Abstract
In mammalian cells, endocytosis of the fluid phase and glycosylphosphatidylinositol-anchored proteins (GPI-APs) forms GEECs (GPI-AP enriched early endosomal compartments) via an Arf1- and Cdc42-mediated, dynamin independent mechanism. Here we use four different fluorescently labeled probes and several markers in combination with quantitative kinetic assays, RNA interference and high resolution imaging to delineate major endocytic routes in Drosophila cultured cells. We find that the hallmarks of the pinocytic GEEC pathway are conserved in Drosophila and identify garz, the fly ortholog of the GTP exchange factor GBF1, as a novel component of this pathway. Live confocal and TIRF imaging reveals that a fraction of GBF1 GFP dynamically associates with ABD RFP (a sensor for activated Arf1 present on nascent pinosomes). Correspondingly, a GTP exchange mutant of GBF1 has altered ABD RFP localization in the evanescent field and is impaired in fluid phase uptake. Furthermore, GBF1 activation is required for the GEEC pathway even in the presence of Brefeldin A, implying that, like Arf1, it has a role in endocytosis that is separable from its role in secretion.
Highlights
Internalization of cargo at the cell surface takes place via multiple mechanisms
Other pathways that do not require clathrin or dynamin for internalization are less well characterized, but are prevalent - the GEEC pathway is one such example [3]. This pathway was originally identified on the basis of the selective internalization of various glycosylphosphatidylinositol-anchored proteins (GPI-APs) such as the folate receptor [4], but has been shown to facilitate the entry of: cholera toxin bound to its ganglioside receptor GM1[5]; the vacuolating toxins aerolysin and VacA[6]; and bulk fluid phase in diverse mammalian cells [4]
A fraction of GPI-APs are organized in nanoclusters which are sensitive to the extraction of cholesterol [7]
Summary
Internalization of cargo at the cell surface takes place via multiple mechanisms. For instance, the well-studied transferrin receptor (TfR) is endocytosed via the dynamin-mediated pinching of clathrin coated vesicles from the plasma membrane [1,2]. Other pathways that do not require clathrin or dynamin for internalization are less well characterized, but are prevalent - the GEEC pathway is one such example [3]. This pathway was originally identified on the basis of the selective internalization of various GPI-APs such as the folate receptor [4], but has been shown to facilitate the entry of: cholera toxin bound to its ganglioside receptor GM1[5]; the vacuolating toxins aerolysin and VacA[6]; and bulk fluid phase (pinocytosis) in diverse mammalian cells [4]. It is not known how Arf activation occurs during GEEC endocytosis
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