Endosome motility defects revealed at super-resolution in live cells using HIDE probes

We report new lipid-based, high-density, environmentally sensitive (HIDE) probes that accurately and selectively image endo-lysosomes and their dynamics at super-resolution for extended times. Treatment of live cells with the small molecules DiIC(16)TCO or DiIC(16’)TCO followed by in situ tetrazine ligation reaction with the silicon-rhodamine dye SiR-Tz generates the HIDE probes DiIC(16)-SiR and DiIC(16’)-SiR in the endo-lysosomal membrane. These new probes support the acquisition of super-resolution videos of organelle dynamics in primary cells for more than 7 minutes with no detectable change in endosome structure or function. Using DiIC(16)-SiR and DiIC(16’)-SiR, we describe the first direct evidence of endosome motility defects in cells from patients with Niemann-Pick Type-C disease. In wild-type fibroblasts, the probes reveal distinct but rare inter-endosome kiss-and-run events that cannot be observed using confocal methods. Our results shed new light on the role of NPC1 in organelle motility and cholesterol trafficking.