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Shameka Shelby, Rachelle Dorvilier; Isolation of MERTK Enriched Phagosomes from RPE-J Cells Challenged with Bovine Outer Segments. Invest. Ophthalmol. Vis. Sci. 2017;58(8):620. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
Phagocytosis of photoreceptor outer segments is essential to the maintenance of retinal structure. Previous studies have implicated Mer receptor tyrosine kinase (MERTK) in the regulation of phagosome trafficking in the retinal pigment epithelium (RPE). As such the aim of this study was to develop a standard protocol for the isolation of MERTK enriched phagosomes from RPE cells to allow for efficient study of phagosome trafficking in vitro.
RPE-J cells cultured post confluence were challenged with photoreceptor outer segments isolated from bovine retinas. The cells were subjected to washes with PBS-EDTA and PBS, followed by mechanical lysis. The resulting homogenate was cleared by low speed centrifugation and subjected to fractionation in a discontinuous sucrose density gradient. Fractions were analyzed via immunoblots against rhodopsin, MERTK, and cathepsin D to identify fractions containing phagosomes. Enriched fractions were further washed and pelleted to obtain concentrated stocks of phagosomes.
MERTK enriched phagosomes containing outer segments were obtained from the interface of 25% and 35% sucrose solutions using the isolation procedure. Expression of rhodopsin was confirmed by western analysis using antibodies that recognize the amino (4D2) and carboxyl termini (1D4) separately. Preprocathepsin D, procathepsin D, and mature cathepsin D expression was also seen in the isolated phagosomes.
The protocol we have developed will allow for the in vitro study of phagosomes from the RPE. Identification of outer segment phagosomes at the interface seen in this study differentiate the protocol from those isolating phagosomes from macrophages that have been challenged with glass beads. Immunoblots using rhodopsin and cathepsin D antibodies indicated that early, maturing, and late phagosomes were isolated. As MERTK signaling has been implicated in the regulation of phagosomal membrane trafficking, isolation of MERTK enriched phagosomes will provide a useful tool in studying protein interactions in the RPE that regulate membrane trafficking and maturation.
This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.
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