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Analyzing Lysosome-Related Organelles by Electron Microscopy

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Lysosomes

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1594))

Abstract

Intracellular organelles have a particular morphological signature that can only be appreciated by ultrastructural analysis at the electron microscopy level. Optical imaging and associated methodologies allow to explore organelle localization and their dynamics at the cellular level. Deciphering the biogenesis and functions of lysosomes and lysosome-related organelles (LROs) and their dysfunctions requires their visualization and detailed characterization at high resolution by electron microscopy. Here, we provide detailed protocols for studying LROs by transmission electron microscopy. While conventional electron microscopy and its recent improvements is the method of choice to investigate organelle morphology, immunoelectron microscopy allows to localize organelle components and description of their molecular make up qualitatively and quantitatively.

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References

  1. Raposo G, Marks MS, Cutler DF (2007) Lysosome-related organelles: driving post-Golgi compartments into specialisation. Curr Opin Cell Biol 19(4):394–401

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Marks MS, Heijnen HF, Raposo G (2013) Lysosome-related organelles: unusual compartments become mainstream. Curr Opin Cell Biol. 25(4):495–505. doi:10.1016/j.ceb.2013.04.008

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Novikoff AB, Albala A, Biempica L (1968) Ultrastructural and cytochemical observations on B-16 and Harding-Passey mouse melanomas. The origin of premelanosomes and compound melanosomes. J Histochem Cytochem 16(5):299–319

    Article  CAS  PubMed  Google Scholar 

  4. Palade GE (1983) Membrane biogenesis: an overview. Methods Enzymol 96:xxix–xxlv

    Article  CAS  PubMed  Google Scholar 

  5. Walther P, Ziegler A (2002) Freeze substitution of high-pressure frozen samples: the visibility of biological membranes is improved when the substitution medium contains water. J Microsc 208(Pt 1):3–10

    Article  CAS  PubMed  Google Scholar 

  6. Studer D, Graber W, Al-Amoudi A, Eggli P (2001) A new approach for cryofixation by high pressure freezing. J Microsc 203:285–294

    Article  CAS  PubMed  Google Scholar 

  7. Hawes P, Netherton CL, Mueller M, Wileman T, Monaghan P (2007) Rapid freeze-substitution preserves membranes in high-pressure frozen tissue culture cells. J Microsc 226(Pt 2):182–189. doi:10.1111/j.1365-2818.2007.01767.x

    Article  CAS  PubMed  Google Scholar 

  8. Shio H, Farquhar MG, de Duve C (1974) Lysosomes of the arterial wall. IV. Cytochemical localization of acid phosphatase and catalase in smooth muscle cells and foam cells from rabbit atheromatous aorta. Am J Pathol 76(1):1–16

    CAS  PubMed  PubMed Central  Google Scholar 

  9. Novikoff AB, Novikoff PM (1977) Cytochemical contributions to differentiating GERL from the Golgi apparatus. Histochem J 9(5):525–551

    Article  CAS  PubMed  Google Scholar 

  10. Slot JW, Geuze HJ (1981) Sizing of protein A-colloidal gold probes for immunoelectron microscopy. J Cell Biol 90:533–536

    Article  CAS  PubMed  Google Scholar 

  11. Geuze HJ, Slot JW, Strous GJ, Lodish HF, Schwartz AL (1983) Intracellular site of asialoglycoprotein receptor-ligand uncoupling: double label immunoelectron microscopy during receptor-mediated endocytosis. Cell 32:277–287

    Article  CAS  PubMed  Google Scholar 

  12. Slot JW, Posthuma G, Chang L, Crapo JD, Geuze HJ (1988b) Quantitative assessment of immuno-gold labeling in cryosections. In: Verleij AJ, Leunissen JLM (eds) Immuno-gold labeling in cell biology. CRC Press Inc., Boca Raton, FL, pp 135–156

    Google Scholar 

  13. Slot JW, Geuze HJ (2007) Cryosectioning and immunolabeling. Nat Protoc 2(10):2480–2491

    Article  CAS  PubMed  Google Scholar 

  14. Raposo G, Kleijmeer MJ, Posthuma G, Slot JW, Geuze HJ (1997) Immunogold labeling of ultrathin cryosections: application in immunology. In: Herzenberg LA, Weir D, Herzenberg LA, Blackwell C (eds) Handbook of experimental immunology, 5th edn, vol 4. Blackwell Science Inc., Cambridge, MA, pp 1–11, Chapter 208

    Google Scholar 

  15. Klumperman J, Raposo G (2014) The complex ultrastructure of the endolysosomal system. Cold Spring Harb Perspect Biol 6(10):a016857. doi:10.1101/cshperspect.a016857

    Article  PubMed  PubMed Central  Google Scholar 

  16. Setty SR, Tenza D, Sviderskaya EV, Bennett DC, Raposo G, Marks MS (2008) Cell-specific ATP7A transport sustains copper-dependent tyrosinase activity in melanosomes. Nature 454(7208):1142–1146

