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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 cells accumulate and fuse at the immune synapse before cytotoxic granules. (A–E) Bead-stimulated human CD8+ T cells were transfected with VAMP8-TFP and perforin-mCherry encoding constructs and imaged 24 h after transfection. (A) Selected live-cell TIRF microscopy images of VAMP8-TFP and perforin-mCherry in a transfected CTLs in contact with an anti-CD3– and anti-CD28–coated coverslip. Bar, 2.5 µm. (B) Mean dwell time of VAMP8-TFP and perforin-mCherry vesicles in the TIRF plane per cell (n = 15, paired t test, ***, P > 0.001). (C) Mean VAMP8-TFP and perforin-mCherry vesicle accumulation over time in the TIRF plane per cell (n = 15). (D) Mean fluorescence dispersion events for VAMP8-TFP and perforin-mCherry vesicles in the TIRF plane per cell (n = 15, paired t test, ***, P > 0.001). (E) Cumulative fluorescence dispersion events for VAMP8-TFP and perforin-mCherry vesicles in the TIRF plane per cell (n = 18). Error bars show means and SDs.
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fig5: VAMP8-carrying cells accumulate and fuse at the immune synapse before cytotoxic granules. (A–E) Bead-stimulated human CD8+ T cells were transfected with VAMP8-TFP and perforin-mCherry encoding constructs and imaged 24 h after transfection. (A) Selected live-cell TIRF microscopy images of VAMP8-TFP and perforin-mCherry in a transfected CTLs in contact with an anti-CD3– and anti-CD28–coated coverslip. Bar, 2.5 µm. (B) Mean dwell time of VAMP8-TFP and perforin-mCherry vesicles in the TIRF plane per cell (n = 15, paired t test, ***, P > 0.001). (C) Mean VAMP8-TFP and perforin-mCherry vesicle accumulation over time in the TIRF plane per cell (n = 15). (D) Mean fluorescence dispersion events for VAMP8-TFP and perforin-mCherry vesicles in the TIRF plane per cell (n = 15, paired t test, ***, P > 0.001). (E) Cumulative fluorescence dispersion events for VAMP8-TFP and perforin-mCherry vesicles in the TIRF plane per cell (n = 18). Error bars show means and SDs.

Mentions: To gain further insights to the relationship between VAMP8-carrying recycling endosome activity and cytotoxic granule exocytosis at human immune synapses, CTLs were cotransfected with VAMP8-TFP and perforin-mCherry and allowed to form a synapse with anti-CD3 and anti-CD28 antibody-coated coverslips. CTLs were imaged in real-time by TIRF microscopy. Interestingly, in live-cell imaging, multiple VAMP8-carrying recycling endosomes rapidly appeared in the TIRF plane followed by fewer perforin-containing vesicles (Fig. 5 A and Video 4). Comparing the dwell times of both VAMP8 and perforin vesicles in the TIRF plane, the former resided significantly shorter than the latter (14.5 ± 0.8 s for VAMP8-carrying vesicles vs. 117.4 ± 18.9 s for perforin-containing vesicles, respectively; Fig. 5 B). The accumulation of VAMP8-carrying recycling endosomes outnumbered that of perforin-containing cytotoxic granules at the immune synapse (Fig. 5 C). At the start of the 6-min recording, VAMP8-carrying vesicles showed rapid accumulation that subsided with time. By comparison, perforin-containing vesicles appeared later in the TIRF plane. Comparing fusion-like events revealed by imaging of VAMP8-TFP and perforin-mCherry, CTLs exhibited many more VAMP8 versus perforin fusion-like events per cell (41.6 ± 7.6 vs. 2.4 ± 0.1, respectively; Fig. 5 D). As previously noted, the VAMP8-carrying vesicle fusion-like events occurred promptly after immune synapse formation and then gradually subsided with time (Fig. 5 E). Similar results were also found upon cotransfection of CTL with VAMP8-TFP and granzyme B–mCherry (Fig. S4 and Video 5). In summary, VAMP8-carrying recycling endosome fusion preceded cytotoxic granule exocytosis, implying that recycling endosome fusion with the plasma membrane might be VAMP8 dependent and represent a prerequisite for cytotoxic granule exocytosis.


