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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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Activation induces up-regulation of VAMP8 expression and polarization of VAMP8 to immune synapses in human CD8+ T cells. (A) Relative expression of VAMP8 transcripts in unstimulated or bead-stimulated (stim.) CD8+ T cells was determined by qRT-PCR. UBC (encoding ubiquitin C) transcripts were used for normalization. Graph depicts data from five individual donors (paired t test, **, P = 0.0035). (B) VAMP8 expression in naive, bead-stimulated for the indicated days, or SEA-stimulated for 5 d CD8+ T cells was determined by Western blot analysis. (C) Expression of VAMP8 relative to GAPDH in CD8+ T cells after various stimulations, as indicated. Graphs represent mean, normalized expression of VAMP8 in five individuals with SD indicated (paired t test, *, P < 0.05; **, P < 0.005; ***, P < 0.001). (D) SIM images of endogenous VAMP8 localization in unconjugated (top) and SEA-target cell-conjugated (bottom) CTLs. (E) SIM images of ectopically expressed VAMP8-TFP localization in unconjugated (top) and SEA-target cell-conjugated (bottom) CTLs. Bars, 2.5 µm.
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fig1: Activation induces up-regulation of VAMP8 expression and polarization of VAMP8 to immune synapses in human CD8+ T cells. (A) Relative expression of VAMP8 transcripts in unstimulated or bead-stimulated (stim.) CD8+ T cells was determined by qRT-PCR. UBC (encoding ubiquitin C) transcripts were used for normalization. Graph depicts data from five individual donors (paired t test, **, P = 0.0035). (B) VAMP8 expression in naive, bead-stimulated for the indicated days, or SEA-stimulated for 5 d CD8+ T cells was determined by Western blot analysis. (C) Expression of VAMP8 relative to GAPDH in CD8+ T cells after various stimulations, as indicated. Graphs represent mean, normalized expression of VAMP8 in five individuals with SD indicated (paired t test, *, P < 0.05; **, P < 0.005; ***, P < 0.001). (D) SIM images of endogenous VAMP8 localization in unconjugated (top) and SEA-target cell-conjugated (bottom) CTLs. (E) SIM images of ectopically expressed VAMP8-TFP localization in unconjugated (top) and SEA-target cell-conjugated (bottom) CTLs. Bars, 2.5 µm.

Mentions: We examined human CTL expression of VAMP8, which from studies of knockout mice has been suggested to represent the vesicular SNARE required for cytotoxic granule exocytosis. VAMP8 transcripts were expressed in human primary CD8+ T cells, and their levels increased upon anti-CD3 and anti-CD28 bead stimulation, as assessed by quantitative real-time PCR (qRT-PCR; Fig. 1 A). Upon anti-CD3 and anti-CD28 bead or superantigen staphylococcal enterotoxin A (SEA) stimulation, VAMP8 protein expression also increased (Fig. 1, B and C). Notably, in our model, increased expression of VAMP8 upon activation coincided with an increased capacity of the stimulated CTL to kill target cells (Pattu et al., 2012). As the proportion of unstimulated, bulk CD8+ T cells expressing perforin and other cytotoxic granule constituents varies from donor to donor depending on the distribution of different cellular subsets (Chiang et al., 2013), we used CD8+ T cells stimulated with beads or SEA that more homogeneously express cytotoxic granule constituents for all further experiments. Such cells are hereafter referred to as CTLs.


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)

Activation induces up-regulation of VAMP8 expression and polarization of VAMP8 to immune synapses in human CD8+ T cells. (A) Relative expression of VAMP8 transcripts in unstimulated or bead-stimulated (stim.) CD8+ T cells was determined by qRT-PCR. UBC (encoding ubiquitin C) transcripts were used for normalization. Graph depicts data from five individual donors (paired t test, **, P = 0.0035). (B) VAMP8 expression in naive, bead-stimulated for the indicated days, or SEA-stimulated for 5 d CD8+ T cells was determined by Western blot analysis. (C) Expression of VAMP8 relative to GAPDH in CD8+ T cells after various stimulations, as indicated. Graphs represent mean, normalized expression of VAMP8 in five individuals with SD indicated (paired t test, *, P < 0.05; **, P < 0.005; ***, P < 0.001). (D) SIM images of endogenous VAMP8 localization in unconjugated (top) and SEA-target cell-conjugated (bottom) CTLs. (E) SIM images of ectopically expressed VAMP8-TFP localization in unconjugated (top) and SEA-target cell-conjugated (bottom) CTLs. Bars, 2.5 µm.
© Copyright Policy - openaccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4493996&req=5

fig1: Activation induces up-regulation of VAMP8 expression and polarization of VAMP8 to immune synapses in human CD8+ T cells. (A) Relative expression of VAMP8 transcripts in unstimulated or bead-stimulated (stim.) CD8+ T cells was determined by qRT-PCR. UBC (encoding ubiquitin C) transcripts were used for normalization. Graph depicts data from five individual donors (paired t test, **, P = 0.0035). (B) VAMP8 expression in naive, bead-stimulated for the indicated days, or SEA-stimulated for 5 d CD8+ T cells was determined by Western blot analysis. (C) Expression of VAMP8 relative to GAPDH in CD8+ T cells after various stimulations, as indicated. Graphs represent mean, normalized expression of VAMP8 in five individuals with SD indicated (paired t test, *, P < 0.05; **, P < 0.005; ***, P < 0.001). (D) SIM images of endogenous VAMP8 localization in unconjugated (top) and SEA-target cell-conjugated (bottom) CTLs. (E) SIM images of ectopically expressed VAMP8-TFP localization in unconjugated (top) and SEA-target cell-conjugated (bottom) CTLs. Bars, 2.5 µm.
Mentions: We examined human CTL expression of VAMP8, which from studies of knockout mice has been suggested to represent the vesicular SNARE required for cytotoxic granule exocytosis. VAMP8 transcripts were expressed in human primary CD8+ T cells, and their levels increased upon anti-CD3 and anti-CD28 bead stimulation, as assessed by quantitative real-time PCR (qRT-PCR; Fig. 1 A). Upon anti-CD3 and anti-CD28 bead or superantigen staphylococcal enterotoxin A (SEA) stimulation, VAMP8 protein expression also increased (Fig. 1, B and C). Notably, in our model, increased expression of VAMP8 upon activation coincided with an increased capacity of the stimulated CTL to kill target cells (Pattu et al., 2012). As the proportion of unstimulated, bulk CD8+ T cells expressing perforin and other cytotoxic granule constituents varies from donor to donor depending on the distribution of different cellular subsets (Chiang et al., 2013), we used CD8+ T cells stimulated with beads or SEA that more homogeneously express cytotoxic granule constituents for all further experiments. Such cells are hereafter referred to as CTLs.

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