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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-carrying recycling endosomes accumulate and fuse at immune synapses. (A–E) Bead-stimulated human CD8+ T cells were transfected with VAMP8-TFP and mCherry-Rab11a encoding constructs and imaged 24 h after transfection. (A) Selected live-cell TIRF microscopy images of VAMP8-TFP and mCherry-Rab11a in a transfected CTL in contact with an anti-CD3– and anti-CD28–coated coverslip. Bar, 2.5 µm. (B) Mean dwell time of VAMP8-TFP and mCherry-Rab11a vesicles in the TIRF plane per cell (n = 15, paired t test, P > 0.05). (C) Mean VAMP8-TFP and mCherry-Rab11a vesicle accumulation over time in the TIRF plane per cell (n = 15). (D) Mean fluorescence dispersion events for VAMP8-TFP and mCherry-Rab11a vesicles in the TIRF plane per cell (n = 16, paired t test, P > 0.05). (E) Mean cumulative fluorescence dispersion events for VAMP8-TFP and mCherry-Rab11a vesicles in the TIRF plane per cell (n = 15). (F and G) Bead-stimulated human CD8+ T cells were transfected with constructs encoding VAMP8-TFP or VAMP8-3×FLAG-TFP and analyzed by flow cytometry 24 h after transfection. CTLs were stimulated with anti-CD3 and anti-CD28 antibodies and then surface stained with the anti-FLAG antibody. (F) Schematic representations of the constructs indicating the SNARE domain (SD) and transmembrane (TM) domains are depicted. Histograms from one representative donor show FLAG staining on TFP+ CTL, as indicated. Data are representative of three independent experiments performed on multiple donors. SBD, syntaxin binding domain. (G) Graphs depict FLAG mean fluorescent intensity (MFI) relative to unstimulated, VAMP8-3×FLAG-TFP–transfected CTLs in five individual donors. Mean and SDs are indicated (paired t test, **, P < 0.01). (H) Bead-stimulated human CD8+ T cells were transfected with VAMP8-pHluorin-mCherry (mCh) encoding constructs and imaged 24 h after transfection. Selected live-cell TIRF microscopy frames of two individual CTL vesicles are shown, as indicated. Graphs below depict respective pHluorin and mCherry vesicle MFI over time (experiments were repeated with three individual donors and n = 10 cells). Bars, 0.15 µm. Error bars show means and SDs.
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fig4: VAMP8-carrying recycling endosomes accumulate and fuse at immune synapses. (A–E) Bead-stimulated human CD8+ T cells were transfected with VAMP8-TFP and mCherry-Rab11a encoding constructs and imaged 24 h after transfection. (A) Selected live-cell TIRF microscopy images of VAMP8-TFP and mCherry-Rab11a in a transfected CTL in contact with an anti-CD3– and anti-CD28–coated coverslip. Bar, 2.5 µm. (B) Mean dwell time of VAMP8-TFP and mCherry-Rab11a vesicles in the TIRF plane per cell (n = 15, paired t test, P > 0.05). (C) Mean VAMP8-TFP and mCherry-Rab11a vesicle accumulation over time in the TIRF plane per cell (n = 15). (D) Mean fluorescence dispersion events for VAMP8-TFP and mCherry-Rab11a vesicles in the TIRF plane per cell (n = 16, paired t test, P > 0.05). (E) Mean cumulative fluorescence dispersion events for VAMP8-TFP and mCherry-Rab11a vesicles in the TIRF plane per cell (n = 15). (F and G) Bead-stimulated human CD8+ T cells were transfected with constructs encoding VAMP8-TFP or VAMP8-3×FLAG-TFP and analyzed by flow cytometry 24 h after transfection. CTLs were stimulated with anti-CD3 and anti-CD28 antibodies and then surface stained with the anti-FLAG antibody. (F) Schematic representations of the constructs indicating the SNARE domain (SD) and transmembrane (TM) domains are depicted. Histograms from one representative donor show FLAG staining on TFP+ CTL, as indicated. Data are representative of three independent experiments performed on multiple donors. SBD, syntaxin binding domain. (G) Graphs depict FLAG mean fluorescent intensity (MFI) relative to unstimulated, VAMP8-3×FLAG-TFP–transfected CTLs in five individual donors. Mean and SDs are indicated (paired t test, **, P < 0.01). (H) Bead-stimulated human CD8+ T cells were transfected with VAMP8-pHluorin-mCherry (mCh) encoding constructs and imaged 24 h after transfection. Selected live-cell TIRF microscopy frames of two individual CTL vesicles are shown, as indicated. Graphs below depict respective pHluorin and mCherry vesicle MFI over time (experiments were repeated with three individual donors and n = 10 cells). Bars, 0.15 µm. Error bars show means and SDs.

