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

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.

Data availability

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

References

  1. 1.

    Maxfield, F. R. & McGraw, T. E. Endocytic recycling. Nat. Rev. Mol. Cell Bio. 5, 121–132 (2004).

    CAS  Google Scholar 

  2. 2.

    Bonifacino, J. S. & Neefjes, J. Moving and positioning the endolysosomal system. Curr. Opin. Cell Biol. 47, 1–8 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  3. 3.

    Sadowski, L., Pilecka, I. & Miaczynska, M. Signaling from endosomes: location makes a difference. Exp. Cell. Res. 315, 1601–1609 (2009).

    CAS  PubMed  Google Scholar 

  4. 4.

    Alabi, A. A. & Tsien, R. W. Perspectives on kiss-and-run: role in exocytosis, endocytosis, and neurotransmission. Annu Rev. Physiol. 75, 393–422 (2013).

    CAS  PubMed  Google Scholar 

  5. 5.

    Gruenberg, J. The endocytic pathway: a mosaic of domains. Nat. Rev. Mol. Cell Bio. 2, 721–730 (2001).

    CAS  Google Scholar 

  6. 6.

    Li, D. et al. Extended-resolution structured illumination imaging of endocytic and cytoskeletal dynamics. Science 349, 6251 (2015).

    Google Scholar 

  7. 7.

    Shim, S. H. et al. Super-resolution fluorescence imaging of organelles in live cells with photoswitchable membrane probes. Proc. Natl Acad. Sci. USA 109, 13978–13983 (2012).

    CAS  PubMed  Google Scholar 

  8. 8.

    Richardson, D. S. et al. SRpHi ratiometric pH biosensors for superresolution microscopy. Nat. Commun. 8, 577 (2017).

    PubMed  PubMed Central  Google Scholar 

  9. 9.

    Wegel, E. et al. Imaging cellular structures in super-resolution with SIM, STED and localisation microscopy: a practical comparison. Sci. Rep. 6, 27290 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  10. 10.

    Das, A. & Barroso, M. M. Endosome-mitochondria interactions are modulated by iron release from transferrin. Mol. Biol. Cell 27, 831–845 (2016).

    Google Scholar 

  11. 11.

    Ba, Q., Raghavan, G., Kiselyov, K. & Yang, G. Whole-cell scale dynamic organization of lysosomes revealed by spatial statistical analysis. Cell Rep. 23, 3591–3606 (2018).

    CAS  PubMed  PubMed Central  Google Scholar 

  12. 12.

    Platt, F. M., Boland, B. & van der Spoel, A. C. Lysosomal storage disorders: the cellular impact of lysosomal dysfunction. J. Cell Biol. 199, 723–734 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  13. 13.

    Futerman, A. H. & van Meer, G. The cell biology of lysosomal storage disorders. Nat. Rev. Mol. Cell Bio. 5, 554–565 (2004).

    CAS  Google Scholar 

  14. 14.

    Pfeffer, S. R. NPC intracellular cholesterol transporter 1 (NPC1)-mediated cholesterol export from lysosomes. J. Biol. Chem. 294, 1706–1709 (2019).

    CAS  PubMed  PubMed Central  Google Scholar 

  15. 15.

    Lim, C. Y. et al. ER-lysosome contacts enable cholesterol sensing by mTORC1 and drive aberrant growth signalling in Niemann–Pick type C. Nat. Cell Biol. 21, 1206–1218 (2019).

    CAS  PubMed  Google Scholar 

  16. 16.

    Leung, K., Chakraborty, K., Saminathan, A., Krishnan, Y. & DNA, A. nanomachine chemically resolves lysosomes in live cells. Nat. Nanotechnol. 14, 176–183 (2019).

    CAS  PubMed  Google Scholar 

  17. 17.

    Vacca, F. et al. Cyclodextrin triggers MCOLN1-dependent endo-lysosome secretion in Niemann–Pick type C cells. J. Lipid Res. 60, 832–843 (2019).

    CAS  PubMed  Google Scholar 

  18. 18.

    Vivas, O. et al. Disease reveals a link between lysosomal cholesterol and PtdIns(4,5)P-2 that regulates neuronal excitability. Cell Rep. 27, 2636–2648 (2019).

    CAS  PubMed  PubMed Central  Google Scholar 

  19. 19.

    Vanier, M. T. & Latour, P. Laboratory diagnosis of Niemann–Pick disease type C: the filipin staining test. Method Cell Biol. 126, 357–375 (2015).

    CAS  Google Scholar 

  20. 20.

    Gelsthorpe, M. E. et al. Niemann–Pick type C1I1061T mutant encodes a functional protein that is selected for endoplasmic reticulum-associated degradation due to protein misfolding. J. Biol. Chem. 283, 8229–8236 (2008).

    CAS  PubMed  PubMed Central  Google Scholar 

  21. 21.

