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 epider moid A431 carcinoma cells through the specific endocytic pathway of tr ansferrin. The FRAP of this fluorescent marker internalized in the lat e compartment of transferrin endocytosis (LCT) was measured in living A431 cells. These experiments showed the presence of an active intrace llular transport of Tf-LRSC which can be interpreted by a mechanism in volving carrier vesicles budding from stationary vacuoles, saltating a long microtubules and fusing with other stationary vacuoles, according to previous video-microscopy observations of a membranous traffic dyn amics in these cells, revealed by a gold complex of an Anti-Transferri n Receptor (ATR) (M. De Brabander, R. Nuygens, H. Geerst, C.R. Hopkins , Cell. Motil. Cystoskel. 9 (1988) 30). When the A431 cells were treat ed with nocodazole or metabolic inhibitors, there remained a residual FRAP which was ascribed to the spontaneous reactivation of the bleache d molecules. According to a theoretical result obtained in the compani on 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-treate d cells from the fractional FRAP of the non-treated cells. This FRAP o f transport was fitted to a formula derived in that companion paper an d 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 mu m(-2) s(-1). The rate value of the fusion of Vesicles with vacuoles was divided by two in c ells treated by AIF(4)(-), and increased to 20% in cells treated with Brefeldin A. These results correspond to an homotypic fusion process r egulated by an heterotrimeric G-protein. Our work suggests that FRAP c an be used to bring information on the transport of membrane component s in living eukaryotic cells. (C) 1997 Elsevier Science B.V.