Abstract
Renewal of rod photoreceptor outer segments in the mammalian eye involves synchronized diurnal shedding after light onset of spent distal outer segment fragments (POS) linked to swift clearance of shed POS from the subretinal space by the adjacent retinal pigment epithelium (RPE). Engulfed POS phagosomes in RPE cells mature to acidified phagolysosomes, which accomplish enzymatic degradation of POS macromolecules. Here, we used an acidophilic fluorophore LysoTracker to label acidic organelles in freshly dissected, live rat RPE tissue flat mounts. We observed that all RPE cells imaged contained numerous acidified POS phagolysosomes whose abundance per cell was dramatically increased 2 h after light onset as compared to either 1 h before or 4 h after light onset. Lack of organelles of similar diameter (of 1–2 μm) in phagocytosis-defective mutant RCS rat RPE confirmed that LysoTracker live imaging detected POS phagolysosomes. Lack of increase in lysosomal membrane protein LAMP-1 in RPE/choroid during the diurnal phagocytic burst suggests that formation of POS phagolysosomes in RPE in situ may not involve extra lysosome membrane biogenesis. Taken together, we report a new imaging approach that directly detects POS phagosome acidification and allows rapid tracking and quantification of POS phagocytosis by live RPE tissue ex situ.
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Abbreviations
- POS:
-
Photoreceptor outer segment fragments
- RPE:
-
Retinal pigment epithelium
References
Andrejewski N, Punnonen EL, Guhde G et al (1999) Normal lysosomal morphology and function in LAMP-1-deficient mice. J Biol Chem 274:12692–12701
Bok D, Hall MO (1971) The role of the pigment epithelium in the etiology of inherited retinal dystrophy in the rat. J Cell Biol 49:664–682
Bosch E, Horwitz J, Bok D (1993) Phagocytosis of outer segments by retinal pigment epithelium: phagosome–lysosome interaction. J Histochem Cytochem 41:253–263
Carlsson SR, Roth J, Piller F et al (1988) Isolation and characterization of human lysosomal membrane glycoproteins, h-lamp-1 and h-lamp-2. Major sialoglycoproteins carrying polylactosaminoglycan. J Biol Chem 263:18911–18919
Esteve-Rudd J, Lopes VS, Jiang M et al (2014) In vivo and in vitro monitoring of phagosome maturation in retinal pigment epithelium cells. Adv Exp Med Biol 801:85–90
Gibbs D, Kitamoto J, Williams DS (2003) Abnormal phagocytosis by retinal pigmented epithelium that lacks myosin VIIa, the Usher syndrome 1B protein. Proc Natl Acad Sci U S A 100:6481–6486
Harter C, Mellman I (1992) Transport of the lysosomal membrane glycoprotein lgp120 (lgp-A) to lysosomes does not require appearance on the plasma membrane. J Cell Biol 117:311–325
LaVail MM (1976) Rod outer segment disk shedding in rat retina: relationship to cyclic lighting. Science 194:1071–1074
Mullen RJ, LaVail MM (1976) Inherited retinal dystrophy: primary defect in pigment epithelium determined with experimental rat chimeras. Science 192:799–801
Nandrot EF, Kim Y, Brodie SE et al (2004) Loss of synchronized retinal phagocytosis and age-related blindness in mice lacking avb5 integrin. J Exp Med 200:1539–1545
Nandrot EF, Anand M, Almeida D et al (2007) Essential role for MFG-E8 as ligand for avb5 integrin in diurnal retinal phagocytosis. Proc Natl Acad Sci U S A 104:12005–12010
Sethna S, Finnemann SC (2013) Analysis of photoreceptor rod outer segment phagocytosis by RPE cells in situ. Methods Mol Biol 935:245–254
Wavre-Shapton ST, Meschede IP, Seabra MC et al (2014) Phagosome maturation during endosome interaction revealed by partial rhodopsin processing in retinal pigment epithelium. J Cell Sci 127(17):3852-3861. doi:10.1242/jcs.154757
Young RW (1967) The renewal of photoreceptor cell outer segments. J Cell Biol 33:61–72
Young RW, Bok D (1969) Participation of the retinal pigment epithelium in the rod outer segment renewal process. J Cell Biol 42:392–403
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This study was supported by NIH grant EY13295.
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Mao, Y., Finnemann, S. (2016). Live Imaging of LysoTracker-Labelled Phagolysosomes Tracks Diurnal Phagocytosis of Photoreceptor Outer Segment Fragments in Rat RPE Tissue Ex Vivo . In: Bowes Rickman, C., LaVail, M., Anderson, R., Grimm, C., Hollyfield, J., Ash, J. (eds) Retinal Degenerative Diseases. Advances in Experimental Medicine and Biology, vol 854. Springer, Cham. https://doi.org/10.1007/978-3-319-17121-0_95
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DOI: https://doi.org/10.1007/978-3-319-17121-0_95
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