    Deffieu, M. S. & Pfeffer, S. R. Niemann–Pick type C 1 function requires lumenal domain residues that mediate cholesterol-dependent NPC2 binding. Proc. Natl Acad. Sci. USA 108, 18932–18936 (2011).

    CAS  PubMed  Google Scholar 

  22. 22.

    Park, W. D. et al. Identification of 58 novel mutations in Niemann–Pick disease Type C: correlation with biochemical phenotype and importance of PTC1-like domains in NPC1. Hum. Mutat. 22, 313–325 (2003).

    CAS  PubMed  Google Scholar 

  23. 23.

    Lu, F. et al. Identification of NPC1 as the target of U18666A, an inhibitor of lysosomal cholesterol export and Ebola infection. eLife 4, e12177 (2015).

    PubMed  PubMed Central  Google Scholar 

  24. 24.

    Ko, D. C., Gordon, M. D., Jin, J. Y. & Scott, M. P. Dynamic movements of organelles containing Niemann–Pick C1 protein: NPC1 involvement in late endocytic events. Mol. Biol. Cell 12, 601–614 (2001).

    CAS  PubMed  PubMed Central  Google Scholar 

  25. 25.

    Millat, G. et al. Niemann–Pick C1 disease: correlations between NPC1 mutations, levels of NPC1 protein, and phenotypes emphasize the functional significance of the putative sterol-sensing domain and of the cysteine-rich luminal loop. Am. J. Hum. Genet. 68, 1373–1385 (2001).

    CAS  PubMed  PubMed Central  Google Scholar 

  26. 26.

    Lebrand, C. et al. Late endosome motility depends on lipids via the small GTPase Rab7. EMBO J. 21, 1289–1300 (2002).

    CAS  PubMed  PubMed Central  Google Scholar 

  27. 27.

    Sobo, K. et al. Late endosomal cholesterol accumulation leads to impaired intra-endosomal trafficking. PLoS ONE 2, e851 (2007).

    PubMed  PubMed Central  Google Scholar 

  28. 28.

    Thompson, A. D., Bewersdorf, J., Toomre, D. & Schepartz, A. HIDE Probes: a new toolkit for visualizing organelle dynamics, longer and at super-resolution. Biochem. 56, 5194–5201 (2017).

    CAS  Google Scholar 

  29. 29.

    Erdmann, R. S. et al. Super-resolution imaging of the Golgi in live cells with a bioorthogonal ceramide probe. Angew. Chem. Int. Ed. 53, 10242–10246 (2014).

    CAS  Google Scholar 

  30. 30.

    Takakura, H. et al. Long time-lapse nanoscopy with spontaneously blinking membrane probes. Nat. Biotechnol. 35, 773–780 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  31. 31.

    Bottanelli, F. et al. A novel physiological role for ARF1 in the formation of bidirectional tubules from the Golgi. Mol. Biol. Cell 28, 1676–1687 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  32. 32.

    Bottanelli, F. et al. Two-colour live-cell nanoscale imaging of intracellular targets. Nat. Commun. 7, 10778 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  33. 33.

    Revelo, N. H. et al. A new probe for super-resolution imaging of membranes elucidates trafficking pathways. J. Cell Biol. 205, 591–606 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  34. 34.

    Blackman, M. L., Royzen, M. & Fox, J. M. Tetrazine ligation: fast bioconjugation based on inverse-electron-demand Diels–Alder reactivity. J. Am. Chem. Soc. 130, 13518–13519 (2008).

    CAS  PubMed  PubMed Central  Google Scholar 

  35. 35.

    Paz, I. et al. Galectin-3, a marker for vacuole lysis by invasive pathogens. Cell Microbiol. 12, 530–544 (2010).

    CAS  PubMed  Google Scholar 

  36. 36.

    Thurston, T. L. M., Wandel, M. P., von Muhlinen, N., Foeglein, A. & Randow, F. Galectin 8 targets damaged vesicles for autophagy to defend cells against bacterial invasion. Nature 482, 414–U1515 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  37. 37.

    Thompson, A. D. et al. Long-term live-cell STED nanoscopy of primary and cultured cells with the plasma membrane HIDE Probe DiI-SiR. Angew. Chem., Int. Ed. Engl. 56, 10408–10412 (2017).

    CAS  Google Scholar 

  38. 38.

    Sun, X. F. et al. Niemann–Pick C variant detection by altered sphingolipid trafficking and correlation with mutations within a specific domain of NPC1. Am. J. Hum. Genet. 68, 1361–1372 (2001).

    CAS  PubMed  PubMed Central  Google Scholar 

  39. 39.

    Salman, A., Cougnoux, A., Farhat, N., Wassif, C. A. & Porter, F. D. Association of NPC1 Variant p. P237S with a pathogenic splice variant in two Niemann–Pick disease type C1 patients. Am. J. Med Genet. A. 173, 1038–1040 (2017).

