Biophysical Society Thematic Meeting| Padova 2019

Quantitative Aspects of Membrane Fusion and Fission

Poster Abstracts

28-POS Board 28 UNRAVELLING THE MOLECULAR DYNAMICS OF ENDOCYTIC ACTORS USING “XENAPSES”, TIRFM-AMENABLE SYNAPSES Sai Krishnan 1 ; Julia Lehrich 1 ; Junxiu Duan 1 ; Natalya Glyvuk 1 ; Yaroslav Tsytsyura 1 ; Ulrike Keller 1 ; Jürgen Klingauf 1 ; 1 Westfälische Wilhelms-Universität, Institute of Medical Physics and Biophysics, Münster, Nordrhein-Westfalen, Germany In the presynapse, endocytosis takes on a specialized role due to the need for synaptic vesicles to be compensated in response to exocytosis. The fission process requires the choreography of a number of endocytic proteins which ensures formation of synaptic vesicles with high fidelity. However, despite decades of research on presynaptic vesicle recycling the precise function of the individual endocytic factors and their molecular dynamics remains hotly debated. To address this we developed “Xenapses”, TIRFM-able synapses, formed by mouse hippocampal neurons cultured on micropatterned host substrates coated with synaptogenic proteins. Xenapses show all the characteristics of a typical synapse and allow us to directly observe the behavior of proteins involved in compensatory endocytosis Our initial investigations followed up on our previous findings on the existence of a pre-sorted and pre-assembled readily retrievable pool (RRetP). In live xenapses, expressed EGFP-clathrin light chain (EGFP-CLC) disappeared seconds after the termination of stimulation pulse followed by slower full recovery. This response profile likely indicates the endocytosis of surface clathrin followed by the nucleation of clathrin RRetP for subsequent round of endocytosis. Xenapses also lend themselves to unroofing, revealing via SEM the EGFP-CLC to stably decorate the clathrin structures on the membrane surface. Together, these findings strengthen the evidence for the RRetP co-polymer and for clathrin to be part of it. Extending this to other endocytic proteins revealed the fission protein dynamin 1-EGFP and the BAR-domain amphiphysin 1-EGFP to be recruited to the pre-synaptic membrane in a stimulus- dependent manner, in contrast to clathrin. EGFP-SNX9 on the other hand exhibits the same response profile as clathrin, suggesting it is part of the RRetP in the presynapse. Future work will focus on delineating with high spatial and temporal resolution the interaction between these and other endocytic proteins.

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