CONICET | Buscador de Institutos y Recursos Humanos

3D orbital tracking of single gold nanoparticles: a new approach to study vesicle trafficking in chromaffin cells.Gabriel M., Moya-Díaz J., Marengo F., Estrada L.Endocytosis and subsequent vesicle recycling serves to keep constant the pools of transmitter-containing vesicles ready for release in neurons and endocrine cells. The study of these processes has been carried out by using different experimental approaches, like electrophysiological measurements and single photon fluorescence microscopy. However, the diverse experimental limitations of these techniques restricted a detailed and high resolved study of the dynamics of vesicle trafficking after endocytosis in the whole cellular volume. Multiphoton microscopy provides optical sectioning for high-resolution imaging. In biological systems, most multiphoton microscopy studies have relied on two-photon excited ﬂuorescence (TPEF) to produce images. Because of their strong brightness and imaging durability, metallic NPs have been recently introduced as labels in fluorescence microscopy. The use of TPEF and metallic nanoparticles in combination provides a noninvasive, spatially localized, in vivo characterization for biological samples. In this work, we tracked single gold nanoparticles after endocytotic internalization in mouse chromaffin cells stimulated with high potassium. We use an orbital-scanning tracking method in a two-photon absorption microscope. In the first place, we compare constitutive and active internalization of gold nanoparticles (AuNPs), assessing the number of internalized AuNPs and evaluating its dynamics in terms of velocity and displacement after stimulation. In the second place, we evaluate these parameters pharmacologically blocking proteins classically involved in the development of endocytotic process. Our results show that endocytosis of AuNPs was much more efficient when exocytosis was induced with high K in comparison with constitutive cycling. In addition, the dynamics of AuNPs had a strong dependence on clathrin dependent endocytosis as well as on cortical actin polymerization. This study shows that the combination of 3D orbital tracking and AuNPs is an interesting tool for the study of vesicle trafficking after endocytosis in live cells.