Background

Accumulating evidence emphasizes the importance of coordinated endosomal recycling in such diverse processes as cell migration, adhesion, morphogenesis, proliferation, cytokinesis, as well as learning and memory (Grant and Donaldson, 2009; Parachoniak and Park, 2012; Wandinger-Ness and Zerial, 2014; Zaoui et al., 2019a and 2019b). There are over 70 Rab GTPases in mammals that have distinct localizations and functions in membrane trafficking. Further compounding complexity, while most Rab GTPases are ubiquitous, some display tissue specificity expression (van der Sluijs et al., 1992; McCaffrey et al., 2001; Grant and Donaldson, 2009; Wandinger-Ness and Zerial, 2014). Among them, the small GTPase Rab4, localized in the early endosome, regulates the trafficking along the endocytic route, from early and recycling endosomes, toward the plasma membrane. Rab4 has been involved in the recycling of receptors such as Tfn and receptor tyrosine kinase (RTK), integrins, ubiquitin ligases, as well as other machinery regulators associated with vesicle formation, budding, transport, and fusion (McCaffrey et al., 2001; Grant and Donaldson, 2009; Stenmark, 2009; Parachoniak et al., 2011; Wandinger-Ness and Zerial, 2014; Zaoui et al., 2019a). We recently reported strict requirement of the MT plus-end tracking proteins CLIP-170 for Rab4-mediated Met RTK recycling at the plasma membrane in response to its ligand, hepatocyte growth factor (HGF). The increased localization of Met/Rab4 vesicles towards the MT plus-ends in response to HGF results in enhanced cell protrusion dynamic and cell migration (Zaoui et al., 2019a).

Therefore, by mediating the compartmentalization of the endocytic pathway into early, recycling, late, and lysosomal tracking routes and by regulating the trafficking from vesicle budding to fusion, Rabs stand as essential integrators of cell signaling. Rabs control the localization, the intensity, and the duration of signaling outputs, to allow spatiotemporal regulation of signal transduction (McCaffrey et al., 2001; Stenmark, 2009; Grant and Donaldson, 2009; Parachoniak and Park, 2012; Wandinger-Ness and Zerial, 2014; Zaoui et al., 2019a). Altered endocytosis is observed in many malignancies and can lead to sustained proliferation, survival, invasiveness, and therapeutic resistance (Parachoniak and Park, 2012; Mellman and Yarden, 2013; Zaoui et al., 2019a and 2019b). Our studies aim at understanding endocytic recycling alterations on cell signaling regulating cytoskeletal rearrangement, cell migration, and invasion in oncogenic contexts.

The protocol described herein permits automated tracking of the endocytic vesicle in three-dimensional live-cell imaging, allowing the study and the quantification of multiple parameters under physiological and tumorigenic conditions. Moreover, this methodology can be easily extended to assess the mobility of other Rab family members or any other cell vesicles.