© 2019. Published by The Company of Biologists Ltd | Development (2019) 146, dev176644. doi:10.1242/dev.176644

TECHNIQUES AND RESOURCES RESEARCH ARTICLE Proximity labeling reveals novel in live Drosophila tissue Katelynn M. Mannix1, Rebecca M. Starble1, Ronit S. Kaufman1 and Lynn Cooley1,2,3,*

ABSTRACT characterized modifications at RCs to date (Hamada-Kawaguchi Gametogenesis is dependent on intercellular communication et al., 2015; Hudson et al., 2019; Kelso et al., 2002; Kline et al., 2018; facilitated by stable intercellular bridges connecting developing Morawe et al., 2011; Yamamoto et al., 2013). germ cells. During Drosophila oogenesis, intercellular bridges Although it is evident that many RC are regulated in (referred to as ring canals; RCs) have a dynamic actin cytoskeleton spatially restricted and temporally defined contexts, we still have an that drives their expansion to a diameter of 10 μm. Although multiple incomplete understanding of how RCs are formed and regulated proteins have been identified as components of RCs, we lack a basic during development. Given their insoluble nature and size understanding of how RC proteins interact together to form and heterogeneity, classical biochemical purification and fractionation regulate the RC cytoskeleton. Thus, here, we optimized a procedure approaches that rely on abundant starting material to isolate and for proximity-dependent in live tissue using the APEX analyze RC proteins in native conditions are not practical. However, enzyme to interrogate the RC . APEX was fused to four proximity labeling approaches have recently been developed (for different RC components (RC-APEX baits) and 55 unique high- reviews, see Chen and Perrimon, 2017; Kim and Roux, 2016) in confidence prey were identified. The RC-APEX baits produced which an enzyme is fused to a or targeted to a subcellular ‘ ’ almost entirely distinct interactomes that included both known RC compartment of interest where it can generate reactive handle ‘ ’ proteins and uncharacterized proteins. A was molecules or tags (usually based) that covalently interact with used to validate close-proximity interactions between the RC-APEX proximal proteins in live cells. The resulting biotin-tagged proteins baits and their respective prey. Furthermore, an RNA interference can then be purified by conventional methods and identified by mass screen revealed functional roles for several high-confidence prey spectrometry (MS). Importantly, these new techniques allow for the genes in RC biology. These findings highlight the utility of enzyme- identification of proteomes and interactomes within the native catalyzed proximity labeling for protein interactome analysis in live environment of the cell. Additionally, the use of biotin tags makes tissue and expand our understanding of RC biology. the interrogation of insoluble cellular compartments possible because denaturing conditions can be used to solubilize, capture and identify KEY WORDS: Drosophila oogenesis, Ring canal, Actin cytoskeleton, previously inaccessible proteins owing to the strength of biotin- -15 Proximity labeling, APEX, Protein-protein interaction, Mass streptavidin noncovalent binding (Kd≈10 M; Green, 1975). spectrometry The two most common classes of enzyme used for proximity labeling approaches are a bacterial biotin ligase (BirA) and INTRODUCTION peroxidase-based enzymes [horseradish peroxidase (HRP) and Ring canals (RCs) are intercellular bridges connecting developing ascorbate peroxidase (APEX)]. BirA (Roux et al., 2012) and its germline cells during both male and female gametogenesis. newer, optimized variants (Branon et al., 2018; Kim et al., 2016) have Unlike other characterized RCs, Drosophila female germline RCs been most commonly used for ‘BioID’ studies of protein-protein accumulate an extensive F-actin cytoskeleton that drives their interactions (PPIs), whereas HRP- and APEX-based applications circumferential expansion during development from a ∼1 μm- have been used almost exclusively to identify subcellular organelle diameter arrested cleavage furrow to an impressive 10 μm-diameter proteomes (for a more comprehensive review, see Kim and Roux, tunnel. Genetic analyses have led to the identification of key proteins 2016). BioID uses membrane-permeable biotin for the substrate, but that are involved in the RC structure (for a recent review, see the relatively low enzymatic activity of BirA requires labeling Yamashita, 2018). Interestingly, proper RC development requires the reaction times of hours to days and an optimal temperature of 37°C, ordered addition and/or activity of key proteins (Cooley, 1998; which is not practical for use in vivo in flies and worms. By contrast, Haglund et al., 2011; Robinson et al., 1994), highlighting the APEX uses a less membrane-permeable substrate, biotin-phenol, but dynamic and complex nature of the RC interactome. Various RC its high enzymatic activity allows for short labeling reaction times proteins are also regulated through post-translational modifications, (seconds to minutes) at a wider range of temperatures. However, the with ubiquitination and phosphorylation being the most well- membrane impermeability of biotin-phenol has posed a significant technical challenge for use of APEX in live tissue.

1Department of Genetics, Yale University School of Medicine, New Haven, Here, we report the application of APEX-mediated proximity CT 06520, USA. 2Department of Cell Biology, Yale University School of Medicine, labeling to the study of PPIs in the highly regulated but largely New Haven, CT 06520, USA. 3Department of Molecular, Cellular and insoluble F-actin cytoskeleton of Drosophila female RCs. We fused Developmental Biology, Yale University, New Haven, CT 06520, USA. APEX to known RC proteins (‘RC-APEX baits’) to probe their *Author for correspondence ([email protected]) interaction partners and identified high-confidence interactors (‘prey’) either unique to each RC-APEX bait or common to K.M.M., 0000-0003-4541-5050; R.M.S., 0000-0002-7724-0761; L.C., 0000- 0003-4665-1258 multiple RC-APEX baits. We used a proximity ligation assay and genetic analysis to validate the high-confidence prey. This work

Received 6 February 2019; Accepted 23 May 2019 demonstrates that APEX-mediated biotinylation can be used to DEVELOPMENT

