Advertisement

pp 1-16 | Cite as

Study of Intracellular Cargo Trafficking and Co-localization in the Phagosome and Autophagy-Lysosomal Pathways of Retinal Pigment Epithelium (RPE) Cells

  • J. Arjuna RatnayakaEmail author
  • Eloise Keeling
  • David S. Chatelet
Protocol
Part of the Methods in Molecular Biology book series

Abstract

The transport and targeting of internalized molecules to distinct intracellular organelles/compartments can prove challenging to visualize clearly, which can contribute to some of the difficulties associated with these studies. By combining several approaches, we show how the trafficking and processing of photoreceptor outer segments in the phagosome and autophagy-lysosomal pathways of the retinal pigment epithelium (RPE) can easily be quantified and visualized as 3D-reconstructed images. This protocol takes advantage of new developments in microscopy and image-analysis software which has the potential to help better understand dynamic intracellular processes that underlie RPE dysfunction associated with irreversible blinding diseases such as age-related macular degeneration. The method described herein can also be used to study the trafficking and co-localization of different intracellular cargos in other cell types and tissues.

Keywords

3D imaging Co-localization Retinal pigment epithelium (RPE) Phagosome Lysosomes Autophagy Confocal Microscopy 

Notes

Acknowledgments

We thank our colleagues Dr. David A. Johnston and Dr. Anton Page (Biomedical Imaging Unit, University of Southampton) for their expertise in light/confocal and ultrastructural microscopy, Ms. Savannah A. Lynn (Faculty of Medicine, University of Southampton) for her expertise in cell culture and imaging, and Dr. David A. Tumbarello (Biological Sciences, University of Southampton) for his expertise in membrane trafficking and cell signaling. This work was funded by support from the Macular Society, UK, and the Gift of Sight Appeal.

