Dissection of the initial steps and dynamics in synaptic vesicle biogenesis using custom-refined nano-affinity tools

Synaptic vesicles (SVs) are among the most complex cellular structures studied in neuroscience. They consist of several different components that must be precisely assembled to perform numerous important functions, not only in neurotransmission, but also in neurodevelopment and cellular trafficking. SV proteins are extensively studied, but while most research has focused on their functions at the synapse, the steps of their formation and assembly remain largely elusive. A limiting factor has been the lack of tools to dissect the behaviour of these intricate but complex organelles without perturbing their integrity. In this thesis, I present an intrabody targeting a key SV protein, synaptotagmin-1 (Syt1), called iNbSyt1, which is highly versatile and offers a wide range of applications, including label-free, minimally invasive live imaging and a highly sensitive synaptic Ca2+ sensor capable of detecting single action potentials. Furthermore, I established an iNbSyt1-driven strategy combining proteomics and automated long-term live imaging to dissect early stages of neuronal development and successfully identified histidine ammonia lyase, microtubule-associated protein 6 and dynein light chain LC8-type 2 as novel early interaction partners of Syt1, potentially involved in its early sorting steps within the neuronal soma.