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J. R. Dunlevy, E. D. Koppelman; Transfection of ARPE-19 Cells With SH3BP4 Fusion Protein Constructs Results in an Increase in Cells With Autophagy Structures. Invest. Ophthalmol. Vis. Sci. 2008;49(13):470.
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SH3BP4 is a protein that contains multiple protein-protein binding domains found in endocytosis and apoptosis protein families. Expression of SH3BP4 fusion proteins has been previously shown to induce nuclear condensation and gross morphological changes such as cell rounding and membrane spiking in ARPE-19 cells. The purpose of this study is to further characterize these morphological changes on the ultrastructural level.
ARPE-19 cells were transiently transfected with either SH3BP4 or control constructs for 48 hours in either the presence of absence of 5 mM 3-methyladenine (3-MA), a class III phosphatidyl inositol-3-kinase inhibitor that also inhibits most autophagy pathways. The cells were then examined by transmission electron microscopy (TEM), incubated with propidium iodide (PI) and examined by live fluorescent microscopy, or fixed, stained with Hoechst 33258 and examined by fluorescent microscopy for nuclear morphology.
ARPE-19 cells that were untreated or transfected with the vector only control constructs were examined by TEM and found to contain mainly small vesicle structures, round nuclei with vast amounts of euchromatin, and very few if any large multiple membrane bound structures. Conversely, cells transfected with SH3BP4 full length or SH3BP4-DD (C-terminal region including the Death Domain) constructs often had convoluted nuclei that had a greatly increased quantity of heterochromatin (but not pyknotic nuclei) and large multiple membrane bound structures that contained intracellular material, sometimes organelles, that resembled autophagosomes. Additionally, large spans of nucleating membranes typically seen prior to autophagosome formation were also observed. Preliminary results showed that addition of 3-MA, an autophagy inhibitor, to SH3BP4-DD transfections resulted in a reversal of the effects seen by TEM as well as a decrease in the number of cells with a condensed nucleus when stained with Hoechst. None of the transfected cell populations were found to substantially incorporate PI indicating that the plasma membrane remains intact despite nuclear and gross morphological changes.
The results of this study show that transfection with SH3BP4 constructs and particularly the SH3BP4-DD construct appear to result in an increase in autophagy within the ARPE-19 cell population. These cell populations do not appear necrotic due to a lack of PI incorporation nor apoptotic due to a lack of pyknotic nuclei in TEM studies.
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