glopips bodipy tmr-ptdins(4,5)p2, c16  (Echelon Biosciences)


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    Echelon Biosciences glopips bodipy tmr-ptdins(4,5)p2, c16
    Glopips Bodipy Tmr Ptdins(4,5)P2, C16, supplied by Echelon Biosciences, used in various techniques. Bioz Stars score: 91/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    glopips bodipy tmr-ptdins(4,5)p2, c16  (Echelon Biosciences)


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    Echelon Biosciences glopips bodipy tmr-ptdins(4,5)p2, c16
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    bodipy tmr phosphatidylinositol 4 5 bisphosphate  (Echelon Biosciences)


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    Echelon Biosciences bodipy tmr phosphatidylinositol 4 5 bisphosphate
    ( a–f ) PI(4,5)P 2 retrieves Fwe to bulk endosomes. ( a–e ) TEM images of the boutons of Flag-Fwe-HA rescue ( nSyb-GAL4/UAS-Flag-Fwe-HA in fwe DB25/DB56 at 25°C, a ), HA-Fwe-APEX2 rescue ( nSyb-GAL4/UAS-HA-Fwe-APEX2 in fwe DB25/DB56 at 25°C, b–c ), or HA-Fwe-APEX2 rescue coexpressing Synj ( nSyb-GAL4/UAS-HA-Fwe-APEX2 / UAS-synj in fwe DB25/DB56 at 25°C, d–e ). After high K + stimulation, the boutons were subjected to DAB labeling. ( a ) The Flag-Fwe-HA rescue boutons presented DAB-negative bulk endosomes (red asterisks) and SVs (yellow arrows). ( b and c ) In the HA-Fwe-APEX2 rescue boutons, DAB signals on bulk endosomes (red asterisks) were higher than those on the SVs (yellow arrows). The views in b and c are from different boutons. ( d and e ) Under the condition of Synj overexpression, perturbation of PI(4,5)P 2 microdomain formation predominantly reduced DAB levels on the bulk endosomes (red asterisks). Views in d and e are from different boutons. ( f ) Quantification data for the DAB staining intensity ratio of bulk endosomes to surrounding SVs. The number of bulk endosomes, NMJ boutons, and larvae counted (Flag-Fwe-HA rescue control): Bulk endosomes (n = 33) derived from 5 NMJ boutons of two different larvae; HA-Fwe-APEX2 rescue: Bulk endosomes (n = 47) derived from 6 NMJ boutons of two different larvae; HA-Fwe-APEX2 rescue expressing Synj: Bulk endosomes (n = 59) derived from 9 NMJ boutons of two different larvae. Individual values were shown in graphs. p values: ns, not significant; ****p<0.0001. Mean ± SEM. Scale bar: 200 nm ( a–e ). Statistics: one-way ANOVA with Tukey’s post hoc test. Figure 6—source data 1. Source data for  .
    Bodipy Tmr Phosphatidylinositol 4 5 Bisphosphate, supplied by Echelon Biosciences, used in various techniques. Bioz Stars score: 91/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "A positive feedback loop between Flower and PI(4,5)P 2 at periactive zones controls bulk endocytosis in Drosophila"

    Article Title: A positive feedback loop between Flower and PI(4,5)P 2 at periactive zones controls bulk endocytosis in Drosophila

    Journal: eLife

    doi: 10.7554/eLife.60125

    ( a–f ) PI(4,5)P 2 retrieves Fwe to bulk endosomes. ( a–e ) TEM images of the boutons of Flag-Fwe-HA rescue ( nSyb-GAL4/UAS-Flag-Fwe-HA in fwe DB25/DB56 at 25°C, a ), HA-Fwe-APEX2 rescue ( nSyb-GAL4/UAS-HA-Fwe-APEX2 in fwe DB25/DB56 at 25°C, b–c ), or HA-Fwe-APEX2 rescue coexpressing Synj ( nSyb-GAL4/UAS-HA-Fwe-APEX2 / UAS-synj in fwe DB25/DB56 at 25°C, d–e ). After high K + stimulation, the boutons were subjected to DAB labeling. ( a ) The Flag-Fwe-HA rescue boutons presented DAB-negative bulk endosomes (red asterisks) and SVs (yellow arrows). ( b and c ) In the HA-Fwe-APEX2 rescue boutons, DAB signals on bulk endosomes (red asterisks) were higher than those on the SVs (yellow arrows). The views in b and c are from different boutons. ( d and e ) Under the condition of Synj overexpression, perturbation of PI(4,5)P 2 microdomain formation predominantly reduced DAB levels on the bulk endosomes (red asterisks). Views in d and e are from different boutons. ( f ) Quantification data for the DAB staining intensity ratio of bulk endosomes to surrounding SVs. The number of bulk endosomes, NMJ boutons, and larvae counted (Flag-Fwe-HA rescue control): Bulk endosomes (n = 33) derived from 5 NMJ boutons of two different larvae; HA-Fwe-APEX2 rescue: Bulk endosomes (n = 47) derived from 6 NMJ boutons of two different larvae; HA-Fwe-APEX2 rescue expressing Synj: Bulk endosomes (n = 59) derived from 9 NMJ boutons of two different larvae. Individual values were shown in graphs. p values: ns, not significant; ****p<0.0001. Mean ± SEM. Scale bar: 200 nm ( a–e ). Statistics: one-way ANOVA with Tukey’s post hoc test. Figure 6—source data 1. Source data for  .
    Figure Legend Snippet: ( a–f ) PI(4,5)P 2 retrieves Fwe to bulk endosomes. ( a–e ) TEM images of the boutons of Flag-Fwe-HA rescue ( nSyb-GAL4/UAS-Flag-Fwe-HA in fwe DB25/DB56 at 25°C, a ), HA-Fwe-APEX2 rescue ( nSyb-GAL4/UAS-HA-Fwe-APEX2 in fwe DB25/DB56 at 25°C, b–c ), or HA-Fwe-APEX2 rescue coexpressing Synj ( nSyb-GAL4/UAS-HA-Fwe-APEX2 / UAS-synj in fwe DB25/DB56 at 25°C, d–e ). After high K + stimulation, the boutons were subjected to DAB labeling. ( a ) The Flag-Fwe-HA rescue boutons presented DAB-negative bulk endosomes (red asterisks) and SVs (yellow arrows). ( b and c ) In the HA-Fwe-APEX2 rescue boutons, DAB signals on bulk endosomes (red asterisks) were higher than those on the SVs (yellow arrows). The views in b and c are from different boutons. ( d and e ) Under the condition of Synj overexpression, perturbation of PI(4,5)P 2 microdomain formation predominantly reduced DAB levels on the bulk endosomes (red asterisks). Views in d and e are from different boutons. ( f ) Quantification data for the DAB staining intensity ratio of bulk endosomes to surrounding SVs. The number of bulk endosomes, NMJ boutons, and larvae counted (Flag-Fwe-HA rescue control): Bulk endosomes (n = 33) derived from 5 NMJ boutons of two different larvae; HA-Fwe-APEX2 rescue: Bulk endosomes (n = 47) derived from 6 NMJ boutons of two different larvae; HA-Fwe-APEX2 rescue expressing Synj: Bulk endosomes (n = 59) derived from 9 NMJ boutons of two different larvae. Individual values were shown in graphs. p values: ns, not significant; ****p<0.0001. Mean ± SEM. Scale bar: 200 nm ( a–e ). Statistics: one-way ANOVA with Tukey’s post hoc test. Figure 6—source data 1. Source data for .

