Kinetic studies of rod outer segment binding and ingestion by cultured rat RPE cells

Hall, Michael O.; Abrams, Toshka A.

doi:10.1016/s0014-4835(87)80105-7

Cited by 76 publications

(55 citation statements)

References 17 publications

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“…Because Mertk only stimulated OS internalization and not binding, the protein must be critical for the ingestion phase. The delayed activation of Mertk is consistent with the observed initial delay in the kinetics of OS ingestion by cultured RPE cells (7,12,15). By 3 h of incubation, however, substantial OS ingestion has occurred (7), and about 70% of total OS are ingested by 4 h (Fig.…”

Section: Discussionsupporting

confidence: 81%

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Mertk Triggers Uptake of Photoreceptor Outer Segments during Phagocytosis by Cultured Retinal Pigment Epithelial Cells

Feng¹,

Yasumura²,

Matthes³

et al. 2002

Journal of Biological Chemistry

207

141

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The RCS rat is a widely studied model of recessively inherited retinal degeneration. The genetic defect, known as rdy (retinal dystrophy), results in failure of the retinal pigment epithelium (RPE) to phagocytize shed photoreceptor outer segment membranes. We previously used positional cloning and in vivo genetic complementation to demonstrate that Mertk is the gene for rdy. We have now used a rat primary RPE cell culture system to demonstrate that the RPE is the site of action of Mertk and to obtain functional evidence for a key role of Mertk in RPE phagocytosis. Phagocytosis is a process by which large particles are internalized by cells to form phagosomes. The process can be divided into three phases: binding, ingestion, and digestion. Retinal pigment epithelial (RPE) 1 cells, which form a polarized epithelium between the photoreceptor cells and the choroid in the outer retina, phagocytize more biomass than any other mammalian cell type (1). The RPE phagocytizes photoreceptor outer segment (OS) membranes (2) that are shed as part of the normal ongoing process of photoreceptor OS renewal (3). Failure of OS membrane uptake leads to photoreceptor cell death (4), as illustrated by the RCS rat, a widely used model for recessively inherited retinal degeneration. The RCS mutation rdy (retinal dystrophy) causes, either directly or indirectly, a defect in RPE phagocytosis (4). This defect leads to an accumulation of shed OS membranes in the subretinal space (4) and a rapid and progressive degeneration of photoreceptor cells (5).The molecular mechanisms of RPE phagocytosis are unclear. Studies of the internalization of exogenous OS by cultured primary RPE cells suggested a receptor-mediated process (6 -8). Inhibition of the RPE cell culture phagocytic assay by antireceptor antibodies or competitive ligands suggested several specific proteins that might play a role in the process, including the mannose receptor (9, 10), CD36 (11), and ␣ v ␤ 5 integrin (12-14). Inhibition of ␣ v ␤ 5 integrin function disrupts the OS binding phase of RPE phagocytosis, whereas the mannose receptor and CD36 have been implicated in both OS binding and ingestion. Cultured RCS RPE cells bind exogenous OS at wildtype levels. However, only a small percentage of bound OS are ingested by RCS RPE cells (15), indicating that the protein encoded by the rdy locus is critical, directly or indirectly, for OS uptake.The gene corresponding to rdy remained unknown until recently. The mannose receptor protein and messenger RNA are present in the RPE of both wild-type and RCS rats from postnatal day (P) 5 to adult (16). CD36 null mice have been reported to have normal electroretinography and retinal histology (17). These data suggest that neither the mannose receptor nor CD36 is the gene mutated in the RCS rat. We used positional cloning to identify a mutation in the receptor tyrosine kinase gene Mertk in the RCS rat. A deletion of RCS genomic DNA results in expression of an aberrant Mertk transcript with a translation termination signal after codon 20 (18),...

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Section: Discussionsupporting

confidence: 81%

“…6, G-I). These data indicate that Mertk gradually accumulates around OS with a time course that is similar to the progressive ingestion of OS (7,15).…”

Section: Resultsmentioning

confidence: 78%

Mertk Triggers Uptake of Photoreceptor Outer Segments during Phagocytosis by Cultured Retinal Pigment Epithelial Cells

Feng¹,

Yasumura²,

Matthes³

et al. 2002

Journal of Biological Chemistry

207

141

View full text Add to dashboard Cite

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“…This was monitored by direct microscopic observation of immunostained phagosomes inside RPE cells (data not shown). Kinetic studies using ROS, compared with nonspecific latex particles, strongly suggest that RPE cells, in this in vitro system, phagocytize ROS through a specific receptor-mediated mechanism (11,38). Resting RPE cells in culture revealed low levels of zif-268 mRNA; c-fos, tis-1, and cox-2 mRNA were undetectable.…”

Section: Resultsmentioning

confidence: 76%

Selective Transcription Factor Induction in Retinal Pigment Epithelial Cells during Photoreceptor Phagocytosis

Ershov¹,

Lukiw²,

Bazán

1996

Journal of Biological Chemistry

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“…The different phases of uptake can also distinguished in vitro by using appropriate experimental procedures 39 . Pulse-chase experiments can be performed using temperature switch: POS binding occurs at 20 °C while internalization needs a temperature of 37 °C to proceed 32,40,7 . This distinction also exists in macrophages, which can recognize POS as well and in which the molecular machinery to eliminate apoptotic cells is very similar to the RPE's machinery 7 .…”

Section: Representative Resultsmentioning

confidence: 99%

Large-Scale Purification of Porcine or Bovine Photoreceptor Outer Segments for Phagocytosis Assays on Retinal Pigment Epithelial Cells

Parinot

Rieu

Chatagnon

et al. 2014

JoVE

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Analysis of one of the vital functions of retinal pigment epithelial (RPE) cells, the phagocytosis of spent aged distal fragments of photoreceptor outer segments (POS) can be performed in vitro. Photoreceptor outer segments with stacks of membranous discs containing the phototransduction machinery are continuously renewed in the retina. Spent POS are eliminated daily by RPE cells. Rodent, porcine/bovine and human RPE cells recognize POS from various species in a similar manner. To facilitate performing large series of experiments with little variability, a large stock of POS can be isolated from porcine eyes and stored frozen in aliquots. This protocol takes advantage of the characteristic of photopigments that display an orange color when kept in the dark. Under dim red light, retinae are collected in a buffer from opened eyecups cut in halves. The retinal cell suspension is homogenized, filtered and loaded onto a continuous sucrose gradient. After centrifugation, POS are located in a discrete band in the upper part of the gradient that has a characteristic orange color. POS are then collected, spun, resuspended sequentially in wash buffers, counted and aliquoted. POS obtained this way can be used for phagocytosis assays and analysis of protein activation, localization or interaction at various times after POS challenge. Alternatively, POS can be labeled with fluorophores, e.g., FITC, before aliquoting for subsequent fluorescence quantification of POS binding or engulfment. Other possible applications include the use of modified POS or POS challenge combined with stress conditions to study the effect of oxidative stress or aging on RPE cells.

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Kinetic studies of rod outer segment binding and ingestion by cultured rat RPE cells

Cited by 76 publications

References 17 publications

Mertk Triggers Uptake of Photoreceptor Outer Segments during Phagocytosis by Cultured Retinal Pigment Epithelial Cells

Mertk Triggers Uptake of Photoreceptor Outer Segments during Phagocytosis by Cultured Retinal Pigment Epithelial Cells

Selective Transcription Factor Induction in Retinal Pigment Epithelial Cells during Photoreceptor Phagocytosis

Large-Scale Purification of Porcine or Bovine Photoreceptor Outer Segments for Phagocytosis Assays on Retinal Pigment Epithelial Cells

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