    Article  CAS  PubMed  Google Scholar 

  17. Theos AC, Tenza D, Martina JA, Hurbain I, Peden AA, Sviderskaya EV, Stewart A, Robinson MS, Bennett DC, Cutler DF, Bonifacino JS, Marks MS, Raposo G (2005) Functions of AP-3 and AP-1 in tyrosinase sorting from endosomes to melanosomes. Mol Biol Cell 16:5356–5372

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Raposo G, Tenza D, Murphy DM, Berson JF, Marks MS (2001) Distinct protein sorting and localization to premelanosomes, melanosomes, and lysosomes in pigmented melanocytic cells. J Cell Biol 152(4):809–824

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Delevoye C, Heiligenstein X, Ripoll L, Gilles-Marsens F, Dennis MK, Linares RA, Derman L, Gokhale A, Morel E, Faundez V, Marks MS, Raposo G (2016) BLOC-1 brings together the actin and microtubule cytoskeletons to generate recycling endosomes. Curr Biol 26(1):1–13. doi:10.1016/j.cub.2015.11.020

    Article  CAS  PubMed  Google Scholar 

  20. Delevoye C, Hurbain I, Tenza D, Sibarita JB, Uzan-Gafsou S, Ohno H, Geerts WJ, Verkleij AJ, Salamero J, Marks MS, Raposo G (2009) AP-1 and KIF13A coordinate endosomal sorting and positioning during melanosome biogenesis. J Cell Biol 187(2):247–264. doi:10.1083/jcb.200907122.

  21. Wasmeier C, Romao M, Plowright L, Bennett DC, Raposo G, Seabra MC (2006) Rab38 and Rab32 control post-Golgi trafficking of melanogenic enzymes. J Cell Biol 175(2):271–281

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. van Niel G, Charrin S, Simoes S, Romao M, Rochin L, Saftig P, Marks MS, Rubinstein E, Raposo G (2011) The tetraspanin CD63 regulates ESCRT-independent and -dependent endosomal sorting during melanogenesis. Dev Cell 21(4):708–721. doi:10.1016/j.devcel.2011.08.019

    Article  PubMed  PubMed Central  Google Scholar 

  23. Koster AJ, Klumperman J (2003) Electron microscopy in cell biology: integrating structure and function. Nat Rev Mol Cell Biol Suppl:SS6–SS10

    Google Scholar 

  24. Daniele T, Hurbain I, Vago R, Casari G, Raposo G, Tacchetti C, Schiaffino MV (2014) Mitochondria and melanosomes establish physical contacts modulated by Mfn2 and involved in organelle biogenesis. Curr Biol 24(4):393–403. doi:10.1016/j.cub.2014.01.007

    Article  CAS  PubMed  Google Scholar 

  25. Hurbain I, Geerts WJ, Boudier T, Marco S, Verkleij AJ, Marks MS, Raposo G (2008) Electron tomography of early melanosomes: Implications for melanogenesis and the generation of fibrillar amyloid sheets. Proc Natl Acad Sci U S A 105(50):19726–19731

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Rochin L, Hurbain I, Serneels L, Fort C, Watt B, Leblanc P, Marks MS, De Strooper B, Raposo G, van Niel G (2013) BACE2 processes PMEL to form the melanosome amyloid matrix in pigment cells. Proc Natl Acad Sci U S A 110(26):10658–10663. doi:10.1073/pnas.1220748110

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Kukulski W, Schorb M, Kaksonen M, Briggs JA (2012) Plasma membrane reshaping during endocytosis is revealed by time-resolved electron tomography. Cell 150(3):508–520. doi:10.1016/j.cell.2012.05.046

    Article  CAS  PubMed  Google Scholar 

  28. Kukulski W, Schorb M, Welsch S, Picco A, Kaksonen M, Briggs JA (2012) Precise, correlated fluorescence microscopy and electron tomography of lowicryl sections using fluorescent fiducial markers. Methods Cell Biol 111:235–257. doi:10.1016/B978-0-12-416026-2.00013-3

    Article  CAS  PubMed  Google Scholar 

  29. Mironov AA, Beznoussenko GV (2012) Correlative light-electron microscopy a potent tool for the imaging of rare or unique cellular and tissue events and structures. Methods Enzymol 504:201–219. doi:10.1016/B978-0-12-391857-4.00010-0

    Article  PubMed  Google Scholar 

  30. Muller-Reichert T, Verkade P (2012) Introduction to correlative light and electron microscopy. Methods Cell Biol 111:xvii–xxix. doi:10.1016/B978-0-12-416026-2.03001-6

    Article  PubMed  Google Scholar 

  31. van Rijnsoever C, Oorschot V, Klumperman J (2008) Correlative light-electron microscopy (CLEM) combining live-cell imaging and immunolabeling of ultrathin cryosections. Nat Methods 5(11):973–980. doi:10.1038/nmeth.1263. doi:nmeth.1263 [pii]

