Behavior and Properties of Mature Lytic Granules at the Immunological Synapse of Human Cytotoxic T Lymphocytes

Abstract

Killing of virally infected cells or tumor cells by cytotoxic T lymphocytes requires targeting of lytic granules to the junction between the CTL and its target. We used whole-cell patch clamp to measure the cell capacitance at fixed intracellular [Ca2+] to study fusion of lytic granules in human CTLs. Expression of a fluorescently labeled human granzyme B construct allowed identification of lytic granule fusion using total internal reflection fluorescence microscopy. In this way capacitance steps due to lytic granule fusion were identified. Our goal was to determine the size of fusing lytic granules and to describe their behavior at the plasma membrane. On average, 5.02 ± 3.09 (mean ± s.d.) lytic granules were released per CTL. The amplitude of lytic granule fusion events was ~ 3.3 fF consistent with a diameter of about 325 nm. Fusion latency was biphasic with time constants of 15.9 and 106 seconds. The dwell time of fusing lytic granules was exponentially distributed with a mean dwell time of 28.5 seconds. Fusion ended in spite of the continued presence of granules at the immune synapse. The mobility of fusing granules at the membrane was indistinguishable from that of lytic granules which failed to fuse. While dwelling at the plasma membrane lytic granules exhibit mobility consistent with docking interspersed with short periods of greater mobility. The failure of lytic granules to fuse when visible in TIRF at the membrane may indicate that a membrane-confined reaction is rate limiting.

Exocytosis in non‐neuronal cells

“…; Ming et al . ), will reveal whether the synapse in the immune system and the synapse in the nervous system operate in similar ways to transmit information (or death) to their postsynaptic counterparts.…”

“…All these molecules like SNARE proteins and priming factors are also known to play an essential role in neurotransmitter release, emphasizing the strong similarities in the molecular mechanisms leading to fusion of these vesicles. Further efforts, including combined TIRFM/membrane capacitance measurements Ming et al 2015), will reveal whether the synapse in the immune system and the synapse in the nervous system operate in similar ways to transmit information (or death) to their postsynaptic counterparts.…”

Cortical actin recovery at the immunological synapse leads to termination of lytic granule secretion in cytotoxic T lymphocytes

“…As such, CTLs need to regulate secretion tightly, both to kill only appropriate target cells on contact and to facilitate their ability to carry out serial responses. Data suggest that each CTL releases only a small number of granules per productive killing outcome (4)(5)(6). However, how CTLs regulate both the initiation and the termination of granule secretion during an individual CTL-target interaction is still not completely understood.…”

Preparing the lethal hit: interplay between exo- and endocytic pathways in cytotoxic T lymphocytes

“…First, the synaptobrevin2-positive CGs were very homogeneous in diameter (about 350 nm), indicating that, in contrast to the postulate by Schmidt and coworkers [63, 64], only one class of mature CGs exists. This finding was supported by recent combined total internal reflection fluorescence (TIRF) microscopy and membrane capacitance measurements that determined a homogeneous diameter of fusing CGs of 312 nm [66]. The second surprising finding of the CLEM experiment was that not all dense-core granules with a diameter of about 350 nm were positive for synaptobrevin2.…”

“…Since CGs are the effector organelles of CTLs, during infection they are transported along the microtubular network of the CTL towards the IS where a low number of CGs (1–5) fuse with the plasma membrane to release their content and kill the infected target cell [66]. Fusion is driven by several SNARE proteins from which synaptobrevin2 was identified as the vesicular SNARE [62] and syntaxin11 [67] as a target SNARE on the plasma membrane.…”