Mertk Is Expressed To The Actin Filament-rich Phagocytic Cup During Retinal Pigment Epithelial Phagocytosis

Abstract

 

Purpose:

 

To determine the filament actin process regulated by Mertk during retinal pigment epithelium uptake of the shedded photoreceptor outer segment in vitro.

 

Methods:

 

Retinal pigment epithelium was isolated from both Mertk gene knockout mice and wild-type mice. Photoreceptor outer segment (POS) was isolated from rat retina and subjected to the cultured retinal pigment epithelium in vitro. Incubation with POS (green) for 3 hours, the cells of phagocytosis were fixed and immuno-stained with anti-Mertk IgG (purple) and phalloidin to stain filament actin (red) to observe the phagocytic cup formation for outer segment internalization. The immuno-fluorescent stained signal was scanned under confocal microscopy. Phagosome was visualized by three dimensional (XYZ) scanning. L1-L5 showed the 3D scanning from bottom to the top of the cultured RPEs. (Obj. = 63i x, Bar=10 micron)

 

Results:

 

Immuno-fluorescent labeling showed differences of the phagocytosis maturation between thewild-type (A) and Mertk mutant (B). When the phagocytosis initiated, the F-actin (red) was concentrated to the apoptotic particle adhesion point of the cells, which would extended out of the cell bodies to form the phagocytotic cup surrounding the particles(L1-L5). The Mertk signal was co-localized with the F-actin surrounding the particle(A). The phagocytic cup was shaped with the POS and growing out of the cell body to capture the POS forming the phagosome(L4-L5). The F-actin polymerization was blocked and the phagocytotic cup formation was disrupted in the Mertk mutant(B).

 

Conclusions:

 

Filament actin played an essential role in RPE phagocytosis mediated by Mertk signal pathway. Upon the Mertk activation, the actin was driven to polymerize and protrude out of the cell body to form the phagocytic cup surrounding the POS particle, which process could be disrupted by the Mertk mutant.  

 

Keywords: retinal pigment epithelium • cytoskeleton • mutations