    Techniques Used: Labeling, Over Expression, Staining, Derivative Assay, Expressing

    Reducing PI(4,5)P 2 availability suppresses ADBE and subsequent SV reformation. ( a ) A schematic for PI(4,5)P 2 suppression by PLC δ1 -PH-EGFP or Synj expression. ( b ) Expression of Synj reduced presynaptic plasma membrane PI(4,5)P 2 upon high K + treatment. The boutons co-expressing UAS-PLC δ1 -PH-EGFP with UAS-RFP (control) or UAS-synj using nSyb-GAL4 were reared at 25°C and subjected to resting condition (10 min incubation of 5 mM K + /0 mM Ca 2+ ) or high K + stimulation (10 min stimulation of 90 mM K + /2 mM Ca 2+ ), followed by α-GFP immunostaining. Single-plane confocal images of the boutons are shown in  . Quantification data for PLC δ1 -PH-EGFP staining intensity are shown, normalized to the value of the resting condition of controls. ( c ) TEM images of the boutons of controls ( nSyb-GAL4/+ at 29°C), mild PLC δ1 -PH-EGFP expression ( nSyb-GAL4/UAS-PLC δ1 -PH-EGFP at 25°C), high PLC δ1 -PH-EGFP expression ( nSyb-GAL4/UAS-PLC δ1 -PH-EGFP at 29°C), high PLC δ1 -PHS39R-EGFP expression ( nSyb-GAL4/UAS-PLC δ1 -PHS39R-EGFP at 29°C), mild Synj expression ( nSyb-GAL4/UAS-synj at 25°C), or high Synj expression ( nSyb-GAL4/UAS-synj at 29°C). At rest (10 min incubation of 5 mM K + /0 mM Ca 2+ ). High K + (10 min stimulation of 90 mM K + /2 mM Ca 2+ ). 10 min recovery (10 min stimulation of 90 mM K + /2 mM Ca 2+ , followed by 10 min incubation of 5 mM K + /0 mM Ca 2+ ). 20 min recovery (10 min stimulation of 90 mM K + /2 mM Ca 2+ , followed by 20 min incubation of 5 mM K + /0 mM Ca 2+ ). Bulk endosomes (>80 nm in diameter, red asterisks). Mitochondria (mt). Quantification data for total number of bulk endosomes per bouton area ( d ). Individual data values are shown in graphs. p values: ns, not significant; *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001. Mean ± SEM. Scale bar: 500 nm. Statistics: one-way ANOVA with Tukey’s post hoc test.  Figure 2—source data 1. Source data for  .
    Figure Legend Snippet: Reducing PI(4,5)P 2 availability suppresses ADBE and subsequent SV reformation. ( a ) A schematic for PI(4,5)P 2 suppression by PLC δ1 -PH-EGFP or Synj expression. ( b ) Expression of Synj reduced presynaptic plasma membrane PI(4,5)P 2 upon high K + treatment. The boutons co-expressing UAS-PLC δ1 -PH-EGFP with UAS-RFP (control) or UAS-synj using nSyb-GAL4 were reared at 25°C and subjected to resting condition (10 min incubation of 5 mM K + /0 mM Ca 2+ ) or high K + stimulation (10 min stimulation of 90 mM K + /2 mM Ca 2+ ), followed by α-GFP immunostaining. Single-plane confocal images of the boutons are shown in . Quantification data for PLC δ1 -PH-EGFP staining intensity are shown, normalized to the value of the resting condition of controls. ( c ) TEM images of the boutons of controls ( nSyb-GAL4/+ at 29°C), mild PLC δ1 -PH-EGFP expression ( nSyb-GAL4/UAS-PLC δ1 -PH-EGFP at 25°C), high PLC δ1 -PH-EGFP expression ( nSyb-GAL4/UAS-PLC δ1 -PH-EGFP at 29°C), high PLC δ1 -PHS39R-EGFP expression ( nSyb-GAL4/UAS-PLC δ1 -PHS39R-EGFP at 29°C), mild Synj expression ( nSyb-GAL4/UAS-synj at 25°C), or high Synj expression ( nSyb-GAL4/UAS-synj at 29°C). At rest (10 min incubation of 5 mM K + /0 mM Ca 2+ ). High K + (10 min stimulation of 90 mM K + /2 mM Ca 2+ ). 10 min recovery (10 min stimulation of 90 mM K + /2 mM Ca 2+ , followed by 10 min incubation of 5 mM K + /0 mM Ca 2+ ). 20 min recovery (10 min stimulation of 90 mM K + /2 mM Ca 2+ , followed by 20 min incubation of 5 mM K + /0 mM Ca 2+ ). Bulk endosomes (>80 nm in diameter, red asterisks). Mitochondria (mt). Quantification data for total number of bulk endosomes per bouton area ( d ). Individual data values are shown in graphs. p values: ns, not significant; *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001. Mean ± SEM. Scale bar: 500 nm. Statistics: one-way ANOVA with Tukey’s post hoc test. Figure 2—source data 1. Source data for .

