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VAMP8-dependent fusion of recycling endosomes with the plasma membrane facilitates T lymphocyte cytotoxicity.

Marshall MR, Pattu V, Halimani M, Maier-Peuschel M, Müller ML, Becherer U, Hong W, Hoth M, Tschernig T, Bryceson YT, Rettig J - J. Cell Biol. (2015)

Bottom Line: Although multiple SNARE proteins have been implicated in cytotoxic granule exocytosis, the role of vesicular SNARE proteins, i.e., vesicle-associated membrane proteins (VAMPs), remains enigmatic.In primary human CTLs, however, VAMP8 colocalized with Rab11a-positive recycling endosomes.Our findings imply that secretory granule exocytosis pathways in other cell types may also be more complex than previously appreciated.

View Article: PubMed Central - HTML - PubMed

Affiliation: Cellular Neurophysiology, Center for Integrative Physiology and Molecular Medicine, Saarland University, 66421 Homburg, Germany Department of Medicine, Center For Infectious Medicine, 14186 Stockholm, Sweden.

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VAMP8 knockdown does not affect proximal T cell signaling, but regulates Stx11 vesicle trafficking and fusion at immune synapses. (A–K) Bead-stimulated human CD8+ T cells were transfected with siRNA, as indicated, as well as LifeAct-GFP (D–F) or Stx11-mCherry (G–K). (A) CTLs were loaded with Ca2+-sensitive Fluo-4 and Fura red dyes and assessed by flow cytometry and stimulated as indicated. iono., ionomycin. (B and C) Western blot of CTL lysates after transfection and stimulation as indicated. (B) Blots for phospho-ERK (pT202/pY204), VAMP8, and loading control GAPDH are shown for one representative donor. (C) Densitometry analysis of phospho-ERK signaling in five individual donors. Mean values are indicated, with bars representing SDs (ANOVA). (D) TIRF microscopy images of CTLs transfected with siRNA, as indicated, and LifeAct-GFP after contact with anti-CD3 and anti-CD28 antibody-coated coverslips for 250 s. (E) Quantification of F-actin clearance based on LifeAct-GFP fluorescence in individual cells (n = 12). (F) Quantification of cell spreading was based on LifeAct-GFP fluorescence in individual cells (n = 15). (G) Selected live-cell TIRF microscopy images of mCherry-Stx11 in representative CTLs transfected with siRNA as indicated. Arrowheads indicate fusion events; vesicle 1 is indicated by closed arrowheads, and vesicle 2 is indicated by open arrowheads. (H) Mean dwell time of mCherry-Stx11 vesicles in the TIRF plane per cell (n = 15, unpaired t test, **, P > 0.01). (I) Mean mCherry-Stx11 vesicle accumulation over time in the TIRF plane per cell (n = 20). (J) Mean overall mCherry-Stx11 accumulation over time in the TIRF plane per cell (n = 20). (K) Mean fluorescence dispersion events for mCherry-Stx11 vesicles in the TIRF plane per cell (n = 15, unpaired t test, **, P > 0.01). (L) Representative TIRF image depicting concentric circle region of interest used for radial analysis and dispersion of VAMP8-TFP and mCherry-Stx11. (M) Graph depicts the distribution of cumulative VAMP8-TFP fluorescence dispersion events on a per cell basis. (N) Graph depicts the distribution of cumulative mCherry-Stx11 fluorescence dispersion events on a per cell basis. (O) Quantification of mCherry-Stx11 radial distribution in TIRF plane of CTLs cotransfected with siRNA, as indicated. siCTRL, control siRNA. Bars: (D and G) 5 µm; (L) 2.5 µm.
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fig7: VAMP8 knockdown does not affect proximal T cell signaling, but regulates Stx11 vesicle trafficking and fusion at immune synapses. (A–K) Bead-stimulated human CD8+ T cells were transfected with siRNA, as indicated, as well as LifeAct-GFP (D–F) or Stx11-mCherry (G–K). (A) CTLs were loaded with Ca2+-sensitive Fluo-4 and Fura red dyes and assessed by flow cytometry and stimulated as indicated. iono., ionomycin. (B and C) Western blot of CTL lysates after transfection and stimulation as indicated. (B) Blots for phospho-ERK (pT202/pY204), VAMP8, and loading control GAPDH are shown for one representative donor. (C) Densitometry analysis of phospho-ERK signaling in five individual donors. Mean values are indicated, with bars representing SDs (ANOVA). (D) TIRF microscopy images of CTLs transfected with siRNA, as indicated, and LifeAct-GFP after contact with anti-CD3 and anti-CD28 antibody-coated coverslips for 250 s. (E) Quantification of F-actin clearance based on LifeAct-GFP fluorescence in individual cells (n = 12). (F) Quantification of cell spreading was based on LifeAct-GFP fluorescence in individual cells (n = 15). (G) Selected live-cell TIRF microscopy images of mCherry-Stx11 in representative CTLs transfected with siRNA as indicated. Arrowheads indicate fusion events; vesicle 1 is indicated by closed arrowheads, and vesicle 2 is indicated by open arrowheads. (H) Mean dwell time of mCherry-Stx11 vesicles in the TIRF plane per cell (n = 15, unpaired t test, **, P > 0.01). (I) Mean mCherry-Stx11 vesicle accumulation over time in the TIRF plane per cell (n = 20). (J) Mean overall mCherry-Stx11 accumulation over time in the TIRF plane per cell (n = 20). (K) Mean fluorescence dispersion events for mCherry-Stx11 vesicles in the TIRF plane per cell (n = 15, unpaired t test, **, P > 0.01). (L) Representative TIRF image depicting concentric circle region of interest used for radial analysis and dispersion of VAMP8-TFP and mCherry-Stx11. (M) Graph depicts the distribution of cumulative VAMP8-TFP fluorescence dispersion events on a per cell basis. (N) Graph depicts the distribution of cumulative mCherry-Stx11 fluorescence dispersion events on a per cell basis. (O) Quantification of mCherry-Stx11 radial distribution in TIRF plane of CTLs cotransfected with siRNA, as indicated. siCTRL, control siRNA. Bars: (D and G) 5 µm; (L) 2.5 µm.