  32. Polishchuk EV, Polishchuk RS, Luini A (2013) Correlative light-electron microscopy as a tool to study in vivo dynamics and ultrastructure of intracellular structures. Methods Mol Biol 931:413–422. doi:10.1007/978-1-62703-056-4_20

    Article  CAS  PubMed  Google Scholar 

  33. Sepulveda FE, Burgess A, Heiligenstein X, Goudin N, Menager MM, Romao M, Cote M, Mahlaoui N, Fischer A, Raposo G, Menasche G, de Saint BG (2015) LYST controls the biogenesis of the endosomal compartment required for secretory lysosome function. Traffic 16(2):191–203. doi:10.1111/tra.12244

    Article  CAS  PubMed  Google Scholar 

  34. Heiligenstein X, Heiligenstein J, Delevoye C, Hurbain I, Bardin S, Paul-Gilloteaux P, Sengmanivong L, Regnier G, Salamero J, Antony C, Raposo G (2014) The CryoCapsule: simplifying correlative light to electron microscopy. Traffic 15(6):700–716. doi:10.1111/tra.12164

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Heiligenstein X, Hurbain I, Delevoye C, Salamero J, Antony C, Raposo G (2014) Step by step manipulation of the CryoCapsule with HPM high pressure freezers. Methods Cell Biol 124:259–274. doi:10.1016/B978-0-12-801075-4.00012-4

    Article  PubMed  Google Scholar 

  36. Markert SM, Britz S, Proppert S, Lang M, Witvliet D, Mulcahy B, Sauer M, Zhen M, Bessereau JL, Stigloher C (2016) Filling the gap: adding super-resolution to array tomography for correlated ultrastructural and molecular identification of electrical synapses at the C. elegans connectome. Neurophotonics 3(4):041802. doi:10.1117/1.NPh.3.4.041802

    Article  PubMed  Google Scholar 

  37. Wolff G, Hagen C, Grunewald K, Kaufmann R (2016) Towards correlative super-resolution fluorescence and electron cryo-microscopy. Biol Cell 108(9):245–258. doi:10.1111/boc.201600008

    Article  CAS  PubMed  Google Scholar 

  38. Liu B, Xue Y, Zhao W, Chen Y, Fan C, Gu L, Zhang Y, Zhang X, Sun L, Huang X, Ding W, Sun F, Ji W, Xu T (2015) Three-dimensional super-resolution protein localization correlated with vitrified cellular context. Sci Rep 5:13017. doi:10.1038/srep13017

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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Acknowledgements

We are grateful to Institut Curie and the BioImaging platform (PICT IBiSA), member of the France-BioImaging national research infrastructure (ANR-10-INSB-04) and CNRS. Research in our group is supported by the Fondation pour la Recherche Médical (Equipes FRM), Association de Recherche pour le Cancer (ARC), Indian French cooperation (CEFIPRA), Clarins and L’Oréal.

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Authors and Affiliations

  1. Institut Curie, PSL Research University, CNRS, UMR 144, F-75005, Paris, France

    Ilse Hurbain, Maryse Romao, Ptissam Bergam, Xavier Heiligenstein & Graça Raposo

  2. Sorbonne Universités, UPMC Univ Paris 06, CNRS, UMR 144, F-75005, Paris, France

    Ilse Hurbain, Maryse Romao, Ptissam Bergam, Xavier Heiligenstein & Graça Raposo

  3. Cell and Tissue Imaging Core Facility PICT-IBiSA, Institut Curie, F-75248, Paris, France

    Ilse Hurbain, Maryse Romao, Ptissam Bergam, Xavier Heiligenstein & Graça Raposo

  4. Characterization Core Lab, King Abdullah University of Science and Technology (KAUST), Thuwal, 23955-6900, Kingdom of Saudi Arabia

    Ptissam Bergam

Authors
  1. Ilse Hurbain
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  2. Maryse Romao
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  3. Ptissam Bergam
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  4. Xavier Heiligenstein
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  5. Graça Raposo
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Corresponding author

Correspondence to Graça Raposo .

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Editors and Affiliations

  1. Experimental Pathology, Department of Clinical and Experimental Medicine, Linköping University, Linköping, Sweden

    Karin Öllinger

  2. Department of Physics, Chemistry and Biology, Linköping University, Linköping, Sweden

    Hanna Appelqvist

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Hurbain, I., Romao, M., Bergam, P., Heiligenstein, X., Raposo, G. (2017). Analyzing Lysosome-Related Organelles by Electron Microscopy. In: Öllinger, K., Appelqvist, H. (eds) Lysosomes. Methods in Molecular Biology, vol 1594. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-6934-0_4

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  • DOI: https://doi.org/10.1007/978-1-4939-6934-0_4

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  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-6932-6

  • Online ISBN: 978-1-4939-6934-0

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