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A Tracking autophagy with RFP-GFP-LC3 marker shows transfer of LC3 to autophagolysomes, visible as red spots due to acidic quenching of GFP. B Numbers of phagophores or autophagosomes per cells, detected as yellow spots are increased in Dnm2 -/- cells. C Numbers of autophagolysosomes, detected as red spots, are dramatically increased in Dnm2 -/- cells treated with CCCP. D Colocalization of RFP-LC3 with GFP-Lamp1 is similarly increased in Dnm2 -/- cells. E, F Colocalization of <t>GFP-VAMP7</t> with RFP-LC3 is increased in Dnm2 -/- cells and increases more after CCCP treatment. G, H Colocalization of GFP-Fip200 with RFP-LC3 is increased in Dnm2 -/- cells and increases more after CCCP treatment. I, J There is also increased colocalization of RFP-Atg9 and GFP-LAMP1 after treatment with CCCP. Cells were incubated with pepstatin and E64d to prevent Atg9 digestion in lysosomes. For panels B, C, F, H and J, 50 cells were counted per experiment, n= 3, unpaired students’ t-test. Scale bar is 10 μm for whole cells and 5 μm for enlarged portions. K Quantification of Atg9 protein levels in wildtype and Dnm2 -/- cells. Levels decrease in Dnm2 -/- cells after treatment with CCCP, but this decrease is partially prevented by Bafilomycin A, suggesting that Atg9 is degraded by autophagy in Dnm2 -/- cells. Band intensities were determined with Licor software, n = 4, unpaired Students’ t-test. L Genetic representation of the proposed roles for Dnm2 and EndoB1 during autophagy. The initial stages of phagophore formation during which EndoB1, as part of the Vps34 complex, promotes the role of Dnm2 in the retrieval of Atg9 through fission from phagophores (1a). At this stage, Dnm2 also plays an inhibitory role, preventing fusion to late endosomes or lysosomes (1b), so that further maturation of the phagophore only occurs when growth of the phagophore is complete. M A later stage during which EndoB1 switches from a positive to a negative function, suppressing the inhibitory effects of Dnm2 on fusion to late endosomes and lysosomes (2).
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Fusion of cytotoxic granules with the plasma membrane is insensitive to Tetanus toxin. (A) Bead-stimulated human CD8+ T cells transfected with GFP or TeNT-GFP as indicated. VAMP2 and <t>VAMP7</t> protein levels were determined by Western blot analysis 12–16 h after transfection. (B) Expression of VAMP2 and VAMP7 protein levels relative to GAPDH in CTLs transfected with GFP or TeNT-GFP as indicated. Graphs represent means [ N = 3, *** p < 0.001 (Student's t -test)]. (C) Bead-stimulated human CTLs co-transfected with either GFP or TeNT-GFP along with granzyme B-mCherry and imaged 12 h after transfection. Representative live-cell TIRFM images of CTLs in contact with an anti-CD3 coated coverslip. Fusion events indicated with open arrowheads. (D) Mean percentage of cytotoxic granule fusion in cells transfected with either GFP ( n = 50) or TeNT-GFP ( n = 40), p = 0.704 (Student's t -test). (E) Mean average number of granules fused per cell over time in the TIRF plane in cells transfected with either GFP ( n = 20) or TeNT-GFP ( n = 15), p = 0.939 (Student's t -test). Bars show mean ± SEM. Scale bar, 5 μm.
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Fusion of cytotoxic granules with the plasma membrane is insensitive to Tetanus toxin. (A) Bead-stimulated human CD8+ T cells transfected with GFP or TeNT-GFP as indicated. VAMP2 and <t>VAMP7</t> protein levels were determined by Western blot analysis 12–16 h after transfection. (B) Expression of VAMP2 and VAMP7 protein levels relative to GAPDH in CTLs transfected with GFP or TeNT-GFP as indicated. Graphs represent means [ N = 3, *** p < 0.001 (Student's t -test)]. (C) Bead-stimulated human CTLs co-transfected with either GFP or TeNT-GFP along with granzyme B-mCherry and imaged 12 h after transfection. Representative live-cell TIRFM images of CTLs in contact with an anti-CD3 coated coverslip. Fusion events indicated with open arrowheads. (D) Mean percentage of cytotoxic granule fusion in cells transfected with either GFP ( n = 50) or TeNT-GFP ( n = 40), p = 0.704 (Student's t -test). (E) Mean average number of granules fused per cell over time in the TIRF plane in cells transfected with either GFP ( n = 20) or TeNT-GFP ( n = 15), p = 0.939 (Student's t -test). Bars show mean ± SEM. Scale bar, 5 μm.
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Autophagosomal Stx17 colocalizes significantly with SNAP29 and <t>VAMP7.</t> a , representative confocal and gSTED images of membrane-bound Stx17 rings ( left ) surrounding LC3-labeled autophagosomes ( center ) and a merge of these images ( right ) from rapamycin-treated HeLa cells. Scale bars , 500 nm. b , intensity line profile through the punctum in a , demonstrating membrane localization of Stx17 resolved by gSTED. c–e , single-channel and merged images of rapamycin-treated HeLa cells expressing Stx17 with either SNAP29 ( c ), VAMP8 ( d ), or VAMP7 ( e ) and a merge of both channels. Scale bars , 10 μm for full field and 3 μm for zoomed regions. f–h , frequency scatter plots of reported single-pixel intensity values in each channel presented in c–e. i , quantification of SNAP29 and Stx17 colocalization using Pearson's correlation coefficient ( PCC ) demonstrating significantly higher correlation than in negative controls generated by single-channel 90° rotation. j and k , quantification of Stx17 puncta colocal with VAMP8 ( j ) or VAMP7 ( k ) puncta using Manders correlation coefficient. All box-and-whisker plots represent the median ( central line ), 25th and 75th quartile ( box ), and the minimum and maximum value ( whiskers ). ns , nonsignificant ( p ≥ 0.05); ***, p = 0.0001–0.0005; ****, p < 0.0001.
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A Tracking autophagy with RFP-GFP-LC3 marker shows transfer of LC3 to autophagolysomes, visible as red spots due to acidic quenching of GFP. B Numbers of phagophores or autophagosomes per cells, detected as yellow spots are increased in Dnm2 -/- cells. C Numbers of autophagolysosomes, detected as red spots, are dramatically increased in Dnm2 -/- cells treated with CCCP. D Colocalization of RFP-LC3 with GFP-Lamp1 is similarly increased in Dnm2 -/- cells. E, F Colocalization of GFP-VAMP7 with RFP-LC3 is increased in Dnm2 -/- cells and increases more after CCCP treatment. G, H Colocalization of GFP-Fip200 with RFP-LC3 is increased in Dnm2 -/- cells and increases more after CCCP treatment. I, J There is also increased colocalization of RFP-Atg9 and GFP-LAMP1 after treatment with CCCP. Cells were incubated with pepstatin and E64d to prevent Atg9 digestion in lysosomes. For panels B, C, F, H and J, 50 cells were counted per experiment, n= 3, unpaired students’ t-test. Scale bar is 10 μm for whole cells and 5 μm for enlarged portions. K Quantification of Atg9 protein levels in wildtype and Dnm2 -/- cells. Levels decrease in Dnm2 -/- cells after treatment with CCCP, but this decrease is partially prevented by Bafilomycin A, suggesting that Atg9 is degraded by autophagy in Dnm2 -/- cells. Band intensities were determined with Licor software, n = 4, unpaired Students’ t-test. L Genetic representation of the proposed roles for Dnm2 and EndoB1 during autophagy. The initial stages of phagophore formation during which EndoB1, as part of the Vps34 complex, promotes the role of Dnm2 in the retrieval of Atg9 through fission from phagophores (1a). At this stage, Dnm2 also plays an inhibitory role, preventing fusion to late endosomes or lysosomes (1b), so that further maturation of the phagophore only occurs when growth of the phagophore is complete. M A later stage during which EndoB1 switches from a positive to a negative function, suppressing the inhibitory effects of Dnm2 on fusion to late endosomes and lysosomes (2).

Journal: bioRxiv

Article Title: Dynamin-2 controls phagophore maturation

doi: 10.1101/241901

Figure Lengend Snippet: A Tracking autophagy with RFP-GFP-LC3 marker shows transfer of LC3 to autophagolysomes, visible as red spots due to acidic quenching of GFP. B Numbers of phagophores or autophagosomes per cells, detected as yellow spots are increased in Dnm2 -/- cells. C Numbers of autophagolysosomes, detected as red spots, are dramatically increased in Dnm2 -/- cells treated with CCCP. D Colocalization of RFP-LC3 with GFP-Lamp1 is similarly increased in Dnm2 -/- cells. E, F Colocalization of GFP-VAMP7 with RFP-LC3 is increased in Dnm2 -/- cells and increases more after CCCP treatment. G, H Colocalization of GFP-Fip200 with RFP-LC3 is increased in Dnm2 -/- cells and increases more after CCCP treatment. I, J There is also increased colocalization of RFP-Atg9 and GFP-LAMP1 after treatment with CCCP. Cells were incubated with pepstatin and E64d to prevent Atg9 digestion in lysosomes. For panels B, C, F, H and J, 50 cells were counted per experiment, n= 3, unpaired students’ t-test. Scale bar is 10 μm for whole cells and 5 μm for enlarged portions. K Quantification of Atg9 protein levels in wildtype and Dnm2 -/- cells. Levels decrease in Dnm2 -/- cells after treatment with CCCP, but this decrease is partially prevented by Bafilomycin A, suggesting that Atg9 is degraded by autophagy in Dnm2 -/- cells. Band intensities were determined with Licor software, n = 4, unpaired Students’ t-test. L Genetic representation of the proposed roles for Dnm2 and EndoB1 during autophagy. The initial stages of phagophore formation during which EndoB1, as part of the Vps34 complex, promotes the role of Dnm2 in the retrieval of Atg9 through fission from phagophores (1a). At this stage, Dnm2 also plays an inhibitory role, preventing fusion to late endosomes or lysosomes (1b), so that further maturation of the phagophore only occurs when growth of the phagophore is complete. M A later stage during which EndoB1 switches from a positive to a negative function, suppressing the inhibitory effects of Dnm2 on fusion to late endosomes and lysosomes (2).

