Single Molecule Imaging and Tracking of Neurotrophins and their Receptors in Living Neuronal Cells

We currently lack a satisfactory understanding of the membrane complexes and internalization routes underpinning the pleiotropic biological outcomes of neurotrophins (NTs), which exert their functions via interlaced binding of three different families of neurotrophin receptors (NRs). We are working to answer several open questions in this field: are NRs membrane movements linked to ligand-specific activation processes? Are different NRs functions linked to different movements at the cell membrane? How does p75NTR enhance NGF-TrkA signalling? Are NGF and its precursor proNGF different signalling molecules as far as NRs binding and internalization is concerned? To address these issues, we developed non-invasive means to covalently fluorolabel with 1:1 stoichiometry both NTs and their receptors. This toolbox was exploited to perform single molecule imaging and tracking (SMIT) at the plasma membrane and inside axons of living neuronal cells using wide-field and TIRF microscopy. We report here results in two different directions. First, we analysed by SMIT the lateral mobility of wt TrkA in comparison to a dead-kinase TrkA and to three other mutants having i) kinase activity, ii) recruitment of intracellular effectors, iii) ubiquitination (and further degradation) separately impaired. Obtained data point to kinase activity as a master regulator of TrkA membrane dynamics and hint at possible mechanisms by which the cell handles the trafficking of kinase-inactive TrkA receptors. Second, we undertook a comparative study about the axonal transport displayed by \u201chomologue\u201d fluorescent proNGF and NGF in compartmented DRG neurons. We demonstrate that proNGF is internalized and retrogradely transported across axons like mature NGF, but the two NTs display remarkable differences both in terms of NTs flux and number of molecules carried per vesicle. Furthermore, we unveiled a competition mechanism favoring NGF transport upon coadministration of the two NTs