mito-v5-apex  (Addgene inc)

Journal: Development (Cambridge, England)

Article Title: Proximity labeling reveals novel interactomes in live Drosophila tissue

doi: 10.1242/dev.176644

Figure Lengend Snippet: General workflow used to identify RC protein interactomes with APEX. (A) Fluorescence images showing localization of indicated proteins at RCs. Pav was visualized by GFP 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 by mat-GAL4 and visualized by anti-V5 immunofluorescence. F-actin was visualized by TRITC-Phalloidin. (B) Scheme of transgenic APEX fusion constructs generated for this study. Cartoons were made based on known structural models of Pav, Kelch and KREP, with APEX positioned according to where it was fused. No structural data are available for HtsRC. (C) (Left) Example workflow of optimized APEX labeling in live ovary tissue with HtsRC::APEX construct. (Right) Overview of streptavidin purification and MS methods used to identify and validate high-confidence prey. Scale bar: 2 μm.

Article Snippet: The final plasmid was injected into BL#24872 into the attP3B site on chr2L at Rainbow Transgenic Flies, Inc. otu-ovhts::V5::APEX1 The V5::APEX1 coding sequence was amplified from pcDNA3 - mito-V5::APEX1 ( Rhee et al., 2013 ; Addgene Plasmid #42607 ) using primers designed to create a V5::APEX1 fragment flanked by 3′ Xho I and 5′ Not I restriction sites. pCOH - ovhts::GFP ( Petrella et al., 2007 ) was digested with Xho I and Not I to excise GFP and the V5::APEX1 fragment was ligated into the plasmid in-frame and in place of GFP .

Techniques: Fluorescence, Immunofluorescence, Transgenic Assay, Construct, Generated, Labeling, Purification

Journal: Development (Cambridge, England)

Article Title: Proximity labeling reveals novel interactomes in live Drosophila tissue

doi: 10.1242/dev.176644

Figure Lengend Snippet: 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) Levels of 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.

Article Snippet: The final plasmid was injected into BL#24872 into the attP3B site on chr2L at Rainbow Transgenic Flies, Inc. otu-ovhts::V5::APEX1 The V5::APEX1 coding sequence was amplified from pcDNA3 - mito-V5::APEX1 ( Rhee et al., 2013 ; Addgene Plasmid #42607 ) using primers designed to create a V5::APEX1 fragment flanked by 3′ Xho I and 5′ Not I restriction sites. pCOH - ovhts::GFP ( Petrella et al., 2007 ) was digested with Xho I and Not I to excise GFP and the V5::APEX1 fragment was ligated into the plasmid in-frame and in place of GFP .

Techniques: Construct, Expressing, Staining, Western Blot, Mutagenesis

Journal: Development (Cambridge, England)

Article Title: Proximity labeling reveals novel interactomes in live Drosophila tissue

doi: 10.1242/dev.176644

Figure Lengend Snippet: RC-APEX baits biotinylate distinct proteins in live egg chambers. (A-F) Ovaries expressing indicated RC-APEX baits were permeabilized and biotinylated, 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 the specific biotin signal at RCs for the RC-APEX fusions (A′-D′), the unlocalized cytoplasmic biotin signal for GBP::APEX (E′) and the absence of signal in the no-APEX control (F′). (A′) Yellow arrows 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 eluates were analyzed by western blotting. (G) Inputs, unbound fractions and eluates were analyzed by western blotting with streptavidin-HRP to confirm that biotinylated proteins were present. (H-K) Western blot analysis was performed on the 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::Kelch was enriched in the APEX::Kelch eluate, whereas a background endogenous Kelch protein band could be detected in all other samples. (J) HtsRC::APEX and endogenous HtsRC were present in the HtsRC::APEX sample, whereas endogenous HtsRC was highly enriched in the APEX::KREP sample. (K) Filamin was observed only in the APEX::KREP sample. Scale bar: 50 μm.