    PubMed  Google Scholar 

  40. 40.

    Pipalia, N. H. et al. Histone deacetylase inhibitors correct the cholesterol storage defect in most Niemann–Pick C1 mutant cells. J. Lipid Res. 58, 695–708 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  41. 41.

    Li, X. C., Saha, P., Li, J., Blobel, G. & Pfeffer, S. R. Clues to the mechanism of cholesterol transfer from the structure of NPC1 middle lumenal domain bound to NPC2. Proc. Natl Acad. Sci. USA 113, 10079–10084 (2016).

    CAS  PubMed  Google Scholar 

  42. 42.

    Eden, E. R. The formation and function of ER-endosome membrane contact sites. BBA-Mol. Cell Biol. L. 1861, 874–879 (2016).

    CAS  Google Scholar 

  43. 43.

    Raiborg, C., Wenzel, E. M. & Stenmark, H. ER-endosome contact sites: molecular compositions and functions. EMBO J. 34, 1848–1858 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  44. 44.

    Watanabe, S. et al. Ultrafast endocytosis at mouse hippocampal synapses. Nature 504, 242–247 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  45. 45.

    Staal, R. G. W., Mosharov, E. V. & Sulzer, D. Dopamine neurons release transmitter via a flickering fusion pore. Nat. Neurosci. 7, 341–346 (2004).

    CAS  PubMed  Google Scholar 

  46. 46.

    Saffi, G. T. & Botelho, R. J. Lysosome fission: planning for an exit. Trends Cell Biol. 29, 635–646 (2019).

    CAS  PubMed  Google Scholar 

  47. 47.

    Vihervaara, T. et al. Sterol binding by OSBP-related protein 1L (ORP1 L) regulates late endosome motility and function. Cell. Mol. Life Sci. 68, 537–551 (2011).

    CAS  PubMed  Google Scholar 

  48. 48.

    Chen, H., Yang, J., Low, P. S. & Cheng, J. X. Cholesterol level regulates endosome motility via Rab proteins. Biophys. J. 94, 1508–1520 (2008).

    CAS  PubMed  Google Scholar 

  49. 49.

    Millard, E. E. et al. The sterol-sensing domain of the Niemann–Pick C1 (NPC1) protein regulates trafficking of low density lipoprotein cholesterol. J. Biol. Chem. 280, 28581–28590 (2005).

    CAS  PubMed  Google Scholar 

  50. 50.

    Li, X. et al. 3.3 A structure of Niemann–Pick C1 protein reveals insights into the function of the C-terminal luminal domain in cholesterol transport. Proc. Natl Acad. Sci. USA 114, 9116–9121 (2017).

    CAS  PubMed  Google Scholar 

  51. 51.

    Mukherjee, S. & Maxfield, F. R. Trafficking of lipid analogs with varying tail lengths and unsaturations in Niemann–Pick C fibroblasts. Mol. Biol. Cell 11, 325a–325a (2000).

    Google Scholar 

  52. 52.

    Devaraj, N. K., Upadhyay, R., Hatin, J. B., Hilderbrand, S. A. & Weissleder, R. Fast and sensitive pretargeted labeling of cancer cells through a tetrazine/trans-cyclooctene cycloaddition. Angew. Chem. Int. Ed. 48, 7013–7016 (2009).

    CAS  Google Scholar 

  53. 53.

    Roberts, R. L., Barbieri, M. A., Pryse, K. M., Chua, M. & Stahl, P. D. Endosome fusion in living cells overexpressing GFP-rab5. J. Cell Sci. 112, 3667–3675 (1999).

    CAS  PubMed  Google Scholar 

  54. 54.

    Humphries, W. H., Szymanski, C. J. & Payne, C. K. Endo-lysosomal vesicles positive for Rab7 and LAMP1 are terminal vesicles for the transport of dextran. PLoS ONE 6, e26626 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  55. 55.

    Steinauer, A. et al. HOPS-dependent endosomal fusion required for efficient cytosolic delivery of therapeutic peptides and small proteins. Proc. Natl Acad. Sci. USA 116, 512–521 (2019).

    CAS  PubMed  Google Scholar 

  56. 56.

    Aits, S. et al. Sensitive detection of lysosomal membrane permeabilization by lysosomal galectin puncta assay. Autophagy 11, 1408–1424 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  57. 57.

    Ibach, J. et al. Single particle tracking reveals that egfr signaling activity is amplified in clathrin-coated pits. PLoS ONE 10, e0143162 (2015).

    PubMed  PubMed Central  Google Scholar 

  58. 58.

    Roepstorff, K. et al. Differential effects of EGFR ligands on endocytic sorting of the receptor. Traffic 10, 1115–1127 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  59. 59.

    Tinevez, J. Y. et al. TrackMate: an open and extensible platform for single-particle tracking. Methods 115, 80–90 (2017).

    CAS  Google Scholar 

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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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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.

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

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