Particle Tracking Of Membrane Vamp2 Within Secretory Cells In The Nanometer Regime

In the past we suggested that a pre-sorted and pre-assembled pool of SV proteins on the presynaptic membrane might support a first wave of clathrin-mediated endocytosis. Recent evidence using IsoSTED nanoscopy of surface vesicular proteins indicates, that such pre-assembled patches exist at the periactive zone in hippocampal boutons. Here we analyzed the diffusion kinetics of the surface-stranded vesicular protein Vamp2 coupled to the photoactivatable proteins Dendra and Eos by 3D particle tracking in combination with photo activation localization microscopy (PALM). The z-position information of the fluorophores was estimated by measuring the widths of the elliptical PSF caused by a cylindrical lens in the detection pathway. The elliptical PSF is translated into axial positions by a least squares fit of the ellipticity. The axial position estimation can then be enhanced by Gaussian image filtering with a resolution enhancement of at least 30%. With 3D particle tracking in living secretory cells like PC12 or hippocampal boutons nanostructures of faster and slower diffusion or transport of a VAMP2-EOS complex were identified. From 3D particle tracking maps we can show highly heterogeneous diffusion behaviour of Vamp2 within PC12 cells and synapses. This enables us to resolve and characterize areas of endocytosis within living cells.