Article Snippet: Addgene provided pcDNA3-mRuby2 (#40260), GFP-C1-PLCdelta-PH (#21179), Dnm2-EGFP (#34686), Dnm2-mCherryN1 (#27689), ptfLC3 (#21074), pMXs-puro-RFP-ATG9A (#60609), pEGFP-LC3 (# 24920), GFP-Rab7A (#61803), mTaqBFP2-ER-5 (#55294), pcDNA3-mRuby2 (#40260), GFP-Atg2 (#36456), GFP-Fip200 (#38192), GFP-VAMP7 (#42316) and pmRFP-LC3 (#21073).

Techniques: Marker, Incubation, Software

Fusion of cytotoxic granules with the plasma membrane is insensitive to Tetanus toxin. (A) Bead-stimulated human CD8+ T cells transfected with GFP or TeNT-GFP as indicated. VAMP2 and VAMP7 protein levels were determined by Western blot analysis 12–16 h after transfection. (B) Expression of VAMP2 and VAMP7 protein levels relative to GAPDH in CTLs transfected with GFP or TeNT-GFP as indicated. Graphs represent means [ N = 3, *** p < 0.001 (Student's t -test)]. (C) Bead-stimulated human CTLs co-transfected with either GFP or TeNT-GFP along with granzyme B-mCherry and imaged 12 h after transfection. Representative live-cell TIRFM images of CTLs in contact with an anti-CD3 coated coverslip. Fusion events indicated with open arrowheads. (D) Mean percentage of cytotoxic granule fusion in cells transfected with either GFP ( n = 50) or TeNT-GFP ( n = 40), p = 0.704 (Student's t -test). (E) Mean average number of granules fused per cell over time in the TIRF plane in cells transfected with either GFP ( n = 20) or TeNT-GFP ( n = 15), p = 0.939 (Student's t -test). Bars show mean ± SEM. Scale bar, 5 μm.

Journal: Frontiers in Immunology

Article Title: Cytotoxic Granule Exocytosis From Human Cytotoxic T Lymphocytes Is Mediated by VAMP7

doi: 10.3389/fimmu.2019.01855

Figure Lengend Snippet: Fusion of cytotoxic granules with the plasma membrane is insensitive to Tetanus toxin. (A) Bead-stimulated human CD8+ T cells transfected with GFP or TeNT-GFP as indicated. VAMP2 and VAMP7 protein levels were determined by Western blot analysis 12–16 h after transfection. (B) Expression of VAMP2 and VAMP7 protein levels relative to GAPDH in CTLs transfected with GFP or TeNT-GFP as indicated. Graphs represent means [ N = 3, *** p < 0.001 (Student's t -test)]. (C) Bead-stimulated human CTLs co-transfected with either GFP or TeNT-GFP along with granzyme B-mCherry and imaged 12 h after transfection. Representative live-cell TIRFM images of CTLs in contact with an anti-CD3 coated coverslip. Fusion events indicated with open arrowheads. (D) Mean percentage of cytotoxic granule fusion in cells transfected with either GFP ( n = 50) or TeNT-GFP ( n = 40), p = 0.704 (Student's t -test). (E) Mean average number of granules fused per cell over time in the TIRF plane in cells transfected with either GFP ( n = 20) or TeNT-GFP ( n = 15), p = 0.939 (Student's t -test). Bars show mean ± SEM. Scale bar, 5 μm.

Article Snippet: VAMP7 coding sequence was amplified from pEGFP-VAMP7 plasmid (42316; Addgene) with forward primer 5′-ATA TAC GGG GTA CCG CCG CCA CCA TGG CGA TTC TTT TTG CT-3′ and reverse primer 5′-ATA TAC CGG AAT TCT TTC TTC ACA CAG CTT GG-3′ and inserted in frame with C-terminal mCherry with forward primer 5′-ATA TAC CCA AGC TTA TGG TGA GCA AGG GCG AG-3′ and reverse primer 5′-ATA TAC GCG GAT CCT TAC TTG TAC AGC TCG TCC AT-3′ or inserted in frame with pHuji with forward primer 5′-ATG TAT ACC CAA GCT TAT GGT GAG CAA GGG CGA G-3′ and reverse primer 5′-ATG TAT ACG CGG ATC CTT ACT TGT ACA GCT CGT C-3′ in pMAX containing GGSGGSGGS linker.

Techniques: Transfection, Western Blot, Expressing

VAMP7 shows a high degree of co-localization with cytotoxic granules. (A) SIM images of bead-stimulated human CD8+ T cells co-transfected with either granzyme B-mCherry or granzyme B-mTFP along with the v-SNAREs mVAMP2, VAMP3, VAMP4, VAMP7, and VAMP8 constructs. Overlay of both channels displayed that only VAMP7 co-localizes with the lytic granule marker granzyme B. (B) Pearson's and (C) Manders' overlap coefficients for co-localization of VAMP3 ( n = 16), VAMP4 ( n = 14), VAMP7 ( n = 13), VAMP8 ( n = 9), and VAMP2 ( n = 14) with granzyme B are given in the text. (D) Bead-stimulated human CD8+ T cells transfected with EGFP-VAMP7 or VAMP7-pHuji and immunolabeled with Alexa647-conjugated anti-granzyme B antibody. (E) The corresponding Pearson's and (F) Manders' overlap coefficients for colocalization of EGFP-VAMP7 ( n = 12) or VAMP7-pHuji ( n = 11) with granzyme B are given in the text. Data are shown as mean ± SEM. Scale bar, 5 μm. (G) Anti CD3/CD28 bead-stimulated human CD8+ T cells transfected with VAMP7-mCherry and incubated on either poly-L-ornithine or anti CD3 antibody coated coverslips for 15 min, fixed and immunolabeled with LAT antibody. (H) The corresponding Pearson's and (I) Manders' overlap coefficients for colocalization of VAMP7-mCherry with LAT on Poly-L-Ornithine coated coverslips ( n = 11) or anti CD3/CD28 antibody coated coverslips ( n = 10) are given in the text. Data are shown as mean ± SEM. Scale bar, 5 μm.

Journal: Frontiers in Immunology

Article Title: Cytotoxic Granule Exocytosis From Human Cytotoxic T Lymphocytes Is Mediated by VAMP7

doi: 10.3389/fimmu.2019.01855

Figure Lengend Snippet: VAMP7 shows a high degree of co-localization with cytotoxic granules. (A) SIM images of bead-stimulated human CD8+ T cells co-transfected with either granzyme B-mCherry or granzyme B-mTFP along with the v-SNAREs mVAMP2, VAMP3, VAMP4, VAMP7, and VAMP8 constructs. Overlay of both channels displayed that only VAMP7 co-localizes with the lytic granule marker granzyme B. (B) Pearson's and (C) Manders' overlap coefficients for co-localization of VAMP3 ( n = 16), VAMP4 ( n = 14), VAMP7 ( n = 13), VAMP8 ( n = 9), and VAMP2 ( n = 14) with granzyme B are given in the text. (D) Bead-stimulated human CD8+ T cells transfected with EGFP-VAMP7 or VAMP7-pHuji and immunolabeled with Alexa647-conjugated anti-granzyme B antibody. (E) The corresponding Pearson's and (F) Manders' overlap coefficients for colocalization of EGFP-VAMP7 ( n = 12) or VAMP7-pHuji ( n = 11) with granzyme B are given in the text. Data are shown as mean ± SEM. Scale bar, 5 μm. (G) Anti CD3/CD28 bead-stimulated human CD8+ T cells transfected with VAMP7-mCherry and incubated on either poly-L-ornithine or anti CD3 antibody coated coverslips for 15 min, fixed and immunolabeled with LAT antibody. (H) The corresponding Pearson's and (I) Manders' overlap coefficients for colocalization of VAMP7-mCherry with LAT on Poly-L-Ornithine coated coverslips ( n = 11) or anti CD3/CD28 antibody coated coverslips ( n = 10) are given in the text. Data are shown as mean ± SEM. Scale bar, 5 μm.

Article Snippet: VAMP7 coding sequence was amplified from pEGFP-VAMP7 plasmid (42316; Addgene) with forward primer 5′-ATA TAC GGG GTA CCG CCG CCA CCA TGG CGA TTC TTT TTG CT-3′ and reverse primer 5′-ATA TAC CGG AAT TCT TTC TTC ACA CAG CTT GG-3′ and inserted in frame with C-terminal mCherry with forward primer 5′-ATA TAC CCA AGC TTA TGG TGA GCA AGG GCG AG-3′ and reverse primer 5′-ATA TAC GCG GAT CCT TAC TTG TAC AGC TCG TCC AT-3′ or inserted in frame with pHuji with forward primer 5′-ATG TAT ACC CAA GCT TAT GGT GAG CAA GGG CGA G-3′ and reverse primer 5′-ATG TAT ACG CGG ATC CTT ACT TGT ACA GCT CGT C-3′ in pMAX containing GGSGGSGGS linker.