1 TECHNIQUES AND RESOURCES Development (2019) 146, dev176644. doi:10.1242/dev.176644 uncover specific PPIs within an intact cytoskeletal structure in Pav localizes to the outer rim of RCs (Minestrini et al., 2002; Ong living cells. and Tan, 2010), whereas HtsRC and Kelch localize to the RC F-actin-rich inner rim (Robinson et al., 1994). Knowing that these RESULTS RC proteins have discrete ‘sublocalizations’ within the RC as well Use of APEX to identify ring canal protein interactomes as unique functions, our goal was to identify the local interactomes in live tissue of each RC-APEX bait protein within the RC using APEX- To interrogate RC protein interactomes, we made constructs in mediated proximity biotinylation as the basis for subsequent which APEX was fused to the RC proteins Pavarotti (Pav), Hu li tai proteomic analysis (see Fig. 1C for general methods workflow shao (Hts)-RC and Kelch, and a substrate-trapping Kelch construct, and proteomic approach). KREP (Fig. 1A,B), and generated transgenic flies expressing these fusion proteins (RC-APEX baits): Pav::APEX, HtsRC::APEX, Localization and expression of RC-APEX bait proteins APEX::Kelch and APEX::KREP (Fig. 1B). We matched the expression of the RC-APEX baits as closely as Pav is a kinesin-like protein that is a component of all characterized possible to their endogenous levels to minimize mislocalization that germline RCs as well as Drosophila somatic cell RCs (Adams et al., could lead to a high background and false-positive results (Hung 1998; Airoldi et al., 2011; Minestrini et al., 2002). Pav and its known et al., 2016; Varnaite and MacNeill, 2016). For Pav::APEX, the binding partner Tumbleweed (Tum), a Rac GTPase-activating protein APEX-coding region was fused to the 3′-end of pav in the context of (RacGAP), constitute the centralspindlin complex as a heterodimeric a bacterial artificial chromosome (BAC) encompassing the pav tetramer (Adams et al., 1998; Tao et al., 2016). HtsRC is a RC- gene; previous C-terminal protein fusions to Pav did not perturb its specific protein required for recruiting the RC actin cytoskeleton function (Goshima and Vale, 2005). To generate HtsRC::APEX, the (Hudson et al., 2019; Petrella et al., 2007). Kelch is the substrate APEX-coding region was fused to the 3′-end of the ovhts cDNA adaptor component of a Cullin3-RING ubiquitin ligase (CRL3) that under control of the otu promoter, which closely matches the targets HtsRC (Hudson and Cooley, 2010; Hudson et al., 2015, expression levels of the endogenous ovhts promoter (Petrella et al., 2019). Given that the KREP protein comprises only the substrate- 2007). APEX::kelch and APEX::KREP were put under UASp targeting domain of Kelch, it cannot bind to Cullin3 and, hence, control and expressed using mat-GAL4 driver, which has been cannot ubiquitinate its substrate, HtsRC, which leads to a dominant- shown previously to be an appropriate GAL4 driver for kelch and negative kelch-like RC phenotype (Hudson and Cooley, 2010; KREP constructs (Hudson and Cooley, 2010; Hudson et al., 2015, Fig. 1A). 2019). We also made a control construct expressing unlocalized APEX by fusing Green Fluorescence Protein (GFP)-Binding Protein (GBP) (Rothbauer et al., 2008) to APEX under UASp control. Epitope tags [FLAG, hemagglutinin (HA), or V5] were included in all five constructs. Immunofluorescence microscopy revealed that the RC-APEX bait constructs localized properly to RCs (Fig. 2A-D and Fig. S1A-D), and the GBP::APEX control was present throughout the cytoplasm of nurse cells (Fig. 2E-E′ and Fig. S1E-E′). As expected, the APEX:: KREP protein induced kelch-like RCs (Fig. 2D″, yellow-boxed inset). Of note, Pav::APEX, HtsRC::APEX and APEX::Kelch all rescued 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 mutant background. We directly compared expression of the RC-APEX constructs with their endogenous counterparts by western blot analysis (Fig. 2G-K). Pav::APEX (Fig. 2G) and HtsRC (Fig. 2H) were expressed at comparable levels to endogenous Pav and HtsRC. APEX::Kelch protein expression was slightly higher than that of endogenous Kelch (Fig. 2I). We also compared expression of different RC-APEX constructs relative to each other using epitope tag antibodies. GBP:: APEX expression driven by mat-GAL4 was notably higher compared with Pav::APEX (Fig. 2J). Detection of HtsRC::APEX, APEX::Kelch and APEX::KREP proteins showed that APEX::KREP was the most Fig. 1. General workflow used to identify RC protein interactomes with highly expressed of the three (Fig. 2K), which is consistent with the APEX. (A) Fluorescence images showing localization of indicated proteins at RCs. Pav was visualized by GFP fluorescence of a (BAC) pav::GFP transgene. high expression previously observed with other KREP-derived HtsRC and Kelch were visualized with anti-HtsRC and anti-Kelch antibodies, constructs (Hudson and Cooley, 2010). Overall, expression of the respectively. KREP was expressed as UASp-APEX::V5::KREP driven by mat- RC-APEX baits was comparable to endogenous levels. GAL4 and visualized by anti-V5 immunofluorescence. F-actin was visualized by TRITC-Phalloidin. (B) Scheme of transgenic APEX fusion constructs Biotinylation of ring canal proteins by RC-APEX baits and generated for this study. Cartoons were made based on known structural streptavidin purification models of Pav, Kelch and KREP, with APEX positioned according to where it Most previous studies using APEX have been in cultured cells that was fused. No structural data are available for HtsRC. (C) (Left) Example workflow of optimized APEX labeling in live ovary tissue with HtsRC::APEX are sufficiently permeable to the biotin-phenol APEX substrate after construct. (Right) Overview of streptavidin purification and MS methods used a 30-min incubation period. The sole Drosophila APEX study to identify and validate high-confidence prey. Scale bar: 2 μm. (Chen et al., 2015) used dissected Drosophila imaginal discs, DEVELOPMENT

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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 expressing various 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) Levelsof APEX::Kelch, expressed in a kelch mutant background, were comparable with endogenous Kelch. (J) mat-GAL4-driven GBP::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. DEVELOPMENT

3 TECHNIQUES AND RESOURCES Development (2019) 146, dev176644. doi:10.1242/dev.176644 salivary glands and larval muscles, all of which were thin enough to lane. A faint band corresponding to endogenous Kelch was allow biotin-phenol entry during the 30-min incubation. However, detectable in the other eluates, probably because of nonspecific the egg chamber is within a muscle sheath and basement membrane, binding to the beads. We found that HtsRC was significantly and the germline cells are surrounded by a layer of somatic follicle enriched in the APEX::KREP eluate (Fig. 3J), which is consistent cells. Our initial experiments using the standard APEX labeling with it being the CRL3Kelch substrate (Hudson et al., 2019). We also protocol developed by the Ting lab (Hung et al., 2016; Rhee et al., detected HtsRC and HtsRC::APEX in the HtsRC::APEX eluate, 2013) for use in cultured cells were unsuccessful. Suspecting a which suggests that HtsRC is able to dimerize. Finally, we checked biotin-phenol permeability issue, we pretreated live egg chambers the eluate samples for the presence of Filamin, a key RC protein. with a small amount of detergent, which resulted in robust biotin Interestingly, Filamin was only detectable in the APEX::KREP labeling of RCs (Fig. S2). sample (Fig. 3K), suggesting that Filamin is also in close proximity After optimizing the biotin-phenol labeling protocol (Fig. 1C, left), to the Kelch substrate-targeting KREP domain. These results gave we were able to achieve consistent and robust RC biotinylation after a us proof of principle that the RC-APEX baits biotinylate unique 3-min incubation in biotin-phenol, followed by 30 s with H2O2. After subsets of proteins, which are probably their native interaction quenching the labeling reaction, we used streptavidin-conjugated partners at the RC. antibodies to visualize biotinylation by immunofluorescence. The RC-APEX fusions specifically biotinylated RCs in all egg chambers, Analysis of purified samples by mass spectrometry including stage 6 or 7 (Fig. 3) and stage 9 or 10 (Fig. S3). As To prepare samples for proteomic analysis, we subjected egg expected, Pav::APEX also biotinylated follicle cell RCs (Fig. 3A′, chambers from 100 flies (≈200 ovaries) of each genotype to our yellow arrows), and GBP::APEX showed cytoplasmic biotin staining biotinylation labeling protocol. Two biological replicates were used (Fig. 3E′). for each of the six samples (Fig. 1B). Samples were subjected to Although treatment with detergent did not affect the structure, liquid chromatography (LC)-MS/MS by MS Bioworks. Spectral stability or apparent composition of RCs (Robinson and Cooley, counts (SpC) were used as a semiquantitative measure of relative 1997b), detergent treatment could pose a problem for studying protein abundance. See Methods for more detailed information on cellular structures that are more labile. Given that APEX was the MS workflow and proteomic data analysis. initially developed for use after fixation (Martell et al., 2012), we As a quality control check, we assessed the correlation of spectral tested whether APEX remained active in egg chambers after counts of each identified protein across the biological replicates for formaldehyde fixation (Fig. S4). APEX retained activity even after a each RC-APEX bait (Fig. S5A). Linear regression analysis showed a 10-min fixation in 2% paraformaldehyde (Fig. S4B,B″), suggesting strong correlation across replicates for each RC-APEX bait, as that APEX-mediated biotinylation will be broadly useful in fixed indicated by the large coefficient of determination (Fig. S5A). To tissues. Nonetheless, to avoid potential fixation-induced crosslinks assess how each RC-APEX bait sample differed in total protein that could obscure peptide identification, we did not use fixative for identification coverage, we generated density plots of the natural log the following experiments. (ln) of the normalized spectral abundance factor (NSAF) values for To capture biotinylated proteins using streptavidin beads, we all proteins identified by MS (Fig. S5B). The density plots for all four needed to verify whether they were solubilized after lysis and present constructs overlapped closely, indicating a similar extent of coverage in the soluble fraction after centrifugation and lysate clarification. of spectral counts between the samples, and eliminating any concerns RCs are known to remain intact after lysis with non-ionic detergents, of instrumentation- and sampling-based artifacts or significant such as TX-100 (Greenbaum et al., 2007; Robinson and Cooley, deviations in sample complexity or content. 1997b). Therefore, we lysed egg chambers in harsh, denaturing lysis buffer containing 8 M urea using a motorized spinning pestle, and the Identification and characterization of RC-APEX high- clarified lysate was incubated with magnetic streptavidin beads (see confidence prey Materials and Methods for more specific details). The magnetic To establish a list of high-confidence prey for each RC-APEX bait streptavidin beads were able to capture the biotinylated proteins from our proteomic data, we used Significance Analysis of (Fig. 3G), as shown by the depletion of the streptavidin-HRP signal in INTeractome (SAINT) (Choi et al., 2011; Morris et al., 2014), a the unbound fractions and the strong signal of biotinylated proteins in common tool used to parse ‘true interactors’ from ‘false interactors’ the eluates. Interestingly, the no-APEX control w1118 sample also in affinity purification-MS data sets. We used a SAINT score cut-off showed a smear of biotinylated proteins by western blot (Fig. 3G), of ≥0.8 (corresponding to a 5% false discovery rate), which resulted possibly because of the presence of endogenously biotinylated in 55 high-confidence prey (Fig. 4A, Table S1). Pav::APEX had proteins or proteins that were biotinylated by other endogenous three high-confidence prey: Pav, Tum and CG43333. HtsRC:: peroxidases. In any event, the biotin signal was stronger in the eluates APEX had five: Muscle-specific protein 300 kDa (Msp300), for the APEX samples compared with the no-APEX w1118 control. Filamin, HtsRC, CG43333 and Chorion protein c at 7F (Cp7Fc). Before proceeding with MS analysis, we checked for the presence APEX::Kelch and APEX::KREP had nine and 43 high-confidence or absence of known RC proteins in the eluates by western blotting prey, respectively. Only a few prey (listed in the Venn diagram in (Fig. 3H-K). The results were encouraging. We observed that only Fig. 4A) were shared between the RC-APEX baits. the Pav::APEX sample showed enrichment over controls for a Pav We used Cytoscape to generate a network map of the RC-APEX protein species (Fig. 3H, Pav::APEX band), which is consistent baits and prey (Fig. 4B). Known RC proteins were among the high- with Pav being dimeric (Minestrini et al., 2002; Sommi et al., 2010; confidence prey (solid line outlining prey gene circle in Fig. 4B). Tao et al., 2016). Similarly, only the APEX::Kelch sample showed Consistent with our western blot analysis of RC-APEX bait eluates enrichment for a Kelch protein species (Fig. 3I, APEX::Kelch (Fig. 3), Pav::APEX identified Pav; HtsRC::APEX and APEX:: band), which is also consistent with Kelch dimerization via its BTB KREP identified HtsRC; APEX::Kelch identified Kelch; and domain (Hudson and Cooley, 2010; Hudson et al., 2015; Robinson APEX::KREP identified Filamin. Interestingly, Filamin was also and Cooley, 1997a). APEX::Kelch was expressed in a kelch mutant identified by HtsRC::APEX, which shows the sensitivity achieved background, which is why endogenous Kelch was absent from that by MS analysis. We mined the Molecular Interaction Search Tool DEVELOPMENT

