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Methodology for Detecting and Tracking Brain-Derived Neurotrophic Factor Complexes in Neurons Using Single Quantum Dots

  • Anke Vermehren-Schmaedick
  • Thomas Jacob
  • Tania Q. VuEmail author
Protocol
Part of the Neuromethods book series (NM, volume 143)

Abstract

We describe a methodological workflow for detecting and tracking single brain-derived neurotrophic factor (BDNF) receptor complexes in primary cultures of neurons. This methodology includes the preparation of BDNF-conjugated quantum dot fluorescent probes for sensitive detection of single BDNF complexes in fixed neurons as well as single-particle tracking (SPT) in live neurons. These methods are valuable for high-resolution localization and quantitation of low-abundance proteins in single neurons and for obtaining nanoscale-resolution, dynamic information of BDNF trafficking for long time durations (min) in live neurons.

Keywords

BDNF Dynamics Growth factor Intracellular trafficking Quantum dot Single-particle tracking TrkB 

Notes

Acknowledgment

This work was supported by NIH-NINDS R01 NS071116 01 and W81XWH-07-2-0107 to T.Q.V.

Supplementary material

Supplementary Movie 1

This movie shows tracking of several quantum dot-labeled brain-derived neurotrophic factors (QD-BDNFs) bound to TrkB receptors moving along distal axons to the cell body. Most of the selected trajectories exhibit the active/pause transport dynamics that characterizes the primary motion observed along axons. Some trajectories are apparently stationary or confined and are shown for comparison to the active behavior. The first half of the movie shows dynamic tracking from a 2D perspective where colors distinguish different particle trajectories, and we also show a 3D flyover of a static rendering of the trajectories in x, y, and time dimensions. Of particular interest is the nature of the pauses in the active transport trajectories which are well resolved at our high spatiotemporal tracking resolutions. (MP4 14001 kb)

