title: In the crosshairs: investigating lytic granules by high-resolution microscopy and electrophysiology.
abstract: Cytotoxic T lymphocytes (CTLs) form an integral part of the adaptive immune system. Their main function is to eliminate bacteria- and virus-infected target cells by releasing perforin and granzymes (the lethal hit) contained within lytic granules (LGs), at the CTL-target-cell interface [the immunological synapse (IS)]. The formation of the IS as well as the final events at the IS leading to target-cell death are both highly complex and dynamic processes. In this review we highlight and discuss three high-resolution techniques that have proven invaluable in the effort to decipher key features of the mechanism of CTL effector function and in particular lytic granule maturation and fusion. Correlative light and electron microscopy allows the correlation between organelle morphology and localization of particular proteins, while total internal reflection fluorescence microscopy (TIRFM) enables the study of lytic granule dynamics at the IS in real time. The combination of TIRFM with patch-clamp membrane capacitance measurements finally provides a tool to quantify the size of fusing LGs at the IS.
authors.affiliation: Institute of Physiology, Saarland University , Homburg, Saar , Germany.
authors.forename: Varsha
authors.initials: V
authors.lastname: Pattu
authors.affiliation: Institute of Physiology, Saarland University , Homburg, Saar , Germany.
authors.forename: Mahantappa
authors.initials: M
authors.lastname: Halimani
authors.affiliation: Institute of Physiology, Saarland University , Homburg, Saar , Germany.
authors.forename: Min
authors.initials: M
authors.lastname: Ming
authors.affiliation: Institute of Physiology, Saarland University , Homburg, Saar , Germany.
authors.forename: Claudia
authors.initials: C
authors.lastname: Schirra
authors.affiliation: Institute of Physiology, Saarland University , Homburg, Saar , Germany.
authors.forename: Ulrike
authors.initials: U
authors.lastname: Hahn
authors.affiliation: Institute of Physiology, Saarland University , Homburg, Saar , Germany.
authors.forename: Hawraa
authors.initials: H
authors.lastname: Bzeih
authors.affiliation: Institute of Physiology, Saarland University , Homburg, Saar , Germany.
authors.forename: Hsin-Fang
authors.initials: HF
authors.lastname: Chang
authors.affiliation: Institute of Physiology, Saarland University , Homburg, Saar , Germany.
authors.forename: Lisa
authors.initials: L
authors.lastname: Weins
authors.affiliation: Institute of Physiology, Saarland University , Homburg, Saar , Germany.
authors.forename: Elmar
authors.initials: E
authors.lastname: Krause
authors.affiliation: Institute of Physiology, Saarland University , Homburg, Saar , Germany.
authors.forename: Jens
authors.initials: J
authors.lastname: Rettig
keywords: SNARE proteins; TIRFM; correlative light and electron microscopy; cytotoxic T cells; lytic granules; structured illumination microscopy