Techniques: Transfection, Construct, Marker, Immunolabeling, Incubation

VAMP7 polarizes to the IS on the same granules as granzyme B and perforin. (A) SIM images of SEA-pulsed human CD8+ T cells transfected with VAMP7-pHuji construct and conjugated with SEA pulsed RAJI cells and fixed after 5, 10, and 15 min, respectively. CTLs were permeabilized and immunolabeled with either Alexa647-conjugated granzyme B or Alexa647-conjugated perforin antibody. (B,C) The corresponding Pearson's overlap coefficients for co-localization of VAMP7-mCherry with granzyme B ( n = 12) and VAMP7-pHuji with perforin ( n = 11) are given in the text. Data are shown as mean ± SEM. Scale bar, 5 μm. (D) SIM images of SEA-pulsed human CD8+ T cells transfected with VAMP2-pHuji, EGFP-VAMP3, and EGFP-VAMP4 constructs and conjugated with SEA pulsed RAJI cells and fixed after 15 min. CTLs were permeabilized and immunolabeled with either Alexa647-conjugated granzyme B or Alexa647-conjugated perforin antibody. (E,F) The corresponding Pearson's overlap coefficients for co-localization of VAMP2, VAMP3, and VAMP4 with granzyme B ( n = 15) or perforin ( n = 16) are given in the text. Data are shown as mean ± SEM. Scale bar, 5 μm.

Journal: Frontiers in Immunology

Article Title: Cytotoxic Granule Exocytosis From Human Cytotoxic T Lymphocytes Is Mediated by VAMP7

doi: 10.3389/fimmu.2019.01855

Figure Lengend Snippet: VAMP7 polarizes to the IS on the same granules as granzyme B and perforin. (A) SIM images of SEA-pulsed human CD8+ T cells transfected with VAMP7-pHuji construct and conjugated with SEA pulsed RAJI cells and fixed after 5, 10, and 15 min, respectively. CTLs were permeabilized and immunolabeled with either Alexa647-conjugated granzyme B or Alexa647-conjugated perforin antibody. (B,C) The corresponding Pearson's overlap coefficients for co-localization of VAMP7-mCherry with granzyme B ( n = 12) and VAMP7-pHuji with perforin ( n = 11) are given in the text. Data are shown as mean ± SEM. Scale bar, 5 μm. (D) SIM images of SEA-pulsed human CD8+ T cells transfected with VAMP2-pHuji, EGFP-VAMP3, and EGFP-VAMP4 constructs and conjugated with SEA pulsed RAJI cells and fixed after 15 min. CTLs were permeabilized and immunolabeled with either Alexa647-conjugated granzyme B or Alexa647-conjugated perforin antibody. (E,F) The corresponding Pearson's overlap coefficients for co-localization of VAMP2, VAMP3, and VAMP4 with granzyme B ( n = 15) or perforin ( n = 16) are given in the text. Data are shown as mean ± SEM. Scale bar, 5 μm.

Article Snippet: VAMP7 coding sequence was amplified from pEGFP-VAMP7 plasmid (42316; Addgene) with forward primer 5′-ATA TAC GGG GTA CCG CCG CCA CCA TGG CGA TTC TTT TTG CT-3′ and reverse primer 5′-ATA TAC CGG AAT TCT TTC TTC ACA CAG CTT GG-3′ and inserted in frame with C-terminal mCherry with forward primer 5′-ATA TAC CCA AGC TTA TGG TGA GCA AGG GCG AG-3′ and reverse primer 5′-ATA TAC GCG GAT CCT TAC TTG TAC AGC TCG TCC AT-3′ or inserted in frame with pHuji with forward primer 5′-ATG TAT ACC CAA GCT TAT GGT GAG CAA GGG CGA G-3′ and reverse primer 5′-ATG TAT ACG CGG ATC CTT ACT TGT ACA GCT CGT C-3′ in pMAX containing GGSGGSGGS linker.

Techniques: Transfection, Construct, Immunolabeling

VAMP7 fuses at the plasma membrane along with granzyme B while VAMP8 fuses earlier and more frequently. (A) Bead stimulated human CTLs co-transfected with granzyme B-mTFP and VAMP7-pHuji constructs and imaged 12 h after transfection. Selected live-cell TIRFM images of granzyme B-mTFP and VAMP7-pHuji in a transfected CTL in contact with an anti-CD3 coated coverslip (GzmB, green, upper panel; VAMP7, magenta, middle panel; merged, lower panel). (B,C) Graphs depict the mean fluorescence intensity of individual mTFP and pHuji vesicles, respectively, over time. Experiments were repeated with three individual donors ( n = 15 cells). Scale bar, 5 μm. (D) Bead stimulated human CTLs co-transfected with VAMP7-mCherry and VAMP8-mTFP constructs and imaged 12 h after transfection. Selected live-cell TIRFM images of VAMP7-mCherry and VAMP8-mTFP in a transfected CTL in contact with an anti-CD3 coated coverslip (VAMP7, magenta, upper panel; VAMP8, green, middle panel; merged, lower panel). (E) Exemplary, individual fusion events (arrows) of VAMP7- (upper panel) and VAMP8-containing vesicles (lower panel) at the indicated time of recording. Acquisition frequency 5 Hz. (F) Mean cumulative fusion events in the TIRF plane per cell [ N = 3, n = 39, *** p < 0.001 ( t -test)]. (G) Mean average number of VAMP7- and VAMP8-containing vesicles in the TIRF plane per cell in the first 3.5 min of measurements [ N = 3, n = 39, *** p < 0.001 (Mann-Whitney U -test)]. Data are shown as mean ± SEM. Scale bar, 5 μm.

Journal: Frontiers in Immunology

Article Title: Cytotoxic Granule Exocytosis From Human Cytotoxic T Lymphocytes Is Mediated by VAMP7

doi: 10.3389/fimmu.2019.01855

Figure Lengend Snippet: VAMP7 fuses at the plasma membrane along with granzyme B while VAMP8 fuses earlier and more frequently. (A) Bead stimulated human CTLs co-transfected with granzyme B-mTFP and VAMP7-pHuji constructs and imaged 12 h after transfection. Selected live-cell TIRFM images of granzyme B-mTFP and VAMP7-pHuji in a transfected CTL in contact with an anti-CD3 coated coverslip (GzmB, green, upper panel; VAMP7, magenta, middle panel; merged, lower panel). (B,C) Graphs depict the mean fluorescence intensity of individual mTFP and pHuji vesicles, respectively, over time. Experiments were repeated with three individual donors ( n = 15 cells). Scale bar, 5 μm. (D) Bead stimulated human CTLs co-transfected with VAMP7-mCherry and VAMP8-mTFP constructs and imaged 12 h after transfection. Selected live-cell TIRFM images of VAMP7-mCherry and VAMP8-mTFP in a transfected CTL in contact with an anti-CD3 coated coverslip (VAMP7, magenta, upper panel; VAMP8, green, middle panel; merged, lower panel). (E) Exemplary, individual fusion events (arrows) of VAMP7- (upper panel) and VAMP8-containing vesicles (lower panel) at the indicated time of recording. Acquisition frequency 5 Hz. (F) Mean cumulative fusion events in the TIRF plane per cell [ N = 3, n = 39, *** p < 0.001 ( t -test)]. (G) Mean average number of VAMP7- and VAMP8-containing vesicles in the TIRF plane per cell in the first 3.5 min of measurements [ N = 3, n = 39, *** p < 0.001 (Mann-Whitney U -test)]. Data are shown as mean ± SEM. Scale bar, 5 μm.

Article Snippet: VAMP7 coding sequence was amplified from pEGFP-VAMP7 plasmid (42316; Addgene) with forward primer 5′-ATA TAC GGG GTA CCG CCG CCA CCA TGG CGA TTC TTT TTG CT-3′ and reverse primer 5′-ATA TAC CGG AAT TCT TTC TTC ACA CAG CTT GG-3′ and inserted in frame with C-terminal mCherry with forward primer 5′-ATA TAC CCA AGC TTA TGG TGA GCA AGG GCG AG-3′ and reverse primer 5′-ATA TAC GCG GAT CCT TAC TTG TAC AGC TCG TCC AT-3′ or inserted in frame with pHuji with forward primer 5′-ATG TAT ACC CAA GCT TAT GGT GAG CAA GGG CGA G-3′ and reverse primer 5′-ATG TAT ACG CGG ATC CTT ACT TGT ACA GCT CGT C-3′ in pMAX containing GGSGGSGGS linker.

Techniques: Transfection, Construct, Fluorescence, MANN-WHITNEY

Knockdown of VAMP7 strongly reduces fusion of cytotoxic granules at the IS. (A) Lysates from bead stimulated human CD8+ T cells transfected with either control or VAMP7 siRNAs (1 or 2, respectively) and blotted for VAMP7 (top) and GAPDH (bottom) as loading control. (B) Quantification of VAMP7 protein expression (in % normalized to control siRNA-treated CTLs) performed by densitometry. Bars indicate SEMs. [VAMP7-siRNA1, N = 3; *** p < 0.001 and VAMP7-siRNA2, N = 3; *** p < 0.001 ( t -test)]. (C) Human CD8+ T cells co-transfected with granzyme B-mCherry along with either ns-siRNA or VAMP7-siRNA1 or VAMP7-siRNA2 and imaged 12 h after transfection. Selected live-cell TIRF microscopy images of granzyme B-mCherry in a transfected CTL in contact with an anti-CD3 coated coverslip. Fusion events are indicated with open circles (three frames shown per granule fused). (D) Mean percentage of cytotoxic granule fusion in cells transfected with either ns-siRNA ( n = 66 and n = 59, respectively) or VAMP7-siRNA1 [ n = 91; ** p < 0.01 ( t -test)] or VAMP7-siRNA2 [ n = 72; *** p < 0.001 ( t -test)]. (E) Mean average number of granules fused over time in the TIRF plane per cell p = 0.206 ( t -test) for VAMP7-siRNA1 and ** p < 0.01 ( t -test) for VAMP7-siRNA2. Bars indicate mean ± SEM. Scale bar, 5 μm. (F) Calcein-based killing assay for CTLs transfected with either ns-siRNA, VAMP7-siRNA1, or VAMP7-siRNA2. Experiments were carried out in duplicate [ N = 4; *** p < 0.001 ( t -test)].