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Fig. 3. See next page for legend.

(MIST; Hu et al., 2018), and imported known interactions into the identified proteins are expressed in the Drosophila ovary. Taken Cytoscape network map (dotted lines connecting prey genes in together, these data indicate that each RC-APEX bait identified Fig. 4B). In addition, we searched publicly available expression data unique interactomes that include previously established interactors, at FlyBase (Thurmond et al., 2018) and found that 51 of the 55 new candidate interactors and known RC proteins. DEVELOPMENT

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Fig. 3. RC-APEX baits biotinylate distinct proteins in live egg chambers. identified Pav and Tum was reassuring, given that Pav dimerizes (A-F) Ovaries expressing indicated RC-APEX baits were permeabilized and (Minestrini et al., 2002; Sommi et al., 2010) and also interacts with biotinylated, then egg chambers were fixed and stained with TRITC-Phalloidin Tum (Adams et al., 1998; Tao et al., 2016). Kelch was the top prey for (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 the APEX::Kelch, consistent with Kelch dimerizing via its BTB domain specific biotin signal at RCs for the RC-APEX fusions (A′-D′), the unlocalized (Hudson and Cooley, 2010; Hudson et al., 2015; Robinson and Kelch cytoplasmic biotin signal for GBP::APEX (E′) and the absence of signal in the no- Cooley, 1997a). HtsRC was identified by the CRL3 substrate- APEX control (F′). (A′) Yellow arrows denote biotinylated follicle cell RCs in (BAC) trapping APEX::KREP construct, supporting the conclusion that the pav::GFP. (G-K) APEX-labeled ovaries were lysed in denaturing conditions, CRL3Kelch substrate is HtsRC (Hudson et al., 2019). HtsRC::APEX biotinylated proteins were captured by magnetic streptavidin beads, and eluates identified HtsRC and Filamin as prey. Although HtsRC and Filamin were analyzed by western blotting. (G) Inputs, unbound fractions and eluates were analyzed by western blotting with streptavidin-HRP to confirm that are known RC proteins that genetically interact (Robinson et al., biotinylated proteins were present. (H-K) Western blot analysis was performed on 1997; Sokol and Cooley, 1999), this is the first evidence of a physical the APEX eluates (labeled on top of lanes) to check for known RC proteins. interaction between them. Additionally, the finding that Filamin was (H) Pav::APEX protein was abundant in the Pav::APEX eluate. (I) APEX::Kelch a top prey for APEX::KREP suggested that Filamin, HtsRC and was enriched in the APEX::Kelch eluate, whereas a background endogenous KREP are all in close proximity at kelch-like RCs. Kelch protein band could be detected in all other samples. (J) HtsRC::APEX and For a more global and quantitative visualization of the prey endogenous HtsRC were present in the HtsRC::APEX sample, whereas identified by each RC-APEX bait, we generated dot plots using the endogenous HtsRC was highly enriched in the APEX::KREP sample. (K) Filamin was observed only in the APEX::KREP sample. Scale bar: 50 μm. ProHits-viz tool (Knight et al., 2017) (Fig. 5A-D). The color of each prey ‘dot’ is dependent on the NSAF log2(FoldChange) for each bait Of the 55 RC-APEX prey genes identified, five were previously compared with the control samples (see key in Fig. 5). The size of known to be involved in RC biology (prey genes with solid outlines each dot is dependent on its relative abundance between bait in Fig. 4B): pav, tum, hts, cheerio (cher)andkel. That Pav::APEX samples, meaning that the maximum-sized dot for each prey is

Fig. 4. Proteomic analysis reveals RC-APEX baits have unique interactomes that include known RC proteins. (A) Venn diagram showing the extent of overlap 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 was dependent on its abundance in terms of spectral counts compared with control samples [log2(FoldChange]). Known RC proteins are outlined in black. Dotted lines indicate previously established interactions mined from MIST (Hu et al., 2018). DEVELOPMENT

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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 CG9336 was 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-confidence prey 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 prey

that had an undefined P-value (e.g. Ef-Ts and Cyp6d4 in APEX::Kelch). DEVELOPMENT

