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: CTLs survey tissues for aberrant cells and can engage and kill target cells within 2 min through regulated exocytosis of specialized secretory lysosomes (Lopez et al., 2013). Exocytosis of cytotoxic proteins needs to be stringently controlled. Remarkably, we found accumulation and exocytosis of numerous VAMP8-carrying, Rab11-positive recycling endosomes within seconds of CTL contact with anti-CD3–coated coverslips. The exocytosis of these vesicles not only outnumbered but also preceded that of perforin- and granzyme B–containing cytotoxic granules. On average, 40 or more recycling endosomes fused with the plasma membrane within 4 min of CTL immune synapse formation. By comparison, less than five fusion-like events of cytotoxic granules were observed per cell. Recycling endosomes have previously been implicated in SNARE-dependent accumulation of TCR clusters to the immune synapse (Das et al., 2004). Specifically, synaptotagmin 7 and VAMP7 mediate fusion of vesicles containing TCR and LAT, respectively (Larghi et al., 2013; Soares et al., 2013). TCR-induced signaling is impaired in VAMP7-deficient CD4+ T cells (Larghi et al., 2013). Whether these vesicles fuse with the plasma membrane or form intracellular signaling platforms is disputed. Our data suggest that VAMP8 mediates recycling endosome fusion with the plasma membrane for delivery of molecules required for cytotoxic granule exocytosis but is not crucial for T cell activation. There is quite possibly heterogeneity in the lymphocyte recycling endosome compartment, where different vesicles may contribute to intracellular signaling versus delivery of cargo to the plasma membrane. Importantly, we find that VAMP8 is required for deposition of Stx11, an effector molecule of cytotoxic granule fusion (Bryceson et al., 2007), at the immune synapse. Thus, we posit a two-step sequential process for lymphocyte cytotoxicity in which VAMP8-mediated recycling endosome fusion brings effector proteins to the plasma membrane for SNARE complex formation and subsequent cytotoxic granule exocytosis (Fig. 8). Our data suggest that recycling endosome fusion is clustered toward the center of the immune synapse, possibly reflecting involvement of the microtubule-organizing complex in both recycling endosome and cytotoxic granule exocytosis. It is possible that additional engagement of integrins at cytotoxic immune synapses would further promote polarization of the recycling endosome compartment to a focal site of exocytosis (Stinchcombe et al., 2006; Liu et al., 2009). Moreover, although our data suggest recycling endosome fusion is critical for delivery of Stx11 to the immune synapse, it is also possible that VAMP8-mediated recycling endosome fusion with the plasma membrane provides other proteins required for cytotoxic granule exocytosis. It is worth noting that Munc13-4 also resides on Rab11-positive recycling endosomes (Ménager et al., 2007). Besides mediating cytotoxic granule to plasma membrane fusion, Munc13-4 has been previously proposed to promote late endosome to lysosome fusion, facilitating cytotoxic granule maturation (Ménager et al., 2007). It will therefore be interesting to address how distinct Munc13-4 isoforms may be involved in regulating exocytosis of recycling endosomes and cytotoxic granules (Cichocki et al., 2014). In summary, our findings unravel a novel component of vesicular fusion for lymphocyte cytotoxicity, providing a new layer of control in the carefully orchestrated process of target cell killing.
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.