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Varsha Pattu:
Endocytosis of lytic granules

Cytotoxic T lymphocytes (CTLs) are part of the adaptive immune system and eliminate bacterial- and virus-infected cells. Once a CTL identified such as cell, it builds a focal contact, the immunological synapse (IS). Within that IS a cluster of activation containing T cell receptors and signaling molecules is localized. IS formation leads to activation and polarization of the CTL. Killing occurs through directed transport and fusion of lytic granules (LG)  which release cytotoxic substances like perforin and granzymes. The molecular machinery executing and regulating these steps is of utmost importance for CTL function.

It is entirely unknown what happens to LGs following fusion at the IS. They may be recycled and reused, or they might be degraded.

We could show that the v-SNARE synaptobrevin2 is mediating the final fusion step of LGs in murine CTLs. Thus, synaptobrevin2 is an ideal marker for mature LGs in CTLs. 

We want to take advantage of a synaptobrevin2-mRFP knock-in mouse and investigate the putative endocytosis of LGs with high-resolution methods like structured illumination microscopy (SIM), total internal reflection fluorescence microscopy (TIRFM) and confocal laser-scanning microscopy.

(A) Synaptobrevin2 co-localizes with granzyme B on lytic granules. CTLs from syb2-mRFP knock-in mice were transfected with granzyme B fused to teal fluorescent protein (TFP). After fixation structured illumination images were obtained, yellow puncta indicate co-localization.

(B) Primary CTLs from syb2-mRFP knock-in mice were incubated with target cells in the presence of Alexa488-coupled anti-mRFP antibody in the medium. Confocal images at the indicated time points show the appearance of endocytosing vesicles (white arrows).