7 TECHNIQUES AND RESOURCES Development (2019) 146, dev176644. doi:10.1242/dev.176644 allocated to the largest SpC value within that bait and the other dots GFP protein trap using rabbit anti-GFP and mouse anti-HtsRC are scaled proportionately. High-confidence prey within each bait antibodies. Compared with the negative control, there were abundant have a yellow outline (see key in Fig. 5). This visualization revealed PLA foci throughout the egg chamber as well as a strong signal at how each RC-APEX bait identified unique sets of prey proteins. For RCs (Fig. 6C). Similarly, we tested potential oligomerization of example, APEX::Kelch identified nine high-confidence prey (third HtsRC in egg chambers expressing the otu-ovhts::V5::APEX column of ‘dots’ all outlined yellow in Fig. 5C) and only one of transgene, which allowed us to use mouse and rabbit antibodies those prey (CG9336) was shared with another RC-APEX bait targeting the same V5 antigen. We detected PLA foci throughout the (APEX::KREP) (yellow outline around CG9336 dot in fourth egg chamber and at RCs (Fig. 6D). Quantification of PLA foci per column for APEX::KREP in Fig. 5C). egg chamber unit area showed that the {HtsRC}→Filamin and To visualize the quantitative proteomic data relative to statistical {HtsRC}→HtsRC samples had significantly more foci than their significance, we generated volcano plots for each RC-APEX bait respective controls (Fig. 6G, right graph). These results validate our (Fig. 5E-H). For each protein identified by MS, a P-value was proteomic results, and support the conclusion that HtsRC can calculated for its spectral counts within the biological replicates for dimerize or oligomerize as well as interact directly with Filamin, both each RC-APEX bait and control samples. For all identified proteins, in the cytosol and at RCs. the -log10(P-value) was plotted against the NSAF log2(FoldChange) HtsRC was a high-confidence prey for the substrate-trapping compared with controls for each RC-APEX bait sample. Proteins APEX::KREP construct, which was expected based on previous that showed a high fold-change compared with controls appear on work (Hudson et al., 2019). We validated this result in egg chambers the right side of the plot, whereas proteins that are statistically expressing APEX::V5::KREP using mouse anti-HtsRC antibodies significant compared with controls (in terms of SpC across and rabbit anti-V5 antibodies. As expected, we observed strong replicates) appear towards the top of the plot. Unsurprisingly, PLA signal throughout the cytoplasm and at RCs (Fig. 6E) with proteins in the top right of the plot generally corresponded to our essentially no foci in the negative control (Fig. 6E, bottom panel; high-confidence prey (gray dots in Fig. 5E-H). For example, the five quantified in Fig. 6G, right). These results provide proof of principle high-confidence prey identified by HtsRC::APEX were the sole that APEX fusion constructs can be used to identify substrates of E3 occupants of the upper-right quadrant (gray-labeled dots in Fig. 5F). ubiquitin ligases in live tissue. Finally, we tested an unexpected interaction between SUMO Validation of RC-APEX high-confidence prey by proximity (encoded by the smt3 gene) and Kelch using Drosophila SUMO ligation assay rabbit antibodies (Abgent) and mouse anti-Kelch antibodies. Given that APEX-mediated biotinylation is proximity dependent, Excitingly, we observed PLA foci throughout the cytoplasm of the top prey are potential direct interactors or binding partners of the egg chambers in the experimental sample but not the negative baits. Confirming physical interactions between these protein pairs control (Fig. 6F; quantification in Fig. 6G, right). The results in their native environments by conventional methods, such as co- suggest that Kelch is in close proximity to the SUMO protein in the , was essentially impossible because of the low cytoplasm of egg chambers, but not at RCs. Future experiments are solubility of the RC cytoskeleton. Therefore, we turned to the needed to test whether Kelch is covalently SUMOylated or Duolink proximity ligation assay (PLA) to test whether RC-APEX interacting with SUMO, perhaps through its predicted SUMO baits were in close proximity (<40 nm) to their prey proteins in egg interaction motif (Zhao et al., 2014). chambers. We were able to test five prey using available antibodies As an additional control, we used the PLA to test interactions and GFP-tagged stocks. Initial PLA experiments using fixed whole- between RC-APEX bait proteins and proteins that were not identified egg chambers were unsuccessful. Suspecting a penetration issue, we as prey. Specifically, we tested for interactions between Pav and performed the PLA in 20-μm ovary cryosections and succeeded in Kelch (Fig. S6A), Kelch and Filamin (Fig. S6B), Pav and HtsRC detecting PLA signals (see Methods for more details). (Fig. S6C), and HtsRC and Kelch (Fig. S6D). All interactions tested To test whether Pav dimerization could be detected using the were negative, and quantification revealed no significant enrichment proximity ligation assay, we used egg chambers expressing the pav:: of PLA foci in the experimental samples compared with their APEX::FLAG BAC transgene. The presence of the FLAG epitope in respective negative controls (Fig. S6E). These results were all the fusion protein allowed us to use FLAG antibodies generated in two consistent with our RC-APEX bait and prey protein pairs and support species (mouse and rabbit) to target the same FLAG antigen. The PLA our conclusion that the PLA was faithfully recapitulating real PPIs. assay confirmed that Pav interacts with itself, as indicated by fluorescent foci throughout egg chambers and at RCs (Fig. 6A, top Identification of novel ring canal regulators through an RNAi panel) compared with the negative control sample in which one of the screen of RC-APEX prey genes antibodies was omitted (Fig. 6A, bottom panel). We detected an To test genetically whether the RC-APEX preys were involved in interaction between Pav and Tum using egg chambers expressing a RC biology, we performed an RNA interference (RNAi) screen GFP::Tum protein trap and the pav::APEX::FLAG BAC transgene. using stocks from the Transgenic RNAi Project (TRiP). We Fluorescence microscopy showed a striking accumulation of PLA foci screened 33 prey gene small hairpin (sh)RNA lines using the throughout the entire egg chamber and at the RCs (Fig. 6B, top panel), mat-GAL4 driver for germline-specific expression. Of the 33 genes, whereas the negative control was devoid of PLA signal (Fig. 6B, 19 had a noticeable phenotype; eight phenotypes were RC specific bottom panel). Quantification of PLA foci showed that there were and 11 had general oogenesis defects (Fig. 7A). Fourteen genes significantly more foci per egg chamber unit area in the {Pav}→Pav showed no phenotype, which could be because: (1) the RNAi line and {Pav}→Tum samples compared with their respective negative did not work; (2) the gene functions in early oogenesis before the controls (Fig. 6G, left graph). These results indicate that the proximity mat-GAL4 driver is expressed; or (3) knocking down the gene has ligation assay can detect true PPIs at their native environment in tissue, no deleterious effects on the egg chamber. and validates Pav and Tum as Pav::APEX prey. Six of the eight RC-specific phenotypes [barentsz (btz), combgap We also tested the previously unknown potential interaction (cg), Protein on ecdysone puffs (Pep), polychaetoid ( pyd), Splicing between HtsRC and Filamin in egg chambers expressing a cher:: factor 2 (SF2) and smallminded (smid)] were significantly enlarged DEVELOPMENT

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Fig. 6. In situ proximity ligation assay confirms close-proximity interactions between RC-APEX bait proteins and respective prey. (A-F) Proximity ligation assays 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 PPIwas indicated by the presence of PLA signal (left panels), which was visualized by fluorescent probes that bind to the PLA DNA product. Antibodies used for each reaction are indicated on the left, and the bottom panels served as negative controls because one antibody was omitted. Boxed insets contain RCs showing F-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 the exception of the {Kelch}→SUMO (F) interaction, which was mostly dispersed throughout the cytoplasm. (G) Positive PLA foci in each egg chamber imaged were 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 the number 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) and μ

5 m (insets). DEVELOPMENT

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Fig. 7. RNAi screen reveals that prey genes are involved in RC biology. (A) Summary of results from RNAi screening of 33 high-confidence prey [with each prey color coded to indicate its respective RC-APEX bait(s)]. pav, hts, cher and kel are included for 37 genes in total. Of the 33 prey genes screened, 19 had a phenotype, eight of which were RC specific. (B-I) Egg chambers of indicated genotypes were stained with TRITC-Phalloidin and HtsRC (B′-I′) to reveal RC size and morphology. Red arrows indicate abnormal F-actin structures. Yellow and blue arrows indicate deformed and collapsed RCs, respectively. (J) RC diameters of the indicated genotypes were measured in FIJI using HtsRC staining as a RC marker, and violin plots were used to represent the data. Thick-dashed white lines show median RC diameter, and thinner upper and lower white lines denote the upper and lower quartiles, respectively. One-way ANOVA was used to compare the mean RC diameter between the RNAi lines and w1118 control. ***P≤0.0001; ****P≤0.00001. (K) RNAi-mediated knockdown of smt3 caused collapsed and deformed RCs, marked by blue and yellow arrows, respectively. (L) Some RCs were positive for SUMO staining (inset). Scale bars: 20 μminL, 25 μm in K and 50 μm in B-I.

RCs (Fig. 7B-H; RC diameter quantified in Fig. 7J). RNAi- possible role for SUMO in RC biology, we stained egg chambers mediated knockdown of Pep also led to abnormal F-actin structures with anti-SUMO antibodies and found occasional RC localization as well as deformed and collapsed RCs (represented by red, yellow, (Fig. 7L, boxed insets). and blue arrows in Fig. 7E′, respectively). Knockdown of pyd also Interestingly, none of the eight genes with RC-specific resulted in deformed RCs (yellow arrows in Fig. 7F′), whereas smid phenotypes were known previously to have functions related to knockdown caused abnormal excess cortical F-actin surrounding RCs. We were particularly surprised to see that five of these genes RCs in stage 10 egg chambers (red arrows in Fig. 7H). Spt5 (btz, cg, Pep, SF2 and Spt5) had established functions involving knockdown led to egg chamber degeneration in mid-to-late-staged DNA and/or RNA binding, processing or regulation, suggesting egg chambers (Fig. 7I) and collapsed RCs (blue arrows in Fig. 7I′). possible ‘new’ roles in RC biology. In summary, this preliminary Finally, RNAi-mediated knockdown of smt3 (the Drosophila RNAi screen revealed that prey genes are involved in RC biology, SUMO gene) led to egg chamber arrest as well as deformed and which provides additional support that the RC-APEX baits collapsed RCs (Fig. 7K, yellow and blue arrows). To examine a identified physiologically relevant protein interactors. DEVELOPMENT

