Detection, localisation and quantification of the interaction between IL-37 and its cell surface receptor in human peripheral blood mononuclear cells via dSTORM super-resolution imaging — ASN Events
  1. Schedule
  2. Poster Session 2
  3. Detection, localisation and quantification of the interaction between IL-37 and its cell surface receptor in human peripheral blood mononuclear cells via dSTORM super-resolution imaging

Detection, localisation and quantification of the interaction between IL-37 and its cell surface receptor in human peripheral blood mononuclear cells via dSTORM super-resolution imaging (#292)

Camden Lo 1 , Kirstin Elgass 2 , Ina Rudloff 1 3 , Claudia Nold 1 3 , Marcel Nold 1 3
  1. MIMR-PHI Institute, Clayton, VIC, Australia
  2. Monash Micro Imaging, Monash University, Melbourne, Victoria, Australia
  3. Department of Paediatrics, Monash University, Melbourne, Victoria, Australia

Aims

The elucidation of protein complexing and their cellular localisation are foundation to cytokine and signalling research. Traditional methodologies such as immunoprecipitation and immunofluorescence cannot show complexing, localisation and abundance of molecules simultaneously. We aim to co-opt super-resolution imaging techniques to study the interactions between the powerful anti-inflammatory cytokine IL-37 and the receptor chains IL-18 receptor alpha (IL-18Ralpha) and SIGIRR. We describe cytokine-receptor binding/complexing, subcellular localisation and molecular numbers of these three proteins in their endogenous, native context, i.e. without exogenous additions, transfections or lysis.

Methods

The basis for our technique is direct stochastic optical reconstruction microscopy (dSTORM, 1). AlexaFluor488-, AlexaFluor568- and AlexaFluor647-tagged secondary antibodies were used to label the ligand (IL-37) and the two subunits of its heterodimeric receptor complex (SIGIRR and IL-18Ralpha) in PBMC from healthy human donors. The fluorophores were induced into a dark state by high intensity laser illumination and reducing buffer conditions. The stochastic return of the fluorophores to an active state is imaged and statistically analysed, allowing detection and localisation at 20-50nm precision. We then derive absolute localisation (positioning) and proximity of all three proteins relative to each other on the cell surface, and infer complexing and interaction based on clustering and co-location at molecular binding distances. As the technique is single molecule sensitive, we further measure and sample the each protein at the cell surface and derive the molecular proportion that is involved in complexing.

Results  and conclusions

IL-37 forms a ligand:receptor complex with SIGIRR and IL-18Ralpha (sub 50nm proximity with each other) on the cell surface of primary, untransfected human PBMC. Thirty min after LPS, approximately 15% of SIGIRR and 6% of IL-18Ralpha molecules are involved in complexing with IL-37 on the cell surface. This is the first demonstration of any super-resolution technique to study three-way protein interactions in primary human samples. 

  1. 1. Super-resolution imaging with small organic fluorophores. Heilemann M, van de Linde S, Mukherjee A, Sauer M. Angew Chem Int Ed Engl. 2009;48(37):6903-8. doi: 10.1002/anie.200902073.