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.
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This work was supported by the NIH (grant nos. R01GM131372-01, A.S. and R01GM118486, D.T.) and the Wellcome Trust (grant no. 095927/A/11/Z), and in part by the NIH (grant no. S10 OD020142 (Leica SP8)). A.G. was in part supported by the NIH (5T32GM06754 3-12). A.G. thanks J. Wolenksi and A. Mennone for assistance with confocal and STED microscopy.
The authors declare no competing interests.
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Supplementary Figs. 1–6 and Note.
HeLa cells labeled with DiIC16-SiR.
HeLa cells labeled with DiIC16’-SiR.
Wild-type (GM05399) fibroblasts labeled with DiIC16-SiR.
I1061T (GM18453) Fibroblasts labeled with DiIC16-SiR.
P237S/I1061T (GM03123) Fibroblasts labeled with DiIC16-SiR.
R404Q (GM18388) Fibroblasts labeled with DiIC16-SiR.
1920delG (GM23945) Fibroblasts labeled with DiIC16-SiR.
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Gupta, A., Rivera-Molina, F., Xi, Z. et al. Endosome motility defects revealed at super-resolution in live cells using HIDE probes. Nat Chem Biol 16, 408–414 (2020). https://doi.org/10.1038/s41589-020-0479-z
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