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DISCUSSION nature of the Kelch-Cullin3 interaction or the relatively low abundance APEX-mediated proximity labeling can be used to identify of Cullin3 present at RCs compared with its strong cytoplasmic protein-protein interactions in live tissue localization (Hudson and Cooley, 2010; Hudson et al., 2015). Since its introduction in 2013, APEX-mediated localized Identification of E3 ubiquitin ligase substrates is challenging biotinylation has been used successfully in several studies to because substrates are short-lived and their interactions with the identify organellar or cellular proteomes, with most of the work ligase are transient. Those challenges, coupled with the insolubility done in cultured cells. Three studies reported APEX-mediated of the RC actin cytoskeleton, made identifying the CRL3Kelch RC labeling in live tissue. Two of these studies were performed in substrate particularly hard. We recently identified HtsRC as the Caenorhabditis elegans in a mutant background that compromised substrate of CRL3Kelch with other methods (Hudson et al., 2019), cuticle integrity, which allowed for successful delivery of the biotin- and of the 40 prey identified by APEX::KREP in this study, only phenol substrate into the worm tissue (Reinke et al., 2017a,b). The one, HtsRC, contains the known Kelch-binding motif (PEAEQ) third labeled the mitochondrial proteome in dissected Drosophila (Schumacher et al., 2014). Thus, our work suggests that APEX is a imaginal discs, salivary glands and larval muscles (Chen et al., 2015). valuable new tool for identifying E3 ubiquitin ligase substrates, These tissues are relatively thin, which permits sufficient entry of particularly in previously inaccessible contexts like in live tissue, or biotin-phenol into the cells without notable intervention. Here, we within insoluble cellular compartments, such as the cytoskeleton. In adapted the use of APEX-mediated proximity labeling to thicker fact, ours is the first study of its kind to use APEX in vivo to identify tissue by using digitonin as a means to sufficiently permeabilize cell an E3 ubiquitin ligase substrate. The advent of substrate-trapping membranes and allow biotin-phenol substrate entry. Our approach constructs that can be fused to APEX and expressed in model could be adapted to other organisms and tissues, live or formaldehyde organisms could lead to future breakthroughs for substrate fixed, to enable the more widespread use of APEX in vivo. identification in tissue-specific and developmental contexts, Of note, the Ting lab recently engineered more catalytically active which will help move the E3 ubiquitin ligase field into mutants of the BirA enzyme used in BioID approaches, referred to as physiologically relevant models. TurboID and miniTurbo (Branon et al., 2018). Essentially, these The APEX::KREP bait identified a surprisingly large number of mutants combine the catalytic efficiency of APEX (i.e. a 10-min high-confidence prey proteins compared with the other three baits. labeling reaction time) with the ease-of-use of BioID (i.e. This could be because of its abundance both in the cytoplasm and straightforward biotin delivery to cells or tissues of interest). RCs, leading to more efficient biotinylation of proteins. Given that However, when TurboID was used in flies and worms, biotin expression of KREP leads to a dominant-negative phenotype of supplementation took hours or days, which limits its potential for kelch-like RCs, it is also possible that protein complexes were capturing more dynamic processes that require fine temporal control caught or trapped in the RC cytoskeleton, which could have and resolution of the labeling reaction. In these cases, our APEX facilitated their biotinylation. Interestingly, many APEX::KREP protocol could be used with dissected tissue to achieve robust prey genes are involved in RNA binding or processing as well as biotinylation on a faster (seconds to minutes) timescale. Further ribonucleoprotein complexes. RNPs are transported along adaptation and optimization of these proximity labeling approaches microtubules, through RCs from the nurse cells into the oocyte will undoubtedly open the door to proteomic experiments in living (Mische et al., 2007); thus, they might indeed have been trapped. organisms. However, RNAi-mediated knockdown of genes for several of these RNP-associated APEX::KREP prey genes led to RC-specific RC-APEX baits identified known binding partners as well as phenotypes, suggesting a role for mRNA regulation and RNP potential new interactors complexes in RC biology. In any event, the smaller number of We set out to test the feasibility of using APEX in vivo to identify proteins identified with HtsRC, Pav and Kelch baits compared with PPIs as opposed to whole-organelle or whole-cell proteomes. KREP suggests that we succeeded in finding specific interactors for Although several RC proteins have been characterized, a full these proteins within intact, morphologically normal RCs. understanding of F-actin regulation in RCs requires more complete information about PPIs in the structure. Given that biotinylation of Proximity labeling revealed new insights into the ring canal nearby proteins is dependent on their proximity to the APEX substructure enzyme, we envisioned that the RC-APEX bait proteins could RCs are massive structures that undergo dramatic and dynamic identify new binding partners. Our results were promising. We were structural and possibly compositional changes during egg chamber especially excited that the two top prey of Pav::APEX were Pav and development. Given the complex nature of the RC cytoskeleton (e.g. Tum, because identifying the Centralspindlin complex (Tao et al., F-actin-dependent membrane and RC expansion, actin recruitment, 2016) provided a powerful positive control and a compelling proof F-actin crosslinking and bundling, and cytoskeleton disassembly in of principle that the RC-APEX baits were capturing real protein the RC lumen), it would make sense that different subcomplexes exist interactors of the respective RC bait proteins. Our results also in RCs to carry out these processes. Evidence already exists for indicated that APEX fused to different domains within proteins can distinct domains within the RC. HtsRC, Kelch and F-actin localize to identify domain-specific interactors. APEX fused to the Kelch N the inner rim of RCs (Robinson et al., 1994), Pav localizes to the outer terminus, which is the site of Kelch homodimerization through rim (Minestrini et al., 2002; Ong and Tan, 2010), and Filamin is its BTB domain (Canning et al., 2013; Errington et al., 2012; Ji present throughout the entire RC cytoskeleton. However, a more and Privé, 2013), labeled distinct proteins compared with APEX:: finely resolved picture of RC protein complex localization is KREP, which contains just the substrate-binding domain of Kelch. emerging from our localized biotinylation results. Kelch itself was a top prey for APEX::Kelch, whereas HtsRC was a Our data showed that different RC-APEX bait constructs top prey for APEX::KREP. Surprisingly, APEX::Kelch did not identified unique subsets of RC proteins suggesting that there are identify Cullin3, a known binding partner of the Kelch BTB domain subcomplexes within the RC that orchestrate mechanical functions, (Canning et al., 2013; Errington et al., 2012; Ji and Privé, 2013; Xu such as nucleating actin filament assembly at the membrane to et al., 2003), as a top prey. This might be because of the transient expand the RC diameter or controlling HtsRC levels to maintain an DEVELOPMENT

