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.

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.

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.

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.

Pituitary adenylate cyclase activating polypeptide (PACAP) is a peptide transmitter released from the sympathetic splanchnic nerve to stimulate neurosecretory chromaffin cells of the adrenal medulla. Previous studies have shown that PACAP is preferentially released under heightened splanchnic firing of the sympathetic stress response. PACAP-dependent stimulation results in an immediate and robust Ca 2þ -dependent catecholamine secretion from chromaffin cells. Yet, PACAP stimulation does not evoke action potential firing in the chromaffin cell. Rather PACAP treatment elicits a sub-threshold membrane depolarization to approximately À50 mV. However, chromaffin cells voltage-clamped at the ''PACAP potential'' (À55mV) do not exhibit the same robust secretion as PACAP-treated cells, suggesting that PACAP may effect voltage-gated calcium entry through more than a simple sub-threshold depolarization. We used perforated patch electrophysiological recordings conducted in adrenal tissue slices to investigate the mechanism by which PACAP evokes rapid and robust Ca 2þ influx and catecholamine secretion. We provide evidence that PACAP excitation includes a facilitation of Ca 2þ influx through low-voltage activated (LVA) Ca 2þ channels. Pharmacological isolation and molecular classification indicate that the target channel is likely CaV3.2. Thus, PACAPevoked adrenal secretion is likely through a parallel membrane depolarization and functional facilitation of LVA calcium channels.