Article Snippet: The final plasmid was injected into BL#24872 into the attP3B site on chr2L at Rainbow Transgenic Flies, Inc. otu-ovhts::V5::APEX1 The V5::APEX1 coding sequence was amplified from pcDNA3 - mito-V5::APEX1 ( Rhee et al., 2013 ; Addgene Plasmid #42607 ) using primers designed to create a V5::APEX1 fragment flanked by 3′ Xho I and 5′ Not I restriction sites. pCOH - ovhts::GFP ( Petrella et al., 2007 ) was digested with Xho I and Not I to excise GFP and the V5::APEX1 fragment was ligated into the plasmid in-frame and in place of GFP .

Techniques: Expressing, Staining, Labeling, Western Blot

Journal: Development (Cambridge, England)

Article Title: Proximity labeling reveals novel interactomes in live Drosophila tissue

doi: 10.1242/dev.176644

Figure Lengend Snippet: 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).

Article Snippet: The final plasmid was injected into BL#24872 into the attP3B site on chr2L at Rainbow Transgenic Flies, Inc. otu-ovhts::V5::APEX1 The V5::APEX1 coding sequence was amplified from pcDNA3 - mito-V5::APEX1 ( Rhee et al., 2013 ; Addgene Plasmid #42607 ) using primers designed to create a V5::APEX1 fragment flanked by 3′ Xho I and 5′ Not I restriction sites. pCOH - ovhts::GFP ( Petrella et al., 2007 ) was digested with Xho I and Not I to excise GFP and the V5::APEX1 fragment was ligated into the plasmid in-frame and in place of GFP .

Techniques: Generated

Journal: Development (Cambridge, England)

Article Title: Proximity labeling reveals novel interactomes in live Drosophila tissue

doi: 10.1242/dev.176644

Figure Lengend Snippet: 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).

Article Snippet: The final plasmid was injected into BL#24872 into the attP3B site on chr2L at Rainbow Transgenic Flies, Inc. otu-ovhts::V5::APEX1 The V5::APEX1 coding sequence was amplified from pcDNA3 - mito-V5::APEX1 ( Rhee et al., 2013 ; Addgene Plasmid #42607 ) using primers designed to create a V5::APEX1 fragment flanked by 3′ Xho I and 5′ Not I restriction sites. pCOH - ovhts::GFP ( Petrella et al., 2007 ) was digested with Xho I and Not I to excise GFP and the V5::APEX1 fragment was ligated into the plasmid in-frame and in place of GFP .

Techniques: Generated

Journal: Development (Cambridge, England)

Article Title: Proximity labeling reveals novel interactomes in live Drosophila tissue

doi: 10.1242/dev.176644

Figure Lengend Snippet: 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 PPI was 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 5 μm (insets).

Article Snippet: The final plasmid was injected into BL#24872 into the attP3B site on chr2L at Rainbow Transgenic Flies, Inc. otu-ovhts::V5::APEX1 The V5::APEX1 coding sequence was amplified from pcDNA3 - mito-V5::APEX1 ( Rhee et al., 2013 ; Addgene Plasmid #42607 ) using primers designed to create a V5::APEX1 fragment flanked by 3′ Xho I and 5′ Not I restriction sites. pCOH - ovhts::GFP ( Petrella et al., 2007 ) was digested with Xho I and Not I to excise GFP and the V5::APEX1 fragment was ligated into the plasmid in-frame and in place of GFP .

Techniques: In Situ, Proximity Ligation Assay, Ligation

Journal: Development (Cambridge, England)

Article Title: Proximity labeling reveals novel interactomes in live Drosophila tissue

doi: 10.1242/dev.176644

Figure Lengend Snippet: 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 μm in L, 25 μm in K and 50 μm in B-I.

Article Snippet: The final plasmid was injected into BL#24872 into the attP3B site on chr2L at Rainbow Transgenic Flies, Inc. otu-ovhts::V5::APEX1 The V5::APEX1 coding sequence was amplified from pcDNA3 - mito-V5::APEX1 ( Rhee et al., 2013 ; Addgene Plasmid #42607 ) using primers designed to create a V5::APEX1 fragment flanked by 3′ Xho I and 5′ Not I restriction sites. pCOH - ovhts::GFP ( Petrella et al., 2007 ) was digested with Xho I and Not I to excise GFP and the V5::APEX1 fragment was ligated into the plasmid in-frame and in place of GFP .

Techniques: Staining, Marker