Abstract
Expansion microscopy (ExM) enables super-resolution fluorescence imaging on standard microscopes by physical expansion of the sample. However, the investigation of interactions between different organisms such as mammalian and fungal cells by ExM remains challenging because different cell types require different expansion protocols to ensure identical, ideally isotropic expansion of both partners. Here, we introduce an ExM method that enables super-resolved visualization of the interaction between NK cells and Aspergillus fumigatus hyphae. 4-fold expansion in combination with confocal fluorescence imaging allows us to resolve details of cytoskeleton rearrangement as well as NK cells’ lytic granules triggered by contact with an RFP-expressing A. fumigatus strain. In particular, subdiffraction-resolution images show polarized degranulation upon contact formation and the presence of LAMP1 surrounding perforin at the NK cell-surface post degranulation. Our data demonstrate that optimized ExM protocols enable the investigation of immunological synapse formation between two different species with so far unmatched spatial resolution.
Highlights
Expansion microscopy (ExM) enables super-resolution fluorescence imaging on standard microscopes by physical expansion of the sample
That the interaction of immune cells with fungi urgently requires detailed investigations with high spatial resolution, we evaluated the possibility of combining all steps that are required for ExM of either fungi or mammalian cells in one mutual procedure using the following workflow (Fig. 1)
Natural killer (NK) cells were cocultured with an A. fumigatus strain constitutively expressing a fluorescent protein located in mitochondria at an multiplicity of infection (MOI) of about 0.5
Summary
Expansion microscopy (ExM) enables super-resolution fluorescence imaging on standard microscopes by physical expansion of the sample. The investigation of interactions between different organisms such as mammalian and fungal cells by ExM remains challenging because different cell types require different expansion protocols to ensure identical, ideally isotropic expansion of both partners. We introduce an ExM method that enables super-resolved visualization of the interaction between NK cells and Aspergillus fumigatus hyphae. Our data demonstrate that optimized ExM protocols enable the investigation of immunological synapse formation between two different species with so far unmatched spatial resolution. The IS has been analyzed in detail for the interaction of NK and cancer cells[15], but little is known for the IS formed between NK cells and Aspergillus fumigatus hyphae[11]. IS formation strongly depends on the polymerization of actin[16] and is impaired in NK cells obtained from allogeneic HSCT recipients, recovering within 180 days post-HSCT17
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