Mentions: Although VAMP8 exhibited strong colocalization with recycling endosomes in our fixed cell experiments, it was unclear whether these VAMP8 vesicles were also trafficking through the recycling endosome compartment and fusing at the immune synapse. To examine the behavior of VAMP8-carrying vesicles, we used TIRF microscopy to visualize vesicle dynamics in the vicinity of the immune synapse. This technique allows real-time visualization of vesicles within 200 nm of the immune synapse (Campi et al., 2005; Douglass and Vale, 2005; Yokosuka et al., 2005). Transfection of CTLs with VAMP8-TFP showed rapid movement and subsequent accumulation of VAMP8-carrying vesicles in the TIRF plane (Video 1). To simultaneously monitor the dynamics of VAMP8 and recycling endosomes, CTLs were cotransfected with VAMP8-TFP and Rab11a-mCherry and allowed to form synapses with anti-CD3 and anti-CD28 antibody-coated coverslips. Shortly after sedimentation, the appearance and subsequent accumulation of both VAMP8- and Rab11a-containing vesicles could be observed at the immune synapse in the TIRF plane (Fig. 4 A and Video 2). VAMP8-TFP and Rab11a-mCherry exhibited a striking colocalization in the TIRF plane (Fig. 4 A). In comparing the dwell times at the immune synapse, we found that there was no statistically significant difference between Rab11a-mCherry vesicles and VAMP8-TFP vesicles, with VAMP8 dwell time of 14.5 ± 0.7 s compared with 15.7 ± 0.9 s of Rab11a (Fig. 4 B). When we examined vesicle accumulation over time, we also found similar numbers of VAMP8 and Rab11a vesicles at immune synapses (Fig. 4 C).


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-carrying recycling endosomes accumulate and fuse at immune synapses. (A–E) Bead-stimulated human CD8+ T cells were transfected with VAMP8-TFP and mCherry-Rab11a encoding constructs and imaged 24 h after transfection. (A) Selected live-cell TIRF microscopy images of VAMP8-TFP and mCherry-Rab11a in a transfected CTL in contact with an anti-CD3– and anti-CD28–coated coverslip. Bar, 2.5 µm. (B) Mean dwell time of VAMP8-TFP and mCherry-Rab11a vesicles in the TIRF plane per cell (n = 15, paired t test, P > 0.05). (C) Mean VAMP8-TFP and mCherry-Rab11a vesicle accumulation over time in the TIRF plane per cell (n = 15). (D) Mean fluorescence dispersion events for VAMP8-TFP and mCherry-Rab11a vesicles in the TIRF plane per cell (n = 16, paired t test, P > 0.05). (E) Mean cumulative fluorescence dispersion events for VAMP8-TFP and mCherry-Rab11a vesicles in the TIRF plane per cell (n = 15). (F and G) Bead-stimulated human CD8+ T cells were transfected with constructs encoding VAMP8-TFP or VAMP8-3×FLAG-TFP and analyzed by flow cytometry 24 h after transfection. CTLs were stimulated with anti-CD3 and anti-CD28 antibodies and then surface stained with the anti-FLAG antibody. (F) Schematic representations of the constructs indicating the SNARE domain (SD) and transmembrane (TM) domains are depicted. Histograms from one representative donor show FLAG staining on TFP+ CTL, as indicated. Data are representative of three independent experiments performed on multiple donors. SBD, syntaxin binding domain. (G) Graphs depict FLAG mean fluorescent intensity (MFI) relative to unstimulated, VAMP8-3×FLAG-TFP–transfected CTLs in five individual donors. Mean and SDs are indicated (paired t test, **, P < 0.01). (H) Bead-stimulated human CD8+ T cells were transfected with VAMP8-pHluorin-mCherry (mCh) encoding constructs and imaged 24 h after transfection. Selected live-cell TIRF microscopy frames of two individual CTL vesicles are shown, as indicated. Graphs below depict respective pHluorin and mCherry vesicle MFI over time (experiments were repeated with three individual donors and n = 10 cells). Bars, 0.15 µm. Error bars show means and SDs.
