Biophysical Society Thematic Meeting| Padova 2019

Quantitative Aspects of Membrane Fusion and Fission

Thursday Speaker Abstracts

VISUALIZING COMPENSATORY ENDOCYTOSIS DYNAMICS IN ‘XENAPSES’, TIRFM-AMENABLE SYNAPSES Jurgen Klingauf 1 ; 1 University of Münster, Institute of Medical Physics and Biophysics, Münster, Nordrhein- Westfalen, Germany Maintaining synaptic transmission requires resorting and retrieval of the fused vesicle components by compensatory endocytosis. We showed in previous studies the existence of a pre- sorted and pre-assembled readily retrievable pool (RRetP) of synaptic vesicle proteins, and identified one of them, synaptophysin 1, to be a main organizer of the RRetP by forming hetero- oligomers with the v-SNARE synaptobrevin. However, are self-assembly forces of SV components sufficient for re-clustering or do endocytic adaptor proteins and clathrin contribute? To address this, we recently developed “Xenapses”, synapses formed by cultured mouse hippocampal neurons on micropatterned host substrates functionalized with synaptogenic proteins. Xenapses show all the hallmarks of a typical synapse both structurally (SV size and cluster, distinct exocytic and endocytic zones) and physiologically (calcium exocytosis coupling, exo-endocytic coupling). The expression of XFP fusion constructs of endocytic proteins allows investigating quantitative aspects of clathrin-mediated endocytosis at room and physiological temperatures. We found that clathrin light chain (EGFP-CLC) stably decorated the RRetP patches on the membrane surface in a punctate form. A fraction of these puncta disappeared seconds after the termination of stimulation pulse followed by slower full recovery of the RRetP. The initial decay correlates with endocytosis of RRetP patches. The slower time constant for recovery suggests RRetP formation to be the rate limiting step in endocytosis. Post-hoc unroofing of these xenapses and subsequent scanning electron microscopy of platinum replicas of the inner membrane surfaces revealed EGFP-CLC to localize to the clathrin-coated structures. Together, these observations constitute the first direct observation of the RRetP dynamics in space and time. In conclusion, clathrin and its adaptors are part of the RRetP co-polymer and provide spatio-temporal control for its formation which is necessary for efficient resorting and retrieval of vesicle components.

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