    Techniques Used: Expressing, Incubation, Immunostaining, Staining

    ( a–c ) PI(4,5)P 2 binds Fwe. ( a ) A schematic of the Fwe structure and a BRET assay. Stars highlight conserved lysine ( K ) and arginine ( R ) in juxta-transmembrane regions: N-terminus ( N ), middle-domain ( M ), and C-terminus ( C ). Drosophila melanogaster (Dm). Mus Musculus (Ms). Human Sapiens (Hs). N-terminal fusion of Nluc to Fwe allows detection of PI(4,5)P 2 binding. A 1D4 epitope was used for protein purification. ( b ) Nluc-Fwe-1D4 in micelles excites BODIPY-TMR-conjugated PI(4,5)P 2 to emit BRET signal, which is decreased by competitive cold PI(4,5)P 2 (1 mM). Alanine substitution of positively-charged residues in all regions (N/M/C > A), both middle-domain and C-terminus (M/C > A), or N-terminus (N > A) reduced BRET signals. ( c ) Corresponding PI(4,5)P 2 binding ability was calculated by subtracting the signal values of N/M/C > A, M/C > A or N > A from that for WT. Quantification data was normalized to the signal value of all mutations (N/M/C). ( d ) PI(4,5)P 2 controls Fwe channel activity. Snapshot Ca 2+ images of the boutons (arrows) expressing lexAop2-GCaMP6f using vglut-lexA. fwe mutant ( fwe DB25/DB56 ). HA-Fwe[WT]-APEX2 rescue ( nSyb-GAL4/UAS-HA-Fwe[WT]-APEX2 in fwe DB25/DB56 ). HA-Fwe[K29A/R33A]-APEX2 rescue ( nSyb-GAL4/UAS-HA-Fwe[K29A/R33A]-APEX2 in fwe DB25/DB56 ). The larvae were reared at 25°C. Images were taken in the fifth minute for one minute after high K + stimulation. ( e ) Quantification data for evoked Ca 2+ level, normalized to the value of HA-Fwe-APEX2 rescue larvae. ( f ) Single-plane confocal images of the boutons expressing UAS-PLC δ1 -PH-EGFP . The larvae were reared at 25°C. After resting conditions or high K + stimulation, the boutons were stained with α-GFP. ( g ) Quantification data for the PLC δ1 -PH-EGFP staining intensity, normalized to the value of the resting condition of HA-Fwe-APEX2 rescue larvae. Individual data values are shown in graphs. p values: ns, not significant; *p<0.05; ***p<0.001; ****p<0.0001. Mean ± SEM. Scale bar: 2 µm ( d, f ). Statistics: Student t -test ( b ). One-way ANOVA with Tukey’s post hoc test ( b, c, e, g ). Figure 4—source data 1. Source data for  .
    Figure Legend Snippet: ( a–c ) PI(4,5)P 2 binds Fwe. ( a ) A schematic of the Fwe structure and a BRET assay. Stars highlight conserved lysine ( K ) and arginine ( R ) in juxta-transmembrane regions: N-terminus ( N ), middle-domain ( M ), and C-terminus ( C ). Drosophila melanogaster (Dm). Mus Musculus (Ms). Human Sapiens (Hs). N-terminal fusion of Nluc to Fwe allows detection of PI(4,5)P 2 binding. A 1D4 epitope was used for protein purification. ( b ) Nluc-Fwe-1D4 in micelles excites BODIPY-TMR-conjugated PI(4,5)P 2 to emit BRET signal, which is decreased by competitive cold PI(4,5)P 2 (1 mM). Alanine substitution of positively-charged residues in all regions (N/M/C > A), both middle-domain and C-terminus (M/C > A), or N-terminus (N > A) reduced BRET signals. ( c ) Corresponding PI(4,5)P 2 binding ability was calculated by subtracting the signal values of N/M/C > A, M/C > A or N > A from that for WT. Quantification data was normalized to the signal value of all mutations (N/M/C). ( d ) PI(4,5)P 2 controls Fwe channel activity. Snapshot Ca 2+ images of the boutons (arrows) expressing lexAop2-GCaMP6f using vglut-lexA. fwe mutant ( fwe DB25/DB56 ). HA-Fwe[WT]-APEX2 rescue ( nSyb-GAL4/UAS-HA-Fwe[WT]-APEX2 in fwe DB25/DB56 ). HA-Fwe[K29A/R33A]-APEX2 rescue ( nSyb-GAL4/UAS-HA-Fwe[K29A/R33A]-APEX2 in fwe DB25/DB56 ). The larvae were reared at 25°C. Images were taken in the fifth minute for one minute after high K + stimulation. ( e ) Quantification data for evoked Ca 2+ level, normalized to the value of HA-Fwe-APEX2 rescue larvae. ( f ) Single-plane confocal images of the boutons expressing UAS-PLC δ1 -PH-EGFP . The larvae were reared at 25°C. After resting conditions or high K + stimulation, the boutons were stained with α-GFP. ( g ) Quantification data for the PLC δ1 -PH-EGFP staining intensity, normalized to the value of the resting condition of HA-Fwe-APEX2 rescue larvae. Individual data values are shown in graphs. p values: ns, not significant; *p<0.05; ***p<0.001; ****p<0.0001. Mean ± SEM. Scale bar: 2 µm ( d, f ). Statistics: Student t -test ( b ). One-way ANOVA with Tukey’s post hoc test ( b, c, e, g ). Figure 4—source data 1. Source data for .

