FLUORESCENCE RECOVERY AFTER PHOTOBLEACHING (FRAP) OF A FLUORESCENT TRANSFERRIN INTERNALIZED IN THE LATE TRANSFERRIN ENDOCYTIC COMPARTMENT OF LIVING A431 CELLS - EXPERIMENTS
F. Azizi et P. Wahl, FLUORESCENCE RECOVERY AFTER PHOTOBLEACHING (FRAP) OF A FLUORESCENT TRANSFERRIN INTERNALIZED IN THE LATE TRANSFERRIN ENDOCYTIC COMPARTMENT OF LIVING A431 CELLS - EXPERIMENTS, Biochimica et biophysica acta. Biomembranes, 1327(1), 1997, pp. 75-88
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.