Quantitative measurement of F-actin accumulation at the NK cell immunological synapse.
NK cells are lymphocytes of the innate immune system that can kill target cells after activation signal-induced directional secretion of lytic granule contents. This process depends upon F-actin polymerization at the NK cell immunological synapse (NKIS), which is the dynamic organization of molecules at the interface between the NK cell and target cell. Although F-actin accumulation at the NKIS is easily visualized, the ability to quantify F-actin at the NKIS is required to understand how F-actin reorganization and accumulation enable NK cell function. Here, we demonstrate several novel algorithms for measuring the content of F-actin accumulated at the NKIS with special emphasis upon actin contributed by the NK cell. These algorithms do not rely upon overexpressing fluorescent proteins or preincubating cells with vital fluorescent dyes. Using models of the activating and inhibitory NKIS as well as NK cells expressing fluorescent protein--cell surface receptor fusion proteins, these algorithms were tested and were used to quantitatively demonstrate that F-actin accumulates at the activating, but not at the inhibitory NKIS. With these approaches, we have also established mathematical formulas that should prove valuable in the comprehensive quantitative evaluation of the NKIS and be more broadly applicable in the measurement of the accumulation of any fluorophore at an intercellular junction.
Banerjee
Pinaki P
PP
The Children's Hospital of Philadelphia Research Institute, 3615 Civic Center Blvd., Philadelphia, PA 19104, USA.
Orange
Jordan S
JS
D000199:Actins / Q000032:analysis* / Q000276:immunology / Q000378:metabolism; D000465:Algorithms; D002478:Cells, Cultured; D005456:Fluorescent Dyes / Q000032:analysis / Q000378:metabolism; D006801:Humans; D054992:Immunological Synapses / Q000276:immunology / Q000378:metabolism*; D007694:Killer Cells, Natural / Q000166:cytology / Q000276:immunology / Q000378:metabolism*; D036641:Microscopy, Fluorescence, Multiphoton / Q000379:methods*; D008954:Models, Biological
D000199:Actins; D005456:Fluorescent Dyes