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

  • Francesco Gobbo
  • Fulvio Bonsignore
  • Rosy Amodeo
  • Antonino Cattaneo
  • Laura Marchetti
Part of the Methods in Molecular Biology book series (MIMB, volume 1727)


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.

Key words

Neurotrophin Nerve growth factor TrkA tyrosine kinase Membrane receptor pool Fluorescence labeling Biotinylation Axonal transport Inducible lentivirus Neuron culture Astrocyte culture 


  1. 1.
    Marchetti L, Luin S, Bonsignore F, de Nadai T, Beltram F, Cattaneo A (2015) Ligand-induced dynamics of neurotrophin receptors investigated by single-molecule imaging approaches. Int J Mol Sci 16:1949–1979CrossRefPubMedPubMedCentralGoogle Scholar
  2. 2.
    De Nadai T, Marchetti L, Di Rienzo C, Calvello M, Signore G, Di Matteo P, Gobbo F, Turturro S, Meucci S, Viegi A, Beltram F, Luin S, Cattaneo A (2016) Precursor and mature NGF live tracking: one versus many at a time in the axons. Sci Rep 6:20272. CrossRefPubMedPubMedCentralGoogle Scholar
  3. 3.
    Di Matteo P, Calvello M, Luin S, Marchetti L, Cattaneo A (2017) An optimized procedure for the site-directed labeling of NGF and proNGF for imaging purposes. Front Mol Biosci 4:4. CrossRefPubMedPubMedCentralGoogle Scholar
  4. 4.
    Jullien J, Guili V, Derrington EA, Darlix JL, Reichardt LF, Rudkin BB (2003) Trafficking of TrkA-green fluorescent protein chimerae during nerve growth factor-induced differentiation. J Biol Chem 278:8706–8716CrossRefPubMedGoogle Scholar
  5. 5.
    Tanaka K, Kelly CE, Goh KY, Lim KB, Ibanez CF (2016) Death domain signaling by disulfide-linked dimers of the p75 neurotrophin receptor mediates neuronal death in the CNS. J Neurosci 36:5587–5595CrossRefPubMedGoogle Scholar
  6. 6.
    Lehigh KM, West KM, Ginty DD (2017) Retrogradely transported TrkA endosomes signal locally within dendrites to maintain sympathetic neuron synapses. Cell Rep 19:86–100CrossRefPubMedPubMedCentralGoogle Scholar
  7. 7.
    Marchetti L, De Nadai T, Bonsignore F, Calvello M, Signore G, Viegi A, Beltram F, Luin S, Cattaneo A (2014) Site-specific labeling of neurotrophins and their receptors via short and versatile peptide tags. PLoS One 9:e113708. CrossRefPubMedPubMedCentralGoogle Scholar
  8. 8.
    Marchetti L, Callegari A, Luin S, Signore G, Viegi A, Beltram F, Cattaneo A (2013) Ligand signature in the membrane dynamics of single TrkA receptor molecules. J Cell Sci 126:4445–4456CrossRefPubMedGoogle Scholar
  9. 9.
    Callegari A, Luin S, Marchetti L, Duci A, Cattaneo A, Beltram F (2012) Single particle tracking of acyl carrier protein (ACP)-tagged TrkA receptors in PC12nnr5 cells. J Neurosci Methods 204:82–86CrossRefPubMedGoogle Scholar
  10. 10.
    Yin J, Lin AJ, Golan DE, Walsh CT (2006) Site-specific protein labeling by Sfp phosphopantetheinyl transferase. Nat Protoc 1:280–285CrossRefPubMedGoogle Scholar
  11. 11.
    Dull T, Zufferey R, Kelly M, Mandel RJ, Nguyen M, Trono D, Naldini L (1998) A third-generation lentivirus vector with a conditional packaging system. J Virol 72:8463–8471PubMedPubMedCentralGoogle Scholar
  12. 12.
    Gobbo F, Marchetti L, Jacob A, Pinto B, Binini N, Pecoraro Bisogni F, Alia C, Luin S, Caleo M, Fellin T, Cancedda L, Cattaneo A (2017) Activity-dependent expression of Channelrhodopsin at neuronal synapses. Nat Commun 8(1):1629. doi:  10.1038/s41467-017-01699-7
  13. 13.
    Capsoni S, Malerba F, Carucci NM, Rizzi C, Criscuolo C, Origlia N, Calvello M, Viegi A, Meli G, Cattaneo A (2017) The chemokine CXCL12 mediates the anti-amyloidogenic action of painless human nerve growth factor. Brain 140:201–217CrossRefPubMedGoogle Scholar
  14. 14.
    Zhou Z, Cironi P, Lin AJ, Xu Y, Hrvatin S, Golan DE, Silver PA, Walsh CT, Yin J (2007) Genetically encoded short peptide tags for orthogonal protein labeling by Sfp and AcpS phosphopantetheinyl transferases. ACS Chem Biol 2:337–246CrossRefPubMedGoogle Scholar
  15. 15.
    Yu T, Calvo L, Anta B, Lopez-Benito S, Southon E, Chao MV, Tessarollo L, Arevalo JC (2011) Regulation of trafficking of activated TrkA is critical for NGF-mediated functions. Traffic 12:521–534CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2018

Authors and Affiliations

  • Francesco Gobbo
    • 1
  • Fulvio Bonsignore
    • 2
  • Rosy Amodeo
    • 2
    • 3
  • Antonino Cattaneo
    • 1
  • Laura Marchetti
    • 1
    • 2
    • 3
  1. 1.Bio@SNS, Scuola Normale SuperiorePisaItaly
  2. 2.NEST, Scuola Normale SuperiorePisaItaly
  3. 3.Center for Nanotechnology Innovation @NEST, Istituto Italiano di TecnologiaPisaItaly

Personalised recommendations