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fig4: VAMP8-carrying recycling endosomes accumulate and fuse at immune synapses. (A–E) Bead-stimulated human CD8+ T cells were transfected with VAMP8-TFP and mCherry-Rab11a encoding constructs and imaged 24 h after transfection. (A) Selected live-cell TIRF microscopy images of VAMP8-TFP and mCherry-Rab11a in a transfected CTL in contact with an anti-CD3– and anti-CD28–coated coverslip. Bar, 2.5 µm. (B) Mean dwell time of VAMP8-TFP and mCherry-Rab11a vesicles in the TIRF plane per cell (n = 15, paired t test, P > 0.05). (C) Mean VAMP8-TFP and mCherry-Rab11a vesicle accumulation over time in the TIRF plane per cell (n = 15). (D) Mean fluorescence dispersion events for VAMP8-TFP and mCherry-Rab11a vesicles in the TIRF plane per cell (n = 16, paired t test, P > 0.05). (E) Mean cumulative fluorescence dispersion events for VAMP8-TFP and mCherry-Rab11a vesicles in the TIRF plane per cell (n = 15). (F and G) Bead-stimulated human CD8+ T cells were transfected with constructs encoding VAMP8-TFP or VAMP8-3×FLAG-TFP and analyzed by flow cytometry 24 h after transfection. CTLs were stimulated with anti-CD3 and anti-CD28 antibodies and then surface stained with the anti-FLAG antibody. (F) Schematic representations of the constructs indicating the SNARE domain (SD) and transmembrane (TM) domains are depicted. Histograms from one representative donor show FLAG staining on TFP+ CTL, as indicated. Data are representative of three independent experiments performed on multiple donors. SBD, syntaxin binding domain. (G) Graphs depict FLAG mean fluorescent intensity (MFI) relative to unstimulated, VAMP8-3×FLAG-TFP–transfected CTLs in five individual donors. Mean and SDs are indicated (paired t test, **, P < 0.01). (H) Bead-stimulated human CD8+ T cells were transfected with VAMP8-pHluorin-mCherry (mCh) encoding constructs and imaged 24 h after transfection. Selected live-cell TIRF microscopy frames of two individual CTL vesicles are shown, as indicated. Graphs below depict respective pHluorin and mCherry vesicle MFI over time (experiments were repeated with three individual donors and n = 10 cells). Bars, 0.15 µm. Error bars show means and SDs.
Mentions: Although VAMP8 exhibited strong colocalization with recycling endosomes in our fixed cell experiments, it was unclear whether these VAMP8 vesicles were also trafficking through the recycling endosome compartment and fusing at the immune synapse. To examine the behavior of VAMP8-carrying vesicles, we used TIRF microscopy to visualize vesicle dynamics in the vicinity of the immune synapse. This technique allows real-time visualization of vesicles within 200 nm of the immune synapse (Campi et al., 2005; Douglass and Vale, 2005; Yokosuka et al., 2005). Transfection of CTLs with VAMP8-TFP showed rapid movement and subsequent accumulation of VAMP8-carrying vesicles in the TIRF plane (Video 1). To simultaneously monitor the dynamics of VAMP8 and recycling endosomes, CTLs were cotransfected with VAMP8-TFP and Rab11a-mCherry and allowed to form synapses with anti-CD3 and anti-CD28 antibody-coated coverslips. Shortly after sedimentation, the appearance and subsequent accumulation of both VAMP8- and Rab11a-containing vesicles could be observed at the immune synapse in the TIRF plane (Fig. 4 A and Video 2). VAMP8-TFP and Rab11a-mCherry exhibited a striking colocalization in the TIRF plane (Fig. 4 A). In comparing the dwell times at the immune synapse, we found that there was no statistically significant difference between Rab11a-mCherry vesicles and VAMP8-TFP vesicles, with VAMP8 dwell time of 14.5 ± 0.7 s compared with 15.7 ± 0.9 s of Rab11a (Fig. 4 B). When we examined vesicle accumulation over time, we also found similar numbers of VAMP8 and Rab11a vesicles at immune synapses (Fig. 4 C).

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