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Measuring Protein Movement, Oligomerization State, and Protein–Protein Interaction in Arabidopsis Roots Using Scanning Fluorescence Correlation Spectroscopy (Scanning FCS)

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Plant Genomics

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1610))

Abstract

Scanning fluorescence correlation spectroscopy (scanning FCS) can be used to determine protein movement, oligomerization state, and protein–protein interaction. Here, we describe how to use the scanning FCS techniques of raster image correlation spectroscopy (RICS) and pair correlation function (pCF) to determine the rate and direction of protein movement. In addition, we detail how number and brightness (N&B) and cross-correlation analyses can be used to determine oligomerization state and binding ratios of protein complexes. We specifically describe how to acquire suitable images for scanning FCS analysis using the model plant Arabidopsis and how to perform the various analyses using the SimFCS software.

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References

  1. Digman MA, Gratton E (2011) Lessons in fluctuation correlation spectroscopy. Annu Rev Phys Chem 62:645–668. doi:10.1146/annurev-physchem-032210-103424.Lessons

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Petrasek Z, Schwille P (2008) Precise measurement of diffusion coefficients using scanning fluorescence correlation spectroscopy. Biophys J 94:1437–1448. doi:10.1529/biophysj.107.108811

    Article  CAS  PubMed  Google Scholar 

  3. Gallagher KL, Paquette AJ, Nakajima K, Benfey PN (2004) Mechanisms regulating SHORT-ROOT intercellular movement. Curr Biol 14:1847–1851. doi:10.1016/j.cub.2004.09.081

    Article  CAS  PubMed  Google Scholar 

  4. Koizumi K, Wu S, MacRae-Crerar A, Gallagher KL (2011) An essential protein that interacts with endosomes and promotes movement of the SHORT-ROOT transcription factor. Curr Biol 21:1559–1564. doi:10.1016/j.cub.2011.08.013

    Article  CAS  PubMed  Google Scholar 

  5. Xu XM, Wang J, Xuan Z et al (2011) Chaperonins facilitate KNOTTED1 cell-to-cell trafficking and stem cell function. Science 333:1141–1144

    Article  CAS  PubMed  Google Scholar 

  6. Nakashima K, Takasaki H, Mizoi J (2012) NAC transcription factors in plant abiotic stress responses. Biochim Biophys Acta 1819:97–103. doi:10.1016/j.bbagrm.2011.10.005

    Article  CAS  PubMed  Google Scholar 

  7. Sornaraj P, Luang S, Lopato S, Hrmova M (2016) Basic leucine zipper (bZIP) transcription factors involved in abiotic stresses: a molecular model of a wheat bZIP factor and implications of its structure in function. Biochim Biophys Acta 1860:46–56. doi:10.1016/j.bbagen.2015.10.014

    Article  CAS  PubMed  Google Scholar 

  8. Digman MA, Brown CM, Sengupta P et al (2005) Measuring fast dynamics in solutions and cells with a laser scanning microscope. Biophys J 89:1317–1327. doi:10.1529/biophysj.105.062836

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Digman MA, Sengupta P, Wiseman PW et al (2005) Fluctuation correlation spectroscopy with a laser-scanning microscope: exploiting the hidden time structure. Biophys J 88:L33–L36. doi:10.1529/biophysj.105.061788

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Digman MA, Gratton E (2009) Analysis of diffusion and binding in cells using the RICS approach. Microsc Res Tech 332:323–332. doi:10.1002/jemt.20655

    Article  Google Scholar 

  11. Digman MA, Wiseman PW, Choi C et al (2009) Stoichiometry of molecular complexes at adhesions in living cells. Proc Natl Acad Sci U S A 106:2170–2175

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Jameson DM, Ross JA, Albanesi JP (2009) Fluorescence fluctuation spectroscopy: ushering in a new age of enlightenment for cellular dynamics. Biophys Rev 1:105–118

