THE KINETIC ASPECTS OF INTRACELLULAR FLUORESCENCE LABELING WITH TMA-DPH SUPPORT THE MATURATION MODEL FOR ENDOCYTOSIS IN L929 CELLS

TMA-DPH 1-(4-trimethylammonium)-6-phenyl-1,3,5-hexatriene) , a hydroph obic fluorescent membrane probe, interacts with living cells by instan taneous incorporation into the plasma membrane, where it becomes fluor escent. It then follows the intracellular constitutive membrane traffi c and acts as a bulk membrane marker of the endocytic pathway (Illinge r, D., P. Poindron, P. Fonteneau, M. Modolell, and J. G. Kuhry. 1990. Biochim. Biophys. Acta. 1030:73-81; Illinger, D., P. Poindron, and J. G. Kuhry. 1991. Biol. Cell. 73:131-138). As such, TMA-DPH displays par ticular properties mainly due to partition between membranes and aqueo us media. From these properties, original arguments can be inferred in favor of the maturation model for the endocytic pathway, against that of pre-existing compartments, in L929 cultured mouse fibroblasts. (a) TMA-DPH labeling is seen to progress from the cell periphery to perin uclear regions during endocytosis without any noticeable loss in fluor escence intensity; with a vesicle shuttle model this evolution would b e accompanied by probe dilution with a decrease in the overall intrace llular fluorescence intensity, and the labeling of the inner (late) co mpartments could in no way become more intense than that of the periph eral (early) ones. (b) From TMA-DPH fluorescence anisotropy assays, it is concluded that membrane fluidity is the same in the successive end ocytic compartments as in the plasma membrane, which probably denotes a similar phospholipidic membrane composition, as might be expected in the maturation model. (c) TMA-DPH internalization and release kinetic s are more easily described with the maturation model.-