Journal: Frontiers in Immunology

Article Title: Cytotoxic Granule Exocytosis From Human Cytotoxic T Lymphocytes Is Mediated by VAMP7

doi: 10.3389/fimmu.2019.01855

Figure Lengend Snippet: Knockdown of VAMP7 strongly reduces fusion of cytotoxic granules at the IS. (A) Lysates from bead stimulated human CD8+ T cells transfected with either control or VAMP7 siRNAs (1 or 2, respectively) and blotted for VAMP7 (top) and GAPDH (bottom) as loading control. (B) Quantification of VAMP7 protein expression (in % normalized to control siRNA-treated CTLs) performed by densitometry. Bars indicate SEMs. [VAMP7-siRNA1, N = 3; *** p < 0.001 and VAMP7-siRNA2, N = 3; *** p < 0.001 ( t -test)]. (C) Human CD8+ T cells co-transfected with granzyme B-mCherry along with either ns-siRNA or VAMP7-siRNA1 or VAMP7-siRNA2 and imaged 12 h after transfection. Selected live-cell TIRF microscopy images of granzyme B-mCherry in a transfected CTL in contact with an anti-CD3 coated coverslip. Fusion events are indicated with open circles (three frames shown per granule fused). (D) Mean percentage of cytotoxic granule fusion in cells transfected with either ns-siRNA ( n = 66 and n = 59, respectively) or VAMP7-siRNA1 [ n = 91; ** p < 0.01 ( t -test)] or VAMP7-siRNA2 [ n = 72; *** p < 0.001 ( t -test)]. (E) Mean average number of granules fused over time in the TIRF plane per cell p = 0.206 ( t -test) for VAMP7-siRNA1 and ** p < 0.01 ( t -test) for VAMP7-siRNA2. Bars indicate mean ± SEM. Scale bar, 5 μm. (F) Calcein-based killing assay for CTLs transfected with either ns-siRNA, VAMP7-siRNA1, or VAMP7-siRNA2. Experiments were carried out in duplicate [ N = 4; *** p < 0.001 ( t -test)].

Article Snippet: VAMP7 coding sequence was amplified from pEGFP-VAMP7 plasmid (42316; Addgene) with forward primer 5′-ATA TAC GGG GTA CCG CCG CCA CCA TGG CGA TTC TTT TTG CT-3′ and reverse primer 5′-ATA TAC CGG AAT TCT TTC TTC ACA CAG CTT GG-3′ and inserted in frame with C-terminal mCherry with forward primer 5′-ATA TAC CCA AGC TTA TGG TGA GCA AGG GCG AG-3′ and reverse primer 5′-ATA TAC GCG GAT CCT TAC TTG TAC AGC TCG TCC AT-3′ or inserted in frame with pHuji with forward primer 5′-ATG TAT ACC CAA GCT TAT GGT GAG CAA GGG CGA G-3′ and reverse primer 5′-ATG TAT ACG CGG ATC CTT ACT TGT ACA GCT CGT C-3′ in pMAX containing GGSGGSGGS linker.

Techniques: Transfection, Expressing, Microscopy

VAMP7 forms a SNARE complex with SNAP-23 and Syntaxin11. (A) Model illustrating the formation of SNARE complex between VAMP7 (v-SNARE), SNAP-23, and Syntaxin11 (t- SNARE) during cytotoxic granule fusion in human CD8+ T cells. (B) Different Twin-Strep-tag fusion constructs used for pulldown assay. Twin-Strep-tag was fused at the C terminus of VAMP7 and at the N terminus of Syntaxin11 with a (GGS)x3 linker. (C) Western blot of bead stimulated human CD8+ T cells transfected with Twin-Strep-tag-tagged VAMP7, immuno-precipitated with anti-FLAG antibody and detected with antibodies against FLAG, SNAP-23, and Syntaxin11. (D) Western blot of bead stimulated human CD8+ T cells transfected with Twin-Strep-tag-tagged Syntaxin11, immuno-precipitated with anti-Syntaxin antibody and detected with antibodies against Strep-tag, SNAP-23, VAMP7, and VAMP2. As control, cells were transfected with Twin-Strep-tag construct. Ten percentage of the lysates were loaded as input. (E,F) Densitometric quantification of the Western blots shown in (C,D) , respectively.

Journal: Frontiers in Immunology

Article Title: Cytotoxic Granule Exocytosis From Human Cytotoxic T Lymphocytes Is Mediated by VAMP7

doi: 10.3389/fimmu.2019.01855

Figure Lengend Snippet: VAMP7 forms a SNARE complex with SNAP-23 and Syntaxin11. (A) Model illustrating the formation of SNARE complex between VAMP7 (v-SNARE), SNAP-23, and Syntaxin11 (t- SNARE) during cytotoxic granule fusion in human CD8+ T cells. (B) Different Twin-Strep-tag fusion constructs used for pulldown assay. Twin-Strep-tag was fused at the C terminus of VAMP7 and at the N terminus of Syntaxin11 with a (GGS)x3 linker. (C) Western blot of bead stimulated human CD8+ T cells transfected with Twin-Strep-tag-tagged VAMP7, immuno-precipitated with anti-FLAG antibody and detected with antibodies against FLAG, SNAP-23, and Syntaxin11. (D) Western blot of bead stimulated human CD8+ T cells transfected with Twin-Strep-tag-tagged Syntaxin11, immuno-precipitated with anti-Syntaxin antibody and detected with antibodies against Strep-tag, SNAP-23, VAMP7, and VAMP2. As control, cells were transfected with Twin-Strep-tag construct. Ten percentage of the lysates were loaded as input. (E,F) Densitometric quantification of the Western blots shown in (C,D) , respectively.

Article Snippet: VAMP7 coding sequence was amplified from pEGFP-VAMP7 plasmid (42316; Addgene) with forward primer 5′-ATA TAC GGG GTA CCG CCG CCA CCA TGG CGA TTC TTT TTG CT-3′ and reverse primer 5′-ATA TAC CGG AAT TCT TTC TTC ACA CAG CTT GG-3′ and inserted in frame with C-terminal mCherry with forward primer 5′-ATA TAC CCA AGC TTA TGG TGA GCA AGG GCG AG-3′ and reverse primer 5′-ATA TAC GCG GAT CCT TAC TTG TAC AGC TCG TCC AT-3′ or inserted in frame with pHuji with forward primer 5′-ATG TAT ACC CAA GCT TAT GGT GAG CAA GGG CGA G-3′ and reverse primer 5′-ATG TAT ACG CGG ATC CTT ACT TGT ACA GCT CGT C-3′ in pMAX containing GGSGGSGGS linker.

Techniques: Strep-tag, Construct, Western Blot, Transfection

Fusion of cytotoxic granules with the plasma membrane is insensitive to Tetanus toxin. (A) Bead-stimulated human CD8+ T cells transfected with GFP or TeNT-GFP as indicated. VAMP2 and VAMP7 protein levels were determined by Western blot analysis 12–16 h after transfection. (B) Expression of VAMP2 and VAMP7 protein levels relative to GAPDH in CTLs transfected with GFP or TeNT-GFP as indicated. Graphs represent means [ N = 3, *** p < 0.001 (Student's t -test)]. (C) Bead-stimulated human CTLs co-transfected with either GFP or TeNT-GFP along with granzyme B-mCherry and imaged 12 h after transfection. Representative live-cell TIRFM images of CTLs in contact with an anti-CD3 coated coverslip. Fusion events indicated with open arrowheads. (D) Mean percentage of cytotoxic granule fusion in cells transfected with either GFP ( n = 50) or TeNT-GFP ( n = 40), p = 0.704 (Student's t -test). (E) Mean average number of granules fused per cell over time in the TIRF plane in cells transfected with either GFP ( n = 20) or TeNT-GFP ( n = 15), p = 0.939 (Student's t -test). Bars show mean ± SEM. Scale bar, 5 μm.

Journal: Frontiers in Immunology

Article Title: Cytotoxic Granule Exocytosis From Human Cytotoxic T Lymphocytes Is Mediated by VAMP7

doi: 10.3389/fimmu.2019.01855

Figure Lengend Snippet: Fusion of cytotoxic granules with the plasma membrane is insensitive to Tetanus toxin. (A) Bead-stimulated human CD8+ T cells transfected with GFP or TeNT-GFP as indicated. VAMP2 and VAMP7 protein levels were determined by Western blot analysis 12–16 h after transfection. (B) Expression of VAMP2 and VAMP7 protein levels relative to GAPDH in CTLs transfected with GFP or TeNT-GFP as indicated. Graphs represent means [ N = 3, *** p < 0.001 (Student's t -test)]. (C) Bead-stimulated human CTLs co-transfected with either GFP or TeNT-GFP along with granzyme B-mCherry and imaged 12 h after transfection. Representative live-cell TIRFM images of CTLs in contact with an anti-CD3 coated coverslip. Fusion events indicated with open arrowheads. (D) Mean percentage of cytotoxic granule fusion in cells transfected with either GFP ( n = 50) or TeNT-GFP ( n = 40), p = 0.704 (Student's t -test). (E) Mean average number of granules fused per cell over time in the TIRF plane in cells transfected with either GFP ( n = 20) or TeNT-GFP ( n = 15), p = 0.939 (Student's t -test). Bars show mean ± SEM. Scale bar, 5 μm.

