We have sought to investigate the responses of Nerve Growth Factor (NGF) receptors TrkA and P75NTR at the plasma membrane of living neuronal cells by single-molecule imaging and tracking. To this purpose we exploit the acyl carrier peptide and some of its shortened versions (A1 and S6 tags, labeled selectively by two different PPTases) to tag human p75NTR and TrkA. These tags were covalently conjugated to the biotin- or fluorophore-substituted arm of a coenzyme A (CoA) substrate. This approach allows: (i) a precise control of stoichiometry and site of biotin conjugation; (ii) versatility of the tags used; (iii) studying two interacting molecules with orthogonal fluorolabels, at the single-molecule or single-interaction-complex level. This experimental toolbox is completed by fast microscopy (e.g. TIRF microscopy with a fast EM-CCD), and by a semi-automatic algorithm for the analysis of the trajectories. This novel algorithm separates self-similar from multimodal trajectories, divides the last ones in subtrajectories, and calculates the combined distributions of parameters measuring the diffusivity, the localization or driftness degree, and/or the number of molecules in tracked spots. We shall present results on the early response of TrkA upon binding different biologically-relevant ligands (including NGF and proNGF): without ligands, TrkA is present mostly as fast-diffusing monomers; ligand binding results in an increasing number of dimers and oligomers, which are typically slower and/or more confined. Each ligand promotes distinct trajectory patterns at the cell membrane, because of different receptor-binding affinities, intracellular effectors recruited and formation of signalling/recycling endosome precursors. We believe that this imaging toolbox and our results pave the way to the quantitative description of the kinetics, dynamics and stoichiometry of any binary or ternary molecular complex formed upon binding of proNGF or NGF to their receptors.
Ligand Fingerprinting in the Membrane Dynamics of Single TrkA and P75NTR Neurotrophin Receptors
Luin Stefano;Marchetti Laura;Cattaneo Antonino
2015-01-01
Abstract
We have sought to investigate the responses of Nerve Growth Factor (NGF) receptors TrkA and P75NTR at the plasma membrane of living neuronal cells by single-molecule imaging and tracking. To this purpose we exploit the acyl carrier peptide and some of its shortened versions (A1 and S6 tags, labeled selectively by two different PPTases) to tag human p75NTR and TrkA. These tags were covalently conjugated to the biotin- or fluorophore-substituted arm of a coenzyme A (CoA) substrate. This approach allows: (i) a precise control of stoichiometry and site of biotin conjugation; (ii) versatility of the tags used; (iii) studying two interacting molecules with orthogonal fluorolabels, at the single-molecule or single-interaction-complex level. This experimental toolbox is completed by fast microscopy (e.g. TIRF microscopy with a fast EM-CCD), and by a semi-automatic algorithm for the analysis of the trajectories. This novel algorithm separates self-similar from multimodal trajectories, divides the last ones in subtrajectories, and calculates the combined distributions of parameters measuring the diffusivity, the localization or driftness degree, and/or the number of molecules in tracked spots. We shall present results on the early response of TrkA upon binding different biologically-relevant ligands (including NGF and proNGF): without ligands, TrkA is present mostly as fast-diffusing monomers; ligand binding results in an increasing number of dimers and oligomers, which are typically slower and/or more confined. Each ligand promotes distinct trajectory patterns at the cell membrane, because of different receptor-binding affinities, intracellular effectors recruited and formation of signalling/recycling endosome precursors. We believe that this imaging toolbox and our results pave the way to the quantitative description of the kinetics, dynamics and stoichiometry of any binary or ternary molecular complex formed upon binding of proNGF or NGF to their receptors.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