    Techniques Used: Bioluminescence Resonance Energy Transfer, Binding Assay, Protein Purification, Activity Assay, Expressing, Mutagenesis, Staining


    Figure Legend Snippet:

    Techniques Used: Mutagenesis, Binding Assay, Stable Transfection, Recombinant, In Situ, Construct, Expressing, Sequencing, Software

    c-45m16a  (Echelon Biosciences)


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    glopips bodipy  (Echelon Biosciences)


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    c16 c45m16a  (Echelon Biosciences)


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    Echelon Biosciences glopips bodipy tmr-ptdins(4,5)p2, c16
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    Echelon Biosciences bodipy tmr phosphatidylinositol 4 5 bisphosphate
    ( a–f ) PI(4,5)P 2 retrieves Fwe to bulk endosomes. ( a–e ) TEM images of the boutons of Flag-Fwe-HA rescue ( nSyb-GAL4/UAS-Flag-Fwe-HA in fwe DB25/DB56 at 25°C, a ), HA-Fwe-APEX2 rescue ( nSyb-GAL4/UAS-HA-Fwe-APEX2 in fwe DB25/DB56 at 25°C, b–c ), or HA-Fwe-APEX2 rescue coexpressing Synj ( nSyb-GAL4/UAS-HA-Fwe-APEX2 / UAS-synj in fwe DB25/DB56 at 25°C, d–e ). After high K + stimulation, the boutons were subjected to DAB labeling. ( a ) The Flag-Fwe-HA rescue boutons presented DAB-negative bulk endosomes (red asterisks) and SVs (yellow arrows). ( b and c ) In the HA-Fwe-APEX2 rescue boutons, DAB signals on bulk endosomes (red asterisks) were higher than those on the SVs (yellow arrows). The views in b and c are from different boutons. ( d and e ) Under the condition of Synj overexpression, perturbation of PI(4,5)P 2 microdomain formation predominantly reduced DAB levels on the bulk endosomes (red asterisks). Views in d and e are from different boutons. ( f ) Quantification data for the DAB staining intensity ratio of bulk endosomes to surrounding SVs. The number of bulk endosomes, NMJ boutons, and larvae counted (Flag-Fwe-HA rescue control): Bulk endosomes (n = 33) derived from 5 NMJ boutons of two different larvae; HA-Fwe-APEX2 rescue: Bulk endosomes (n = 47) derived from 6 NMJ boutons of two different larvae; HA-Fwe-APEX2 rescue expressing Synj: Bulk endosomes (n = 59) derived from 9 NMJ boutons of two different larvae. Individual values were shown in graphs. p values: ns, not significant; ****p<0.0001. Mean ± SEM. Scale bar: 200 nm ( a–e ). Statistics: one-way ANOVA with Tukey’s post hoc test. Figure 6—source data 1. Source data for  .
    Bodipy Tmr Phosphatidylinositol 4 5 Bisphosphate, supplied by Echelon Biosciences, used in various techniques. Bioz Stars score: 91/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Echelon Biosciences c-45m16a
    ( a–f ) PI(4,5)P 2 retrieves Fwe to bulk endosomes. ( a–e ) TEM images of the boutons of Flag-Fwe-HA rescue ( nSyb-GAL4/UAS-Flag-Fwe-HA in fwe DB25/DB56 at 25°C, a ), HA-Fwe-APEX2 rescue ( nSyb-GAL4/UAS-HA-Fwe-APEX2 in fwe DB25/DB56 at 25°C, b–c ), or HA-Fwe-APEX2 rescue coexpressing Synj ( nSyb-GAL4/UAS-HA-Fwe-APEX2 / UAS-synj in fwe DB25/DB56 at 25°C, d–e ). After high K + stimulation, the boutons were subjected to DAB labeling. ( a ) The Flag-Fwe-HA rescue boutons presented DAB-negative bulk endosomes (red asterisks) and SVs (yellow arrows). ( b and c ) In the HA-Fwe-APEX2 rescue boutons, DAB signals on bulk endosomes (red asterisks) were higher than those on the SVs (yellow arrows). The views in b and c are from different boutons. ( d and e ) Under the condition of Synj overexpression, perturbation of PI(4,5)P 2 microdomain formation predominantly reduced DAB levels on the bulk endosomes (red asterisks). Views in d and e are from different boutons. ( f ) Quantification data for the DAB staining intensity ratio of bulk endosomes to surrounding SVs. The number of bulk endosomes, NMJ boutons, and larvae counted (Flag-Fwe-HA rescue control): Bulk endosomes (n = 33) derived from 5 NMJ boutons of two different larvae; HA-Fwe-APEX2 rescue: Bulk endosomes (n = 47) derived from 6 NMJ boutons of two different larvae; HA-Fwe-APEX2 rescue expressing Synj: Bulk endosomes (n = 59) derived from 9 NMJ boutons of two different larvae. Individual values were shown in graphs. p values: ns, not significant; ****p<0.0001. Mean ± SEM. Scale bar: 200 nm ( a–e ). Statistics: one-way ANOVA with Tukey’s post hoc test. Figure 6—source data 1. Source data for  .
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    Echelon Biosciences glopips bodipy
    ( a–f ) PI(4,5)P 2 retrieves Fwe to bulk endosomes. ( a–e ) TEM images of the boutons of Flag-Fwe-HA rescue ( nSyb-GAL4/UAS-Flag-Fwe-HA in fwe DB25/DB56 at 25°C, a ), HA-Fwe-APEX2 rescue ( nSyb-GAL4/UAS-HA-Fwe-APEX2 in fwe DB25/DB56 at 25°C, b–c ), or HA-Fwe-APEX2 rescue coexpressing Synj ( nSyb-GAL4/UAS-HA-Fwe-APEX2 / UAS-synj in fwe DB25/DB56 at 25°C, d–e ). After high K + stimulation, the boutons were subjected to DAB labeling. ( a ) The Flag-Fwe-HA rescue boutons presented DAB-negative bulk endosomes (red asterisks) and SVs (yellow arrows). ( b and c ) In the HA-Fwe-APEX2 rescue boutons, DAB signals on bulk endosomes (red asterisks) were higher than those on the SVs (yellow arrows). The views in b and c are from different boutons. ( d and e ) Under the condition of Synj overexpression, perturbation of PI(4,5)P 2 microdomain formation predominantly reduced DAB levels on the bulk endosomes (red asterisks). Views in d and e are from different boutons. ( f ) Quantification data for the DAB staining intensity ratio of bulk endosomes to surrounding SVs. The number of bulk endosomes, NMJ boutons, and larvae counted (Flag-Fwe-HA rescue control): Bulk endosomes (n = 33) derived from 5 NMJ boutons of two different larvae; HA-Fwe-APEX2 rescue: Bulk endosomes (n = 47) derived from 6 NMJ boutons of two different larvae; HA-Fwe-APEX2 rescue expressing Synj: Bulk endosomes (n = 59) derived from 9 NMJ boutons of two different larvae. Individual values were shown in graphs. p values: ns, not significant; ****p<0.0001. Mean ± SEM. Scale bar: 200 nm ( a–e ). Statistics: one-way ANOVA with Tukey’s post hoc test. Figure 6—source data 1. Source data for  .
