 min. Cryoblocks were stored at-80°C until sectioning. Before sectioning, blockswere equilibrated at -20°C for5 min. Then, 20-μm slices were sectioned, transferred to Superfrost Pluscharged slides (Fisher Scientific), and stored at -80°C until future use. For thePLA, theDuolink® In SituRed Starter KitMouse/Rabbit (Sigma-Aldrich) wasused with minor protocol modifications. Slides containing ovary cryosectionswere equilibrated at room temperature for 5 min, then blocked for 1 h in PLAblocking buffer (1× PBS, 0.3%TritonX-100, 1%BSA). AnAqua Hold II penwas used to form hydrophobic barriers around tissue on the slide to contain thesmall volumes of solutions used in this protocol. Slides were placed in ahumidity chamber and tissue was incubated with 40 μl primary antibodysolution and FITC-Phalloidin in PLA blocking buffer for 2 h at roomtemperature. See Table S2 for a list of antibodies and their concentrations usedin this assay. Tissuewaswashed twice for 5 min with PLA blocking buffer andincubated in 40 μl PLA probe solution (8 μl PLUS probe, 8 μl MINUS probe,24 μl antibody diluent) for 1.5 h at 37°C in the humidity chamber. Slides werewashed twice for 5 min in wash buffer A then incubated for 45 min in thehumidity chamber at 37°C in 40 μl ligation solution (8 μl ligation buffer, 31 μlddH2O, 1 μl ligase). Slides were washed twice for 5 min in wash buffer A andincubated for 2 h in the humidity chamber at 37°C in 40 μl amplificationsolution (8 μl amplification buffer, 31.5 μl ddH2O, 0.5 μl polymerase)supplemented with FITC-Phalloidin. Slides were washed twice for 10 minin wash buffer B, rinsed for 1 min with 0.01× wash buffer B, and mounted in30 μl PLA mounting medium overnight in the humidity chamber at 4°C.Slides were imaged on a Leica SP8 laser scanning confocalmicroscope using a40×1.3 NA oil-immersion objective lens and all laser settings were equalbetween each slide and its respective control to allow for quantitativecomparison of PLA foci. Positive PLA foci were counted in FIJI using theAuto-thresholding and Particle Analysis tools, with theminimum area of a fociset to 2 μm2. Egg chamber area was calculated in FIJI using the Freehand andArea Measure tools.

Validation of prey genes by RNAi screeningSee Table S2 for a list of fly stocks used for the RNAi screen. Ovaries weredissected from fattened females, fixed and stained with TRITC-Phalloidinand anti-HtsRC antibodies to check for any abnormalities in egg chamberdevelopment or RC morphology.

AcknowledgementsWe thank Peter McLean for help with the (BAC) pav::APEX transgene. We thankTian Xu (Yale University, Westlake University) for providing access to the LeicaSP8 confocal microscope. We are grateful to Ellen LeMosy (Augusta University) aswell as members of the Weatherbee lab (Yale University) for help with thecryosectioning protocol. We thank Alice Ting for helpful comments and access toprotocols for APEX-mediated biotinylation. We are grateful to Michael Ford and theMS Bioworks team for their services. Stocks obtained from the BloomingtonDrosophila Stock Center (NIH P40OD018537) were used in this study. We thankthe TRiP at Harvard Medical School for providing transgenic RNAi fly stocks usedin this study.

Competing interestsThe authors declare no competing or financial interests.

Author contributionsConceptualization: K.M.M., L.C.; Methodology: K.M.M., R.M.S., R.S.K., L.C.;Investigation: K.M.M., R.M.S., R.S.K., L.C.; Resources: K.M.M., R.M.S., R.S.K.,L.C.; Writing - original draft: K.M.M.; Writing - review & editing: K.M.M., R.M.S.,R.S.K., L.C.; Visualization: K.M.M., R.M.S., L.C.; Supervision: L.C.; Fundingacquisition: L.C.

FundingK.M.M. was aColdSpring Harbor Proteomics Course participant. K.M.M. andR.S.K.were supported in part by a National Institute of General Medical Sciences NationalInstitutes of Health training grant T32 GM007223 and a Gruber Science Fellowship.This work was funded by National Institutes of Health grants R01 GM043301 andRC1 GM091791. Deposited in PMC for release after 12 months.

Data availabilityA Scaffold file containing all proteomics data, including peptide spectral matches(PSM), is available from the Dryad Digital Repository (Mannix et al., 2019): dryad.9p9c594.

Supplementary informationSupplementary information available online athttp://dev.biologists.org/lookup/doi/10.1242/dev.176644.supplemental

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