Abstract
Purpose: :
7a5 is an uncharacterized gene which encodes for an 852 amino acid protein. The 7a5 protein contains domains belonging to both the EH (Eps15–Homology)–network family of endocytosis proteins and the death domain family of apoptosis proteins. The only other known protein to contain this unique combination of domains is a 7a5–related protein, SH3BP4. The presence of both endocytosis and death domain moieties within the same protein promises that research into the role of 7a5 will be of fundamental significance to cellular structure and function particularly in retinal pigment epithelial cells. The purpose of this study is to determine the effects of 7a5 expression on ARPE–19 cells.
Methods: :
7a5 cDNA encoding for the open reading frame was amplified using RT–PCR, subcloned into pBluescript and DNA sequenced. 7a5 was then ligated in–frame with enhanced–green fluorescent protein in the pEGFP–C2 vector. ARPE–19 grown on glass coverslips were transiently transfected with either GFP–7a5 or GFP–7a5 and myc–SH3BP4. Fixed and live cells were examined by confocal microscopy to examine expression patterns and subcellular localization.
Results: :
ARPE–19 transfected for 24 or 72 hours were found to localize GFP–7a5 to both the cell periphery and vesicle structures. At 24 hours, APRE–19 localized GFP–7a5 mainly to the basolateral surface of the cells, including the cell periphery and membrane spikes. At 72 hours, there was an increase in the number of cells with GFP–7a5 vesicle structures as well as an increase in the number of vesicles present in individual cells. Many of the larger vesicles appeared to have a tubular structure with GFP–7a5 labeling the periphery of the vesicles and tubes. Additionally, the GFP–7a5 vesicle structures were often located within the apical portion of the cell where there was little if any GFP–7a5 staining at the cell periphery. Live confocal microscopy at 72 hours also showed GFP–7a5 vesicle fusion and migration. Transfected ARPE–19 cells expressing both GFP–7a5 and myc–tagged SH3BP4 showed co–localization of these two related proteins along the basolateral surface of the cell including cell projections and membrane spikes.
Conclusions: :
7a5 is a newly identified protein that is related to SH3BP4. Both of these proteins have domains belonging to both the EH–network family of endocytosis proteins and the death domain family of apoptosis proteins. This first study of the 7a5 protein shows that GFP–7a5 localizes mainly to the cell periphery basolaterally and in vesicle structures apically in ARPE–19 cells. GFP–7a5 was also found to have partial co–localization with its related protein, SH3BP4 particularly at the cell periphery.
Keywords: retinal pigment epithelium • cell membrane/membrane specializations • microscopy: confocal/tunneling