    Glopips Bodipy, supplied by Echelon Biosciences, used in various techniques. Bioz Stars score: 91/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Echelon Biosciences c16 c45m16a
    ( a–f ) PI(4,5)P 2 retrieves Fwe to bulk endosomes. ( a–e ) TEM images of the boutons of Flag-Fwe-HA rescue ( nSyb-GAL4/UAS-Flag-Fwe-HA in fwe DB25/DB56 at 25°C, a ), HA-Fwe-APEX2 rescue ( nSyb-GAL4/UAS-HA-Fwe-APEX2 in fwe DB25/DB56 at 25°C, b–c ), or HA-Fwe-APEX2 rescue coexpressing Synj ( nSyb-GAL4/UAS-HA-Fwe-APEX2 / UAS-synj in fwe DB25/DB56 at 25°C, d–e ). After high K + stimulation, the boutons were subjected to DAB labeling. ( a ) The Flag-Fwe-HA rescue boutons presented DAB-negative bulk endosomes (red asterisks) and SVs (yellow arrows). ( b and c ) In the HA-Fwe-APEX2 rescue boutons, DAB signals on bulk endosomes (red asterisks) were higher than those on the SVs (yellow arrows). The views in b and c are from different boutons. ( d and e ) Under the condition of Synj overexpression, perturbation of PI(4,5)P 2 microdomain formation predominantly reduced DAB levels on the bulk endosomes (red asterisks). Views in d and e are from different boutons. ( f ) Quantification data for the DAB staining intensity ratio of bulk endosomes to surrounding SVs. The number of bulk endosomes, NMJ boutons, and larvae counted (Flag-Fwe-HA rescue control): Bulk endosomes (n = 33) derived from 5 NMJ boutons of two different larvae; HA-Fwe-APEX2 rescue: Bulk endosomes (n = 47) derived from 6 NMJ boutons of two different larvae; HA-Fwe-APEX2 rescue expressing Synj: Bulk endosomes (n = 59) derived from 9 NMJ boutons of two different larvae. Individual values were shown in graphs. p values: ns, not significant; ****p<0.0001. Mean ± SEM. Scale bar: 200 nm ( a–e ). Statistics: one-way ANOVA with Tukey’s post hoc test. Figure 6—source data 1. Source data for  .
    C16 C45m16a, supplied by Echelon Biosciences, used in various techniques. Bioz Stars score: 91/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    ( a–f ) PI(4,5)P 2 retrieves Fwe to bulk endosomes. ( a–e ) TEM images of the boutons of Flag-Fwe-HA rescue ( nSyb-GAL4/UAS-Flag-Fwe-HA in fwe DB25/DB56 at 25°C, a ), HA-Fwe-APEX2 rescue ( nSyb-GAL4/UAS-HA-Fwe-APEX2 in fwe DB25/DB56 at 25°C, b–c ), or HA-Fwe-APEX2 rescue coexpressing Synj ( nSyb-GAL4/UAS-HA-Fwe-APEX2 / UAS-synj in fwe DB25/DB56 at 25°C, d–e ). After high K + stimulation, the boutons were subjected to DAB labeling. ( a ) The Flag-Fwe-HA rescue boutons presented DAB-negative bulk endosomes (red asterisks) and SVs (yellow arrows). ( b and c ) In the HA-Fwe-APEX2 rescue boutons, DAB signals on bulk endosomes (red asterisks) were higher than those on the SVs (yellow arrows). The views in b and c are from different boutons. ( d and e ) Under the condition of Synj overexpression, perturbation of PI(4,5)P 2 microdomain formation predominantly reduced DAB levels on the bulk endosomes (red asterisks). Views in d and e are from different boutons. ( f ) Quantification data for the DAB staining intensity ratio of bulk endosomes to surrounding SVs. The number of bulk endosomes, NMJ boutons, and larvae counted (Flag-Fwe-HA rescue control): Bulk endosomes (n = 33) derived from 5 NMJ boutons of two different larvae; HA-Fwe-APEX2 rescue: Bulk endosomes (n = 47) derived from 6 NMJ boutons of two different larvae; HA-Fwe-APEX2 rescue expressing Synj: Bulk endosomes (n = 59) derived from 9 NMJ boutons of two different larvae. Individual values were shown in graphs. p values: ns, not significant; ****p<0.0001. Mean ± SEM. Scale bar: 200 nm ( a–e ). Statistics: one-way ANOVA with Tukey’s post hoc test. Figure 6—source data 1. Source data for  .
    Labeling With Glopip, supplied by Echelon Biosciences, used in various techniques. Bioz Stars score: 91/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    ( a–f ) PI(4,5)P 2 retrieves Fwe to bulk endosomes. ( a–e ) TEM images of the boutons of Flag-Fwe-HA rescue ( nSyb-GAL4/UAS-Flag-Fwe-HA in fwe DB25/DB56 at 25°C, a ), HA-Fwe-APEX2 rescue ( nSyb-GAL4/UAS-HA-Fwe-APEX2 in fwe DB25/DB56 at 25°C, b–c ), or HA-Fwe-APEX2 rescue coexpressing Synj ( nSyb-GAL4/UAS-HA-Fwe-APEX2 / UAS-synj in fwe DB25/DB56 at 25°C, d–e ). After high K + stimulation, the boutons were subjected to DAB labeling. ( a ) The Flag-Fwe-HA rescue boutons presented DAB-negative bulk endosomes (red asterisks) and SVs (yellow arrows). ( b and c ) In the HA-Fwe-APEX2 rescue boutons, DAB signals on bulk endosomes (red asterisks) were higher than those on the SVs (yellow arrows). The views in b and c are from different boutons. ( d and e ) Under the condition of Synj overexpression, perturbation of PI(4,5)P 2 microdomain formation predominantly reduced DAB levels on the bulk endosomes (red asterisks). Views in d and e are from different boutons. ( f ) Quantification data for the DAB staining intensity ratio of bulk endosomes to surrounding SVs. The number of bulk endosomes, NMJ boutons, and larvae counted (Flag-Fwe-HA rescue control): Bulk endosomes (n = 33) derived from 5 NMJ boutons of two different larvae; HA-Fwe-APEX2 rescue: Bulk endosomes (n = 47) derived from 6 NMJ boutons of two different larvae; HA-Fwe-APEX2 rescue expressing Synj: Bulk endosomes (n = 59) derived from 9 NMJ boutons of two different larvae. Individual values were shown in graphs. p values: ns, not significant; ****p<0.0001. Mean ± SEM. Scale bar: 200 nm ( a–e ). Statistics: one-way ANOVA with Tukey’s post hoc test. Figure 6—source data 1. Source data for  .
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    Image Search Results


