Super-resolution microscopy at the site of neuronal communication

QBI houses the Advanced Microimaging and Analysis Facility, which contains state-of-the art microscopes. Our lab has been instrumental in the inclusion of several super-resolution microscopes, through successive ARC LIEF grants. This has allowed us to probe the nanoscopic environment of neurons and neurosecretory cells undergoing communication. Our most recent publications have used super-resolution microscopy extensively.  

Deciphering the intra- and intermolecular steps via which prepare secretory vesicles for fusion is key to understanding neuronal and hormonal communication. We demonstrated that Munc18-1 and syntaxin-1A and are organised in nanodomains on the plasma membrane of neurons and neurosecretory cells that control SNARE-dependent neuroexocytosis through lateral trapping in these nanoclusters. In Bademosi et al. 2017 (Nature Communications), we combined super-resolution with opto- and thermogenetic neuronal stimulation in living neurons in the fruit fly.  We were able to characterise the changes in mobility of the tSNARE syntaxin1a and how they regulate neurotransmitter release. In Kasula et al. 2016 (Journal of Cell Biology) we found that the Munc18-1 domain 3a hinge-loop controls syntaxin-1A engagement into the SNARE complex during priming. 

We also developed a new technique sdTIM, Subdiffractional tracking of internalised molecules, to be able to visualise small synaptic vesicles in living hippocampal nerve terminals (Joensuu et al. 2016 Journal of Cell Biology).  This super-resolution technique was able to capture, and subsequently analyse, the dynamics of thousands of individual synaptic vesicle trajectories, to uncover the dynamics of synaptic vesicle pool mobility.  It revealed diffusive and transport states of synaptic vesicles in resting and stimulated conditions. Our aim is to apply sdTIM to study the mechanism of neuronal communication under disease conditions in future studies.  

Munc18 and synucleopathies

Because of the importance of Munc18-1 in vesicle fusion, human mutations are linked to epilepsy and other neurological symptoms. Recent genetic studies in humans have uncovered a variety of mutations in Munc18 that lead to EIEE (early infantile epileptic encephalopathy), a severe and often fatal developmental condition with no treatment, and little knowledge of the underlying cause. We showed that the human disease- linked mutation C180Y, which is unstable at body temperature, leads to the formation of intracellular aggregates (Martin et al. 2014 Cell Reports). In a follow up study, we revealed that both C180Y and other human EIEE-linked missense mutations controlled the aggregation of α-synuclein, in a similar fashion to Parkinson's disease pathology (Chai et al. 2016 Journal of Cell Biology). This provides the first evidence for a communal mode of action between an epileptic syndrome and neurodegenerative synucleopathies. This is an ongoing field of research in our laboratory.