Article Snippet: VAMP7 coding sequence was amplified from pEGFP-VAMP7 plasmid (42316; Addgene) with forward primer 5′-ATA TAC GGG GTA CCG CCG CCA CCA TGG CGA TTC TTT TTG CT-3′ and reverse primer 5′-ATA TAC CGG AAT TCT TTC TTC ACA CAG CTT GG-3′ and inserted in frame with C-terminal mCherry with forward primer 5′-ATA TAC CCA AGC TTA TGG TGA GCA AGG GCG AG-3′ and reverse primer 5′-ATA TAC GCG GAT CCT TAC TTG TAC AGC TCG TCC AT-3′ or inserted in frame with pHuji with forward primer 5′-ATG TAT ACC CAA GCT TAT GGT GAG CAA GGG CGA G-3′ and reverse primer 5′-ATG TAT ACG CGG ATC CTT ACT TGT ACA GCT CGT C-3′ in pMAX containing GGSGGSGGS linker.

Techniques: Transfection, Western Blot, Expressing

VAMP7 shows a high degree of co-localization with cytotoxic granules. (A) SIM images of bead-stimulated human CD8+ T cells co-transfected with either granzyme B-mCherry or granzyme B-mTFP along with the v-SNAREs mVAMP2, VAMP3, VAMP4, VAMP7, and VAMP8 constructs. Overlay of both channels displayed that only VAMP7 co-localizes with the lytic granule marker granzyme B. (B) Pearson's and (C) Manders' overlap coefficients for co-localization of VAMP3 ( n = 16), VAMP4 ( n = 14), VAMP7 ( n = 13), VAMP8 ( n = 9), and VAMP2 ( n = 14) with granzyme B are given in the text. (D) Bead-stimulated human CD8+ T cells transfected with EGFP-VAMP7 or VAMP7-pHuji and immunolabeled with Alexa647-conjugated anti-granzyme B antibody. (E) The corresponding Pearson's and (F) Manders' overlap coefficients for colocalization of EGFP-VAMP7 ( n = 12) or VAMP7-pHuji ( n = 11) with granzyme B are given in the text. Data are shown as mean ± SEM. Scale bar, 5 μm. (G) Anti CD3/CD28 bead-stimulated human CD8+ T cells transfected with VAMP7-mCherry and incubated on either poly-L-ornithine or anti CD3 antibody coated coverslips for 15 min, fixed and immunolabeled with LAT antibody. (H) The corresponding Pearson's and (I) Manders' overlap coefficients for colocalization of VAMP7-mCherry with LAT on Poly-L-Ornithine coated coverslips ( n = 11) or anti CD3/CD28 antibody coated coverslips ( n = 10) are given in the text. Data are shown as mean ± SEM. Scale bar, 5 μm.

Journal: Frontiers in Immunology

Article Title: Cytotoxic Granule Exocytosis From Human Cytotoxic T Lymphocytes Is Mediated by VAMP7

doi: 10.3389/fimmu.2019.01855

Figure Lengend Snippet: VAMP7 shows a high degree of co-localization with cytotoxic granules. (A) SIM images of bead-stimulated human CD8+ T cells co-transfected with either granzyme B-mCherry or granzyme B-mTFP along with the v-SNAREs mVAMP2, VAMP3, VAMP4, VAMP7, and VAMP8 constructs. Overlay of both channels displayed that only VAMP7 co-localizes with the lytic granule marker granzyme B. (B) Pearson's and (C) Manders' overlap coefficients for co-localization of VAMP3 ( n = 16), VAMP4 ( n = 14), VAMP7 ( n = 13), VAMP8 ( n = 9), and VAMP2 ( n = 14) with granzyme B are given in the text. (D) Bead-stimulated human CD8+ T cells transfected with EGFP-VAMP7 or VAMP7-pHuji and immunolabeled with Alexa647-conjugated anti-granzyme B antibody. (E) The corresponding Pearson's and (F) Manders' overlap coefficients for colocalization of EGFP-VAMP7 ( n = 12) or VAMP7-pHuji ( n = 11) with granzyme B are given in the text. Data are shown as mean ± SEM. Scale bar, 5 μm. (G) Anti CD3/CD28 bead-stimulated human CD8+ T cells transfected with VAMP7-mCherry and incubated on either poly-L-ornithine or anti CD3 antibody coated coverslips for 15 min, fixed and immunolabeled with LAT antibody. (H) The corresponding Pearson's and (I) Manders' overlap coefficients for colocalization of VAMP7-mCherry with LAT on Poly-L-Ornithine coated coverslips ( n = 11) or anti CD3/CD28 antibody coated coverslips ( n = 10) are given in the text. Data are shown as mean ± SEM. Scale bar, 5 μm.

Article Snippet: VAMP7 coding sequence was amplified from pEGFP-VAMP7 plasmid (42316; Addgene) with forward primer 5′-ATA TAC GGG GTA CCG CCG CCA CCA TGG CGA TTC TTT TTG CT-3′ and reverse primer 5′-ATA TAC CGG AAT TCT TTC TTC ACA CAG CTT GG-3′ and inserted in frame with C-terminal mCherry with forward primer 5′-ATA TAC CCA AGC TTA TGG TGA GCA AGG GCG AG-3′ and reverse primer 5′-ATA TAC GCG GAT CCT TAC TTG TAC AGC TCG TCC AT-3′ or inserted in frame with pHuji with forward primer 5′-ATG TAT ACC CAA GCT TAT GGT GAG CAA GGG CGA G-3′ and reverse primer 5′-ATG TAT ACG CGG ATC CTT ACT TGT ACA GCT CGT C-3′ in pMAX containing GGSGGSGGS linker.

Techniques: Transfection, Construct, Marker, Immunolabeling, Incubation

VAMP7 polarizes to the IS on the same granules as granzyme B and perforin. (A) SIM images of SEA-pulsed human CD8+ T cells transfected with VAMP7-pHuji construct and conjugated with SEA pulsed RAJI cells and fixed after 5, 10, and 15 min, respectively. CTLs were permeabilized and immunolabeled with either Alexa647-conjugated granzyme B or Alexa647-conjugated perforin antibody. (B,C) The corresponding Pearson's overlap coefficients for co-localization of VAMP7-mCherry with granzyme B ( n = 12) and VAMP7-pHuji with perforin ( n = 11) are given in the text. Data are shown as mean ± SEM. Scale bar, 5 μm. (D) SIM images of SEA-pulsed human CD8+ T cells transfected with VAMP2-pHuji, EGFP-VAMP3, and EGFP-VAMP4 constructs and conjugated with SEA pulsed RAJI cells and fixed after 15 min. CTLs were permeabilized and immunolabeled with either Alexa647-conjugated granzyme B or Alexa647-conjugated perforin antibody. (E,F) The corresponding Pearson's overlap coefficients for co-localization of VAMP2, VAMP3, and VAMP4 with granzyme B ( n = 15) or perforin ( n = 16) are given in the text. Data are shown as mean ± SEM. Scale bar, 5 μm.

Journal: Frontiers in Immunology

Article Title: Cytotoxic Granule Exocytosis From Human Cytotoxic T Lymphocytes Is Mediated by VAMP7

doi: 10.3389/fimmu.2019.01855

Figure Lengend Snippet: VAMP7 polarizes to the IS on the same granules as granzyme B and perforin. (A) SIM images of SEA-pulsed human CD8+ T cells transfected with VAMP7-pHuji construct and conjugated with SEA pulsed RAJI cells and fixed after 5, 10, and 15 min, respectively. CTLs were permeabilized and immunolabeled with either Alexa647-conjugated granzyme B or Alexa647-conjugated perforin antibody. (B,C) The corresponding Pearson's overlap coefficients for co-localization of VAMP7-mCherry with granzyme B ( n = 12) and VAMP7-pHuji with perforin ( n = 11) are given in the text. Data are shown as mean ± SEM. Scale bar, 5 μm. (D) SIM images of SEA-pulsed human CD8+ T cells transfected with VAMP2-pHuji, EGFP-VAMP3, and EGFP-VAMP4 constructs and conjugated with SEA pulsed RAJI cells and fixed after 15 min. CTLs were permeabilized and immunolabeled with either Alexa647-conjugated granzyme B or Alexa647-conjugated perforin antibody. (E,F) The corresponding Pearson's overlap coefficients for co-localization of VAMP2, VAMP3, and VAMP4 with granzyme B ( n = 15) or perforin ( n = 16) are given in the text. Data are shown as mean ± SEM. Scale bar, 5 μm.

Article Snippet: VAMP7 coding sequence was amplified from pEGFP-VAMP7 plasmid (42316; Addgene) with forward primer 5′-ATA TAC GGG GTA CCG CCG CCA CCA TGG CGA TTC TTT TTG CT-3′ and reverse primer 5′-ATA TAC CGG AAT TCT TTC TTC ACA CAG CTT GG-3′ and inserted in frame with C-terminal mCherry with forward primer 5′-ATA TAC CCA AGC TTA TGG TGA GCA AGG GCG AG-3′ and reverse primer 5′-ATA TAC GCG GAT CCT TAC TTG TAC AGC TCG TCC AT-3′ or inserted in frame with pHuji with forward primer 5′-ATG TAT ACC CAA GCT TAT GGT GAG CAA GGG CGA G-3′ and reverse primer 5′-ATG TAT ACG CGG ATC CTT ACT TGT ACA GCT CGT C-3′ in pMAX containing GGSGGSGGS linker.

