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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.

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Fig. 1: DiIC16-SiR and DiIC16’-SiR are new HIDE probes for late endosomal imaging.
Fig. 2: DiIC16-SiR and DiIC16’-SiR selectively label late endosomes.
Fig. 3: DiIC16-SiR and DiIC16’-SiR do not damage endosomal membranes or alter endocytic trafficking.
Fig. 4: DiIC16-SiR enables long time-lapse STED imaging of rare events in live HeLa cells and fibroblasts.
Fig. 5: Endosome motility defects in NPD disease are only visible with HIDE probes and STED microscopy.

Data availability

The materials and data reported in this study are available upon reasonable request from the corresponding author.


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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.

Author information




A.G., D.T., and A.S. conceived the project. A.G., F.R.-M, Z.X., D.T., and A.S. designed experiments. A.G. and F.R.-M. performed imaging experiments. A.G. designed and synthesized DiIC16TCO and DiIC16’TCO. A.G., F.R.-M., and Z.X. prepared HeLa and fibroblast samples for microscopy. A.G. and A.S. wrote the manuscript.

Corresponding authors

Correspondence to Derek Toomre or Alanna Schepartz.

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The authors declare no competing interests.

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Supplementary information

Supplementary Information

Supplementary Figs. 1–6 and Note.

Reporting Summary

Supplementary Video 1

HeLa cells labeled with DiIC16-SiR.

Supplementary Video 2

HeLa cells labeled with DiIC16’-SiR.

Supplementary Video 3

Wild-type (GM05399) fibroblasts labeled with DiIC16-SiR.

Supplementary Video 4

I1061T (GM18453) Fibroblasts labeled with DiIC16-SiR.

Supplementary Video 5

P237S/I1061T (GM03123) Fibroblasts labeled with DiIC16-SiR.

Supplementary Video 6

R404Q (GM18388) Fibroblasts labeled with DiIC16-SiR.

Supplementary Video 7

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).

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