Fluorescence Anisotropy for Monitoring cis- and trans-Membrane Interactions of Synaptotagmin-1

Vesicle fusion induces neurotransmitter release, orchestrated by synaptotagmin-1 (Syt-1) as a Ca²⁺ sensor. However, the precise molecular mechanisms of Syt-1 remain controversial, with various and competing models proposed based on different ionic strengths. Syt-1, residing on the vesicle membrane alongside anionic phospholipids such as phosphatidylserine (PS), undergoes Ca²⁺-induced binding to its own vesicle membrane, known as the cis-interaction, which prevents the trans-interaction of Syt-1 with the plasma membrane. Fluorescence anisotropy offers a methodological advantage for studying protein-membrane interactions. This protocol outlines a method utilizing fluorescence anisotropy to monitor the cis- and trans-membrane interactions of Syt-1, employing both purified native vesicles and plasma membrane-mimicking liposomes (PM-liposomes).