Presynaptic targeting of botulinum neurotoxin type A requires a tripartite PSG‐Syt1‐SV2 plasma membrane nanocluster for synaptic vesicle entry
Merja Joensuu, Parnayan Syed, Saber H. Saber, Vanessa Lanoue, Tristan P. Wallis, James Rae, Ailisa Blum, Rachel S. Gormal, Christopher Small, Shanley Sanders, Anmin Jiang, Stefan Mahrhold, Nadja Krez, Michael A. Cousin, Ruby Cooper‐White, Justin J. Cooper‐White, Brett M. Collins, Robert G. Parton, Giuseppe Balistreri, Andreas Rummel, Frédéric A. Meunier
Abstract
The unique nerve terminal targeting of botulinum neurotoxin type A (BoNT/A) is due to its capacity to bind two receptors on the neuronal plasma membrane: polysialoganglioside (PSG) and synaptic vesicle glycoprotein 2 (SV2). Whether and how PSGs and SV2 may coordinate other proteins for BoNT/A recruitment and internalization remains unknown. Here, we demonstrate that the targeted endocytosis of BoNT/A into synaptic vesicles (SVs) requires a tripartite surface nanocluster. Live-cell super-resolution imaging and electron microscopy of catalytically inactivated BoNT/A wildtype and receptor-binding-deficient mutants in cultured hippocampal neurons demonstrated that BoNT/A must bind coincidentally to a PSG and SV2 to target synaptic vesicles. We reveal that BoNT/A simultaneously interacts with a preassembled PSG-synaptotagmin-1 (Syt1) complex and SV2 on the neuronal plasma membrane, facilitating Syt1-SV2 nanoclustering that controls endocytic sorting of the toxin into synaptic vesicles. Syt1 CRISPRi knockdown suppressed BoNT/A- and BoNT/E-induced neurointoxication as quantified by SNAP-25 cleavage, suggesting that this tripartite nanocluster may be a unifying entry point for selected botulinum neurotoxins that hijack this for synaptic vesicle targeting.