    ( a–f ) PI(4,5)P 2 retrieves Fwe to bulk endosomes. ( a–e ) TEM images of the boutons of Flag-Fwe-HA rescue ( nSyb-GAL4/UAS-Flag-Fwe-HA in fwe DB25/DB56 at 25°C, a ), HA-Fwe-APEX2 rescue ( nSyb-GAL4/UAS-HA-Fwe-APEX2 in fwe DB25/DB56 at 25°C, b–c ), or HA-Fwe-APEX2 rescue coexpressing Synj ( nSyb-GAL4/UAS-HA-Fwe-APEX2 / UAS-synj in fwe DB25/DB56 at 25°C, d–e ). After high K + stimulation, the boutons were subjected to DAB labeling. ( a ) The Flag-Fwe-HA rescue boutons presented DAB-negative bulk endosomes (red asterisks) and SVs (yellow arrows). ( b and c ) In the HA-Fwe-APEX2 rescue boutons, DAB signals on bulk endosomes (red asterisks) were higher than those on the SVs (yellow arrows). The views in b and c are from different boutons. ( d and e ) Under the condition of Synj overexpression, perturbation of PI(4,5)P 2 microdomain formation predominantly reduced DAB levels on the bulk endosomes (red asterisks). Views in d and e are from different boutons. ( f ) Quantification data for the DAB staining intensity ratio of bulk endosomes to surrounding SVs. The number of bulk endosomes, NMJ boutons, and larvae counted (Flag-Fwe-HA rescue control): Bulk endosomes (n = 33) derived from 5 NMJ boutons of two different larvae; HA-Fwe-APEX2 rescue: Bulk endosomes (n = 47) derived from 6 NMJ boutons of two different larvae; HA-Fwe-APEX2 rescue expressing Synj: Bulk endosomes (n = 59) derived from 9 NMJ boutons of two different larvae. Individual values were shown in graphs. p values: ns, not significant; ****p<0.0001. Mean ± SEM. Scale bar: 200 nm ( a–e ). Statistics: one-way ANOVA with Tukey’s post hoc test. Figure 6—source data 1. Source data for  .

    Journal: eLife

    Article Title: A positive feedback loop between Flower and PI(4,5)P 2 at periactive zones controls bulk endocytosis in Drosophila

    doi: 10.7554/eLife.60125

    Figure Lengend Snippet: ( a–f ) PI(4,5)P 2 retrieves Fwe to bulk endosomes. ( a–e ) TEM images of the boutons of Flag-Fwe-HA rescue ( nSyb-GAL4/UAS-Flag-Fwe-HA in fwe DB25/DB56 at 25°C, a ), HA-Fwe-APEX2 rescue ( nSyb-GAL4/UAS-HA-Fwe-APEX2 in fwe DB25/DB56 at 25°C, b–c ), or HA-Fwe-APEX2 rescue coexpressing Synj ( nSyb-GAL4/UAS-HA-Fwe-APEX2 / UAS-synj in fwe DB25/DB56 at 25°C, d–e ). After high K + stimulation, the boutons were subjected to DAB labeling. ( a ) The Flag-Fwe-HA rescue boutons presented DAB-negative bulk endosomes (red asterisks) and SVs (yellow arrows). ( b and c ) In the HA-Fwe-APEX2 rescue boutons, DAB signals on bulk endosomes (red asterisks) were higher than those on the SVs (yellow arrows). The views in b and c are from different boutons. ( d and e ) Under the condition of Synj overexpression, perturbation of PI(4,5)P 2 microdomain formation predominantly reduced DAB levels on the bulk endosomes (red asterisks). Views in d and e are from different boutons. ( f ) Quantification data for the DAB staining intensity ratio of bulk endosomes to surrounding SVs. The number of bulk endosomes, NMJ boutons, and larvae counted (Flag-Fwe-HA rescue control): Bulk endosomes (n = 33) derived from 5 NMJ boutons of two different larvae; HA-Fwe-APEX2 rescue: Bulk endosomes (n = 47) derived from 6 NMJ boutons of two different larvae; HA-Fwe-APEX2 rescue expressing Synj: Bulk endosomes (n = 59) derived from 9 NMJ boutons of two different larvae. Individual values were shown in graphs. p values: ns, not significant; ****p<0.0001. Mean ± SEM. Scale bar: 200 nm ( a–e ). Statistics: one-way ANOVA with Tukey’s post hoc test. Figure 6—source data 1. Source data for .

