Dual channel STED nanoscopy of lytic granules on actin filaments in natural killer cells

Abstract

Natural killer (NK) cells are innate immune effectors that eliminate diseased and tumorigenic targets through the directed secretion of specialized secretory lysosomes, termed lytic granules. This directed secretion is triggered following the formation of an immunological synapse (IS), which is characterized by actin re-modeling and receptor organization at the interface between the NK cell and its susceptible target. Actin at the IS has been described to be permissive to secretion by forming a large central clearance through which lytic granules are released. However we, and others, have recently shown that the actin network in NK cells at the IS is dynamic yet pervasive. These efforts used multiple high resolution imaging techniques to demonstrate that the actin network does not act as a barrier to secretion, but instead enables the secretion of lytic granules through minimally sized clearances. In our recent publication we visualized actin using continuous wave stimulated emission depletion (CW-STED) and lytic granules using the confocal modality. Here we report for the first time dual channel STED nanoscopy of NK cell lytic granules on actin filaments.

Keywords: STED microscopy; actin; immunological synapse; lytic granules; natural killer cells.

Figure 1. Visualization of lytic granules imaged by CW-STED and confocal on F-actin. NK92 cells were adhered to glass coated with antibody to activating (NKp30) and adhesion (CD18) receptor then fixed, permeabilized and stained for perforin and actin. Cells were imaged using CW-STED (actin, green) and either CW-STED or confocal (perforin, red). Shown is the same cell with granules detected by STED (A) or confocal (B). A region of interest is enlarged to show greater resolution of granules (center panel). (C) Full width half maximum (FWHM) measurements of confocal (green line) and STED images (red line). Horizontal dashed lines show half maxima, vertical dashed lines show width at half maxima. (D) Representative line profile of pixel intensities of actin (green line) and perforin (red line) taken from a line bisecting a single granule (shown in white in STED image enlargement). AU, arbitrary units.