Purchase this article with an account.
A.–L. Law, M.J. Hayes, J. Greenwood, S.E. Moss; Recruitment of Annexin 2 to the Nascent Phagosome is an Essential Step in the Phagocytosis of Outer Segments by RPE Cells . Invest. Ophthalmol. Vis. Sci. 2006;47(13):5881.
Download citation file:
© ARVO (1962-2015); The Authors (2016-present)
To examine the role of annexin 2 in the formation and internalisation of phagosomes in retinal pigment epithelial (RPE) cells.
Human ARPE19 cells were cultured on the glass inserts of Matek dishes for immunofluorescence and live imaging, or plastic Nunc dishes for biochemical analysis. In some studies ARPE19 cells were exposed to siRNA for annexin 2 for three days prior to experimentation. Outer segments were prepared from porcine retina using standard protocols (typically 50 – 100 eyes per prep), and either used fresh in biochemical experiments, or labelled with an appropriate fluorescent marker prior to imaging experiments.
Depletion of annexin 2 using siRNA led to a significant quantitative reduction in the phagocytosis of rod outer segments in cultured RPE cells. In cells expressing annexin 2, imaged by confocal microscopy, a striking enrichment of annexin 2 and F–actin was observed at the phagocytic cup immediately upon engagement of the apical cell surface by the fluorescently labeled outer segments. Annexin 2 and F–actin were concomitantly lost from the phagocytosed material during internalization. Biochemical studies revealed that the kinetics of tyrosine phosphorylation of annexin 2 coincided with recruitment of the protein to nascent phagosomes, and that the loss of annexin 2 from maturing phagosomes was temporally coincident with the disappearance of tyrosine phosphorylation.
These results identify annexin 2 as an essential component of the phagocytic machinery in RPE cells, and suggest that its activity may be regulated by tyrosine kinases such as FAK and Mer that are known to have key roles in this process.
This PDF is available to Subscribers Only