11 TECHNIQUES AND RESOURCES Development (2019) 146, dev176644. doi:10.1242/dev.176644 open RC lumen. Of particular interest is the finding that HtsRC:: UASp-APEX2::V5::KREP APEX identified Filamin and HtsRC as top prey. Given that The APEX2 coding sequence was amplified from pcDNA3-APEX2-NES localization of HtsRC to RCs is genetically dependent on the cher (Addgene Plasmid #49386) and the KREP coding sequence of kelch was gene (Robinson et al., 1997; Sokol and Cooley, 1999), we can now amplified with primers designed to contain an N-terminal V5 tag sequence. speculate that Filamin directly recruits HtsRC to the RC, where Through overlap-extension PCR, the APEX2::V5::KREP coding sequence was assembled with flanking attB1 and attB2 sites and recombined first into HtsRC functions to recruit the abundant F-actin cytoskeleton pDONR201 and then into pPW-attB (Table S2) in BP Clonase II and LR (Hudson et al., 2019). This also suggests that HtsRC has the ability Clonase II Gateway recombination reactions, respectively. The pPW- to dimerize or oligomerize, possibly through its C-terminal coiled- APEX2::V5::KREP plasmid was injected into a strain carrying the attP2 coil domain (predicted using Phyre2; Kelley et al., 2015). It will be phiC31 landing site on chr3L at Rainbow Transgenic Flies. interesting to test whether the oligomerization status of HtsRC differs based on its localization in the cytoplasm or at RCs and UASp-GBP::FLAG::APEX2 whether this affects its actin-regulating activity. The GBP (Rothbauer et al., 2008) coding sequence was amplified with It is also possible that many of the RC-APEX bait-prey interactions primers designed to contain 3′ and 5′ SfiI restriction sites. The FLAG::APEX2 we identified occurred in the cytoplasm rather than the RC. The sequence was amplified from pcDNA3-APEX2-NES (Addgene Plasmid proximity ligation assay showed RC-specific signals for most of the #49386) with primers designed to contain a 3′ SfiIsiteanda5′ BamHIsite. interactions tested, but there were also positive foci present in the The fragments were digested with their respective restriction enzymes and cytoplasm. Cytoplasmic interactions could be important for ligated into a pUASp-attB plasmid modified to contain a polylinker sequence downstream of UASp site and P-element transposons. Transgenic flies were assembling protein complexes destined for RCs; for example, generated via P-element-mediated insertion at Rainbow Transgenic Flies. HtsRC could bind to Filamin in the cytoplasm before localizing to RCs. We expect that additional analysis of the RC-APEX prey will Tissue fixation, fluorescence microscopy and imaging lead to new insights into RC formation, maintenance and growth See Table S2 for more information regarding the antibodies and reagents used during development. in this study. Ovaries were dissected in ionically matched Drosophila saline (IMADS) buffer (Singleton and Woodruff, 1994) and fixed for 10 min in MATERIALS AND METHODS either 6% formaldehyde, 75 mM KCl, 25 mM NaCl, 3 mM MgCl2,and Drosophila husbandry 17 mM potassium phosphate, pH 6.8 (Verheyen and Cooley, 1994) or 4% Drosophila were maintained at 25°C on standard fly food medium. Before formaldehyde in PBT [PBS with 0.3% Triton X-100 and 0.5% bovine serum ovary dissections, females were fattened on wet yeast paste overnight at albumin (BSA)]. Fixed tissue was washed in PBT and incubated with the 25°C. See Table S2 for a detailed list of the fly stocks used in this study. indicated primary antibodies in PBT for ∼1-2 h at room temperature or overnight at 4°C. Following washes with PBT, tissue was incubated with Molecular cloning and Drosophila transgenesis secondary antibodies conjugated to Alexa Fluor and DAPI in PBT for ∼1-2 h (BAC) pav::HA::APEX2::FLAG at room temperature. F-actin was labeled with Phalloidin conjugated to APEX2 was recombineered into a BAC containing the pav gene at its C Tetramethylrhodamine (TRITC). Samples were washed in PBT and mounted terminus (BAC ID 322-102N3) to create pav::HA::APEX2::FLAG.This on slides in ProLong Diamond (Thermo Fisher Scientific). Samples were BAC contains the entire pav locus on a 21-kb genomic fragment imaged with a Leica SP8 laser scanning confocal microscope using a 40×1.3 (chr3L:4,229,286…4,250,505, FlyBase release 6). Briefly, we used a two- NA oil-immersion objective lens or a Zeiss Axiovert 200 m equipped with step BAC recombineering protocol to first insert a Kanamycin resistance either a CARV II spinning disc confocal imager or a CrEST X-light spinning cassette (Wang et al., 2006), which was subsequently replaced by HA:: disc system and a Photometrics CoolSNAP HQ2 camera using a 40×1.2 NA APEX2::FLAG through streptomycin selection. The HA::APEX2::FLAG water-immersion objective. sequence was made through PCR amplification of APEX2 from pcDNA3- APEX2-NES (Addgene Plasmid #49386) with primers designed to add an Western blot analysis of ovary lysates N-terminal HA epitope tag sequence and a C-terminal FLAG epitope tag Ovary lysates were generated by homogenizing dissected ovaries in sequence. The APEX2 sequence contained a C-terminal nuclear export signal SDS-PAGE sample buffer. One ovary equivalent was loaded per lane and (NES), which was incorporated in an attempt to keep Pav::APEX localized separated by SDS-PAGE. Proteins were transferred to a nitrocellulose exclusively to RCs. The final plasmid was injected into BL#24872 into the membrane, stained with amido black to visualize total protein, blocked in attP3B site on chr2L at Rainbow Transgenic Flies, Inc. 5% milk in Tris-buffered saline with 0.1% Tween-20 (TBST) for 30 min at room temperature, and probed with the indicated primary antibodies for otu-ovhts::V5::APEX1 ∼2 h at room temperature in 5% milk/TBST. After washing the blots three The V5::APEX1 coding sequence was amplified from pcDNA3-mito-V5:: times in TBST for 5 min, the blots were incubated with HRP-conjugated APEX1 (Rhee et al., 2013; Addgene Plasmid #42607) using primers secondary antibodies for ∼1 h at room temperature followed by enhanced designed to create a V5::APEX1 fragment flanked by 3′ XhoI and 5′ NotI chemiluminescence (ECL) development for 5 min. A CCD camera was used restriction sites. pCOH-ovhts::GFP (Petrella et al., 2007) was digested with for imaging. See Table S2 for more information on the antibodies used in XhoI and NotI to excise GFP and the V5::APEX1 fragment was ligated into this study. the plasmid in-frame and in place of GFP. Transgenic flies were generated via P-element-mediated insertion at GenetiVision. Proteomic analysis of APEX samples In vivo biotinylation of proteins using RC-APEX baits UASp-APEX2::V5::kelch Before dissection, female flies were fattened overnight on wet yeast paste at The APEX2 coding sequence was amplified from pcDNA3-APEX2-NES 25°C in a vial with males. For small-scale pilot experiments or experiments (Addgene Plasmid #49386) and the entire kelch coding sequence was in which fluorescence microscopy was the final experimental readout, amplified with primers designed to contain an N-terminal V5 tag sequence. ovaries from 10-20 flies were used for biotin-phenol proximity labeling. For Through overlap-extension PCR, the APEX2::V5::kelch coding sequence large-scale proteomic experiments, three batches of labeling were performed was assembled with flanking attB1 and attB2 sites and recombined first into with ovaries from 30-35 flies in each batch, yielding a total of 100 flies used pDONR201 and then into pPW-attB (Table S2) in BP Clonase II and LR per sample. Ovaries were dissected in 1× PBS, pH 8.0 and gently pipetted up Clonase II Gateway recombination reactions, respectively. The pPW- and down ∼ten times to splay open the ovaries. The pH of all solutions was APEX2::V5::kelch plasmid was injected into a strain carrying the attP2 kept above 7.5 to prevent precipitation of the biotin-phenol. Egg chambers phiC31 landing site on chr3L at Rainbow Transgenic Flies. were incubated in 500 μl biotin-phenol labeling solution (1 mM biotin- DEVELOPMENT

