Fluorescence recovery after photobleaching (FRAP) of a fluorescent transferrin internalized in the late transferrin endocytic compartment of living A431 cells: experiments.

In this work, we verified that transferrin fluorescently labelled with lissamine rhodamine sulfochloride (Tf-LRSC) is internalized in epidermoid A431 carcinoma cells through the specific endocytic pathway of transferrin. The FRAP of this fluorescent marker internalized in the late compartment of transferrin endocytosis (LCT) was measured in living A431 cells. These experiments showed the presence of an active intracellular transport of Tf-LRSC which can be interpreted by a mechanism involving carrier vesicles budding from stationary vacuoles, saltating along microtubules and fusing with other stationary vacuoles, according to previous video-microscopy observations of a membranous traffic dynamics in these cells, revealed by a gold complex of an Anti-Transferrin Receptor (ATR) (M. De Brabander, R. Nuygens, H. Geerst, C.R. Hopkins, Cell. Motil. Cystoskel. 9 (1988) 30). When the A431 cells were treated with nocodazole or metabolic inhibitors, there remained a residual FRAP which was ascribed to the spontaneous reactivation of the bleached molecules. According to a theoretical result obtained in the companion paper (P. Wahl, F. Azizi, Biochim. Biophys. Acta 1327 (1997) 69-74), we derived the fractional FRAP characterizing the transport process of Tf-LRSC by subtracting the fractional FRAP of the nocodazole-treated cells from the fractional FRAP of the non-treated cells. This FRAP of transport was fitted to a formula derived in that companion paper and based on the mechanism outlined above. From the time constant value determined by this fit, the number of vesicles which fused with a unit of vacuole surface was calculated to be 0.15 microm(-2) s(-1). The rate value of the fusion of vesicles with vacuoles was divided by two in cells treated by AlF4-, and increased to 20% in cells treated with Brefeldin A. These results correspond to an homotypic fusion process regulated by an heterotrimeric G-protein. Our work suggests that FRAP can be used to bring information on the transport of membrane components in living eukaryotic cells.