ALFA-PRF: a novel approach to detect murine perforin release from CTLs into the immune synapse
- Author(s)
- Rudd-Schmidt, JA; Laine, RF; Noori, T; Brennan, AJ; Voskoboinik, I;
- Journal Title
- Frontiers in Immunology
- Publication Type
- Research article
- Abstract
- When killing through the granule exocytosis pathway, cytotoxic lymphocytes release key effector molecules into the immune synapse, perforin and granzymes, to initiate target cell killing. The pore-forming perforin is essential for the function of cytotoxic lymphocytes, as its pores disrupt the target cell membrane and allow diffusion of pro-apoptotic serine proteases, granzyme, into the target cell, where they initiate various cell death cascades. Unlike human perforin, the detection of its murine counterpart in a live cell system has been problematic due its relatively low expression level and the lack of sensitive antibodies. The lack of a suitable methodology to visualise murine perforin secretion into the synapse hinders the study of the cytotoxic lymphocyte secretory machinery in murine models of human disease. Here, we describe a novel recombinant technology, whereby a short ALFA-tag sequence has been fused with the amino-terminus of a mature murine perforin, and this allowed its detection by the highly specific FluoTag((R))-X2 anti-ALFA nanobodies using both Total Internal Reflection Fluorescence (TIRF) microscopy of an artificial synapse, and confocal microscopy of the physiological immune synapse with a target cell. This methodology can have broad application in the field of cytotoxic lymphocyte biology and for the many models of human disease.
- Keywords
- Animals; Mice; Cell Death; Cell Membrane/metabolism; Granzymes/metabolism; *Perforin/metabolism; *T-Lymphocytes, Cytotoxic; *Immunological Synapses; cytotoxic granules; cytotoxic lymphocyte; granzyme; immunological synapse; microscopy; trafficking
- Department(s)
- Laboratory Research
- PubMed ID
- 36618385
- Publisher's Version
- https://doi.org/10.3389/fimmu.2022.931820
- Open Access at Publisher's Site
https://doi.org/10.3389/fimmu.2022.931820- Terms of Use/Rights Notice
- Refer to copyright notice on published article.
Creation Date: 2025-03-07 05:57:25
Last Modified: 2025-03-07 06:00:21