High-speed imaging of Rab family small GTPases reveals rare events in nanoparticle trafficking in living cells.

Despite the increased application of nanomaterials in diagnostics and therapeutics, methods to study the interactions of nanoparticles with subcellular structures in living cells remain relatively undeveloped. Here we describe a robust and quantitative method that allows for the precise tracking of all cell-associated nanoparticles as they pass through endocytic compartments in a living cell. Using rapid multicolor 3D live cell confocal fluorescence microscopy, combined with transient overexpression of small GTPases marking various endocytic membranes, our studies reveal the kinetics of nanoparticle trafficking through early endosomes to late endosomes and lysosomes. We show that, following internalization, 40 nm polystyrene nanoparticles first pass through an early endosome intermediate decorated with Rab5, but that these nanoparticles rapidly transfer to late endosomes and ultimately lysosomes labeled with Rab9 and Rab7, respectively. Larger nanoparticles of 100 nm diameter also reach acidic Rab9- and Rab7-positive compartments although at a slower rate compared to the smaller 40 nm nanoparticles. Our work also reveals that relatively few nanoparticles are able to access endocytic recycling pathways, as judged by lack of significant colocalization with Rab11. Finally, we demonstrate that this quantitative approach is sufficiently sensitive to be able to detect rare events in nanoparticle trafficking, specifically the presence of nanoparticles in Rab1A-labeled structures, thereby revealing the wide range of intracellular interactions between nanoparticles and the intracellular environment.