References

  1. 1.
    Bibel M, Barde Y (2000) Neurotrophins: key regulators of cell fate and cell shape in the vertebrate nervous system. Genes Dev 14:2919–2937CrossRefGoogle Scholar
  2. 2.
    Huang E, Reichardt L (2001) Neurotrophins: roles in neuronal development and function. Annu Rev Neurosci 24:677–736CrossRefGoogle Scholar
  3. 3.
    Binder D, Scharfman H (2004) Brain-derived neurotrophic factor. Growth Factors 22:123–131CrossRefGoogle Scholar
  4. 4.
    Mannion R, Costigan M, Decosterd I, Amaya F, Ma Q, Holstege J, Ji R, Acheson A, Lindsay R, Wilkinson G, Woolf CJ (1999) Neurotrophins: peripherally and centrally acting modulators of tactile stimulus-induced inflammatory pain hypersensitivity. Proc Natl Acad Sci U S A 3:9385–9390CrossRefGoogle Scholar
  5. 5.
    Clark C, Hasser E, Kunze D, Katz D, Kline D (2011) Endogenous brain-derived neurotrophic factor in the nucleus tractus solitarius tonically regulates synaptic and autonomic function. J Neurosci 24:12318–12329CrossRefGoogle Scholar
  6. 6.
    Cunha C, Brambilla R, Thomas KL (2010) A simple role for BDNF in learning and memory? Front Mol Neurosci 3:1–14PubMedPubMedCentralGoogle Scholar
  7. 7.
    Cohen-Cory S, Kidane A, Shirkey N, Marshak S (2010) Brain-derived neurotrophic factor and the development of structural neuronal connectivity. Dev Neurobiol 70:271–288PubMedPubMedCentralGoogle Scholar
  8. 8.
    Murphy JE, Padilla BE, Hasdemir B, Cottrell GS, Bunnett NW (2009) Endosomes: a legitimate platform for the signaling train. Proc Natl Acad Sci U S A 106:17615–17622CrossRefGoogle Scholar
  9. 9.
    Sorkin A, von Zastrow M (2009) Endocytosis and signaling: intertwining molecular networks. Nat Rev Mol Cell Biol 10:609–622CrossRefGoogle Scholar
  10. 10.
    Sadowski L, Pilecka I, Miaczynska M (2009) Signaling from endosomes: location makes a difference. Exp Res 315:1601–1609CrossRefGoogle Scholar
  11. 11.
    Lazo O, Gonzalez A, Ascaño M, Kuruvilla R, Couve A, Bronfman F (2013) BDNF regulates Rab11-mediated recycling endosome dynamics to induce dendritic branching. J Neurosci 33:6112–6122CrossRefGoogle Scholar
  12. 12.
    Huang S-H, Wang J, Sui W-H, Chen B, Zhang X-Y, Yan J, Geng Z, Chen Z-Y (2013) BDNF-dependent recycling facilitates TrkB translocation to postsynaptic density during LTP via a Rab11-dependent pathway. J Neurosci 33:9214–9230CrossRefGoogle Scholar
  13. 13.
    Ouyang Q, Lizarraga SB, Schmidt M, Yang U, Gong J, Ellisor D, Kauer JA, Morrow EM (2013) Christianson syndrome protein NHE6 modulates TrkB endosomal signaling required for neuronal circuit development. Neuron  https://doi.org/10.1016/j.neuron.2013.07.043 CrossRefGoogle Scholar
  14. 14.
    Salinas S, Bilsland L, Schiavo G (2008) Molecular landmarks along the axonal route: axonal transport in health and disease. Curr Opin Cell Biol 20:445–453CrossRefGoogle Scholar
  15. 15.
    Poon WW, Carlos AJ, Aguilar BL, Berchtold NC, Kawano CK, Zograbyan V, Yaopruke T, Shelanski M, Cotman CW (2013) β-Amyloid (Aβ) oligomers impair brain-derived neurotrophic factor retrograde trafficking by down-regulating ubiquitin C-terminal hydrolase, UCH-L1. J Biol Chem 288:16937–16948CrossRefGoogle Scholar
  16. 16.
    Allen SJ, Dawbarn D (2006) Clinical relevance of the neurotrophins and their receptors. Clin Sci 110:175–191CrossRefGoogle Scholar
  17. 17.
    Chao MV, Rajagopal R, Lee FS (2006) Neurotrophin signalling in health and disease. Clin Sci 110:167–173CrossRefGoogle Scholar
  18. 18.
    Sah D, Ossipov M, Porreca F (2003) Neurotrophic factors as novel therapeutics for neuropathic pain. Nature 2:460–472Google Scholar
  19. 19.
    Chang Y-P, Pinaud F, Antelman J, Weiss S (2008) Tracking bio-molecules in live cells using quantum dots. J Biophotonics 1:287–298CrossRefGoogle Scholar
  20. 20.
    Vu TQ, Lam W, Hatch E, Lidke D (2015) Quantum dots for quantitative imaging: from single molecules to tissue. Cell Tissue Res 360:71–86CrossRefGoogle Scholar
  21. 21.
    Poon WW, Blurton-Jones M, Tu CH, Feinberg LM, Chabrier MA, Harris JW, Jeon NL, Cotman CW (2011) β-amyloid impairs axonal BDNF retrograde trafficking. Neurobiol Aging 32:821–833CrossRefGoogle Scholar
  22. 22.
    Zhou B, Cai Q, Xie Y, Sheng Z (2012) Snapin recruits dynein to BDNF-TrkB signaling endosomes for retrograde axonal transport and is essential for dendrite growth of cortical neurons. Cell Rep 2:42–51CrossRefGoogle Scholar
  23. 23.
    Matusica D, EJ C (2014) Local versus long-range neurotrophin receptor signalling: endosomes are not just carriers for axonal transport. Semin Cell Dev Biol 31:57–63CrossRefGoogle Scholar
  24. 24.
    Bronfman F, Lazo O, Flores C, Escudero C (2014) Spatiotemporal intracellular dynamics of neurotrophin and its receptors. Implications for neurotrophin signaling and neuronal function. Handb Exp Pharmacol 220:33–65CrossRefGoogle Scholar
  25. 25.
