Observing single dynamin-mediated fission events in TIRFM-amenable synapses.

Abstract

The presynapse features a unique tight coupling of exo- and endocytosis resulting in the formation of synaptic vesicles with uniform morphology. The difficulty in accessing the presynapse has hampered attempts to directly visualize the functionality of presynaptic proteins which enable such exquisite pairing. To this end our lab has 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 synapse and allow us to directly observe the behavior of proteins involved in compensatory endocytosis. Xenapses lend themselves to unroofing, revealing via SEM the membrane surface to be decorated with preassembled clathrin structures, concomitant with a pre-sorted and pre-assembled readily retrievable pool (RRetP) of endocytic structures. We have further combined this preparation with CRISPR-Cas9 to endogenously label dynamin I. This has enabled the study of triggered compensatory endocytosis at an unprecedented spatial and temporal resolution. Dynamin-dependent fission occurred as discreet, quantal events, demonstrating the rate of endocytosis to be determined by the number of vesicles retrieved within a unit of time rather than by an intrinsic value. While dynamin was recruited within subsecond timescale following a single action potential, the subsequent assembly and fission, denoted by gradual increase in fluorescence followed by rapid collapse, occurred over many seconds with a peak at 5 seconds at physiological temperature. This casts a shadow over the dynamin-dependent ultrafast endocytic mode proposed to predominate under such conditions. These observations advance dynamin fission as the rate-limiting step in endocytosis, with the preassembled RRetP providing an element of speed advantage for the rapid, high fidelity retrieval of synaptic vesicles. The full potential of our system will be realized by investigating the choreography of the key endocytic proteins relative to dynamin fission.