Ligand-Induced Dynamics of Neurotrophin Receptors Investigated by Single-Molecule Imaging Approaches

Abstract

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.

Lysosome Dynamic Properties during Neuronal Stem Cell Differentiation Studied by Spatiotemporal Fluctuation Spectroscopy and Organelle Tracking

“…When the cytoskeleton reached a highly organized structure (DIV14, Figure S1G-I), the D m dropped to lower values, indicating a confinement exerted by the bundles of actin filaments on the organelles. A similar role of actin has been reported for the regulation of membrane mobility of selected membrane receptors [15]. The standard error (SE) associated with both the size and the local diffusivity becomes sensibly smaller during differentiation, probably reflecting an ongoing process by which the highly heterogeneous (both in size and dynamics) population of lysosomes at DIV0 progressively becomes more homogeneous and acquires its final structural/dynamic identity in the fully differentiated cell.…”

“…The former is a method based on the spatiotemporal correlation analysis of fluorescence fluctuation; it allows fast and robust extraction of the average dynamic features of moving objects (from molecules to entire organelles) directly from stacks of images, with no need of calculating the single trajectories [12][13][14]. The latter, through localization, makes it possible to retrieve information on single molecules or vesicles [10,[15][16][17], which are averaged out by the iMSD method. The mean square displacement (MSD) calculated in both methods (averaged in a movie, in the former, and for each trajectory, in the latter) can be analyzed for extracting quantitative dynamics parameters and possibly their distribution and changes with time [18].…”

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

“…Ideally, a quantitative comparison between the axonal transport of NGF and proNGF requires the two molecules to be fluoro-labeled at the same molecular site and with the same stoichiometry. So far, the mature forms of NTs have been chemically coupled to organic fluorophores 12 13 14 15 16 or to biotin 17 18 19 20 21 . While this has allowed obtaining valuable information about NGF trafficking, it has not been possible to control the exact number and site of conjugated probes, so that mixed and not fully reproducible labeled protein populations are obtained.…”

An Optimized Procedure for the Site-Directed Labeling of NGF and proNGF for Imaging Purposes

“…The lack of a standardized protocol for the production and purification of fluorolabeled neurotrophins has led to the development of a multitude of different strategies for this purpose (Marchetti et al, 2015 ; De Nadai et al, 2016 ). The chemical conjugation of organic dyes to NTs amino- or carboxy- residues (Rosenberg et al, 1986 ) is maybe the most straightforward of these approaches, but suffers of poor reproducibility.…”