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

Nature Chemical Biology volume 16, pages 408–414 (2020)Cite this article

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Abstract

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.

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Data availability

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

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Acknowledgements

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.

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Author notes

  1. Alanna Schepartz

    Present address: Department of Chemistry, University of California, Berkeley, CA, USA

Authors and Affiliations

  1. Department of Chemistry, Yale University, New Haven, CT, USA

    Aarushi Gupta & Alanna Schepartz

  2. Department of Cell Biology, Yale University School of Medicine, New Haven, CT, USA

    Felix Rivera-Molina, Zhiqun Xi & Derek Toomre

  3. Department of Molecular, Cellular, and Developmental Biology, Yale University, New Haven, CT, USA

    Alanna Schepartz

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Contributions

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.

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Correspondence to Derek Toomre or Alanna Schepartz.

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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). https://doi.org/10.1038/s41589-020-0479-z

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