Techniques: Transfection, Construct, Immunolabeling

VAMP7 fuses at the plasma membrane along with granzyme B while VAMP8 fuses earlier and more frequently. (A) Bead stimulated human CTLs co-transfected with granzyme B-mTFP and VAMP7-pHuji constructs and imaged 12 h after transfection. Selected live-cell TIRFM images of granzyme B-mTFP and VAMP7-pHuji in a transfected CTL in contact with an anti-CD3 coated coverslip (GzmB, green, upper panel; VAMP7, magenta, middle panel; merged, lower panel). (B,C) Graphs depict the mean fluorescence intensity of individual mTFP and pHuji vesicles, respectively, over time. Experiments were repeated with three individual donors ( n = 15 cells). Scale bar, 5 μm. (D) Bead stimulated human CTLs co-transfected with VAMP7-mCherry and VAMP8-mTFP constructs and imaged 12 h after transfection. Selected live-cell TIRFM images of VAMP7-mCherry and VAMP8-mTFP in a transfected CTL in contact with an anti-CD3 coated coverslip (VAMP7, magenta, upper panel; VAMP8, green, middle panel; merged, lower panel). (E) Exemplary, individual fusion events (arrows) of VAMP7- (upper panel) and VAMP8-containing vesicles (lower panel) at the indicated time of recording. Acquisition frequency 5 Hz. (F) Mean cumulative fusion events in the TIRF plane per cell [ N = 3, n = 39, *** p < 0.001 ( t -test)]. (G) Mean average number of VAMP7- and VAMP8-containing vesicles in the TIRF plane per cell in the first 3.5 min of measurements [ N = 3, n = 39, *** p < 0.001 (Mann-Whitney U -test)]. Data are shown as mean ± SEM. Scale bar, 5 μm.

Journal: Frontiers in Immunology

Article Title: Cytotoxic Granule Exocytosis From Human Cytotoxic T Lymphocytes Is Mediated by VAMP7

doi: 10.3389/fimmu.2019.01855

Figure Lengend Snippet: VAMP7 fuses at the plasma membrane along with granzyme B while VAMP8 fuses earlier and more frequently. (A) Bead stimulated human CTLs co-transfected with granzyme B-mTFP and VAMP7-pHuji constructs and imaged 12 h after transfection. Selected live-cell TIRFM images of granzyme B-mTFP and VAMP7-pHuji in a transfected CTL in contact with an anti-CD3 coated coverslip (GzmB, green, upper panel; VAMP7, magenta, middle panel; merged, lower panel). (B,C) Graphs depict the mean fluorescence intensity of individual mTFP and pHuji vesicles, respectively, over time. Experiments were repeated with three individual donors ( n = 15 cells). Scale bar, 5 μm. (D) Bead stimulated human CTLs co-transfected with VAMP7-mCherry and VAMP8-mTFP constructs and imaged 12 h after transfection. Selected live-cell TIRFM images of VAMP7-mCherry and VAMP8-mTFP in a transfected CTL in contact with an anti-CD3 coated coverslip (VAMP7, magenta, upper panel; VAMP8, green, middle panel; merged, lower panel). (E) Exemplary, individual fusion events (arrows) of VAMP7- (upper panel) and VAMP8-containing vesicles (lower panel) at the indicated time of recording. Acquisition frequency 5 Hz. (F) Mean cumulative fusion events in the TIRF plane per cell [ N = 3, n = 39, *** p < 0.001 ( t -test)]. (G) Mean average number of VAMP7- and VAMP8-containing vesicles in the TIRF plane per cell in the first 3.5 min of measurements [ N = 3, n = 39, *** p < 0.001 (Mann-Whitney U -test)]. Data are shown as mean ± SEM. Scale bar, 5 μm.

Article Snippet: VAMP7 coding sequence was amplified from pEGFP-VAMP7 plasmid (42316; Addgene) with forward primer 5′-ATA TAC GGG GTA CCG CCG CCA CCA TGG CGA TTC TTT TTG CT-3′ and reverse primer 5′-ATA TAC CGG AAT TCT TTC TTC ACA CAG CTT GG-3′ and inserted in frame with C-terminal mCherry with forward primer 5′-ATA TAC CCA AGC TTA TGG TGA GCA AGG GCG AG-3′ and reverse primer 5′-ATA TAC GCG GAT CCT TAC TTG TAC AGC TCG TCC AT-3′ or inserted in frame with pHuji with forward primer 5′-ATG TAT ACC CAA GCT TAT GGT GAG CAA GGG CGA G-3′ and reverse primer 5′-ATG TAT ACG CGG ATC CTT ACT TGT ACA GCT CGT C-3′ in pMAX containing GGSGGSGGS linker.

Techniques: Transfection, Construct, Fluorescence, MANN-WHITNEY

Knockdown of VAMP7 strongly reduces fusion of cytotoxic granules at the IS. (A) Lysates from bead stimulated human CD8+ T cells transfected with either control or VAMP7 siRNAs (1 or 2, respectively) and blotted for VAMP7 (top) and GAPDH (bottom) as loading control. (B) Quantification of VAMP7 protein expression (in % normalized to control siRNA-treated CTLs) performed by densitometry. Bars indicate SEMs. [VAMP7-siRNA1, N = 3; *** p < 0.001 and VAMP7-siRNA2, N = 3; *** p < 0.001 ( t -test)]. (C) Human CD8+ T cells co-transfected with granzyme B-mCherry along with either ns-siRNA or VAMP7-siRNA1 or VAMP7-siRNA2 and imaged 12 h after transfection. Selected live-cell TIRF microscopy images of granzyme B-mCherry in a transfected CTL in contact with an anti-CD3 coated coverslip. Fusion events are indicated with open circles (three frames shown per granule fused). (D) Mean percentage of cytotoxic granule fusion in cells transfected with either ns-siRNA ( n = 66 and n = 59, respectively) or VAMP7-siRNA1 [ n = 91; ** p < 0.01 ( t -test)] or VAMP7-siRNA2 [ n = 72; *** p < 0.001 ( t -test)]. (E) Mean average number of granules fused over time in the TIRF plane per cell p = 0.206 ( t -test) for VAMP7-siRNA1 and ** p < 0.01 ( t -test) for VAMP7-siRNA2. Bars indicate mean ± SEM. Scale bar, 5 μm. (F) Calcein-based killing assay for CTLs transfected with either ns-siRNA, VAMP7-siRNA1, or VAMP7-siRNA2. Experiments were carried out in duplicate [ N = 4; *** p < 0.001 ( t -test)].

Journal: Frontiers in Immunology

Article Title: Cytotoxic Granule Exocytosis From Human Cytotoxic T Lymphocytes Is Mediated by VAMP7

doi: 10.3389/fimmu.2019.01855

Figure Lengend Snippet: Knockdown of VAMP7 strongly reduces fusion of cytotoxic granules at the IS. (A) Lysates from bead stimulated human CD8+ T cells transfected with either control or VAMP7 siRNAs (1 or 2, respectively) and blotted for VAMP7 (top) and GAPDH (bottom) as loading control. (B) Quantification of VAMP7 protein expression (in % normalized to control siRNA-treated CTLs) performed by densitometry. Bars indicate SEMs. [VAMP7-siRNA1, N = 3; *** p < 0.001 and VAMP7-siRNA2, N = 3; *** p < 0.001 ( t -test)]. (C) Human CD8+ T cells co-transfected with granzyme B-mCherry along with either ns-siRNA or VAMP7-siRNA1 or VAMP7-siRNA2 and imaged 12 h after transfection. Selected live-cell TIRF microscopy images of granzyme B-mCherry in a transfected CTL in contact with an anti-CD3 coated coverslip. Fusion events are indicated with open circles (three frames shown per granule fused). (D) Mean percentage of cytotoxic granule fusion in cells transfected with either ns-siRNA ( n = 66 and n = 59, respectively) or VAMP7-siRNA1 [ n = 91; ** p < 0.01 ( t -test)] or VAMP7-siRNA2 [ n = 72; *** p < 0.001 ( t -test)]. (E) Mean average number of granules fused over time in the TIRF plane per cell p = 0.206 ( t -test) for VAMP7-siRNA1 and ** p < 0.01 ( t -test) for VAMP7-siRNA2. Bars indicate mean ± SEM. Scale bar, 5 μm. (F) Calcein-based killing assay for CTLs transfected with either ns-siRNA, VAMP7-siRNA1, or VAMP7-siRNA2. Experiments were carried out in duplicate [ N = 4; *** p < 0.001 ( t -test)].

Article Snippet: VAMP7 coding sequence was amplified from pEGFP-VAMP7 plasmid (42316; Addgene) with forward primer 5′-ATA TAC GGG GTA CCG CCG CCA CCA TGG CGA TTC TTT TTG CT-3′ and reverse primer 5′-ATA TAC CGG AAT TCT TTC TTC ACA CAG CTT GG-3′ and inserted in frame with C-terminal mCherry with forward primer 5′-ATA TAC CCA AGC TTA TGG TGA GCA AGG GCG AG-3′ and reverse primer 5′-ATA TAC GCG GAT CCT TAC TTG TAC AGC TCG TCC AT-3′ or inserted in frame with pHuji with forward primer 5′-ATG TAT ACC CAA GCT TAT GGT GAG CAA GGG CGA G-3′ and reverse primer 5′-ATG TAT ACG CGG ATC CTT ACT TGT ACA GCT CGT C-3′ in pMAX containing GGSGGSGGS linker.

Techniques: Transfection, Expressing, Microscopy

VAMP7 forms a SNARE complex with SNAP-23 and Syntaxin11. (A) Model illustrating the formation of SNARE complex between VAMP7 (v-SNARE), SNAP-23, and Syntaxin11 (t- SNARE) during cytotoxic granule fusion in human CD8+ T cells. (B) Different Twin-Strep-tag fusion constructs used for pulldown assay. Twin-Strep-tag was fused at the C terminus of VAMP7 and at the N terminus of Syntaxin11 with a (GGS)x3 linker. (C) Western blot of bead stimulated human CD8+ T cells transfected with Twin-Strep-tag-tagged VAMP7, immuno-precipitated with anti-FLAG antibody and detected with antibodies against FLAG, SNAP-23, and Syntaxin11. (D) Western blot of bead stimulated human CD8+ T cells transfected with Twin-Strep-tag-tagged Syntaxin11, immuno-precipitated with anti-Syntaxin antibody and detected with antibodies against Strep-tag, SNAP-23, VAMP7, and VAMP2. As control, cells were transfected with Twin-Strep-tag construct. Ten percentage of the lysates were loaded as input. (E,F) Densitometric quantification of the Western blots shown in (C,D) , respectively.