    Dahan M, Levi S, Luccardini C, Rostaing P, Riveau B, Triller A (2003) Diffusion dynamics of glycine receptors revealed by single-quantum dot tracking. Science 302:442–445CrossRefGoogle Scholar
  26. 26.
    Groc L, Lafourcade M, Heine M, Renner M, Racine V, Sibarita J-B, Lounis B, Choquet D, Cognet L (2007) Surface trafficking of neurotransmitter receptor: comparison between single-molecule/quantum dot strategies. J Neurosci 27:12433–12437CrossRefGoogle Scholar
  27. 27.
    Rosenthal SJ, Chang JC, Kovtun O, McBride JR, Tomlinson ID (2011) Biocompatible quantum dots for biological applications. Chem Biol 18:10–24CrossRefGoogle Scholar
  28. 28.
    Pinaud F, Clarke S, Stittner A, Dahan M (2010) Probing cellular events, one quantum dot at a time. Nat Methods 7(4):275–285CrossRefGoogle Scholar
  29. 29.
    Michalet X, Pinaud FF, Bentolila LA, Tsay JM, Doose S, Li JJ, Sundaresan G, Wu AM, Gambhir SS, Weiss S (2005) Quantum dots for live cells, in vivo imaging, and diagnosis. Science 307:538–544CrossRefGoogle Scholar
  30. 30.
    Fichter KM, Flajolet M, Greengard P, Vu TQ (2010) Kinetics of G-protein-coupled receptor endosomal trafficking pathways revealed by single quantum dots. Proc Natl Acad Sci U S A 107:18658–18663 doi: 1013763107 [pii] 10.1073/pnas.1013763107CrossRefGoogle Scholar
  31. 31.
    Zhang K, Osakada Y, Vrljic M, Chen L, Mudrakola H, Cui B (2010) Single molecule imaging of NGF axonal transport in microfluidic devices. Lab Chip 10:2566–2573CrossRefGoogle Scholar
  32. 32.
    Cui B, Wu C, Chen L, Ramirez A, Bearer E, Li W-P, Mobley W, Chu S (2007) One at a time, live tracking of NGF axonal transport using quantum dots. Proc Natl Acad Sci U S A 104:13666–13671CrossRefGoogle Scholar
  33. 33.
    Sung K, Maloney M, Yang J, Wu C (2011) A novel method for producing mono-biotinylated, biologically active neurotrophic factors: an essential reagent for single molecule study of axonal transport. J Neurosci Methods 200:121–128CrossRefGoogle Scholar
  34. 34.
    Xie W, Zhang K, Cui B (2012) Functional characterization and axonal transport of quantum dot labeled BDNF. Integr Biol 4:953–960CrossRefGoogle Scholar
  35. 35.
    Zhao X, Zhou Y, Weissmiller A, Pearn M, Mobley W, Wu C (2014) Real-time imaging of axonal transport of quantum dot-labeled BDNF in primary neurons. J Vis Exp (91):51899Google Scholar
  36. 36.
    Sundara Rajan S, Vu TQ (2006) Quantum dots monitor TrkA receptor dynamics in the interior of neural PC12 cells. Nano Lett 6:2049–2059CrossRefGoogle Scholar
  37. 37.
    Sundara Rajan S, Yan Kiu H, Vu T (2008) Ligand-bound quantum dot probes for studying the molecular scale dynamics of receptor endocytic trafficking in live cells. ACS Nano 2:1153–1166CrossRefGoogle Scholar
  38. 38.
    Vu T, Maddipati R, Blute TA, Nehilla BJ, Nusblat L, Desai TA (2005) Peptide-conjugated quantum dots activate neuronal receptors and initiate downstream signaling of neurite growth. Nano Lett 5:603–607CrossRefGoogle Scholar
  39. 39.
    Vermehren-Schmaedick A, Wesley K, Jacob T, Ramunno-Johnson D, Balkowiec A, Lidke KA, Vu T (2014) Heterogeneous intracellular trafficking dynamics of brain-derived neurotrophic factor complexes in the neuronal Soma revealed by single quantum dot tracking. PLoS One 9:1–13CrossRefGoogle Scholar
  40. 40.
    Edelstein A, Amodaj N, Hoover K, Vale R, Stuurman N (2010) Computer control of microscopes using micro manager. Curr Protoc Mol Biol Chapter 14:Unit 14 20. doi:  https://doi.org/10.1002/0471142727.mb1420s92
  41. 41.
    Kaech S, Banker G (2007) Culturing hippocampal neurons. Nat Protoc 1:2406–2415CrossRefGoogle Scholar
  42. 42.
    Sbalzarini I, Koumoutsakos P (2005) Feature point tracking and trajectory analysis for video imaging in cell biology. J Struct Biol 151:182–195CrossRefGoogle Scholar
  43. 43.
    Jaqaman K, Loerke D, Mettlen M, Kuwata H, Grinstein S, Schmid SL, Danuser G (2008) Robust single-particle tracking in live-cell time-lapse sequences. Nat Methods 5:695–702CrossRefGoogle Scholar
  44. 44.
    Luengo Hendriks C, Rieger B, van Ginkel M, van Kempen G, van Vliet L (1999) DIPimage: a scientific image processing toolbox for MATLAB. Delft University of Technology, Delft, The NetherlandsGoogle Scholar
  45. 45.
    Zhongming L, Piechowicz M, Qiu S (2016) A simplified method for ultra-low density, long-term primary hippocampal neuron culture. J Vis Exp (109):1–8Google Scholar

Copyright information

© Springer Science+Business Media New York 2018

Authors and Affiliations

  • Anke Vermehren-Schmaedick
    • 1
  • Thomas Jacob
    • 2
  • Tania Q. Vu
    • 2
    Email author
  1. 1.Department of Physiology and PharmacologyOregon Health and Science UniversityPortlandUSA
  2. 2.Department of Biomedical Engineering, Oregon Center for Spatial Systems BiomedicineOregon Health and Science UniversityPortlandUSA

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