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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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Vesicular VAMP8 colocalizes with Rab11a. (A) SIM images of ectopically expressed VAMP8-TFP and mCherry-Rab5a, mCherry-Rab7a, or mCherry-Rab11a localization in unconjugated (top) and SEA-target cell-conjugated (bottom) CTLs as indicated. Bars, 2.5 µm. (B) Pearson’s coefficients for VAMP8 and markers of the various endosomal compartments (n = 11, 11, and 12 CTLs, respectively). (C) Mander’s coefficients between VAMP8 (M1) and markers of the various endosomal compartments (M2; n = 11). Error bars are means and SDs. Rest., resting; Conj., conjugated.
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fig3: Vesicular VAMP8 colocalizes with Rab11a. (A) SIM images of ectopically expressed VAMP8-TFP and mCherry-Rab5a, mCherry-Rab7a, or mCherry-Rab11a localization in unconjugated (top) and SEA-target cell-conjugated (bottom) CTLs as indicated. Bars, 2.5 µm. (B) Pearson’s coefficients for VAMP8 and markers of the various endosomal compartments (n = 11, 11, and 12 CTLs, respectively). (C) Mander’s coefficients between VAMP8 (M1) and markers of the various endosomal compartments (M2; n = 11). Error bars are means and SDs. Rest., resting; Conj., conjugated.

Mentions: Having established that VAMP8 did not colocalize with cytotoxic granules, we sought the identity of the endosomal compartment that did contain VAMP8. CTLs were transfected with VAMP8-TFP and various endosomal markers coupled to mCherry. CTLs were thereafter mixed with SEA-pulsed target cells, fixed, and imaged using SIM. Imaging revealed limited colocalization between VAMP8 and Rab5a, a marker of early endosomes (Rybin et al., 1996; Barysch et al., 2009), or Rab7a, a marker of late endosomes (Pearson’s coefficient r of 0.34/0.39 and 0.36/0.36, respectively; Figs. 3 and S2; Chavrier et al., 1990). However, a substantial degree of colocalization was observed between VAMP8 and Rab11a, a marker of recycling endosomes (Ullrich et al., 1996), as indicated by a Pearson’s coefficient of 0.68/0.72 in unconjugated and conjugated CTLs, respectively (Figs. 3 and S2). Overall, VAMP8 preferentially localized to recycling endosomes in CTLs, with its distribution not being drastically affected by stimulation. These findings raise questions of how VAMP8 may facilitate human lymphocyte cytotoxicity.


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)

Vesicular VAMP8 colocalizes with Rab11a. (A) SIM images of ectopically expressed VAMP8-TFP and mCherry-Rab5a, mCherry-Rab7a, or mCherry-Rab11a localization in unconjugated (top) and SEA-target cell-conjugated (bottom) CTLs as indicated. Bars, 2.5 µm. (B) Pearson’s coefficients for VAMP8 and markers of the various endosomal compartments (n = 11, 11, and 12 CTLs, respectively). (C) Mander’s coefficients between VAMP8 (M1) and markers of the various endosomal compartments (M2; n = 11). Error bars are means and SDs. Rest., resting; Conj., conjugated.
© Copyright Policy - openaccess
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4493996&req=5

fig3: Vesicular VAMP8 colocalizes with Rab11a. (A) SIM images of ectopically expressed VAMP8-TFP and mCherry-Rab5a, mCherry-Rab7a, or mCherry-Rab11a localization in unconjugated (top) and SEA-target cell-conjugated (bottom) CTLs as indicated. Bars, 2.5 µm. (B) Pearson’s coefficients for VAMP8 and markers of the various endosomal compartments (n = 11, 11, and 12 CTLs, respectively). (C) Mander’s coefficients between VAMP8 (M1) and markers of the various endosomal compartments (M2; n = 11). Error bars are means and SDs. Rest., resting; Conj., conjugated.
Mentions: Having established that VAMP8 did not colocalize with cytotoxic granules, we sought the identity of the endosomal compartment that did contain VAMP8. CTLs were transfected with VAMP8-TFP and various endosomal markers coupled to mCherry. CTLs were thereafter mixed with SEA-pulsed target cells, fixed, and imaged using SIM. Imaging revealed limited colocalization between VAMP8 and Rab5a, a marker of early endosomes (Rybin et al., 1996; Barysch et al., 2009), or Rab7a, a marker of late endosomes (Pearson’s coefficient r of 0.34/0.39 and 0.36/0.36, respectively; Figs. 3 and S2; Chavrier et al., 1990). However, a substantial degree of colocalization was observed between VAMP8 and Rab11a, a marker of recycling endosomes (Ullrich et al., 1996), as indicated by a Pearson’s coefficient of 0.68/0.72 in unconjugated and conjugated CTLs, respectively (Figs. 3 and S2). Overall, VAMP8 preferentially localized to recycling endosomes in CTLs, with its distribution not being drastically affected by stimulation. These findings raise questions of how VAMP8 may facilitate human lymphocyte cytotoxicity.

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