Skip to main content
SpringerLink
Log in

Light Microscopy and Dynamic Light Scattering to Study Liquid-Liquid Phase Separation of Tau Proteins In Vitro

  • Protocol
  • First Online:
Protein Aggregation

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2551))

Abstract

Tau is an intrinsically disordered protein that binds and stabilizes axonal microtubules (MTs) in neurons of the central nervous system. The binding of Tau to MTs is mediated by its repeat domain and flanking proline-rich domains. The positively charged (basic) C-terminal half of Tau also mediates the assembly Tau into fibrillar aggregates in Alzheimer’s disease (AD) and tauopathy brains. In recent years, another assembly form of Tau has been identified: Tau can undergo liquid-liquid phase separation (LLPS), which leads to its condensation into liquid-dense phases, either by complex coacervation with polyanions like heparin or RNA or through “self-coacervation” at high Tau concentrations. Condensation of Tau in the absence of polyanions can be enhanced by the presence of molecular crowding agents in a dilute Tau solution. In vitro experiments using recombinant purified Tau are helpful to study the physicochemical determinants of Tau LLPS, which can then be extrapolated into the cellular context. Tau condensation is a new aspect of Tau biology that may play a role for the initiation of Tau aggregation, but also for its physiological function(s), for example, the binding to microtubules. Here we describe how to study the condensation of Tau in vitro using light microscopy, including fluorescence recovery after photobleaching (FRAP), to assess the shape and molecular diffusion in the condensates, a proxy for the degree of condensate percolation. We also describe turbidity measurements of condensate-containing solutions to assess the overall amount of LLPS and time-resolved dynamic light scattering (trDLS) to study the formation and size of Tau condensates.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Log in via an institution

Subscribe and save

Springer+ Basic
$34.99 /Month
  • Get 10 units per month
  • Download Article/Chapter or eBook
  • 1 Unit = 1 Article or 1 Chapter
  • Cancel anytime
Subscribe now

Buy Now

Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 149.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 199.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 279.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

Similar content being viewed by others

References

  1. Goedert M, Hasegawa M, Jakes R et al (1997) Phosphorylation of microtubule-associated protein tau by stress-activated protein kinases. FEBS Lett 409:57–62. https://doi.org/10.1016/S0014-5793(97)00483-3

    Article  CAS  PubMed  Google Scholar 

  2. Mukrasch MD, Von Bergen M, Biernat J et al (2007) The “jaws” of the tau-microtubule interaction. J Biol Chem 282:12230–12239. https://doi.org/10.1074/jbc.M607159200

    Article  CAS  PubMed  Google Scholar 

  3. Gustke N, Trinczek B, Biernat J et al (1994) Domains of τ protein and interactions with microtubules. Biochemistry 33:9511–9522. https://doi.org/10.1021/bi00198a017

    Article  CAS  PubMed  Google Scholar 

  4. Schwalbe M, Kadavath H, Biernat J et al (2015) Structural impact of tau phosphorylation at threonine 231. Structure 23:1448–1458. https://doi.org/10.1016/j.str.2015.06.002

    Article  CAS  PubMed  Google Scholar 

  5. Iqbal K, Liu F, Gong C-X, Grundke-Iqbal I (2010) Tau in Alzheimer disease and related tauopathies. Curr Alzheimer Res 7:656–664. https://doi.org/10.2174/156720510793611592

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Wegmann S, Biernat J, Mandelkow E (2021) A current view on tau protein phosphorylation in Alzheimer’s disease. Curr Opin Neurobiol 69:131–138. https://doi.org/10.1016/j.conb.2021.03.003

    Article  CAS  PubMed  Google Scholar 

  7. Goedert M, Jakes R, Spillantini MG et al (1996) Assembly of microtubule-associated protein tau into Alzheimer-like filaments induced by sulphated glycosaminoglycans. Nature 383:550–553

    Article  CAS  PubMed  Google Scholar 

  8. Kampers T, Friedhoff P, Biernat J et al (1996) RNA stimulates aggregation of microtubule-associated protein tau into Alzheimer-like paired helical filaments. FEBS Lett 399:344–349. https://doi.org/10.1016/S0014-5793(96)01386-5

    Article  CAS  PubMed  Google Scholar 

  9. Von Bergen M, Barghorn S, Biernat J et al (2005) Tau aggregation is driven by a transition from random coil to beta sheet structure. Biochim Biophys Acta Mol basis Dis 1739:158–166

    Article  Google Scholar 

  10. Akoury E, Gajda M, Pickhardt M et al (2013) Inhibition of tau filament formation by conformational modulation. J Am Chem Soc 135:2853–2862. https://doi.org/10.1021/ja312471h

    Article  CAS  PubMed  Google Scholar 

  11. Wegmann S, Eftekharzadeh B, Tepper K et al (2018) Tau protein liquid–liquid phase separation can initiate tau aggregation. EMBO J 37:e98049. https://doi.org/10.15252/embj.201798049

