Poster Pitches Australian Microbial Ecology 2022

Correlative light and electron microscopy to detect protein-protein interactions in microbial symbiosis (#64)

Yunke Jia 1 , Torsten Thomas 1 , Joanna Biazik-Richmond 2 , Renee Whan 3
  1. Centre for Marine Science and Innovation, School of Biological, Earth and Environmental Sciences, UNSW, Sydney, NSW, Australia
  2. Electron Microscope Unit, UNSW, Sydney, NSW, Australia
  3. Katharina Gaus Light Microscopy Facility, UNSW, Sydney, NSW, Australia

Fluorescence in situ hybridization (FISH) and immunofluorescence (Immuno) microscopy are powerful techniques to detect specific bacteria and proteins, respectively. Combining these techniques with electron microscopy in the advanced technique called correlative light and electron microscopy (CLEM) has the potential to detect specific bacteria or molecules with the high-resolution (ultrastructure) information. However, FISH-CLEM and Immuno-CLEM are often challenging due to the low fluorescent signals in the ultrathin sections that are required for electron microscopy. Here, we optimized FISH-CLEM and Immuno-CLEM with respect to ultrathin section preparation, probe hybridization and image acquisition, which resulted in a significant improvement in fluorescent signal strength and detection sensitivity. This optimized protocol was applied to visualize the interactions of eukaryotic-like proteins (ELPs), which are produced by bacterial symbionts of sponges. ELPs have been postulated to be key molecular mediators of bacteria-sponge symbiosis and have been in vitro shown to interact with specific binding partner proteins (BPPs) that the sponge produces.  The optimized method enabled the visualization of specific ELPs within specific bacteria hosted in the tissue of the sponge Cymbastela concentrica and furthermore revealed patterns of co-localisation with BPP. The technique therefore enables to put protein-protein interactions with the spatial context of bacteria-host interactions and therefore gives new insight into the molecular mechanisms of symbiosis.