Fluorolabeling of the PPTase-Related Chemical Tags: Comparative Study of Different Membrane Receptors and Different Fluorophores in the Labeling Reactions.

The set-up of an advanced imaging experiment requires a careful selection of suitable labeling strategies and fluorophores for the tagging of the molecules of interest. Here we provide an experimental workflow to allow evaluation of fluorolabeling performance of the chemical tags target of phosphopantetheinyl transferase enzymes (PPTases), once inserted in the sequence of different proteins of interest. First, S6 peptide tag was fused to three different single-pass transmembrane proteins (the tyrosine receptor kinases TrkA and VEGFR2 and the tumor necrosis factor receptor p75NTR), providing evidence that all of them can be conveniently albeit differently labeled. Moreover, we chose the S6-tagged TrkA construct to test eight different organic fluorophores for the PPTase labeling of membrane receptors in living cells. We systematically compared their non-specific internalization when added to a S6-tag negative cell culture, the percentage of S6-TrkA expressing cells effectively labeled and the relative mean fluorescence intensity, their photostability upon conjugation, and ratio of specific (cellular) versus background (glass-adhered) signal. This allowed to identify which fluorophores are actually recommended for these labeling reactions. Finally, we compared the PPTase labeling of a purified, YBBR-tagged Nerve Growth Factor with two differently charged organic dyes. We detected some batch-to-batch variability in the labeling yield, regardless of the fluorophore used. However, upon purification of the fluorescent species and incubation with living primary DRG neurons, no significant difference could be appreciated in both internalization and axonal transport of the labeled neurotrophins.

Fast-diffusing p75NTR monomers support apoptosis and growth cone collapse by neurotrophin ligands.

The p75 neurotrophin (NT) receptor (p75NTR) plays a crucial role in balancing survival-versus-death decisions in the nervous system. Yet, despite 2 decades of structural and biochemical studies, a comprehensive, accepted model for p75NTR activation by NT ligands is still missing. Here, we present a single-molecule study of membrane p75NTR in living cells, demonstrating that the vast majority of receptors are monomers before and after NT activation. Interestingly, the stoichiometry and diffusion properties of the wild-type (wt) p75NTR are almost identical to those of a receptor mutant lacking residues previously believed to induce oligomerization. The wt p75NTR and mutated (mut) p75NTR differ in their partitioning in cholesterol-rich membrane regions upon nerve growth factor (NGF) stimulation: We argue that this is the origin of the ability of wt p75NTR , but not of mut p75NTR, to mediate immature NT (proNT)-induced apoptosis. Both p75NTR forms support proNT-induced growth cone retraction: We show that receptor surface accumulation is the driving force for cone collapse. Overall, our data unveil the multifaceted activity of the p75NTR monomer and let us provide a coherent interpretative frame of existing conflicting data in the literature.

Site-Specific Direct Labeling of Neurotrophins and Their Receptors: From Biochemistry to Advanced Imaging Applications.

We describe here a versatile methodological platform to achieve site-directed and stoichiometry-controlled labeling of neurotrophins and their receptors with various probes, ranging from biotin to small organic dyes. This labeling method works in vitro on purified neurotrophins as well as in a living cell context, where it achieves selective labeling of surface-exposed neurotrophin receptors. Here, we list all experimental details of our labeling protocols, along with examples of the wide range of applications in which these can be used.

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.

Site-specific labeling of neurotrophins and their receptors via short and versatile peptide tags.

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.