Abstract
Abstract: :
Purpose: The integrin receptor αvß5 plays an important role in the phagocytosis of photoreceptor outer segment fragments (OS) by RPE cells. Here, we determined expression pattern and activity of a fluorescent ß5 integrin fusion protein in RPE cells. These are the first studies that directly observe the redistribution of a phagocytic receptor in the RPE during OS uptake. Methods: We generated an expression plasmid encoding a fusion protein of full–length human ß5 integrin with the green fluorescent protein (GFP) tag at its intracellular C–terminus. We transiently transfected RPE and fibroblast cells originally derived from mouse, rat and human tissue with the construct. To study ß5–GFP fusion protein expression, subcellular localization and αvß5–GFP receptor formation we used laser scanning confocal microscopy and double immunofluorescence labeling. To determine whether ß5–GFP expression had an effect on the phagocytic activity of transfectants towards isolated OS, we used fluorescence scanning to quantify OS binding and internalization by cells expressing GFP alone or the ß5–GFP fusion protein. Results: Transfection of ß5–GFP encoding plasmids resulted in fluorescent protein expression in all cell lines tested. At steady–state, the fluorescent fusion protein localized to the plasma membrane and to reticular compartments of the biosynthetic machinery. In contrast, soluble GFP distributed diffusely in the nucleus and in the cytosol of control transfectants. Plasma membrane ß5–GFP fluorescence co–localized with αvß5 heterodimer–specific immunofluorescence signals at the surface of transfected cells. Furthermore, phagocytosis assays showed that ß5–GFP expression increased both OS binding and OS internalization by transfected cells. Conclusion: ß5–GFP forms heterodimers at the cell surface with mouse, rat, and human αv subunits confirming that the C–terminal GFP tag impedes neither assembly of integrin receptors nor their transport to the plasma membrane. Expression of ß5–GFP promotes OS phagocytosis indicating that surface αvß5–GFP integrin receptors are fully functional. For the first time, this ß5–GFP construct allows us to use fluorescence imaging to record live the mobilization of both particles and receptors during phagocytosis by RPE cells.
Keywords: retinal pigment epithelium • microscopy: light/fluorescence/immunohistochemistry