References

  1. 1.
    Khandhadia S, Cherry J, Lotery AJ (2012) Age-related macular degeneration. Adv Exp Med Biol 724:15–36.  https://doi.org/10.1007/978-1-4614-0653-2_2CrossRefGoogle Scholar
  2. 2.
    Bhutto I, Lutty G (2012) Understanding age-related macular degeneration (AMD): relationships between the photoreceptor/retinal pigment epithelium/Bruch’s membrane/choriocapillaris complex. Mol Aspects Med 33(4):295–317. S0098-2997(12)00045-3 [pii];  https://doi.org/10.1016/j.mam.2012.04.005Google Scholar
  3. 3.
    Wong WL, Su X, Li X, Cheung CM, Klein R, Cheng CY, Wong TY (2014) Global prevalence of age-related macular degeneration and disease burden projection for 2020 and 2040: a systematic review and meta-analysis. Lancet Glob Health 2(2):e106–e116.  https://doi.org/10.1016/s2214-109x(13)70145-1CrossRefGoogle Scholar
  4. 4.
    Christensen DRG, Brown FE, Cree AJ, Ratnayaka JA, Lotery AJ (2017) Sorsby fundus dystrophy—a review of pathology and disease mechanisms. Exp Eye Res 165:35–46.  https://doi.org/10.1016/j.exer.2017.08.014CrossRefGoogle Scholar
  5. 5.
    Tanna P, Strauss RW, Fujinami K, Michaelides M (2017) Stargardt disease: clinical features, molecular genetics, animal models and therapeutic options. Br J Ophthalmol 101(1):25–30.  https://doi.org/10.1136/bjophthalmol-2016-308823CrossRefGoogle Scholar
  6. 6.
    Keeling E, Lotery AJ, Tumbarello DA, Ratnayaka JA (2018) Impaired cargo clearance in the retinal pigment epithelium (RPE) underlies irreversible blinding diseases. Cell 7(2).  https://doi.org/10.3390/cells7020016Google Scholar
  7. 7.
    Keeling E, Johnston A, Chatelet D, Tumbarello D, Lotery A, Ratnayaka JA (2018) Lysosomal impairment in the retinal pigment epithelium (RPE)—a pathway of damage in the ageing retina. Invest Ophthalmol Vis Sci 59(9):4487Google Scholar
  8. 8.
    Lynn SA, Ward G, Keeling E, Scott JA, Cree AJ, Johnston DA, Page A, Cuan-Urquizo E, Bhaskar A, Grossel MC, Tumbarello DA, Newman TA, Lotery AJ, Ratnayaka JA (2017) Ex-vivo models of the retinal pigment epithelium (RPE) in long-term culture faithfully recapitulate key structural and physiological features of native RPE. Tissue Cell 49(4):447–460.  https://doi.org/10.1016/j.tice.2017.06.003CrossRefGoogle Scholar
  9. 9.
    Lynn SA, Keeling E, Dewing JM, Johnston DA, Page A, Cree AJ, Tumbarello DA, Newman TA, Lotery AJ, Ratnayaka JA (2018) A convenient protocol for establishing a human cell culture model of the outer retina. F1000Research 7:1107.  https://doi.org/10.12688/f1000research.15409.1CrossRefGoogle Scholar
  10. 10.
    Ratnayaka JA, Lynn SA, Griffiths H, Scott J, Cree A, Lotery AJ (2015) An ex-vivo platform for manipulation and study of retinal pigment epithelial (RPE) cells in long-term culture. Invest Ophthalmol Vis Sci 56(7):2332–2332Google Scholar
  11. 11.
    Costes SV, Daelemans D, Cho EH, Dobbin Z, Pavlakis G, Lockett S (2004) Automatic and quantitative measurement of protein-protein colocalization in live cells. Biophys J 86(6):3993–4003.  https://doi.org/10.1529/biophysj.103.038422CrossRefGoogle Scholar
  12. 12.
    Nixon RA, Cataldo AM, Mathews PM (2000) The endosomal-lysosomal system of neurons in Alzheimer’s disease pathogenesis: a review. Neurochem Res 25(9–10):1161–1172Google Scholar
  13. 13.
    Lakowski J, Welby E, Budinger D, Di Marco F, Di Foggia V, Bainbridge JWB, Wallace K, Gamm DM, Ali RR, Sowden JC (2018) Isolation of human photoreceptor precursors via a cell surface marker panel from stem cell-derived retinal organoids and fetal retinae. Stem Cells.  https://doi.org/10.1002/stem.2775Google Scholar
  14. 14.
    Marmorstein AD, Johnson AA, Bachman LA, Andrews-Pfannkoch C, Knudsen T, Gilles BJ, Hill M, Gandhi JK, Marmorstein LY, Pulido JS (2018) Mutant Best1 expression and impaired phagocytosis in an iPSC model of autosomal recessive bestrophinopathy. Sci Rep 8(1):4487.  https://doi.org/10.1038/s41598-018-21651-zCrossRefGoogle Scholar
  15. 15.
    Hallam D, Collin J, Bojic S, Chichagova V, Buskin A, Xu Y, Lafage L, Otten EG, Anyfantis G, Mellough C, Przyborski S, Alharthi S, Korolchuk V, Lotery A, Saretzki G, McKibbin M, Armstrong L, Steel D, Kavanagh D, Lako M (2017) An induced pluripotent stem cell patient specific model of complement factor H (Y402H) polymorphism displays characteristic features of age-related macular degeneration and indicates a beneficial role for UV light exposure. Stem Cells 35(11):2305–2320.  https://doi.org/10.1002/stem.2708CrossRefGoogle Scholar
  16. 16.
    Schraermeyer U, Enzmann V, Kohen L, Addicks K, Wiedemann P, Heimann K (1997) Porcine iris pigment epithelial cells can take up retinal outer segments. Exp Eye Res 65(2):277–287Google Scholar
  17. 17.
    Mao Y, Finnemann SC (2013) Analysis of photoreceptor outer segment phagocytosis by RPE cells in culture. Methods Mol Biol 935:285–295.  https://doi.org/10.1007/978-1-62703-080-9_20CrossRefGoogle Scholar
  18. 18.
    Krohne TU, Stratmann NK, Kopitz J, Holz FG (2010) Effects of lipid peroxidation products on lipofuscinogenesis and autophagy in human retinal pigment epithelial cells. Exp Eye Res 90 (3):465-471. S0014-4835(10)00002-3 [pii];  https://doi.org/10.1016/j.exer.2009.12.011Google Scholar
  19. 19.
    Pfeffer BA, Philp NJ (2014) Cell culture of retinal pigment epithelium: special issue. Exp Eye Res 126:1–4. S0014-4835(14)00192-4 [pii];  https://doi.org/10.1016/j.exer.2014.07.010Google Scholar
  20. 20.
    Mazzoni F, Safa H, Finnemann SC (2014) Understanding photoreceptor outer segment phagocytosis: use and utility of RPE cells in culture. Exp Eye Res 126:51–60.  https://doi.org/10.1016/j.exer.2014.01.010CrossRefGoogle Scholar
  21. 21.
    Hall MO, Abrams T (1987) Kinetic studies of rod outer segment binding and ingestion by cultured rat RPE cells. Exp Eye Res 45(6):907–922Google Scholar

Copyright information

© Springer Science+Business Media New York 2019

Authors and Affiliations

  • J. Arjuna Ratnayaka
    • 1
    Email author
  • Eloise Keeling
    • 1
  • David S. Chatelet
    • 2
  1. 1.Clinical and Experimental Sciences, Faculty of MedicineUniversity of SouthamptonSouthamptonUK
  2. 2.Biomedical Imaging UnitUniversity of SouthamptonSouthamptonUK

Personalised recommendations