    Article  PubMed  PubMed Central  Google Scholar 

  12. Hernández-Vega A, Braun M, Scharrel L et al (2017) Local nucleation of microtubule bundles through tubulin concentration into a condensed tau phase. Cell Rep 20:2304–2312. https://doi.org/10.1016/j.celrep.2017.08.042

    Article  PubMed  PubMed Central  Google Scholar 

  13. Ambadipudi S, Biernat J, Riedel D et al (2017) Liquid–liquid phase separation of the microtubule-binding repeats of the Alzheimer-related protein Tau. Nat Commun 8:275. https://doi.org/10.1038/s41467-017-00480-0

    Article  PubMed  PubMed Central  Google Scholar 

  14. Boyko S, Qi X, Chen TH et al (2019) Liquid-liquid phase separation of tau protein: the crucial role of electrostatic interactions. J Biol Chem 294:11054–11059. https://doi.org/10.1074/jbc.AC119.009198

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Kanaan NM, Hamel C, Grabinski T et al (2020) Liquid-liquid phase separation induces pathogenic tau conformations in vitro. Nat Commun 11:1. https://doi.org/10.1038/s41467-020-16580-3

    Article  Google Scholar 

  16. Ukmar-Godec T, Hutten S, Grieshop MP et al (2019) Lysine/RNA-interactions drive and regulate biomolecular condensation. Nat Commun 10:2909. https://doi.org/10.1038/s41467-019-10792-y

    Article  PubMed  PubMed Central  Google Scholar 

  17. Zhang X, Lin Y, Eschmann NA et al (2017) RNA stores tau reversibly in complex coacervates. PLoS Biol 15(7):e2002183. https://doi.org/10.1371/journal.pbio.2002183

    Article  PubMed  PubMed Central  Google Scholar 

  18. Lin Y, Fichou Y, Zeng Z et al (2020) Electrostatically driven complex coacervation and amyloid aggregation of tau are independent processes with overlapping conditions. ACS Chem Neurosci 11:615–627. https://doi.org/10.1021/acschemneuro.9b00627

    Article  CAS  PubMed  Google Scholar 

  19. Lin Y, McCarty J, Rauch JN et al (2019) Narrow equilibrium window for complex coacervation of tau and RNA under cellular conditions. elife 8:1–21. https://doi.org/10.7554/eLife.42571

    Article  Google Scholar 

  20. Chandupatla RR, Flatley A, Feederle R et al (2020) Novel antibody against low-n oligomers of tau protein promotes clearance of tau in cells via lysosomes. Alzheimers Dement Transl Res Clin Interv 6:1–18. https://doi.org/10.1002/trc2.12097

    Google Scholar 

  21. Siahaan V, Krattenmacher J, Hyman AA et al (2019) Kinetically distinct phases of tau on microtubules regulate kinesin motors and severing enzymes. Nat Cell Biol 21:1086–1092

    Article  CAS  PubMed  Google Scholar 

  22. Tan R, Lam AJ, Tan T et al (2019) Microtubules gate tau condensation to spatially regulate microtubule functions. Nat Cell Biol 21:1078–1085. https://doi.org/10.1038/s41556-019-0375-5

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Zhang X, Vigers M, McCarty J et al (2020) The proline-rich domain promotes Tau liquid-liquid phase separation in cells. J Cell Biol 219:11. https://doi.org/10.1083/JCB.202006054

    Article  Google Scholar 

  24. Konzack S, Thies E, Marx A et al (2007) Swimming against the tide: mobility of the microtubule-associated protein tau in neurons. J Neurosci 27:9916–9927. https://doi.org/10.1523/JNEUROSCI.0927-07.2007

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Janning D, Igaev M, Sündermann F et al (2014) Single-molecule tracking of tau reveals fast kiss-and-hop interaction with microtubules in living neurons. Mol Biol Cell 25:3541–3551. https://doi.org/10.1091/mbc.E14-06-1099

    Article  PubMed  PubMed Central  Google Scholar 

  26. Brangwynne CP, Tompa P, Pappu RV (2015) Polymer physics of intracellular phase transitions. Nat Phys 11:899–904

    Article  CAS  Google Scholar 

  27. Brangwynne CP, Eckmann CR, Courson DS et al (2009) Germline P granules are liquid droplets that localize by controlled dissolution/condensation. Science 324:1729–1732. https://doi.org/10.1126/science.1172046

    Article  CAS  PubMed  Google Scholar 

  28. Molliex A, Temirov J, Lee J et al (2015) Phase separation by low complexity domains promotes stress granule assembly and drives pathological fibrillization. Cell 163:123–133. https://doi.org/10.1016/j.cell.2015.09.015

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Gibson BA, Doolittle LK, Schneider MWG et al (2019) Organization of chromatin by intrinsic and regulated phase separation. Cell 179(2):470–484.e21. https://doi.org/10.1016/j.cell.2019.08.037

