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Cytotoxic granule endocytosis depends on the Flower protein.

ABSTRACT: Cytotoxic T lymphocytes (CTLs) kill target cells by the regulated release of cytotoxic substances from granules at the immunological synapse. To kill multiple target cells, CTLs use endocytosis of membrane components of cytotoxic granules. We studied the potential calcium dependence of endocytosis in mouse CTLs on Flower, which mediates the calcium dependence of synaptic vesicle endocytosis in Drosophila melanogaster Flower is predominantly localized on intracellular vesicles that move to the synapse on target cell contact. Endocytosis is entirely blocked at an early stage in Flower-deficient CTLs and is rescued to wild-type level by reintroducing Flower or by raising extracellular calcium. A Flower mutant lacking binding sites for the endocytic adaptor AP-2 proteins fails to rescue endocytosis, indicating that Flower interacts with proteins of the endocytic machinery to mediate granule endocytosis. Thus, our data identify Flower as a key protein mediating granule endocytosis.

SUBMITTER: Chang HF

PROVIDER: S-EPMC5800809 | biostudies-literature | 2018 Feb

REPOSITORIES: biostudies-literature

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Cytotoxic granule endocytosis depends on the Flower protein.

Chang Hsin-Fang HF Mannebach Stefanie S Beck Andreas A Ravichandran Keerthana K Krause Elmar E Frohnweiler Katja K Fecher-Trost Claudia C Schirra Claudia C Pattu Varsha V Flockerzi Veit V Rettig Jens J

The Journal of cell biology 20171229 2

Cytotoxic T lymphocytes (CTLs) kill target cells by the regulated release of cytotoxic substances from granules at the immunological synapse. To kill multiple target cells, CTLs use endocytosis of membrane components of cytotoxic granules. We studied the potential calcium dependence of endocytosis in mouse CTLs on Flower, which mediates the calcium dependence of synaptic vesicle endocytosis in <i>Drosophila melanogaster</i> Flower is predominantly localized on intracellular vesicles that move to ...[more]

PMID: 29288152

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Project description:Granules of cytotoxic T lymphocytes (CTL) are derived from the lysosomal compartment. Synaptotagmin7 (Syt7) appears to be the calcium sensor triggering fusion of lysosomes in fibroblasts. Syt7 has been proposed to control cytotoxic granule (CG) fusion in lymphocytes and mice lacking Syt7 have reduced ability to clear infections. However, fusion of CG persists in the absence of Syt7. To clarify the role of Syt7 in CTL function, we have examined the fusion of cytotoxic granules of CD8+ T-lymphocytes from Syt7 knock-out mice. We have recorded granule fusion in living CTL, using total internal reflection microscopy. Since Syt7 is considered a high affinity calcium-sensor specialized for fusion under low calcium conditions, we have compared cytotoxic granule fusion under low and high calcium conditions in the same CTL. There was no difference in latencies or numbers of fusion events per CTL under low-calcium conditions, indicating that Syt7 is not required for cytotoxic granule fusion. A deficit of fusion in Syt7 KO CTL was seen when a high-calcium solution was introduced. Expressing wild type Syt7 in Syt7 KO lymphocytes reversed this deficit, confirming its Syt7-dependence. Mutations of Syt7 which disrupt calcium binding to its C2A domain reduced the efficacy of this rescue. We counted the cytotoxic granules present at the plasma membrane to determine if the lack of fusion events in the Syt7 KO CTL was due to a lack of granules. In low calcium there were no differences in fusion events per CTL, and granule numbers were similar. In high calcium, granule number was similar though wild type CTL exhibited significantly more fusion than Syt7 KO CTL. The modest differences in granule counts do not account for the lack of fusion in high calcium in Syt7 KO CTL. In Syt7 KO CTL expressing wild type Syt7, delivery of cytotoxic granules to the plasma membrane was comparable to that of wild type CTL. Syt7 KO CTL expressing Syt7 with deficient calcium binding in the C2A domain had significantly less fusion and fewer CG at the plasma membrane. These results indicate that Syt7 is involved in trafficking of CG to the plasma membrane.

Dataset Information

Cytotoxic granule endocytosis depends on the Flower protein.

Publications

Cytotoxic granule endocytosis depends on the Flower protein.

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