Biophysical Society Thematic Meeting| Padova 2019

Quantitative Aspects of Membrane Fusion and Fission

Poster Abstracts

13-POS Board 13 XENAPSES GROWN ON QUANTIFOIL-SUPPORTED TEM GRIDS: TOWARDS CRYO-ELECTRON TOMOGRAPHY OF EXOCYTIC AND ENDOCYTIC STRUCTURES Junxiu Duan 1,2 ; Sai Krishnan 1 ; Yaroslav Tsytsyura 1 ; Nataliya Glyvuk 1 ; Ulrike Keller 1 ; Jürgen Klingauf 1 ; 1 University of Münster, Institute of Medical Physics and Biophysics, Münster, Nordrhein- Westfalen, Germany 2 University of Münster, CiM-IMPRS Graduate Program, Münster, Nordrhein-Westfalen, Germany For studying quantitative aspects of synaptic exo- and endocytosis at the molecular level, we have developed cultured ‘xenapses’, TIRFM-amenable purely pre-synaptic boutons. These are formed by murine hippocampal neurons cultured on microstructured glass coverslips which have been functionalized with synaptogenic proteins. In order to correlate results from fluorescence microscopy (TIRFM and dSTORM/PALM) with the endocytic ultrastructure we here developed methods for micropatterning and functionalization of Quantifoil films on transmission electron microscopy (TEM) grids. While on glass coverslips micropatterning is easily achieved by micro- contact printing of the functionalized polymer, for the thin and brittle Quantifoils we developed a bottom up approach involving click chemistry and UV-photolithography. Xenapses are formed on TEM grids within a few days. Their functionality was confirmed by calcium and pHluorin imaging experiments. At xenapses expressing synptotagmin-pHluorin we observed robust exocytosis and subsequent compensatory endocytosis upon stimulation. Xenapses, cryo-fixed by high-pressure freezing, displayed well-preserved ultrastructure, with large numbers of synaptic vesicles at or near the pre-synaptic membrane. Our results show that structure and function of xenapses on TEM grids do not differ from their counterparts on glass coverslips.Thus, xenapses grown on Quantifoil-supported TEM grids enable cryo-electron tomography of endocytic structures at different time points after stimulation.

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