Mentions: To further dissect the mechanism whereby VAMP8 facilitates lymphocyte cytotoxicity, we first studied the impact of VAMP8 knockdown on TCR signaling in human CTLs. CTLs were transfected with control siRNA or VAMP8 siRNA, labeled with Fluo-4 and Fura red Ca2+-sensitive dyes, and assessed by ratiometric flow cytometry. Knockdown of VAMP8 did not significantly alter intracellular Ca2+ mobilization after TCR stimulation (Figs. 7 A and S5 D), which is crucial for lymphocyte cytotoxicity (Maul-Pavicic et al., 2011). Similarly, knockdown of VAMP8 did not reduce phosphorylation of the MAPK extracellular signal–regulated kinase (ERK) after TCR stimulation (Fig. 7, B and C; and Fig. S5 E). ERK phosphorylation has been implicated in cytotoxic lymphocyte granule polarization (Jenkins et al., 2009). Thus, VAMP8 was not required for proximal TCR signals leading to intracellular Ca2+ mobilization and phosphorylation of ERK. To study actin dynamics, CTLs were cotransfected with recombinant, fluorescently tagged actin (LifeAct-GFP) and control siRNA or VAMP8 siRNA and subsequently imaged by TIRF microscopy (Fig. 7, D and E; and Fig. S5 F). In line with the experiments negating a role for VAMP8 in proximal TCR-induced Ca2+ mobilization and phosphorylation of ERK, knockdown of VAMP8 did not affect established F-actin architectural phases during immune synapse formation (Rak et al., 2011). An even distribution of F-actin was initially observed upon contact with the coverslip. After cell spreading and within ∼40 s, distribution of F-actin around the cell perimeter was observed. This annular distribution was accompanied by the appearance of granule-sized clearance in the actin meshwork and normal cell spreading (Fig. 7, E and F). In conclusion, our results indicated that VAMP8-mediated fusion with the plasma membrane is not required for proximal TCR-induced signaling and actin reorganization.


