Synaptotagmin 1 oligomerization via the juxtamembrane linker regulates spontaneous and evoked neurotransmitter release

• Published in: Proceedings of the National Academy of Sciences - PNAS 2021-11, Vol.118 (48), p.1-12
• Main Authors: Courtney, Kevin C., Vevea, Jason D., Li, Yueqi, Wu, Zhenyong, Zhang, Zhao, Chapman, Edwin R.
• Format: Article
• Language: English
• Subjects: Animals; Atomic force microscopy; Biological Sciences; Calcium - metabolism; Calcium ions; Cytoplasm - metabolism; Electron microscopy; Electrophysiology; Exocytosis; In Vitro Techniques; Light; Light scattering; Lipid bilayers; Lipid Bilayers - chemistry; Lipids; Lipids - chemistry; Lysine; Lysine - chemistry; Membrane Fusion; Microscopy; Microscopy, Atomic Force; Neurons - metabolism; Neurotransmitter Agents - metabolism; Neurotransmitter release; Neurotransmitters; Neutralization; Oligomerization; Phospholipids; Phospholipids - chemistry; Photon correlation spectroscopy; Presynaptic Terminals - metabolism; Protein Domains; Protein Multimerization; Recombinant Proteins - metabolism; Residues; Scattering, Radiation; Self-association; Synaptic Vesicles - metabolism; Synaptotagmin; Synaptotagmin I - metabolism; Synchronism
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.2113859118

Synaptotagmin 1 (syt1) is a Ca2+ sensor that regulates synaptic vesicle exocytosis. Cell-based experiments suggest that syt1 functions as a multimer; however, biochemical and electron microscopy studies have yielded contradictory findings regarding putative self-association. Here, we performed dynamic light scattering on syt1 in solution, followed by electron microscopy, and we used atomic force microscopy to study syt1 self-association on supported lipid bilayers under aqueous conditions. Ring-like multimers were clearly observed. Multimerization was enhanced by Ca2+ and required anionic phospholipids. Large ring-like structures (∼180 nm) were reduced to smaller rings (∼30 nm) upon neutralization of a cluster of juxtamembrane lysine residues; further substitution of residues in the second C2-domain completely abolished self-association. When expressed in neurons, syt1 mutants with graded reductions in self-association activity exhibited concomitant reductions in 1) clamping spontaneous release and 2) triggering and synchronizing evoked release. Thus, the juxtamembrane linker of syt1 plays a crucial role in exocytosis by mediating multimerization.