Enzymostaining : re-engineering enzymes to super-resolve the molecular machinery of exocytosis

Abstract

Synaptic vesicle exocytosis, which is a fundamental process to life, leads to neurotransmitter release at nerve terminals. Dysfunctional exocytosis has been associated with various conditions, including schizophrenia and Obsessive-Compulsive Disorder (OCD). SNARE proteins are the machinery that drives exocytosis, which makes them particularly good targets for studying what happens at the cell level using various standard, diffraction-limited and super-resolution microscopy techniques. While classical antibody-based approaches have been used to fluorescently label SNAREs for standard confocal microscopy, the inherent properties of antibodies impose limits on super-resolution microscopy techniques required for studying the SNAREs. Meanwhile, Botulinum Neurotoxins (BoNTs), also known as Botox, are among some of the most lethal substances known to man. Their toxicity is due to their natural ability to bind and specifically cleave SNARE proteins. This causes Botulism, a condition characterised by impairment of neurotransmission and paralysis. Since BoNTs naturally bind and specifically target neuronal SNAREs, my work has focused on re-engineering BoNTs in a way that these may still bind while not cleaving their target SNAREs. I propose that botulinum neurotoxins, and re-engineered enzymes in general, may provide, upon optimisation, a good platform for the next-generation of detection agents.