Mapping EGFR1 sorting domains in endosomes with a calibrated 3D expansion microscopy toolkit

Endosomes are nanoscale intracellular compartments that sort and recycle cell surface receptors such as epidermal growth factor receptor 1 (EGFR1). Nanometre-scale interactions and co-clustering of signalling proteins, cargo, and the membrane are critical to this process. Direct visualisation of these interactions has been hindered by the limited 3D resolution achievable with conventional and super-resolution microscopies. Here, we present the adaptation of expansion microscopy (ExM) to visualise, and quantify nanoclusters of endosomal proteins of human retinal pigment epithelial (RPE-1) cells. A 3D distortion analysis was developed leveraging the Farneback optical flow principle for detecting anisotropies in the hydrogel expansion. Analysis of pre- and post- ExM image volumes for 3D anisotropies revealed under-expansion of cytoplasmic regions within ExM hydrogels, often leading to over-estimation of size and distance measurements of small compartments such as endosomes. A self-assembling protein nanocage that reports the true local and nanoscale expansion factor was genetically introduced into the cells to calibrate ExM images of cytoplasmic regions containing endosomes. To stimulate and visualise the internalisation and sorting of EGFR1 in mammalian cells, a pulse-chase protocol was carried out with fluorescently-tagged EGF. The cells were subsequently fixed at 15- and 30- minute time points and subjected to 10-fold ExM and multiplexed 3D Airyscan microscopy to map cargo and EGFR1 vs other endosomal proteins. A volume tracing pipeline was developed to visualise the changes in the labelled EGF and EGFR1 densities at the limiting membrane of the endosomes. With multiplexed 3D ExM image volumes, we observed the enrichment of both EGF and EGFR1 in the endosomal interior and the accumulation of endosomal protein Rab5a near the limiting membrane during this early maturation of the endosomes. Taken together, the multiplexed 3D ExM toolkit offers a quantitative framework for visualising and measuring the intrinsic biology of small sub-cellular organelles like endosomes at true molecular-scale resolution.