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Optogenetic Control of Microtubule Dynamics

  • Jeffrey van Haren
  • Lauren S. Adachi
  • Torsten WittmannEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 2101)

Abstract

Light can be controlled with high spatial and temporal accuracy. Therefore, optogenetics is an attractive experimental approach to modulate intracellular cytoskeleton dynamics at much faster timescales than by genetic modification. For example, in mammalian cells, microtubules (MTs) grow tens of micrometers per minute and many intracellular MT functions are mediated by a complex of +TIP proteins that dynamically associate with growing MT plus ends. EB1 is a central component of this +TIP protein network, and we recently developed a photo-inactivated π-EB1 by inserting a blue light-sensitive LOV2/Zdk1 module between the EB1 MT-binding domain and the +TIP adaptor domain. Blue light-induced π-EB1 photodissociation results in disassembly of the +TIP complex and strongly attenuates MT growth in mammalian cells.

In this chapter, we discuss theoretical and practical aspects of how to perform high-resolution live-cell microscopy in combination with π-EB1 photodissociation. However, these techniques are broadly applicable to other LOV2-based and likely other blue light-sensitive optogenetics. In addition to being a tool to investigate +TIP functions acutely and with subcellular resolution, because of its dramatic and rapid change in intracellular localization, π-EB1 can serve as a powerful tool to test and characterize optogenetic illumination setups. We describe protocols on how to achieve micrometer-scale intracellular control of π-EB1 activity using patterned illumination, and we introduce a do-it-yourself LED cube design compatible with transmitted light microscopy in multiwell plates.

Key words

Optogenetics π-EB1 Photodissociation Microtubules EB1 +TIP LOV2 LOVTRAP Zdk1 Live-cell microscopy 

Notes

Acknowledgments

This work was supported by NIH grants R01 GM094819, R01 NS107480, and R21 CA224194 to T.W. We would also like to thank Dylan Romero and Jenny Tai from The Makers Lab at UCSF for valuable advice and assistance with 3D printing.