Journal: Frontiers in Immunology

Article Title: Cytotoxic Granule Exocytosis From Human Cytotoxic T Lymphocytes Is Mediated by VAMP7

doi: 10.3389/fimmu.2019.01855

Figure Lengend Snippet: VAMP7 forms a SNARE complex with SNAP-23 and Syntaxin11. (A) Model illustrating the formation of SNARE complex between VAMP7 (v-SNARE), SNAP-23, and Syntaxin11 (t- SNARE) during cytotoxic granule fusion in human CD8+ T cells. (B) Different Twin-Strep-tag fusion constructs used for pulldown assay. Twin-Strep-tag was fused at the C terminus of VAMP7 and at the N terminus of Syntaxin11 with a (GGS)x3 linker. (C) Western blot of bead stimulated human CD8+ T cells transfected with Twin-Strep-tag-tagged VAMP7, immuno-precipitated with anti-FLAG antibody and detected with antibodies against FLAG, SNAP-23, and Syntaxin11. (D) Western blot of bead stimulated human CD8+ T cells transfected with Twin-Strep-tag-tagged Syntaxin11, immuno-precipitated with anti-Syntaxin antibody and detected with antibodies against Strep-tag, SNAP-23, VAMP7, and VAMP2. As control, cells were transfected with Twin-Strep-tag construct. Ten percentage of the lysates were loaded as input. (E,F) Densitometric quantification of the Western blots shown in (C,D) , respectively.

Article Snippet: VAMP7 coding sequence was amplified from pEGFP-VAMP7 plasmid (42316; Addgene) with forward primer 5′-ATA TAC GGG GTA CCG CCG CCA CCA TGG CGA TTC TTT TTG CT-3′ and reverse primer 5′-ATA TAC CGG AAT TCT TTC TTC ACA CAG CTT GG-3′ and inserted in frame with C-terminal mCherry with forward primer 5′-ATA TAC CCA AGC TTA TGG TGA GCA AGG GCG AG-3′ and reverse primer 5′-ATA TAC GCG GAT CCT TAC TTG TAC AGC TCG TCC AT-3′ or inserted in frame with pHuji with forward primer 5′-ATG TAT ACC CAA GCT TAT GGT GAG CAA GGG CGA G-3′ and reverse primer 5′-ATG TAT ACG CGG ATC CTT ACT TGT ACA GCT CGT C-3′ in pMAX containing GGSGGSGGS linker.

Techniques: Strep-tag, Construct, Western Blot, Transfection

Autophagosomal Stx17 colocalizes significantly with SNAP29 and VAMP7. a , representative confocal and gSTED images of membrane-bound Stx17 rings ( left ) surrounding LC3-labeled autophagosomes ( center ) and a merge of these images ( right ) from rapamycin-treated HeLa cells. Scale bars , 500 nm. b , intensity line profile through the punctum in a , demonstrating membrane localization of Stx17 resolved by gSTED. c–e , single-channel and merged images of rapamycin-treated HeLa cells expressing Stx17 with either SNAP29 ( c ), VAMP8 ( d ), or VAMP7 ( e ) and a merge of both channels. Scale bars , 10 μm for full field and 3 μm for zoomed regions. f–h , frequency scatter plots of reported single-pixel intensity values in each channel presented in c–e. i , quantification of SNAP29 and Stx17 colocalization using Pearson's correlation coefficient ( PCC ) demonstrating significantly higher correlation than in negative controls generated by single-channel 90° rotation. j and k , quantification of Stx17 puncta colocal with VAMP8 ( j ) or VAMP7 ( k ) puncta using Manders correlation coefficient. All box-and-whisker plots represent the median ( central line ), 25th and 75th quartile ( box ), and the minimum and maximum value ( whiskers ). ns , nonsignificant ( p ≥ 0.05); ***, p = 0.0001–0.0005; ****, p < 0.0001.

Journal: The Journal of Biological Chemistry

Article Title: A VPS33A-binding motif on syntaxin 17 controls autophagy completion in mammalian cells

doi: 10.1074/jbc.RA118.005947

Figure Lengend Snippet: Autophagosomal Stx17 colocalizes significantly with SNAP29 and VAMP7. a , representative confocal and gSTED images of membrane-bound Stx17 rings ( left ) surrounding LC3-labeled autophagosomes ( center ) and a merge of these images ( right ) from rapamycin-treated HeLa cells. Scale bars , 500 nm. b , intensity line profile through the punctum in a , demonstrating membrane localization of Stx17 resolved by gSTED. c–e , single-channel and merged images of rapamycin-treated HeLa cells expressing Stx17 with either SNAP29 ( c ), VAMP8 ( d ), or VAMP7 ( e ) and a merge of both channels. Scale bars , 10 μm for full field and 3 μm for zoomed regions. f–h , frequency scatter plots of reported single-pixel intensity values in each channel presented in c–e. i , quantification of SNAP29 and Stx17 colocalization using Pearson's correlation coefficient ( PCC ) demonstrating significantly higher correlation than in negative controls generated by single-channel 90° rotation. j and k , quantification of Stx17 puncta colocal with VAMP8 ( j ) or VAMP7 ( k ) puncta using Manders correlation coefficient. All box-and-whisker plots represent the median ( central line ), 25th and 75th quartile ( box ), and the minimum and maximum value ( whiskers ). ns , nonsignificant ( p ≥ 0.05); ***, p = 0.0001–0.0005; ****, p < 0.0001.

Article Snippet: The mutant variants of pmCherry-C1-Stx17 (S2A, S2E, and Q196G) were obtained by site-directed mutagenesis using the QuikChange II site-directed mutagenesis kit (Agilent). pEGFP-C2-LC3 ( ) (Addgene, 24920), pEGFP-C2-Atg14 ( ) (Addgene, 21635), and pEGFP-C1-VAMP7 ( ) (Addgene, 42316) were obtained from other groups, and the former was used to generate pmCherry-C2-LC3 and pEGFP-mCherry-C2-LC3 by restriction and ligation. pEGFP-N3 and pmCherry-N1 (Clontech, discontinued) were used for FLIM-FRET control work along with pCDNA3.1-EGFP-mCherry, a fusion of two fluorescent proteins separated by a 12-amino-acid linker, which was generated by isolation of the fusion protein from previously described pGEX-KG_EGFP-mCherry ( ) and ligation into the pCDNA3.1 vector backbone.

Techniques: Labeling, Expressing, Generated, Whisker Assay

In situ FLIM-FRET identifies VAMP7 as a heterodimer-dependent interaction partner of Stx17. a and b , intensity images and their corresponding fluorescence lifetime maps probing changes in lifetime and proximity between donor-only or donor and acceptor samples in rapamycin-treated cells. Scale bars , 10 μm. Shown is EGFP-VAMP8 ( a ) and EGFP-VAMP7 ( b ) expressed alone or co-expressed with either WT mCherry-Stx17 or mCherry-Stx17[Q196G]. c and d , mean single-pixel FRET efficiency histograms, enveloped by S.E., derived from the data sets presented in a and b , respectively. Statistical significance was tested using a Mann–Whitney test to compare integral values > 0.1 ( n = 7–10 cells). e , schematic demonstrating the differing R-SNARE compositions, which have an unknown impact on complex structure and fluorophore separation distance. ns , nonsignificant ( p ≥ 0.05); **, p = 0.0005–0.005.

Journal: The Journal of Biological Chemistry

Article Title: A VPS33A-binding motif on syntaxin 17 controls autophagy completion in mammalian cells

doi: 10.1074/jbc.RA118.005947

Figure Lengend Snippet: In situ FLIM-FRET identifies VAMP7 as a heterodimer-dependent interaction partner of Stx17. a and b , intensity images and their corresponding fluorescence lifetime maps probing changes in lifetime and proximity between donor-only or donor and acceptor samples in rapamycin-treated cells. Scale bars , 10 μm. Shown is EGFP-VAMP8 ( a ) and EGFP-VAMP7 ( b ) expressed alone or co-expressed with either WT mCherry-Stx17 or mCherry-Stx17[Q196G]. c and d , mean single-pixel FRET efficiency histograms, enveloped by S.E., derived from the data sets presented in a and b , respectively. Statistical significance was tested using a Mann–Whitney test to compare integral values > 0.1 ( n = 7–10 cells). e , schematic demonstrating the differing R-SNARE compositions, which have an unknown impact on complex structure and fluorophore separation distance. ns , nonsignificant ( p ≥ 0.05); **, p = 0.0005–0.005.

Article Snippet: The mutant variants of pmCherry-C1-Stx17 (S2A, S2E, and Q196G) were obtained by site-directed mutagenesis using the QuikChange II site-directed mutagenesis kit (Agilent). pEGFP-C2-LC3 ( ) (Addgene, 24920), pEGFP-C2-Atg14 ( ) (Addgene, 21635), and pEGFP-C1-VAMP7 ( ) (Addgene, 42316) were obtained from other groups, and the former was used to generate pmCherry-C2-LC3 and pEGFP-mCherry-C2-LC3 by restriction and ligation. pEGFP-N3 and pmCherry-N1 (Clontech, discontinued) were used for FLIM-FRET control work along with pCDNA3.1-EGFP-mCherry, a fusion of two fluorescent proteins separated by a 12-amino-acid linker, which was generated by isolation of the fusion protein from previously described pGEX-KG_EGFP-mCherry ( ) and ligation into the pCDNA3.1 vector backbone.

