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

AbstractIn previous works, other authors characterized a compartment (LCT) of A431 carcinoma cells in which markers of transferrin endocytose had accumulated during a long chase period. This compartment, was essentially formed by large stationary vacuoles. A few small vesicles budded from these vacuoles, rapidly saltated along microtubules and eventually fused with other vacuoles, causing an intracellular transport of the marker bound to the limiting membrane (M. De Brabander, R. Nuygens, H. Geerts, C.R. Hopkins, Cell. Mot. Cytoskel. 9 (1988) 30). In the present paper, we derived the fluorescence recovery after photobleaching (FRAP) of a fluorescent marker of LCT. We assumed that the rate of the intracellular transport of the marker was controlled by the fission–fusion process between vesicles and vacuoles. We showed that the concentration of a bleached fluorescent marker was a decreasing exponential function of the time elapsed from the beginning of the recovery phase. The rate constant of this exponential was equal to the product of the vesicle surface by the number of vesicles which fused with a unit of vacuole surface during one second. If a fraction of the marker spontaneously reactivated itself with a much higher rate constant of reaction than the rate constant of the transport process, the fractional FRAP of the marker was the sum of the fractional FRAP of both processes occurring separately. In a companion paper (F. Azizi, P. Wahl, Biochim. Biophys. Acta 1327 (1997) 75–88), our FRAP experiments will be described and analysed with the mathematical expressions derived in the present paper