References

  1. 1.
    Losi A, Gardner KH, Möglich A (2018) Blue-light receptors for optogenetics. Chem Rev 118:10659–10709.  https://doi.org/10.1021/acs.chemrev.8b00163CrossRefPubMedPubMedCentralGoogle Scholar
  2. 2.
    Goglia AG, Toettcher JE (2019) A bright future: optogenetics to dissect the spatiotemporal control of cell behavior. Curr Opin Chem Biol 48:106–113.  https://doi.org/10.1016/j.cbpa.2018.11.010CrossRefPubMedGoogle Scholar
  3. 3.
    Huala E, Oeller PW, Liscum E et al (1997) Arabidopsis NPH1: a protein kinase with a putative redox-sensing domain. Science 278:2120–2123.  https://doi.org/10.1126/science.278.5346.2120CrossRefPubMedGoogle Scholar
  4. 4.
    Christie JM, Salomon M, Nozue K et al (1999) LOV (light, oxygen, or voltage) domains of the blue-light photoreceptor phototropin (nph1): binding sites for the chromophore flavin mononucleotide. Proc Natl Acad Sci U S A 96:8779–8783.  https://doi.org/10.1073/pnas.96.15.8779CrossRefPubMedPubMedCentralGoogle Scholar
  5. 5.
    Christie JM (2007) Phototropin blue-light receptors. Annu Rev Plant Biol 58:21–45.  https://doi.org/10.1146/annurev.arplant.58.032806.103951CrossRefPubMedGoogle Scholar
  6. 6.
    Salomon M, Christie JM, Knieb E et al (2000) Photochemical and mutational analysis of the FMN-binding domains of the plant blue light receptor, phototropin. Biochemistry 39:9401–9410CrossRefGoogle Scholar
  7. 7.
    Harper SM, Neil LC, Gardner KH (2003) Structural basis of a phototropin light switch. Science 301:1541–1544.  https://doi.org/10.1126/science.1086810CrossRefPubMedGoogle Scholar
  8. 8.
    Konold PE, Mathes T, Weienborn J et al (2016) Unfolding of the C-terminal Jα Helix in the LOV2 photoreceptor domain observed by time-resolved vibrational spectroscopy. J Phys Chem Lett 7:3472–3476.  https://doi.org/10.1021/acs.jpclett.6b01484CrossRefPubMedGoogle Scholar
  9. 9.
    Zayner JP, Antoniou C, Sosnick TR (2012) The amino-terminal helix modulates light-activated conformational changes in AsLOV2. J Mol Biol 419:61–74.  https://doi.org/10.1016/j.jmb.2012.02.037CrossRefPubMedPubMedCentralGoogle Scholar
  10. 10.
    Kottke T, Xie A, Larsen DS, Hoff WD (2018) Photoreceptors take charge: emerging principles for light sensing. Annu Rev Biophys 47:291–313.  https://doi.org/10.1146/annurev-biophys-070317-033047CrossRefGoogle Scholar
  11. 11.
    Yao X, Rosen MK, Gardner KH (2008) Estimation of the available free energy in a LOV2-Jα photoswitch. Nat Chem Biol 4:491–497.  https://doi.org/10.1038/nchembio.99CrossRefPubMedPubMedCentralGoogle Scholar
  12. 12.
    Wu YI, Frey D, Lungu OI et al (2009) A genetically encoded photoactivatable Rac controls the motility of living cells. Nature 461:104–108.  https://doi.org/10.1038/nature08241CrossRefPubMedPubMedCentralGoogle Scholar
  13. 13.
    Lungu OI, Hallett RA, Choi EJ et al (2012) Designing Photoswitchable peptides using the AsLOV2 domain. Chem Biol 19:507–517.  https://doi.org/10.1016/j.chembiol.2012.02.006CrossRefPubMedPubMedCentralGoogle Scholar
  14. 14.
    Dagliyan O, Tarnawski M, Chu PH et al (2016) Engineering extrinsic disorder to control protein activity in living cells. Science 354:1441–1444.  https://doi.org/10.1126/science.aah3404CrossRefPubMedPubMedCentralGoogle Scholar
  15. 15.
    Reynolds KA, McLaughlin RN, Ranganathan R (2011) Hot spots for allosteric regulation on protein surfaces. Cell 147:1564–1575.  https://doi.org/10.1016/j.cell.2011.10.049CrossRefPubMedPubMedCentralGoogle Scholar
  16. 16.
    Wang H, Vilela M, Winkler A et al (2016) LOVTRAP: an optogenetic system for photoinduced protein dissociation. Nat Methods 13:755–758.  https://doi.org/10.1038/nmeth.3926CrossRefPubMedPubMedCentralGoogle Scholar
  17. 17.
    Guntas G, Hallett RA, Zimmerman SP et al (2015) Engineering an improved light-induced dimer (iLID) for controlling the localization and activity of signaling proteins. Proc Natl Acad Sci U S A 112:112–117.  https://doi.org/10.1073/pnas.1417910112CrossRefPubMedGoogle Scholar
  18. 18.
    Strickland D, Lin Y, Wagner E et al (2012) TULIPs: tunable, light-controlled interacting protein tags for cell biology. Nat Methods 9:379–384.  https://doi.org/10.1038/nmeth.1904CrossRefPubMedPubMedCentralGoogle Scholar
  19. 19.
    Kumar P, Wittmann T (2012) +TIPs: SxIPping along microtubule ends. Trends Cell Biol 22:418–428CrossRefGoogle Scholar
  20. 20.
    van Haren J, Wittmann T (2019) Microtubule plus end dynamics—do we know how microtubules grow? BioEssays 41:e1800194.  https://doi.org/10.1002/bies.201800194CrossRefPubMedGoogle Scholar
