Overexpression generates aberrant distribution of endocytic regulators - the case of the Rab11/LAMP1 compartment.

While the uptake of cargos via endocytosis and the subsequent trafficking through the cell is crucial for normal cellular function and tightly regulated, the study of this bears challenges. Most studies of Rab GTPases, the primary coordinators of endocytic progression, rely on ectopic expression of fluorescently tagged proteins via transient transfection. Previous studies already showed that the design of the fluorescent tag as well as the unpredictable nature of transient transfection can cause problems. Even though the pitfalls of overexpression have been reported for several research fields, the consequences of overexpression on endocytic trafficking are under-reported. To highlight the importance of working with endogenous levels of proteins to draw conclusions about endosome colocalization and identity, we present an example where the colocalization of two endosomal regulators/markers, Rab11 and LAMP1, varied drastically when these proteins were analyzed at their endogenous levels or following ectopic expression. When both proteins were ectopically expressed, up to 90% colocalization was observed. However, when analyzed at the endogenous level no colocalization was detectable. This study shows how important vesicular trafficking perturbation can occur following ectopic expression of endosomal proteins.