    Article Snippet: Chemical compound, drug , BODIPY-TMR Phosphatidylinositol 4,5-bisphosphate , Echelon Bioscience , Cat#C-45M16A , Used in .

    Techniques: Labeling, Over Expression, Staining, Derivative Assay, Expressing

    Reducing PI(4,5)P 2 availability suppresses ADBE and subsequent SV reformation. ( a ) A schematic for PI(4,5)P 2 suppression by PLC δ1 -PH-EGFP or Synj expression. ( b ) Expression of Synj reduced presynaptic plasma membrane PI(4,5)P 2 upon high K + treatment. The boutons co-expressing UAS-PLC δ1 -PH-EGFP with UAS-RFP (control) or UAS-synj using nSyb-GAL4 were reared at 25°C and subjected to resting condition (10 min incubation of 5 mM K + /0 mM Ca 2+ ) or high K + stimulation (10 min stimulation of 90 mM K + /2 mM Ca 2+ ), followed by α-GFP immunostaining. Single-plane confocal images of the boutons are shown in  . Quantification data for PLC δ1 -PH-EGFP staining intensity are shown, normalized to the value of the resting condition of controls. ( c ) TEM images of the boutons of controls ( nSyb-GAL4/+ at 29°C), mild PLC δ1 -PH-EGFP expression ( nSyb-GAL4/UAS-PLC δ1 -PH-EGFP at 25°C), high PLC δ1 -PH-EGFP expression ( nSyb-GAL4/UAS-PLC δ1 -PH-EGFP at 29°C), high PLC δ1 -PHS39R-EGFP expression ( nSyb-GAL4/UAS-PLC δ1 -PHS39R-EGFP at 29°C), mild Synj expression ( nSyb-GAL4/UAS-synj at 25°C), or high Synj expression ( nSyb-GAL4/UAS-synj at 29°C). At rest (10 min incubation of 5 mM K + /0 mM Ca 2+ ). High K + (10 min stimulation of 90 mM K + /2 mM Ca 2+ ). 10 min recovery (10 min stimulation of 90 mM K + /2 mM Ca 2+ , followed by 10 min incubation of 5 mM K + /0 mM Ca 2+ ). 20 min recovery (10 min stimulation of 90 mM K + /2 mM Ca 2+ , followed by 20 min incubation of 5 mM K + /0 mM Ca 2+ ). Bulk endosomes (>80 nm in diameter, red asterisks). Mitochondria (mt). Quantification data for total number of bulk endosomes per bouton area ( d ). Individual data values are shown in graphs. p values: ns, not significant; *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001. Mean ± SEM. Scale bar: 500 nm. Statistics: one-way ANOVA with Tukey’s post hoc test.  Figure 2—source data 1. Source data for  .

    Journal: eLife

    Article Title: A positive feedback loop between Flower and PI(4,5)P 2 at periactive zones controls bulk endocytosis in Drosophila

    doi: 10.7554/eLife.60125

    Figure Lengend Snippet: Reducing PI(4,5)P 2 availability suppresses ADBE and subsequent SV reformation. ( a ) A schematic for PI(4,5)P 2 suppression by PLC δ1 -PH-EGFP or Synj expression. ( b ) Expression of Synj reduced presynaptic plasma membrane PI(4,5)P 2 upon high K + treatment. The boutons co-expressing UAS-PLC δ1 -PH-EGFP with UAS-RFP (control) or UAS-synj using nSyb-GAL4 were reared at 25°C and subjected to resting condition (10 min incubation of 5 mM K + /0 mM Ca 2+ ) or high K + stimulation (10 min stimulation of 90 mM K + /2 mM Ca 2+ ), followed by α-GFP immunostaining. Single-plane confocal images of the boutons are shown in . Quantification data for PLC δ1 -PH-EGFP staining intensity are shown, normalized to the value of the resting condition of controls. ( c ) TEM images of the boutons of controls ( nSyb-GAL4/+ at 29°C), mild PLC δ1 -PH-EGFP expression ( nSyb-GAL4/UAS-PLC δ1 -PH-EGFP at 25°C), high PLC δ1 -PH-EGFP expression ( nSyb-GAL4/UAS-PLC δ1 -PH-EGFP at 29°C), high PLC δ1 -PHS39R-EGFP expression ( nSyb-GAL4/UAS-PLC δ1 -PHS39R-EGFP at 29°C), mild Synj expression ( nSyb-GAL4/UAS-synj at 25°C), or high Synj expression ( nSyb-GAL4/UAS-synj at 29°C). At rest (10 min incubation of 5 mM K + /0 mM Ca 2+ ). High K + (10 min stimulation of 90 mM K + /2 mM Ca 2+ ). 10 min recovery (10 min stimulation of 90 mM K + /2 mM Ca 2+ , followed by 10 min incubation of 5 mM K + /0 mM Ca 2+ ). 20 min recovery (10 min stimulation of 90 mM K + /2 mM Ca 2+ , followed by 20 min incubation of 5 mM K + /0 mM Ca 2+ ). Bulk endosomes (>80 nm in diameter, red asterisks). Mitochondria (mt). Quantification data for total number of bulk endosomes per bouton area ( d ). Individual data values are shown in graphs. p values: ns, not significant; *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001. Mean ± SEM. Scale bar: 500 nm. Statistics: one-way ANOVA with Tukey’s post hoc test. Figure 2—source data 1. Source data for .

    Article Snippet: Chemical compound, drug , BODIPY-TMR Phosphatidylinositol 4,5-bisphosphate , Echelon Bioscience , Cat#C-45M16A , Used in .