  21. 21.
    van Haren J, Charafeddine RA, Ettinger A et al (2018) Local control of intracellular microtubule dynamics by EB1 photodissociation. Nat Cell Biol 20:252–261.  https://doi.org/10.1038/s41556-017-0028-5CrossRefPubMedPubMedCentralGoogle Scholar
  22. 22.
    Wittmann T, van Haren J (2018) Generation of cell lines with light-controlled microtubule dynamics. Protoc Exchange.  https://doi.org/10.1038/protex.2017.155
  23. 23.
    Grimm JB, Muthusamy AK, Liang Y et al (2017) A general method to fine-tune fluorophores for live-cell and in vivo imaging. Nat Methods 14:987–994.  https://doi.org/10.1038/nmeth.4403CrossRefPubMedPubMedCentralGoogle Scholar
  24. 24.
    Swartz TE, Corchnoy SB, Christie JM et al (2001) The photocycle of a flavin-binding domain of the blue light photoreceptor phototropin. J Biol Chem 276:36493–36500.  https://doi.org/10.1074/jbc.M103114200CrossRefPubMedGoogle Scholar
  25. 25.
    Kennis JTM, Crosson S, Gauden M et al (2003) Primary reactions of the LOV2 domain of phototropin, a plant blue-light photoreceptor. Biochemistry 42:3385–3392.  https://doi.org/10.1021/bi034022kCrossRefPubMedGoogle Scholar
  26. 26.
    Eitoku T, Nakasone Y, Matsuoka D et al (2005) Conformational dynamics of phototropin 2 LOV2 domain with the linker upon photoexcitation. J Am Chem Soc 127:13238–13244.  https://doi.org/10.1021/ja052523iCrossRefPubMedGoogle Scholar
  27. 27.
    Kottke T, Heberle J, Hehn D et al (2003) Phot-LOV1: photocycle of a blue-light receptor domain from the green alga Chlamydomonas reinhardtii. Biophys J 84:1192–1201.  https://doi.org/10.1016/S0006-3495(03)74933-9CrossRefPubMedPubMedCentralGoogle Scholar
  28. 28.
    Laissue PP, Alghamdi RA, Tomancak P et al (2017) Assessing phototoxicity in live fluorescence imaging. Nat Methods 14:657–661.  https://doi.org/10.1038/nmeth.4344CrossRefPubMedGoogle Scholar
  29. 29.
    Ettinger A, Wittmann T (2014) Fluorescence live cell imaging. Methods Cell Biol 123:77–94.  https://doi.org/10.1016/B978-0-12-420138-5.00005-7CrossRefPubMedPubMedCentralGoogle Scholar
  30. 30.
    Zayner JP, Sosnick TR (2014) Factors that control the chemistry of the LOV domain photocycle. PLoS One 9:e87074.  https://doi.org/10.1371/journal.pone.0087074CrossRefPubMedPubMedCentralGoogle Scholar
  31. 31.
    Kawano F, Aono Y, Suzuki H, Sato M (2013) Fluorescence imaging-based high-throughput screening of fast- and slow-cycling LOV proteins. PLoS One 8:e82693.  https://doi.org/10.1371/journal.pone.0082693CrossRefPubMedPubMedCentralGoogle Scholar
  32. 32.
    Grzelak A, Rychlik B, Bartosz G (2001) Light-dependent generation of reactive oxygen species in cell culture media. Free Radic Biol Med 30:1418–1425.  https://doi.org/10.1016/S0891-5849(01)00545-7CrossRefPubMedGoogle Scholar
  33. 33.
    Zigler JS, Lepe-Zuniga JL, Vistica B, Gery I (1985) Analysis of the cytotoxic effects of light-exposed HEPES-containing culture medium. In Vitro Cell Dev Biol 21:282–287.  https://doi.org/10.1007/BF02620943CrossRefPubMedGoogle Scholar
  34. 34.
    Halliwell B, Butt VS (1972) Flavin mononucleotide-sensitized photo-oxidation of glyoxylate in Good’s buffers. (Short Communications). Biochem J 129:1157–1158.  https://doi.org/10.1042/bj1291157CrossRefPubMedPubMedCentralGoogle Scholar
  35. 35.
    Stockley JH, Evans K, Matthey M et al (2017) Surpassing light-induced cell damage in vitro with novel cell culture media. Sci Rep 7:849.  https://doi.org/10.1038/s41598-017-00829-xCrossRefPubMedPubMedCentralGoogle Scholar
  36. 36.
    Stehbens S, Pemble H, Murrow L, Wittmann T (2012) Imaging intracellular protein dynamics by spinning disk confocal microscopy. Methods Enzymol 504:293–313.  https://doi.org/10.1016/B978-0-12-391857-4.00015-XCrossRefPubMedPubMedCentralGoogle Scholar
  37. 37.
    Christie JM, Corchnoy SB, Swartz TE et al (2007) Steric interactions stabilize the signaling state of the LOV2 domain of phototropin 1. Biochemistry 46:9310–9319.  https://doi.org/10.1021/bi700852wCrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2020

Authors and Affiliations

  • Jeffrey van Haren
    • 1
    • 2
  • Lauren S. Adachi
    • 1
  • Torsten Wittmann
    • 1
    Email author
  1. 1.Department of Cell and Tissue BiologyUniversity of California San FranciscoSan FranciscoUSA
  2. 2.Department of Cell BiologyErasmus MCRotterdamThe Netherlands

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