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 DiIC16TCO or DiIC16'TCO followed by in situ tetrazine ligation reaction with the silicon-rhodamine dye SiR-Tz generates the HIDE probes DiIC16-SiR and DiIC16'-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 min with no detectable change in endosome structure or function. Using DiIC16-SiR and DiIC16'-SiR, we describe 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. HIDE probes were developed for long time-lapse imaging of endolysosomes and their dynamics at super-resolution. These probes enabled analyses of endosome motility in primary fibroblasts from patients with Niemann–Pick C with distinct mutations in NPC1.