Biophysical Society Thematic Meeting| Padova 2019

Quantitative Aspects of Membrane Fusion and Fission

Poster Abstracts

24-POS Board 24 DYNAMICS OF THE READILY RETRIEVABLE POOL OF SURFACE SYNAPTIC VESICLE PROTEINS ANALYSED BY FRAP Martin Kahms 1 ; Debora C Moschner 1 ; Juergen Klingauf 1 ; 1 University of Muenster, Institute of Medical Physics and Biophysics, Muenster, Nordrhein- Westfalen, Germany During synaptic transmission, synaptic vesicles (SVs) fuse with the plasma membrane at the active zone and are subsequently retrieved at the peri-active zone by compensatory endocytosis with high fidelity. However, the mechanisms of SV protein re-sorting and organization into patches of readily retrievable pool (RRetP) of SVs in the peri-active zone prior to endocytosis remain poorly understood. Here, we analyzed the dynamic organization of SV proteins of the RRetP and investigated their sorting determinants. We deleted and swapped domains of various SV proteins and demonstrated trans-membrane domains of these proteins to be dispensable for correct sorting into budding SVs. This suggests that retrieval efficacy is mainly determined by the cytosolic domains of SV proteins. Furthermore, we performed fluorescence recovery after photobleaching (FRAP) experiments at presynaptic boutons expressing pHluorin-tagged synaptic proteins. This enables the selective bleaching of the surface fraction of SV proteins and analysis of their mobility as well as their affinity towards the RRetP. We found that most SV proteins can readily diffuse within axonal and presynaptic membranes, but are enriched at the RRetP, albeit with rather low affinity. In contrast, we observed that two major large SV proteins, the vacuolar ATPase (v-ATPase) and the vesicular glutamate transporter (v-Glut), display limited mobility with their diffusion restricted to the pre-synaptic compartment, and are mostly part of the RRetP. Thus, we hypothesize that these molecules serve as central hubs, which recruit other endocytic synaptic cargo molecules at the peri-active zone and orchestrate the molecular composition of the readily retrievable pool.

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