    Techniques: Expressing, Incubation, Immunostaining, Staining

    ( a–c ) PI(4,5)P 2 binds Fwe. ( a ) A schematic of the Fwe structure and a BRET assay. Stars highlight conserved lysine ( K ) and arginine ( R ) in juxta-transmembrane regions: N-terminus ( N ), middle-domain ( M ), and C-terminus ( C ). Drosophila melanogaster (Dm). Mus Musculus (Ms). Human Sapiens (Hs). N-terminal fusion of Nluc to Fwe allows detection of PI(4,5)P 2 binding. A 1D4 epitope was used for protein purification. ( b ) Nluc-Fwe-1D4 in micelles excites BODIPY-TMR-conjugated PI(4,5)P 2 to emit BRET signal, which is decreased by competitive cold PI(4,5)P 2 (1 mM). Alanine substitution of positively-charged residues in all regions (N/M/C > A), both middle-domain and C-terminus (M/C > A), or N-terminus (N > A) reduced BRET signals. ( c ) Corresponding PI(4,5)P 2 binding ability was calculated by subtracting the signal values of N/M/C > A, M/C > A or N > A from that for WT. Quantification data was normalized to the signal value of all mutations (N/M/C). ( d ) PI(4,5)P 2 controls Fwe channel activity. Snapshot Ca 2+ images of the boutons (arrows) expressing lexAop2-GCaMP6f using vglut-lexA. fwe mutant ( fwe DB25/DB56 ). HA-Fwe[WT]-APEX2 rescue ( nSyb-GAL4/UAS-HA-Fwe[WT]-APEX2 in fwe DB25/DB56 ). HA-Fwe[K29A/R33A]-APEX2 rescue ( nSyb-GAL4/UAS-HA-Fwe[K29A/R33A]-APEX2 in fwe DB25/DB56 ). The larvae were reared at 25°C. Images were taken in the fifth minute for one minute after high K + stimulation. ( e ) Quantification data for evoked Ca 2+ level, normalized to the value of HA-Fwe-APEX2 rescue larvae. ( f ) Single-plane confocal images of the boutons expressing UAS-PLC δ1 -PH-EGFP . The larvae were reared at 25°C. After resting conditions or high K + stimulation, the boutons were stained with α-GFP. ( g ) Quantification data for the PLC δ1 -PH-EGFP staining intensity, normalized to the value of the resting condition of HA-Fwe-APEX2 rescue larvae. Individual data values are shown in graphs. p values: ns, not significant; *p<0.05; ***p<0.001; ****p<0.0001. Mean ± SEM. Scale bar: 2 µm ( d, f ). Statistics: Student t -test ( b ). One-way ANOVA with Tukey’s post hoc test ( b, c, e, g ). Figure 4—source data 1. Source data for  .

    Journal: eLife

    Article Title: A positive feedback loop between Flower and PI(4,5)P 2 at periactive zones controls bulk endocytosis in Drosophila

    doi: 10.7554/eLife.60125

    Figure Lengend Snippet: ( a–c ) PI(4,5)P 2 binds Fwe. ( a ) A schematic of the Fwe structure and a BRET assay. Stars highlight conserved lysine ( K ) and arginine ( R ) in juxta-transmembrane regions: N-terminus ( N ), middle-domain ( M ), and C-terminus ( C ). Drosophila melanogaster (Dm). Mus Musculus (Ms). Human Sapiens (Hs). N-terminal fusion of Nluc to Fwe allows detection of PI(4,5)P 2 binding. A 1D4 epitope was used for protein purification. ( b ) Nluc-Fwe-1D4 in micelles excites BODIPY-TMR-conjugated PI(4,5)P 2 to emit BRET signal, which is decreased by competitive cold PI(4,5)P 2 (1 mM). Alanine substitution of positively-charged residues in all regions (N/M/C > A), both middle-domain and C-terminus (M/C > A), or N-terminus (N > A) reduced BRET signals. ( c ) Corresponding PI(4,5)P 2 binding ability was calculated by subtracting the signal values of N/M/C > A, M/C > A or N > A from that for WT. Quantification data was normalized to the signal value of all mutations (N/M/C). ( d ) PI(4,5)P 2 controls Fwe channel activity. Snapshot Ca 2+ images of the boutons (arrows) expressing lexAop2-GCaMP6f using vglut-lexA. fwe mutant ( fwe DB25/DB56 ). HA-Fwe[WT]-APEX2 rescue ( nSyb-GAL4/UAS-HA-Fwe[WT]-APEX2 in fwe DB25/DB56 ). HA-Fwe[K29A/R33A]-APEX2 rescue ( nSyb-GAL4/UAS-HA-Fwe[K29A/R33A]-APEX2 in fwe DB25/DB56 ). The larvae were reared at 25°C. Images were taken in the fifth minute for one minute after high K + stimulation. ( e ) Quantification data for evoked Ca 2+ level, normalized to the value of HA-Fwe-APEX2 rescue larvae. ( f ) Single-plane confocal images of the boutons expressing UAS-PLC δ1 -PH-EGFP . The larvae were reared at 25°C. After resting conditions or high K + stimulation, the boutons were stained with α-GFP. ( g ) Quantification data for the PLC δ1 -PH-EGFP staining intensity, normalized to the value of the resting condition of HA-Fwe-APEX2 rescue larvae. Individual data values are shown in graphs. p values: ns, not significant; *p<0.05; ***p<0.001; ****p<0.0001. Mean ± SEM. Scale bar: 2 µm ( d, f ). Statistics: Student t -test ( b ). One-way ANOVA with Tukey’s post hoc test ( b, c, e, g ). Figure 4—source data 1. Source data for .

    Article Snippet: Chemical compound, drug , BODIPY-TMR Phosphatidylinositol 4,5-bisphosphate , Echelon Bioscience , Cat#C-45M16A , Used in .

    Techniques: Bioluminescence Resonance Energy Transfer, Binding Assay, Protein Purification, Activity Assay, Expressing, Mutagenesis, Staining

    Journal: eLife

    Article Title: A positive feedback loop between Flower and PI(4,5)P 2 at periactive zones controls bulk endocytosis in Drosophila

    doi: 10.7554/eLife.60125

    Figure Lengend Snippet:

    Article Snippet: Chemical compound, drug , BODIPY-TMR Phosphatidylinositol 4,5-bisphosphate , Echelon Bioscience , Cat#C-45M16A , Used in .

    Techniques: Mutagenesis, Binding Assay, Stable Transfection, Recombinant, In Situ, Construct, Expressing, Sequencing, Software