VAMP8-dependent fusion of recycling endosomes with the plasma membrane facilitates T lymphocyte cytotoxicity.

Marshall MR, Pattu V, Halimani M, Maier-Peuschel M, Müller ML, Becherer U, Hong W, Hoth M, Tschernig T, Bryceson YT, Rettig J - J. Cell Biol. (2015)

VAMP8 knockdown does not affect proximal T cell signaling, but regulates Stx11 vesicle trafficking and fusion at immune synapses. (A–K) Bead-stimulated human CD8+ T cells were transfected with siRNA, as indicated, as well as LifeAct-GFP (D–F) or Stx11-mCherry (G–K). (A) CTLs were loaded with Ca2+-sensitive Fluo-4 and Fura red dyes and assessed by flow cytometry and stimulated as indicated. iono., ionomycin. (B and C) Western blot of CTL lysates after transfection and stimulation as indicated. (B) Blots for phospho-ERK (pT202/pY204), VAMP8, and loading control GAPDH are shown for one representative donor. (C) Densitometry analysis of phospho-ERK signaling in five individual donors. Mean values are indicated, with bars representing SDs (ANOVA). (D) TIRF microscopy images of CTLs transfected with siRNA, as indicated, and LifeAct-GFP after contact with anti-CD3 and anti-CD28 antibody-coated coverslips for 250 s. (E) Quantification of F-actin clearance based on LifeAct-GFP fluorescence in individual cells (n = 12). (F) Quantification of cell spreading was based on LifeAct-GFP fluorescence in individual cells (n = 15). (G) Selected live-cell TIRF microscopy images of mCherry-Stx11 in representative CTLs transfected with siRNA as indicated. Arrowheads indicate fusion events; vesicle 1 is indicated by closed arrowheads, and vesicle 2 is indicated by open arrowheads. (H) Mean dwell time of mCherry-Stx11 vesicles in the TIRF plane per cell (n = 15, unpaired t test, **, P > 0.01). (I) Mean mCherry-Stx11 vesicle accumulation over time in the TIRF plane per cell (n = 20). (J) Mean overall mCherry-Stx11 accumulation over time in the TIRF plane per cell (n = 20). (K) Mean fluorescence dispersion events for mCherry-Stx11 vesicles in the TIRF plane per cell (n = 15, unpaired t test, **, P > 0.01). (L) Representative TIRF image depicting concentric circle region of interest used for radial analysis and dispersion of VAMP8-TFP and mCherry-Stx11. (M) Graph depicts the distribution of cumulative VAMP8-TFP fluorescence dispersion events on a per cell basis. (N) Graph depicts the distribution of cumulative mCherry-Stx11 fluorescence dispersion events on a per cell basis. (O) Quantification of mCherry-Stx11 radial distribution in TIRF plane of CTLs cotransfected with siRNA, as indicated. siCTRL, control siRNA. Bars: (D and G) 5 µm; (L) 2.5 µm.
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fig7: VAMP8 knockdown does not affect proximal T cell signaling, but regulates Stx11 vesicle trafficking and fusion at immune synapses. (A–K) Bead-stimulated human CD8+ T cells were transfected with siRNA, as indicated, as well as LifeAct-GFP (D–F) or Stx11-mCherry (G–K). (A) CTLs were loaded with Ca2+-sensitive Fluo-4 and Fura red dyes and assessed by flow cytometry and stimulated as indicated. iono., ionomycin. (B and C) Western blot of CTL lysates after transfection and stimulation as indicated. (B) Blots for phospho-ERK (pT202/pY204), VAMP8, and loading control GAPDH are shown for one representative donor. (C) Densitometry analysis of phospho-ERK signaling in five individual donors. Mean values are indicated, with bars representing SDs (ANOVA). (D) TIRF microscopy images of CTLs transfected with siRNA, as indicated, and LifeAct-GFP after contact with anti-CD3 and anti-CD28 antibody-coated coverslips for 250 s. (E) Quantification of F-actin clearance based on LifeAct-GFP fluorescence in individual cells (n = 12). (F) Quantification of cell spreading was based on LifeAct-GFP fluorescence in individual cells (n = 15). (G) Selected live-cell TIRF microscopy