Apical sorting pathways in Retinal Pigment Epithelial (RPE) cells

Purpose. In a previous study We were able to show that the polarized cell line RPE-J delivers the the influenza hemmaglutinin (HA) viral glycoprotein to the apical surface via an indirect pathway. Here we compare the sorting of HA in primary cultures of Long Evans rat RPE, explants, and RPE-J cells.Methods. We used the temperature sensitive influenza mutant ts061 and a temperature blockade (20°C) to trap the proteins in the Trans-Golgi-Network (TGN). Removal of the temperature block was then used to follow the delivery of HA to the cell surface. Primary RPE and RPE-J cells were plated onto Matrigel coated Transwell filters. Explants were prepared from 2 month old rat eyes,after enzymatic removal of neural retina. RPE were infected with influenza and kept at 39.5°C to allow HA accumulation in the rough Endoplasmic Reticulum (rER) and then switched to 20°C for 3h to allow accumulation in the TGN. Additional incubation of infected monolayers at 32°C (RPE-J) and 37°C (primary RPE and explants) was used to assess the appearance of HA on the surface of infected cells. Infected monolayers were washed, fixed and processed for laser scanning confocal microscopy (LSCM). Results. Cultured cells were optimally infected when incubated at 39.5*C for 10h. Shift ing the infected monolayers to 20°C for 3h resulted in accumulation of HA in a juxtanuclear compartment that could be stained with antibodies to TGN-38, a marker for the TGN. Data obtained show that the kinetics of the surface delivery of HA in primary cultures of RPE was quicker (1h) than in RPE-J cells (4h).The steps of the surface delivery are under investigation. Conclusions. The use of the temperature sensitive influenza mutant ts061 has been a powerful tool to study the biosynthetic machinery of a variety of cell types. The conditions to accomplish this in RPE are established here.