Fig 1.

(a) Schematic of the fluorescent model cargo without (top-left, eGFP-RTA^E177D) or with (top-right, eGFP-RTA^E177D-HDEL) the ER-retention motif HDEL, which ensures the physical interaction with KDELRs. eGFP-RTA^E177D lacking the HDEL motif serves as negative control and is unable to bind KDELRs. An additional mammalian enhanced GFP tag was introduced at the N-terminus to monitor the binding/uptake and interacellular transport of the model cargo in living cells. Furthermore, a less toxic variant of the cytotoxic A subunit of ricin (RTA^E177D) serves as a second marker to determine the cargo uptake via cell viability. The integrated His-tag ((His)₆-Tag) is used for affinity purification of the fluorescent model cargos from E. coli lysates. (bottom row) Schematics of cargo-KDELR interaction at the mammalian cell surface in the presence/absence of the HDEL motif. (b) Confocal laser scanning microscopy of human cell lines treated with eGFP-RTA^E177D-HDEL or eGFP-RTA^E177D (negative control). In the latter case, the images represent the steady-state regime of receptor cluster development (t ≥ 150 min). Scale bars, 20 μm. (c) Similar to panel (b) but for mouse cell lines.