images of mCherry-Stx11 in representative CTLs transfected with siRNA as indicated. Arrowheads indicate fusion events; vesicle 1 is indicated by closed arrowheads, and vesicle 2 is indicated by open arrowheads. (H) Mean dwell time of mCherry-Stx11 vesicles in the TIRF plane per cell (n = 15, unpaired t test, **, P > 0.01). (I) Mean mCherry-Stx11 vesicle accumulation over time in the TIRF plane per cell (n = 20). (J) Mean overall mCherry-Stx11 accumulation over time in the TIRF plane per cell (n = 20). (K) Mean fluorescence dispersion events for mCherry-Stx11 vesicles in the TIRF plane per cell (n = 15, unpaired t test, **, P > 0.01). (L) Representative TIRF image depicting concentric circle region of interest used for radial analysis and dispersion of VAMP8-TFP and mCherry-Stx11. (M) Graph depicts the distribution of cumulative VAMP8-TFP fluorescence dispersion events on a per cell basis. (N) Graph depicts the distribution of cumulative mCherry-Stx11 fluorescence dispersion events on a per cell basis. (O) Quantification of mCherry-Stx11 radial distribution in TIRF plane of CTLs cotransfected with siRNA, as indicated. siCTRL, control siRNA. Bars: (D and G) 5 µm; (L) 2.5 µm.
Mentions: To further dissect the mechanism whereby VAMP8 facilitates lymphocyte cytotoxicity, we first studied the impact of VAMP8 knockdown on TCR signaling in human CTLs. CTLs were transfected with control siRNA or VAMP8 siRNA, labeled with Fluo-4 and Fura red Ca2+-sensitive dyes, and assessed by ratiometric flow cytometry. Knockdown of VAMP8 did not significantly alter intracellular Ca2+ mobilization after TCR stimulation (Figs. 7 A and S5 D), which is crucial for lymphocyte cytotoxicity (Maul-Pavicic et al., 2011). Similarly, knockdown of VAMP8 did not reduce phosphorylation of the MAPK extracellular signal–regulated kinase (ERK) after TCR stimulation (Fig. 7, B and C; and Fig. S5 E). ERK phosphorylation has been implicated in cytotoxic lymphocyte granule polarization (Jenkins et al., 2009). Thus, VAMP8 was not required for proximal TCR signals leading to intracellular Ca2+ mobilization and phosphorylation of ERK. To study actin dynamics, CTLs were cotransfected with recombinant, fluorescently tagged actin (LifeAct-GFP) and control siRNA or VAMP8 siRNA and subsequently imaged by TIRF microscopy (Fig. 7, D and E; and Fig. S5 F). In line with the experiments negating a role for VAMP8 in proximal TCR-induced Ca2+ mobilization and phosphorylation of ERK, knockdown of VAMP8 did not affect established F-actin architectural phases during immune synapse formation (Rak et al., 2011). An even distribution of F-actin was initially observed upon contact with the coverslip. After cell spreading and within ∼40 s, distribution of F-actin around the cell perimeter was observed. This annular distribution was accompanied by the appearance of granule-sized clearance in the actin meshwork and normal cell spreading (Fig. 7, E and F). In conclusion, our results indicated that VAMP8-mediated fusion with the plasma membrane is not required for proximal TCR-induced signaling and actin reorganization.

Bottom Line: Although multiple SNARE proteins have been implicated in cytotoxic granule exocytosis, the role of vesicular SNARE proteins, i.e., vesicle-associated membrane proteins (VAMPs), remains enigmatic.In primary human CTLs, however, VAMP8 colocalized with Rab11a-positive recycling endosomes.Our findings imply that secretory granule exocytosis pathways in other cell types may also be more complex than previously appreciated.

View Article: PubMed Central - HTML - PubMed

Affiliation: Cellular Neurophysiology, Center for Integrative Physiology and Molecular Medicine, Saarland University, 66421 Homburg, Germany Department of Medicine, Center For Infectious Medicine, 14186 Stockholm, Sweden.

Show MeSH
Related in: MedlinePlus