CONICET | Buscador de Institutos y Recursos Humanos

Endocytosis and subsequent vesicle recycling serves to keep constant thepools of transmitter-containing vesicles ready for release in neurons andendocrine cells. The study of these processes has been carried out by using different experimental approaches, like electrophysiological measurementsand single photon fluorescence microscopy. However, the diverse experimentallimitations of these techniques restricted a detailed and high resolvedstudy of the dynamics of vesicle trafficking after endocytosis in the wholecellular volume. Multiphoton microscopy provides optical sectioning forhigh-resolution imaging. In biological systems, most multiphoton microscopystudies have relied on two-photon excited fluorescence (TPEF) to produceimages. 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 anoninvasive, spatially localized, in vivo characterization for biological samples.In this work, we tracked single gold nanoparticles after endocytoticinternalization 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 internalizationof gold nanoparticles (AuNPs), assessing the number of internalized AuNPsand evaluating its dynamics in terms of velocity and displacement after stimulation.In the second place, we evaluate these parameters pharmacologicallyblocking proteins classically involved in the development of endocytotic process.Our results show that endocytosis of AuNPs was much more efficientwhen exocytosis was induced with high K in comparison with constitutivecycling. In addition, the dynamics of AuNPs had a strong dependence on clathrindependent endocytosis as well as on cortical actin polymerization. Thisstudy shows that the combination of 3D orbital tracking and AuNPs is aninteresting tool for the study of vesicle trafficking after endocytosis in livecells.