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.
Highlights
The advent of GFP technology and fluorescent light microscopy has significantly changed cell biology [1,2]
Cells cultured on solid surfaces are exposed to an environment that is different from the one surrounding cells in a living multicellular organism
In order to determine whether environment and spatial organization affect the architecture of subcellular compartments, we compared cells grown either on glass or in a 3D matrix with cells in intact tissues
Summary
The advent of GFP technology and fluorescent light microscopy has significantly changed cell biology [1,2]. Our group has developed an experimental model based on the use of the salivary glands (SGs) as a model organ, which makes possible imaging at a subcellular resolution by minimizing the motion artifacts and extending the imaging time (see [24] for a detailed protocol) Using this experimental model we have investigated several aspect of membrane trafficking such as, regulated exocytosis of large secretory granules [20,25], endocytosis and trafficking of molecules through the endo-lysosomal system [17,26], and stimulated uptake of plasmid DNAs from the apical plasma membrane of the SG epithelium [18,27]. We imaged and compared the kinetics of uptake of transferrin (Tfn) and dextran in a population of stromal cells both in the SGs of live rats and after the cells were explanted and grown on glass
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