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Blackman LD, Gunatillake PA, Cass P et al (2019) An introduction to zwitterionic polymer behavior and applications in solution and at surfaces. Chem Soc Rev 48:757–770

    Article  CAS  PubMed  Google Scholar 

  31. Madinya JJ, Chang LW, Perry SL et al (2020) Sequence-dependent self-coacervation in high charge-density polyampholytes. Mol Syst Des Eng 5:632–644. https://doi.org/10.1039/c9me00074g

    Article  CAS  Google Scholar 

  32. Von Bergen M, Friedhoff P, Biernat J et al (2000) Assembly of τ protein into Alzheimer paired helical filaments depends on a local sequence motif (306VQIVYK311) forming β structure. Proc Natl Acad Sci U S A 97:5129–5134. https://doi.org/10.1073/pnas.97.10.5129

    Article  Google Scholar 

  33. Wegmann S, Yu JJ, Chinnathambi S et al (2010) Human tau isoforms assemble into ribbon-like fibrils that display polymorphic structure and stability. J Biol Chem 285:27302–27313. https://doi.org/10.1074/jbc.M110.145318

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Alberti S, Saha S, Woodruff JB et al (2018) A User’s guide for phase separation assays with purified proteins. J Mol Biol 430:4806–4820. https://doi.org/10.1016/j.jmb.2018.06.038

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Carisey A, Stroud M, Tsang R et al (2011) Fluorescence recovery after photobleaching. Methods Mol Biol 769:387–402. https://doi.org/10.1007/978-1-61779-207-6_26

    Article  CAS  PubMed  Google Scholar 

  36. Xu R (2015) Light scattering: a review of particle characterization applications. Particuology 18:11–21. https://doi.org/10.1016/j.partic.2014.05.002

    Article  Google Scholar 

  37. Barghorn S, Biernat J, Mandelkow E (2005) Purification of recombinant tau protein and preparation of alzheimer-paired helical filaments in vitro. Methods Mol Biol 299:35–51. https://doi.org/10.1385/1-59259-874-9:035

    CAS  PubMed  Google Scholar 

  38. Brown RE, Jarvis KL, Hyland KJ (1989) Protein measurement using bicinchoninic acid: elimination of interfering substances. Anal Biochem 180:136–139. https://doi.org/10.1016/0003-2697(89)90101-2

    Article  CAS  PubMed  Google Scholar 

  39. Ceballos AV, McDonald CJ, Elbaum-Garfinkle S (2018) Methods and strategies to quantify phase separation of disordered proteins, 1st edn. Elsevier Inc.

    Google Scholar 

  40. Baul U, Chakraborty D, Mugnai ML et al (2019) Sequence effects on size, shape, and structural heterogeneity in intrinsically disordered proteins. J Phys Chem B 123:3462–3474. https://doi.org/10.1021/acs.jpcb.9b02575

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. Wegmann S, Medalsy ID, Mandelkow E et al (2012) The fuzzy coat of pathological human Tau fibrils is a two-layered polyelectrolyte brush. Proc Natl Acad Sci U S A 110:E313–E321. https://doi.org/10.1073/pnas.1212100110

    PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgments

This work was funded by the Helmholtz Society (S.W. and E.M.), the Hertie Foundation (S.W.), and the German Research Foundation (DFG) in the priority program SPP2191 (C.B., E.M, S.W.).

Author information

Authors and Affiliations

  1. German Center for Neurodegenerative Diseases (DZNE), Berlin, Germany

    Janine Hochmair & Susanne Wegmann

  2. University Hamburg, Institute for Biochemistry and Molecular Biology, Laboratory for Structural Biology of Infection and Inflammation, Hamburg, Germany

    Christian Exner & Christian Betzel

  3. German Center for Neurodegenerative Diseases (DZNE), Bonn, Germany

    Eckhard Mandelkow

  4. Research Center CAESAR, Bonn, Germany

    Eckhard Mandelkow

Authors
  1. Janine Hochmair
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Christian Exner
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Christian Betzel
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Eckhard Mandelkow
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Susanne Wegmann
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Susanne Wegmann .

Editor information

Editors and Affiliations

  1. Cambridge, MA, USA

    Andrzej Stanisław Cieplak

Rights and permissions

Reprints and permissions

Copyright information

© 2023 Springer Science+Business Media, LLC, part of Springer Nature

About this protocol

Check for updates. Verify currency and authenticity via CrossMark

Cite this protocol

Hochmair, J., Exner, C., Betzel, C., Mandelkow, E., Wegmann, S. (2023). Light Microscopy and Dynamic Light Scattering to Study Liquid-Liquid Phase Separation of Tau Proteins In Vitro. In: Cieplak, A.S. (eds) Protein Aggregation. Methods in Molecular Biology, vol 2551. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-2597-2_15

Download citation

  • DOI: https://doi.org/10.1007/978-1-0716-2597-2_15

  • Published:

  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-2596-5

  • Online ISBN: 978-1-0716-2597-2

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation