Intravital Microscopy Reveals Differences in the Kinetics of Endocytic Pathways between Cell Cultures and Live Animals

Abstract

Intravital microscopy has enabled imaging of the dynamics of subcellular structures in live animals, thus opening the door to investigating membrane trafficking under physiological conditions. Here, we sought to determine whether the architecture and the environment of a fully developed tissue influences the dynamics of endocytic processes. To this aim, we imaged endocytosis in the stromal cells of rat salivary glands both in situ and after they were isolated and cultured on a solid surface. We found that the internalization of transferrin and dextran, two molecules that traffic via distinct mechanisms, is substantially altered in cultured cells, supporting the idea that the three dimensional organization of the tissue and the cues generated by the surrounding environment strongly affect membrane trafficking events.

Imaging cell biology in live animals: Ready for prime time

“…Moreover, the trafficking of these molecules through the endo-lysosomal system was documented, providing interesting insights on early endosomal fusion (Fig. 4 E; Masedunskas and Weigert, 2008; Masedunskas et al, 2012b). Notably, significant differences were observed in the kinetics of internalization of transferrin and dextran.…”

“…However, in freshly explanted stromal cells adherent on glass, transferrin was internalized within 1 min, whereas dextran appeared in endosomal structures after 10–15 min. Although the reasons for this difference were not addressed, it is clear that the environment in vivo has profound effects on the regulation of intracellular processes (Masedunskas et al, 2012b). Similar differences have been reported for the caveolae that in vivo are more dynamic than in cell cultures (Thomsen et al, 2002; Oh et al, 2007).…”

Imaging membrane remodeling during regulated exocytosis in live mice

“…For example, various aspects of endocytosis, such as the trafficking of molecules throughout the endo-lysosomal system were investigated, revealing that the kinetics of the endocytic pathways in vivo differ from what was previously reported in cultured cells [5, 44]. Moreover, a novel modality of basal mitochondrial metabolism that occurs in vivo in the salivary epithelium was recently described, and for the first time, the coordination of mitochondria, and its regulation through gap junctions was unraveled [45].…”

Imaging the pharmacology of nanomaterials by intravital microscopy: Toward understanding their biological behavior

“…As invading tumor cells extend pseudopodia during migration and intravasation, protruding membranes become increasingly fluid, and fluidity reaches a maximum once cells actually enter circulation [51]. Receptor endocytosis is also complex; at the simplest level, imaging shows that the model compounds dextran and transferrin both exhibit measurable differences in internalization kinetics in vivo compared to in cell culture [74]. IVM of HER2-targeted Qdots has revealed a more complete and dynamic model of NP binding and receptor trafficking: once targeted NPs have extravasated into the tumor tissue, they bind to surface receptor and traffic to perinuclear endocytic vesicles in a discrete stepwise fashion characterized by periods of relative immobility followed by spurts of directional transport [75].…”