Super-resolving botulinum neurotoxin type A molecules: from surface landing to internalization in synaptic vesicles

Botulinum neurotoxin type-A (BoNT/A) is internalized into motor nerve terminals as part of its intoxication strategy to incapacitate nerve-muscle communication. The “dual receptor” model explained how BoNT/A initially interacts with GT1b gangliosides, thereby concentrating the toxin on the presynaptic membrane to foster the subsequent interaction with a proteinaceous co-receptor SV2 which triggers receptor-mediated endocytosis. I will revisit the “dual receptor” concept using two single-molecule imaging strategies,1-3 allowing tracking of single Atto647N-labeled BoNT/A molecules upon (i) landing on the plasma membrane (by uPAINT) and (ii) internalization in synaptic vesicles (by sdTIM) of living mature hippocampal neurons. With 30 to 40 nm localization precision, we revealed that once internalized in synaptic vesicles, Atto647N-tagged BoNT/A exhibits a markedly lower mobility than on the plasma membrane. I will discuss how individual genetic inactivation of the GT1b and SV2 binding sites of the neurotoxin affects the diffusion states of BoNT/A mutants on the plasma membrane and axonal trafficking. Single neurotoxin super-resolution imaging uncovers an updated dual receptor model taking into consideration the diffusive patterns generated by each of the co-receptors and leading to defined nanoscale dynamic organizations at key steps of the intoxication journey.