12 TECHNIQUES AND RESOURCES Development (2019) 146, dev176644. doi:10.1242/dev.176644 phenol, 0.5% digitonin, 1× PBS pH 8.0) for 3 min with rotation at room MS/MS. Data were searched using a local copy of Mascot (Version 2.6.0; temperature. To initiate APEX-mediated biotinylation, H2O2 was added to a Matrix Science) using the UniProt Drosophila melanogaster database with final concentration of 0.05 mM and the solution was gently inverted to mix common laboratory contaminants added as well as custom protein sequences for 30 s. After 30 s, 10 mM NaN3 was added to quench the reaction. Egg for HtsRC that included known single nucleotide polymorphisms (SNPs) and chambers were washed 3× in 500 μl quenching buffer (10 mM NaN3 in 1x an N-terminal cleavage site at V693, and the APEX1 and APEX2 protein PBS, pH 8.0). For every labeling reaction performed, egg chambers were sequences. Trypsin was selected as the enzyme and two missed cleavages always fixed and analyzed by fluorescence microscopy to ensure that the were allowed. Cysteine carbamidomethylation was selected as a fixed labeling reaction was successful (i.e. that RCs were biotinylated). Standard modification. Methionine oxidation, N-terminal acetylation, N-terminal fixation and fluorescence protocols were used, as described earlier. To pyroglutamylation, asparagine or glutamine deamidation, and tyrosine visualize biotin signal in egg chambers, fixed and permeabilized egg biotinylation by biotin-tyramide (chemical formula: C18H23N3O3S) were chambers were incubated with streptavidin-AF488 or streptavidin-AF568 at selected as variable modifications. A peptide mass tolerance of 10 ppm and a a 1:500 dilution in PBT for ∼1 h at room temperature. For large-scale fragment mass tolerance of 0.02 Da were used. The resultant Mascot DAT labeling reactions, a very small amount of egg chambers was removed after files were parsed using Scaffold (Version 4.8.4; Proteome Software) for the last wash in quenching buffer and analyzed by fluorescence microscopy. validation, filtering, and to create a nonredundant protein list per sample. Immediately after the last wash, the remaining quenching buffer was Experiment-wide grouping with binary peptide-protein weights was selected removed from the egg chambers, and the tissue was frozen at -80°C. as the Protein Grouping Strategy. Peptide and Protein Thresholds were set to 95% and 99%, respectively, with a 1 peptide minimum. These settings Purification of biotinylated proteins from ovary lysates resulted in a 0.0% peptide false discovery rate (FDR) and a 0.4% protein FDR Labeled and quenched egg chambers were thawed on ice and the tissue from as measured by Decoy matches. The Scaffold Samples report was exported as the three batches of labeling was combined into one tube for each sample. an Excel spreadsheet. Contaminant proteins were removed from the Tissue was ground in 100-200 μl urea lysis buffer (8 M urea, 10 mM Tris spreadsheet data set. Spectral peptide matches for the Ovhts polyprotein pH 8.0, 50 mM NaPi, 300 mM NaCl, 5 μg/ml each of chymostatin, encoded by the ovhts transcript were manually parsed into its two leupeptin, antipain and pepstatin, 10 mM NaN3,1μM bortezomib, 10 mM corresponding protein products: HtsF (AA 1-658) and HtsRC (AA 659- N-ethylmaleimide, 5 mM Trolox, 10 mM sodium ascorbate) with a 1156). Thus, spectral counts were manually entered into the spreadsheet for mechanical homogenizer. After grinding, additional lysis buffer was the HtsF and HtsRC proteins across each sample. added to a final volume of 1 ml and the lysate was incubated for an additional 30 min at room temperature to ensure sufficient lysis and Data analysis using SAINT solubilization. The crude lysate was centrifuged at 13,200 rpm (16,100 g) The Automated Processing of SAINT Templated Layouts (APOSTL) for 10 min at room temperature, and the clarified lysate was transferred to a (Kuenzi et al., 2016) tool was used to preprocess the MS data to generate new tube with 500 μl pre-equilibrated magnetic streptavidin beads and corresponding data files that were compatible with analysis by SAINTexpress incubated for 1 h at room temperature with rotation. The unbound fraction (Teo et al., 2014). The resultant SAINTexpress data set was processed further was removed from the beads and the beads were washed three times in 1 ml using APOSTL; namely, normalized spectral abundance factor (NSAF) urea wash buffer (2 M urea, 10 mM Tris pH 8.0, 50 mM NaPi, 300 mM values were calculated for each prey based on its respective SpC observed in NaCl, 5 μg/ml each of chymostatin, leupeptin, antipain, pepstatin, each bait as well as the log2 fold-change compared with control 10 mM NaN3,1μM bortezomib, 10 mM N-ethylmaleimide, 5 mM [log2(FoldChange)]. Trolox, 10 mM sodium ascorbate). To analyze the purified fraction by western blotting, 100 μl bead/wash solution was removed during the last Interactome network visualization with Cytoscape wash (i.e. 10% of bead fraction). Beads were eluted with vigorous mixing at Cytoscape was used to display the ‘interactomes’ of the RC-APEX baits and 95°C in 50 μl elution buffer [SDS sample buffer (0.1 M Tris pH 8.0, 4% their respective high-confidence prey. RC-APEX baits and their prey genes SDS, 0.01% Bromophenol Blue, 5% β-mercaptoethanol) with 10 mM were indicated by colored squares and circles, respectively. The size of the prey biotin]. Following the last wash, the remaining wash solution was removed gene circle was scaled based on its average abundance compared with control from the rest of the beads and the beads were frozen immediately at -80°C or samples [log2(FoldChange) NSAF]. Known RC genes were manually denoted on dry ice. Beads were shipped on dry ice overnight to MS Bioworks for MS with a solid outline. The Molecular Interaction Search Tool (MIST) (Hu et al., analysis using their IP-works platform. 2018) was used to search for previously established interactions between all high-confidence prey genes, and positive interactions were indicated with a Sample preparation for mass spectrometry dotted line. Proteins were eluted from streptavidin beads by incubation with 1.5× lithium dodecyl sulfate (LDS) buffer for 15 min at 100°C. Eluted proteins Generation of dot plots and heatmaps with ProHits-viz were separated from the beads by centrifugation and magnetic separation. The ProHits-viz tool (Knight et al., 2017) was used to generate dot plots to Then, 50% of each eluate was processed by SDS-PAGE with a 10% Bis-Tris visualize proteomics data for any high-confidence prey across all four NuPAGE gel using the MES buffer system (Invitrogen). The gel lanes were RC-APEX bait samples. The color of each prey ‘dot’ was dependent on the excised, sliced into ten equal-sized fragments, and subjected to in-gel NSAF log2(FoldChange) for each bait compared with the control samples. trypsin digestion using a robot (ProGest, DigiLab). For the in-gel trypsin The size of each dot was dependent on its relative abundance between bait digestion, gel slices were washed with 25 mM ammonium bicarbonate samples, meaning that the maximum-sized dot for each prey was allocated to followed by acetonitrile, reduced with 10 mM dithiothreitol at 60°C, the largest SpC value within that bait and the other dots were scaled alkylated with 50 mM iodoacetamide at room temperature, digested with proportionately. High-confidence prey within each bait had a yellow outline, sequencing grade trypsin (Promega) at 37°C for 4 h, and quenched with corresponding to a SAINT score ≥0.8, whereas prey with a SAINT score formic acid. The resultant supernatant was analyzed by MS. <0.8 for that bait had a faint outline.

LC-MS/MS data acquisition and processing Generation of volcano plots Half of each digested sample was analyzed by nano LC-MS/MS with a Waters Volcano plots were generated for each RC-APEX to visualize the NanoAcquity HPLC system interfaced to a Thermo Fisher Scientific Q quantitative proteomic data relative to their statistical significance. For Exactive. Peptides were loaded on a trapping column and eluted over a 75-μm each protein identified by MS, a P-value was calculated for its spectral analytical column at a flow rate of 350 nl/min. Both columns were packed counts within the biological replicates for each RC-APEX bait and control with Luna C18 resin (Phenomenex). The MS instrument was operated in data- samples. For all identified proteins, the -log10(P-value) was plotted against dependent mode with the Orbitrap operating at 780,000 FWHM for MS and the NSAF log2(FoldChange) compared with controls for each RC-APEX 17,500 FWHM for MS/MS. The 15 most abundant ions were selected for bait sample. DEVELOPMENT

