Project A8 – Quantitative mapping of synaptic molecules within single neurons using correlated super-resolution and electron microscopy

Working on this project:

Mike Heilemann & Erin Schuman & Teams

Synaptic activity is coupled to protein copy numbers, which vary in individual synapses and are dependent on and regulated by the presence of mRNA and miRNA. We developed nano-scale imaging of proteins with molecular resolution in neurons and novel labels to target mRNA with super-resolution microscopy. We will now establish a microscopy platform that will allow a comprehensive analysis of the protein and mRNA “localizome”, by visualizing multiple proteins and mRNA together with miRNA in the same cell using single-molecule super-resolution imaging with excellent multiplexing capabilities. We will localize and quantify proteins and mRNA in synapses, we will study their local regulation by miRNA, and we will develop single-molecule imaging of newly transcribed proteins. In the second project, we will develop a nano-analytical platform based on DNA orgimai to study the binding mechanism of RNA-binding proteins to their target RNA sequence with single-molecule resolution. This novel platform will be compatible with endogenous, unmodified proteins, and provide information on binding equilibria and kinetics, sequence specificity, interaction with co-factors and the influence of mutations. The platform is modular, and can integrate many different mRNA targets at the same time. It will further be used to study the kinetics of the Adenine binding riboswitch.