The role of PI(4,5)P₂ and synaptotagmin in membrane fusion - an in vitro study

Abstract

Synaptotagmin-1 (syt-1) is known to trigger fusion of neuronal synaptic vesicles with the presynaptic membrane by recognizing acidic membrane lipids. In particular, binding to PI(4,5)P2 is believed to be crucial for its function as a calcium sensor. We propose a mechanism for syt-1 to interact with anionic bilayers and promote fusion in the presence of SNARE proteins. We found that in the absence of Ca2+ the binding of syt-1 to membranes depends on the PI(4,5)P2 content. Addition of Ca2+ switches the interaction forces from weak to strong eventually exceeding the cohesion of the C2A domain, while the interaction between PI(4,5)P2 and the C2B domain was preserved even in the absence of Ca2+ or phosphatidylserine. Fusion of large unilamellar vesicles equipped with syt-1 and synaptobrevin with freestanding target membranes composed of PS/PI(4,5)P2 show an increased fusion speed, and by effective suppression of stalled intermediate states, a larger number of full fusion events. Fusion efficiency could be maximized when irreversible docking is additionally prevented by addition of multivalent anions. The picture that emerges is that syt-1 remodels the membrane in the presence of calcium and PIP2, thereby substantially increasing the efficiency of membrane fusion by avoiding stalled intermediate states.

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“…However, E-Syt senses a decrease in intracellular or endoplasmic reticulum calcium ion concentration in MCSs, which in turn anchors with PI (Porter and Palade, 1957;Okeke et al, 2016) P2. (Cortés et al, 2019;Dietz et al, 2022). The local concentration of PI (Porter and Palade, 1957;Okeke et al, 2016) P2 increases, and after decomposition, more IP3 is generated (Ehrlich et al, 2011;Chang and Liou, 2016).…”