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