Folate reception by vegetative Dictyostelium discoideum amoebae: distribution of receptors and trafficking of ligand.

Abstract

We report the first explicit demonstration of post-reception processing of a Dictyostelium chemoattractant. Folic acid stimulates reorganization of the cytoskeleton of vegetative amoebae of D. discoideum. In particular, folic acid is a potent chemoattractant and it causes enlargement of the filopodial array. The distribution of folic acid receptors and the fate of bound folate were investigated by presenting an agonist consisting of the conjugate, folic acid-lactalbumin-FITC (Folate*), to these amoebae. This novel probe was specifically bound to folic acid receptors of these amoebae and it stimulated chemotaxis and enlargement of their filopodial array. Hence, Folate* is a physiologically competent probe. The probe sans-folate moiety was not bound anywhere to living or fixed amoebae. Since Folate* did not bind to amoebae after incubation with equimolar folic acid, this probe is a receptor-specific agonist. We report here the first description, by confocal visualization of a competent agonist, of the distribution of folate receptors of D. discoideum vegetative amoebae and of the fate of this ligand. Examination of fixed amoebae revealed that bound Folate* was distributed generally over their entire surface including their filopodia. However, in living amoebae, Folate* was bound only at the cell body and this bound Folate* was almost completely internalized as concentrated packets into vacuoles. This endocytosis of the probe and the clustering of endocytosed Folate* is consistent with receptor-mediated internalization of a ligand. Possible routes for internalization of the folate probe and the implications of this endocytosis for signal molecule processing and temporal sensing are discussed.