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 cells accumulate and fuse at the immune synapse before cytotoxic granules. (A–E) Bead-stimulated human CD8+ T cells were transfected with VAMP8-TFP and perforin-mCherry encoding constructs and imaged 24 h after transfection. (A) Selected live-cell TIRF microscopy images of VAMP8-TFP and perforin-mCherry in a transfected CTLs in contact with an anti-CD3– and anti-CD28–coated coverslip. Bar, 2.5 µm. (B) Mean dwell time of VAMP8-TFP and perforin-mCherry vesicles in the TIRF plane per cell (n = 15, paired t test, ***, P > 0.001). (C) Mean VAMP8-TFP and perforin-mCherry vesicle accumulation over time in the TIRF plane per cell (n = 15). (D) Mean fluorescence dispersion events for VAMP8-TFP and perforin-mCherry vesicles in the TIRF plane per cell (n = 15, paired t test, ***, P > 0.001). (E) Cumulative fluorescence dispersion events for VAMP8-TFP and perforin-mCherry vesicles in the TIRF plane per cell (n = 18). Error bars show means and SDs.
© Copyright Policy - openaccess
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fig5: VAMP8-carrying cells accumulate and fuse at the immune synapse before cytotoxic granules. (A–E) Bead-stimulated human CD8+ T cells were transfected with VAMP8-TFP and perforin-mCherry encoding constructs and imaged 24 h after transfection. (A) Selected live-cell TIRF microscopy images of VAMP8-TFP and perforin-mCherry in a transfected CTLs in contact with an anti-CD3– and anti-CD28–coated coverslip. Bar, 2.5 µm. (B) Mean dwell time of VAMP8-TFP and perforin-mCherry vesicles in the TIRF plane per cell (n = 15, paired t test, ***, P > 0.001). (C) Mean VAMP8-TFP and perforin-mCherry vesicle accumulation over time in the TIRF plane per cell (n = 15). (D) Mean fluorescence dispersion events for VAMP8-TFP and perforin-mCherry vesicles in the TIRF plane per cell (n = 15, paired t test, ***, P > 0.001). (E) Cumulative fluorescence dispersion events for VAMP8-TFP and perforin-mCherry vesicles in the TIRF plane per cell (n = 18). Error bars show means and SDs.
Mentions: To gain further insights to the relationship between VAMP8-carrying recycling endosome activity and cytotoxic granule exocytosis at human immune synapses, CTLs were cotransfected with VAMP8-TFP and perforin-mCherry and allowed to form a synapse with anti-CD3 and anti-CD28 antibody-coated coverslips. CTLs were imaged in real-time by TIRF microscopy. Interestingly, in live-cell imaging, multiple VAMP8-carrying recycling endosomes rapidly appeared in the TIRF plane followed by fewer perforin-containing vesicles (Fig. 5 A and Video 4). Comparing the dwell times of both VAMP8 and perforin vesicles in the TIRF plane, the former resided significantly shorter than the latter (14.5 ± 0.8 s for VAMP8-carrying vesicles vs. 117.4 ± 18.9 s for perforin-containing vesicles, respectively; Fig. 5 B). The accumulation of VAMP8-carrying recycling endosomes outnumbered that of perforin-containing cytotoxic granules at the immune synapse (Fig. 5 C). At the start of the 6-min recording, VAMP8-carrying vesicles showed rapid accumulation that subsided with time. By comparison, perforin-containing vesicles appeared later in the TIRF plane. Comparing fusion-like events revealed by imaging of VAMP8-TFP and perforin-mCherry, CTLs exhibited many more VAMP8 versus perforin fusion-like events per cell (41.6 ± 7.6 vs. 2.4 ± 0.1, respectively; Fig. 5 D). As previously noted, the VAMP8-carrying vesicle fusion-like events occurred promptly after immune synapse formation and then gradually subsided with time (Fig. 5 E). Similar results were also found upon cotransfection of CTL with VAMP8-TFP and granzyme B–mCherry (Fig. S4 and Video 5). In summary, VAMP8-carrying recycling endosome fusion preceded cytotoxic granule exocytosis, implying that recycling endosome fusion with the plasma membrane might be VAMP8 dependent and represent a prerequisite for cytotoxic granule exocytosis.

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