Techniques: In Situ, Fluorescence, Derivative Assay, MANN-WHITNEY

Stx17 N-terminal phosphoserine modulates SNARE complex formation. a , an alignment of the N-peptide sequences of human syntaxin family proteins; negatively and positively charged residues are indicated in red and blue , respectively. Stx17 ( gray ), encodes a highly negative N terminus. b , NetPhos 3.1 phosphorylation site prediction scores for each residue of Stx17 indicates consensus for serine 2 phosphorylation. c–e , intensity and fluorescence lifetime maps of rapamycin-treated HeLa cells expressing EGFP-SNAP29 ( c ), EGFP-VAMP7 ( d ), or EGFP-VAMP8 ( e ) alone or alongside mCherry-fused Stx17[WT], Stx17[S2A], or Stx17[S2E] as indicated. Scale bars , 10 μm. f–h , mean single-pixel FRET efficiency histograms, enveloped by their S.E., derived from the data sets presented in c–e respectively; integral values describe efficiencies between 0.1 and 1. Statistical significance was tested using a Mann–Whitney test to compare single-cell integral values > 0.1 ( n = 9–14 cells). ns , nonsignificant ( p ≥ 0.05); *, p = 0.005–0.05; **, p = 0.0005–0.005.

Journal: The Journal of Biological Chemistry

Article Title: A VPS33A-binding motif on syntaxin 17 controls autophagy completion in mammalian cells

doi: 10.1074/jbc.RA118.005947

Figure Lengend Snippet: Stx17 N-terminal phosphoserine modulates SNARE complex formation. a , an alignment of the N-peptide sequences of human syntaxin family proteins; negatively and positively charged residues are indicated in red and blue , respectively. Stx17 ( gray ), encodes a highly negative N terminus. b , NetPhos 3.1 phosphorylation site prediction scores for each residue of Stx17 indicates consensus for serine 2 phosphorylation. c–e , intensity and fluorescence lifetime maps of rapamycin-treated HeLa cells expressing EGFP-SNAP29 ( c ), EGFP-VAMP7 ( d ), or EGFP-VAMP8 ( e ) alone or alongside mCherry-fused Stx17[WT], Stx17[S2A], or Stx17[S2E] as indicated. Scale bars , 10 μm. f–h , mean single-pixel FRET efficiency histograms, enveloped by their S.E., derived from the data sets presented in c–e respectively; integral values describe efficiencies between 0.1 and 1. Statistical significance was tested using a Mann–Whitney test to compare single-cell integral values > 0.1 ( n = 9–14 cells). ns , nonsignificant ( p ≥ 0.05); *, p = 0.005–0.05; **, p = 0.0005–0.005.

Article Snippet: The mutant variants of pmCherry-C1-Stx17 (S2A, S2E, and Q196G) were obtained by site-directed mutagenesis using the QuikChange II site-directed mutagenesis kit (Agilent). pEGFP-C2-LC3 ( ) (Addgene, 24920), pEGFP-C2-Atg14 ( ) (Addgene, 21635), and pEGFP-C1-VAMP7 ( ) (Addgene, 42316) were obtained from other groups, and the former was used to generate pmCherry-C2-LC3 and pEGFP-mCherry-C2-LC3 by restriction and ligation. pEGFP-N3 and pmCherry-N1 (Clontech, discontinued) were used for FLIM-FRET control work along with pCDNA3.1-EGFP-mCherry, a fusion of two fluorescent proteins separated by a 12-amino-acid linker, which was generated by isolation of the fusion protein from previously described pGEX-KG_EGFP-mCherry ( ) and ligation into the pCDNA3.1 vector backbone.

Techniques: Fluorescence, Expressing, Derivative Assay, MANN-WHITNEY

VPS33A promotes fusion by stabilizing the SNARE bundle. a , box plot of Stx17-positive puncta concentration per μm 2 in rapamycin-treated HeLa cells, demonstrating accumulation of autophagosomes upon VPS33A knockdown (VPS33A siRNA) when compared with a nontargeting negative control knockdown (control siRNA). Significance was tested using an unpaired two-sample t test ( n = 4). b–d , FLIM-FRET analysis of rapamycin-treated HeLa cells expressing EGFP-VAMP7 alone or alongside either mCherry-Stx17 or mCherry-SNAP29. Mean single-pixel FRET efficiency histograms for VPS33A siRNA–treated samples ( b ) and negative control siRNA-treated samples ( c ), both enveloped by their S.E. FRET efficiency integral values >0.1 were tested for statistical significance using a Mann–Whitney test ( n = 4 cells). d , representative intensity and fluorescence lifetime maps. Scale bars , 10 μm. ns , nonsignificant ( p ≥ 0.05); *, p = 0.005–0.05.

Journal: The Journal of Biological Chemistry

Article Title: A VPS33A-binding motif on syntaxin 17 controls autophagy completion in mammalian cells

doi: 10.1074/jbc.RA118.005947

Figure Lengend Snippet: VPS33A promotes fusion by stabilizing the SNARE bundle. a , box plot of Stx17-positive puncta concentration per μm 2 in rapamycin-treated HeLa cells, demonstrating accumulation of autophagosomes upon VPS33A knockdown (VPS33A siRNA) when compared with a nontargeting negative control knockdown (control siRNA). Significance was tested using an unpaired two-sample t test ( n = 4). b–d , FLIM-FRET analysis of rapamycin-treated HeLa cells expressing EGFP-VAMP7 alone or alongside either mCherry-Stx17 or mCherry-SNAP29. Mean single-pixel FRET efficiency histograms for VPS33A siRNA–treated samples ( b ) and negative control siRNA-treated samples ( c ), both enveloped by their S.E. FRET efficiency integral values >0.1 were tested for statistical significance using a Mann–Whitney test ( n = 4 cells). d , representative intensity and fluorescence lifetime maps. Scale bars , 10 μm. ns , nonsignificant ( p ≥ 0.05); *, p = 0.005–0.05.

Article Snippet: The mutant variants of pmCherry-C1-Stx17 (S2A, S2E, and Q196G) were obtained by site-directed mutagenesis using the QuikChange II site-directed mutagenesis kit (Agilent). pEGFP-C2-LC3 ( ) (Addgene, 24920), pEGFP-C2-Atg14 ( ) (Addgene, 21635), and pEGFP-C1-VAMP7 ( ) (Addgene, 42316) were obtained from other groups, and the former was used to generate pmCherry-C2-LC3 and pEGFP-mCherry-C2-LC3 by restriction and ligation. pEGFP-N3 and pmCherry-N1 (Clontech, discontinued) were used for FLIM-FRET control work along with pCDNA3.1-EGFP-mCherry, a fusion of two fluorescent proteins separated by a 12-amino-acid linker, which was generated by isolation of the fusion protein from previously described pGEX-KG_EGFP-mCherry ( ) and ligation into the pCDNA3.1 vector backbone.

Techniques: Concentration Assay, Negative Control, Expressing, MANN-WHITNEY, Fluorescence

Proposed model of Stx17 interaction dynamics with VPS33A. Syntaxin 17 ( green ) and VPS33A ( purple ) interact via two distinct binding modes switched by the phosphorylation status of Stx17 serine 2. We propose that prior to fusion, VPS33A domain 1 associates with the phosphorylated Stx17 N-peptide ( i ); the Stx17 N-peptide is locally dephosphorylated at the fusion site, which alters VPS33A binding and enables SNARE bundle formation, providing a final stage regulatory mechanism ( ii ); and SNAP29 and VAMP7 may now associate with Stx17, forming a SNARE bundle that is stabilized by an alternate VPS33A association and that drives membrane fusion ( iii ). Following fusion, the cis -SNARE bundle persists until actively disassembled ( iv ), following which, we suspect that Stx17 serine 2 is phosphorylated once more, allowing re-engagement of the VPS33A domain 1 with the Stx17 N-peptide to deter further fusion events ( v ).

Journal: The Journal of Biological Chemistry

Article Title: A VPS33A-binding motif on syntaxin 17 controls autophagy completion in mammalian cells

doi: 10.1074/jbc.RA118.005947

Figure Lengend Snippet: Proposed model of Stx17 interaction dynamics with VPS33A. Syntaxin 17 ( green ) and VPS33A ( purple ) interact via two distinct binding modes switched by the phosphorylation status of Stx17 serine 2. We propose that prior to fusion, VPS33A domain 1 associates with the phosphorylated Stx17 N-peptide ( i ); the Stx17 N-peptide is locally dephosphorylated at the fusion site, which alters VPS33A binding and enables SNARE bundle formation, providing a final stage regulatory mechanism ( ii ); and SNAP29 and VAMP7 may now associate with Stx17, forming a SNARE bundle that is stabilized by an alternate VPS33A association and that drives membrane fusion ( iii ). Following fusion, the cis -SNARE bundle persists until actively disassembled ( iv ), following which, we suspect that Stx17 serine 2 is phosphorylated once more, allowing re-engagement of the VPS33A domain 1 with the Stx17 N-peptide to deter further fusion events ( v ).

Article Snippet: The mutant variants of pmCherry-C1-Stx17 (S2A, S2E, and Q196G) were obtained by site-directed mutagenesis using the QuikChange II site-directed mutagenesis kit (Agilent). pEGFP-C2-LC3 ( ) (Addgene, 24920), pEGFP-C2-Atg14 ( ) (Addgene, 21635), and pEGFP-C1-VAMP7 ( ) (Addgene, 42316) were obtained from other groups, and the former was used to generate pmCherry-C2-LC3 and pEGFP-mCherry-C2-LC3 by restriction and ligation. pEGFP-N3 and pmCherry-N1 (Clontech, discontinued) were used for FLIM-FRET control work along with pCDNA3.1-EGFP-mCherry, a fusion of two fluorescent proteins separated by a 12-amino-acid linker, which was generated by isolation of the fusion protein from previously described pGEX-KG_EGFP-mCherry ( ) and ligation into the pCDNA3.1 vector backbone.

Techniques: Binding Assay