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Validation of RC-APEX high-confidence prey using proximity Funding ligation assay K.M.M. was a Cold Spring Harbor Proteomics Course participant. K.M.M. and R.S.K. Ovaries from ∼50 flies were dissected and fixed in 6% formaldehyde, 75 mM were supported in part by a National Institute of General Medical Sciences National KCl, 25 mM NaCl, 3 mM MgCl and 17 mM potassium phosphate, pH 6.8 Institutes of Health training grant T32 GM007223 and a Gruber Science Fellowship. 2 This work was funded by National Institutes of Health grants R01 GM043301 and with heptane for 8 min. After washing three times for 10 min in PBT, the tissue RC1 GM091791. Deposited in PMC for release after 12 months. was incubated with 500 μl 30% sucrose solution with gentle rotation for 30 min, at which point the ovaries settled to the bottom of the tube. Tissue was Data availability rinsed with fresh sucrose solution and transferred to an Eppendorf tube cap, A Scaffold file containing all proteomics data, including peptide spectral matches where it was fully submerged in cold optimal cutting temperature (OCT) (PSM), is available from the Dryad Digital Repository (Mannix et al., 2019): dryad. compound. After a 1-2 h incubation, the tissue was embedded in a cryoblock 9p9c594. mold in OCT by incubating on dry ice for 10 min. Cryoblocks were stored at -80°C until sectioning. Before sectioning, blocks were equilibrated at -20°C for Supplementary information 5 min. Then, 20-μm slices were sectioned, transferred to Superfrost Plus Supplementary information available online at charged slides (Fisher Scientific), and stored at -80°C until future use. For the http://dev.biologists.org/lookup/doi/10.1242/dev.176644.supplemental PLA, the Duolink® In Situ Red Starter Kit Mouse/Rabbit (Sigma-Aldrich) was used with minor protocol modifications. Slides containing ovary cryosections References were equilibrated at room temperature for 5 min, then blocked for 1 h in PLA Adams, R. R., Tavares, A. A. M., Salzberg, A., Bellen, H. J. and Glover, D. M. (1998). Pavarotti encodes a kinesin-like protein required to organize the central blocking buffer (1× PBS, 0.3% Triton X-100, 1% BSA). An Aqua Hold II pen spindle and contractile ring for cytokinesis. Genes Dev. 12, 1483-1494. doi:10. was used to form hydrophobic barriers around tissue on the slide to contain the 1101/gad.12.10.1483 small volumes of solutions used in this protocol. Slides were placed in a Airoldi, S. J., McLean, P. F., Shimada, Y. and Cooley, L. (2011). Intercellular humidity chamber and tissue was incubated with 40 μl primary antibody protein movement in syncytial Drosophila follicle cells. J. Cell Sci. 124, 4077-4086. solution and FITC-Phalloidin in PLA blocking buffer for 2 h at room doi:10.1242/jcs.090456 temperature. See Table S2 for a list of antibodies and their concentrations used Branon, T. C., Bosch, J. A., Sanchez, A. D., Udeshi, N. D., Svinkina, T., Carr, in this assay. Tissue was washed twice for 5 min with PLA blocking buffer and S. A., Feldman, J. L., Perrimon, N. and Ting, A. Y. (2018). Efficient proximity μ μ μ labeling in living cells and organisms with TurboID. Nat. Biotechnol. 36, 880-887. incubated in 40 l PLA probe solution (8 lPLUSprobe,8 lMINUSprobe, doi:0.1038/nbt.4201 24 μl antibody diluent) for 1.5 h at 37°C in the humidity chamber. Slides were Canning, P., Cooper, C. D. O., Krojer, T., Murray, J. W., Pike, A. C. W., Chaikuad, washed twice for 5 min in wash buffer A then incubated for 45 min in the A., Keates, T., Thangaratnarajah, C., Hojzan, V., Ayinampudi, V. et al. (2013). humidity chamber at 37°C in 40 μl ligation solution (8 μl ligation buffer, 31 μl Structural basis for Cul3 protein assembly with the BTB-Kelch family of E3 ubiquitin ligases. J. Biol. Chem. 288, 7803-7814. doi:10.1074/jbc.M112.437996 ddH2O, 1 μl ligase). Slides were washed twice for 5 min in wash buffer A and incubated for 2 h in the humidity chamber at 37°C in 40 μl amplification Chen, C.-L. and Perrimon, N. (2017). Proximity-dependent labeling methods for solution (8 μl amplification buffer, 31.5 μlddHO, 0.5 μl polymerase) proteomic profiling in living cells: Proximity-dependent labeling methods. Wiley 2 Interdiscip. Rev. Dev. Biol. 6.4, e272. doi:10.1002/wdev.272 supplemented with FITC-Phalloidin. Slides were washed twice for 10 min Chen, C.-L., Hu, Y., Udeshi, N. D., Lau, T. Y., Wirtz-Peitz, F., He, L., Ting, A. Y., in wash buffer B, rinsed for 1 min with 0.01× wash buffer B, and mounted in Carr, S. A. and Perrimon, N. (2015). Proteomic mapping in live Drosophila 30 μl PLA mounting medium overnight in the humidity chamber at 4°C. tissues using an engineered ascorbate peroxidase. Proc. Natl. Acad. Sci. USA Slides were imaged on a Leica SP8 laser scanning confocal microscope using a 112, 12093-12098. doi:10.1073/pnas.1515623112 40×1.3 NA oil-immersion objective lens and all laser settings were equal Choi, H., Larsen, B., Lin, Z.-Y., Breitkreutz, A., Mellacheruvu, D., Fermin, D., between each slide and its respective control to allow for quantitative Qin, Z. S., Tyers, M., Gingras, A.-C. and Nesvizhskii, A. I. (2011). SAINT: – comparison of PLA foci. Positive PLA foci were counted in FIJI using the probabilistic scoring of affinity purification mass spectrometry data. Nat. Methods 8, 70-73. doi:10.1038/nmeth.1541 Auto-thresholding and Particle Analysis tools, with the minimum area of a foci Cooley, L. (1998). Drosophila ring canal growth requires Src and Tec kinases. Cell 2 set to 2 μm . Egg chamber area was calculated in FIJI using the Freehand and 93, 913-915. doi:10.1016/S0092-8674(00)81196-4 Area Measure tools. Errington, W. J., Khan, M. Q., Bueler, S. A., Rubinstein, J. L., Chakrabartty, A. and Prive, G. G. (2012). Adaptor protein self-assembly drives the control of a cullin-RING ubiquitin ligase. Structure 20, 1141-1153. doi:10.1016/j.str.2012.04. Validation of prey genes by RNAi screening 009 See Table S2 for a list of fly stocks used for the RNAi screen. Ovaries were Goshima, G. and Vale, R. D. (2005). Cell cycle-dependent dynamics and regulation dissected from fattened females, fixed and stained with TRITC-Phalloidin of mitotic kinesins in Drosophila S2 cells. Mol. Biol. Cell 16, 3896-3907. doi:10. and anti-HtsRC antibodies to check for any abnormalities in egg chamber 1091/mbc.e05-02-0118 “ ” development or RC morphology. Green, N. M. (1975). Avidin . Advances in Protein Chemistry, Vol. 29, pp. 85-133. Academic Press. Greenbaum, M. P., Ma, L. and Matzuk, M. M. (2007). Conversion of midbodies into Acknowledgements germ cell intercellular bridges. Dev. Biol. 305, 389-396. doi:10.1016/j.ydbio.2007. We thank Peter McLean for help with the (BAC) pav::APEX transgene. We thank 02.025 Tian Xu (Yale University, Westlake University) for providing access to the Leica Haglund, K., Nezis, I. P. and Stenmark, H. (2011). Structure and functions of stable SP8 confocal microscope. We are grateful to Ellen LeMosy (Augusta University) as intercellular bridges formed by incomplete cytokinesis during development. well as members of the Weatherbee lab (Yale University) for help with the Commun. Integr. Biol. 4, 1-9. doi:10.4161/ cib.4.1.13550 cryosectioning protocol. We thank Alice Ting for helpful comments and access to Hamada-Kawaguchi, N., Nishida, Y. and Yamamoto, D. (2015). Btk29A-mediated protocols for APEX-mediated biotinylation. We are grateful to Michael Ford and the tyrosine phosphorylation of armadillo/β-catenin promotes ring canal growth in MS Bioworks team for their services. Stocks obtained from the Bloomington drosophila oogenesis. PLOS ONE 10, e0121484. doi:10.1371/journal.pone. Drosophila Stock Center (NIH P40OD018537) were used in this study. We thank 0121484 the TRiP at Harvard Medical School for providing transgenic RNAi fly stocks used Hu, Y., Vinayagam, A., Nand, A., Comjean, A., Chung, V., Hao, T., Mohr, S. E. in this study. and Perrimon, N. (2018). Molecular Interaction Search Tool (MIST): an integrated resource for mining gene and protein interaction data. Nucleic Acids Res. 46, D567-D574. doi:10.1093/nar/gkx1116 Competing interests Hudson, A. M. and Cooley, L. (2010). Drosophila Kelch functions with Cullin-3 to The authors declare no competing or financial interests. organize the ring canal actin cytoskeleton. J. Cell Biol. 188, 29-37. doi:10.1083/ jcb.200909017 Author contributions Hudson, A. M., Mannix, K. M. and Cooley, L. (2015). Actin Cytoskeletal Conceptualization: K.M.M., L.C.; Methodology: K.M.M., R.M.S., R.S.K., L.C.; organization in drosophila germline ring canals depends on Kelch function in a Investigation: K.M.M., R.M.S., R.S.K., L.C.; Resources: K.M.M., R.M.S., R.S.K., Cullin-RING E3 ligase. Genetics 201, 1117-1131. doi:10.1534/genetics.115. L.C.; Writing - original draft: K.M.M.; Writing - review & editing: K.M.M., R.M.S., 181289 R.S.K., L.C.; Visualization: K.M.M., R.M.S., L.C.; Supervision: L.C.; Funding Hudson, A. M., Mannix, K. M., Gerdes, J. A., Kottemann, M. C. and Cooley, L. acquisition: L.C. (2019). Targeted substrate degradation by Kelch controls the actin cytoskeleton DEVELOPMENT

14 TECHNIQUES AND RESOURCES Development (2019) 146, dev176644. doi:10.1242/dev.176644

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