Figure 2: Influence of bafilomycin, nocodazole, and low temperature on endocytic pathways in HeLa cells. The recycling pathway (transferrin, LDLR) and the transport of ligands (LDL) and fluid-phase marker to lysosomes are shown. Iron-loaded transferrin binds to its receptor at the plasma membrane. The complex is internalized via clathrin-coated vesicles (CCV) and delivered into early endosomes within 2–5 minutes, where the iron is released and transferred into the cytoplasm. Apotransferrin remains bound to the receptor and recycles via a fast and a slow pathway. At the plasma membrane, apotransferrin is released at the neutral pH. Similarly, internalized LDL is released from its receptor in early compartments allowing for LDLR to return to the plasma membrane via the same pathways as apotransferrin. Although a major portion of fluid-phase marker (e.g., dextran) is recycled, the remaining fluid and released ligands (LDL) are delivered from early endosomes (within 5 min), via endosomal carrier vesicles (ECV) and late endosomes (within 15 min), to lysosomes (within 25 min). Transferrin transport to and recycling via the perinuclear recycling compartment is blocked by nocodazole, whereas bafilomycin and lowering the temperature to 20°C are without effect. In contrast, bafilomycin arrests fluid-phase markers in early endosomes by preventing budding of ECV, whereas nocodazole leads to accumulation of cargo in ECV. Finally, incubation at 20°C prevents delivery of markers from late endosomes to lysosomes. For further details and endosomal pH determination see [105, 108]. Adapted from Fuchs and Blaas [112].