Highlights Method enables investigation of amyloid-β oligomer stoichiometry without requiring extrinsic fluorescent probes. Uses single-molecule stepwise photobleaching in vitro . Unveils heterogeneity within populations of oligomers. Assays oligomer-induced dysregulation of intracellular Ca 2+ homeostasis in living cells.

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SummaryThe ability of tumors to establish a pro-tumorigenic microenvironment is becoming an important point of investigation in the search for new therapeutics. Tumors form microenvironments in part by the “education” of immune cells attracted via chemotactic axes such as that of CCR5-CCL5. Further, CCR5 upregulation by cancer cells, coupled with its association with pro-tumorigenic features such as drug-resistance and metastasis, has suggested CCR5 as a target for therapeutic inhibition. However, with several conformational ‘pools’ being reported, phenotypic investigations must be capable of unveiling heterogeneity. Addressing this challenge, we performed structured illumination (SIM) and Partially TIRF coupled HILO (PaTCH) microscopy for super-resolution imaging and single-molecule imaging of CCR5 in fixed cells. Determining the positions of super-resolved CCR5 assemblies revealed a non-random spatial orientation. Further, intensity-tracking of assemblies revealed a distribution of molecular stoichiometries indicative of dimeric sub-units independent of CCL5 perturbation. These biophysical methods can provide important insights into the structure and function of onco-immunogenic receptors and a plethora of other biomolecules.HighlightsWe use SIM and novel PaTCH microscopy for precise bioimaging and single-molecule tracking of receptor protein CCR5 in model cell linesBy tracking the position of CCR5 assemblies we conclude that they are clustered in the plasma membrane beyond a level expected from a random distributionWe use these high precision data to determine molecular stoichiometries of CCR5 assemblies

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