Ligand-induced dynamics of neurotrophin receptors investigated by single-molecule imaging approaches

Neurotrophins are secreted proteins that regulate neuronal development and survival, as well as maintenance and plasticity of the adult nervous system. The biological activity of neurotrophins stems from their binding to two membrane receptor types, the tropomyosin receptor kinase and the p75 neurotrophin receptors (NRs). The intracellular signalling cascades thereby activated have been extensively investigated. Nevertheless, a comprehensive description of the ligand-induced nanoscale details of NRs dynamics and interactions spanning from the initial lateral movements triggered at the plasma membrane to the internalization and transport processes is still missing. Recent advances in high spatio-temporal resolution imaging techniques have yielded new insight on the dynamics of NRs upon ligand binding. Here we discuss requirements, potential and practical implementation of these novel approaches for the study of neurotrophin trafficking and signalling, in the framework of current knowledge available also for other ligand-receptor systems. We shall especially highlight the correlation between the receptor dynamics activated by different neurotrophins and the respective signalling outcome, as recently revealed by single-molecule tracking of NRs in living neuronal cells

Precursor and mature NGF live tracking: one versus many at a time in the axons

The classical view of nerve growth factor (NGF) action in the nervous system is linked to its retrograde axonal transport. However, almost nothing is known on the trafficking properties of its unprocessed precursor proNGF, characterized by different and generally opposite biological functions with respect to its mature counterpart. Here we developed a strategy to fluorolabel both purified precursor and mature neurotrophins (NTs) with a controlled stoichiometry and insertion site. Using a single particle tracking approach, we characterized the axonal transport of proNGF versus mature NGF in living dorsal root ganglion neurons grown in compartmentalized microfluidic devices. We demonstrate that proNGF is retrogradely transported as NGF, but with a lower flux and a different distribution of numbers of neurotrophins per vesicle. Moreover, exploiting a dual-color labelling technique, we analysed the transport of both NT forms when simultaneously administered to the axon tips

Precursor and mature NGF live tracking: one versus many at a time in the axons

The classical view of nerve growth factor (NGF) action in the nervous system is linked to its retrograde axonal transport. However, almost nothing is known on the trafficking properties of its unprocessed precursor proNGF, characterized by different and generally opposite biological functions with respect to its mature counterpart. Here we developed a strategy to fluorolabel both purified precursor and mature neurotrophins (NTs) with a controlled stoichiometry and insertion site. Using a single particle tracking approach, we characterized the axonal transport of proNGF versus mature NGF in living dorsal root ganglion neurons grown in compartmentalized microfluidic devices. We demonstrate that proNGF is retrogradely transported as NGF, but with a lower flux and a different distribution of numbers of neurotrophins per vesicle. Moreover, exploiting a dual-color labelling technique, we analysed the transport of both NT forms when simultaneously administered to the axon tips

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

Site-Specific Labeling of Neurotrophins and Their Receptors via Short and Versatile Peptide Tags

<div><p>We present a toolbox for the study of molecular interactions occurring between NGF and its receptors. By means of a suitable insertional mutagenesis method we show the insertion of an 8 amino acid tag (A4) into the sequence of NGF and of 12 amino acid tags (A1 and S6) into the sequence of TrkA and P75NTR NGF-receptors. These tags are shortened versions of the acyl and peptidyl carrier proteins; they are here covalently conjugated to the biotin-substituted arm of a coenzyme A (coA) substrate by phosphopantetheinyl transferase enzymes (PPTases). We demonstrate site-specific biotinylation of the purified recombinant tagged neurotrophin, in both the immature proNGF and mature NGF forms. The resulting tagged NGF is fully functional: it can signal and promote PC12 cells differentiation similarly to recombinant wild-type NGF. Furthermore, we show that the insertion of A1 and S6 tags into human TrkA and P75NTR sequences leads to the site-specific biotinylation of these receptors at the cell surface of living cells. Crucially, the two tags are labeled selectively by two different PPTases: this is exploited to reach orthogonal fluorolabeling of the two receptors co-expressed at low density in living cells. We describe the protocols to obtain the enzymatic, site-specific biotinylation of neurotrophins and their receptors as an alternative to their chemical, nonspecific biotinylation. The present strategy has three main advantages: i) it yields precise control of stoichiometry and site of biotin conjugation; ii) the tags used can be functionalized with virtually any small probe that can be carried by coA substrates, besides (and in addition to) biotin; iii) above all it makes possible to image and track interacting molecules at the single-molecule level in living systems.</p></div

Corrigendum: Precursor and mature NGF live tracking: one versus many at a time in the axons

Scientific Reports 6: Article number: 20272; published online: 01 February 2016; updated: 30 March 2016 This Article contains errors in Affiliation 3. The correct affiliation is listed below: Center for Nanotechnology Innovation @NEST, Istituto Italiano di Tecnologia, Piazza San Silvestro 12-56127 Pisa, Italy.</jats:p

Site-specific biotinylation of TrkA and P75NTR receptors.

<p>Western blot for the analysis of the biotinylation reaction in living cells of A1/S6-TrkA-EGFP (<b>A</b>) and A1/S6-P75NTR-EGFP constructs (<b>B</b>) using CoA-biotin substrate and AcpS or SfpS PPTases. The same biotinylation reaction is performed in parallel using untagged TrkA-EGFP (<b>A</b>) and P75NTR-EGFP (<b>B</b>) as negative controls, and ACP-TrkA (<b>A</b>) as positive control. Streptavidin-HRP is used for detection of biotin. Anti-TrkA (<b>A</b>) and anti-P75NTR (<b>B</b>) blots are loading controls together with anti-GFP (both panels). The anti-TrkA blot contains an unspecific band running over TrkA, as already shown <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0113708#pone.0113708-Callegari1" target="_blank">[4]</a>; the actual TrkA band in each lane is highlighted by a star. At the bottom of each panel the densitometric analysis of the blot bands is reported. The biotin signal was normalized to the content of GFP (for TrkA-EGFP, A1-TrkA-EGFP, S6-TrkA-EGFP lanes), TrkA (for ACP-TrkA lanes), and P75NTR (for P75NTR-EGFP, A1-P75NTR-EGFP, S6-P75NTR-EGFP lanes), with the higher value normalized to 1. Results reported are mean±sem from 3 (panel A) and 2 (panel B) independent blots.</p

Schematic overview of the insertional mutagenesis method.

<p><b>A</b>) Cartoon depicting NGF, TrkA, and P75NTR constructs prepared for this study. The site of tag insertion is indicated in each case by a red arrow. On top of the arrow the complete amino acidic tag sequence is reported. The promoters used for expression of the receptors are depicted upstream each receptor construct. On the extracellular domain of the receptors, sites of interaction with NGF are highlighted; on the intracellular domain of the receptors, sites of receptor activity are highlighted. Abbreviations: CMV = Cytomegalovirus promoter; TRE = Tet-Responsive-Element promoter; SP = signal peptide; EC = extracellular domain; IC = intracellular domain; D5 = proximal immunoglobulin-like domain; I = NGF-interaction site 1; II = NGF-interaction site 2; DD = death domain. <b>B</b>) Crystal structure of NGF (left; PBD. n.1BET), NGF-TrkA (EC) (middle; PBD. n.2IFG), NGF-P75NTR(EC) (right; PBD. n.1SG1). The color code is the same as in panel A. Red spots highlight the sites of tag insertion in each protein. <b>C</b>) Scheme of the insertional mutagenesis procedure used in this study. Details are provided in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0113708#pone.0113708.s006" target="_blank">Text S1</a>.</p