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Rossow MJ, Sasaki JM, Digman MA, Gratton E (2010) Raster image correlation spectroscopy in live cells. Nat Protoc 5:1761–1774. doi:10.1038/nprot.2010.122

  14. Hinde E, Cardarelli F, Digman MA, Gratton E (2010) In vivo pair correlation analysis of EGFP intranuclear diffusion reveals DNA-dependent molecular flow. PNAS 107:16560–16565. doi:10.1073/pnas.1006731107

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Hinde E, Cardarelli F (2011) Measuring the flow of molecules in cells. Biophys Rev 3:119–129

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Vetri V, Ossato G, Militello V et al (2011) Fluctuation methods to study protein aggregation in live cells: Concanavalin A oligomers formation. Biophys J 100:774–783. doi:10.1016/j.bpj.2010.11.089

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Clark NM, Hinde E, Winter CM et al (2016) Tracking transcription factor mobility and interaction in Arabidopsis roots with fluorescence correlation spectroscopy. Elife. doi:10.7554/eLife.14770

    Google Scholar 

  18. Brown CM, Dalal RB, Hebert B et al (2008) Raster image correlation spectroscopy (RICS) for measuring fast protein dynamics and concentrations with a commercial laser scanning confocal microscope. J Microsc 229:78–91

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Digman MA, Gratton E (2009) Imaging barriers to diffusion by pair correlation functions. Biophys J 97:665–673. doi:10.1016/j.bpj.2009.04.048

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Digman MA, Dalal R, Horwitz AF, Gratton E (2008) Mapping the number of molecules and brightness in the laser scanning microscope. Biophys J 94:2320–2332. doi:10.1529/biophysj.107.114645

    Article  CAS  PubMed  Google Scholar 

  21. Digman MA, Wiseman PW, Horwitz AR, Gratton E (2009) Detecting protein complexes in living cells from laser scanning confocal image sequences by the cross correlation raster image spectroscopy method. Biophys J 96:707–716. doi:10.1016/j.bpj.2008.09.051

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Supatto W, Mcmahon A, Fraser S, Stathopoulos A (2009) Quantitative imaging of collective cell migration during Drosophila gastrulation: multiphoton microscopy and computational analysis. Nat Protoc 4:1397–1412. doi:10.1038/nprot.2009.130

    Article  PubMed  PubMed Central  Google Scholar 

  23. Chen Y, Mu JD, Ruan Q, Gratton E (2002) Molecular brightness characterization of EGFP in vivo by fluorescence fluctuation spectroscopy. Biophys J 82:133–144. doi:10.1016/S0006-3495(02)75380-0

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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Acknowledgments

N. M. C. is supported by a NSF GRF (DGE-1252376). This work was funded by a NSF CAREER grant (MCB-1453130) to R. S.

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Authors and Affiliations

  1. Department of Plant and Microbial Biology, North Carolina State University, Raleigh, NC, 27695, USA

    Natalie M. Clark & Rosangela Sozzani

  2. Biomathematics Graduate Program, North Carolina State University, Raleigh, NC, 27695, USA

    Natalie M. Clark & Rosangela Sozzani

Authors
  1. Natalie M. Clark
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  2. Rosangela Sozzani
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Corresponding author

Correspondence to Rosangela Sozzani .

Editor information

Editors and Affiliations

  1. Gregor Mendel Institute (GMI), Austrian Academy of Sciences, Vienna Biocenter (VBC), Vienna, Austria

    Wolfgang Busch

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Clark, N.M., Sozzani, R. (2017). Measuring Protein Movement, Oligomerization State, and Protein–Protein Interaction in Arabidopsis Roots Using Scanning Fluorescence Correlation Spectroscopy (Scanning FCS). In: Busch, W. (eds) Plant Genomics. Methods in Molecular Biology, vol 1610. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7003-2_16

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  • DOI: https://doi.org/10.1007/978-1-4939-7003-2_16

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  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-7001-8

  • Online ISBN: 978-1-4939-7003-2

  • eBook Packages: Springer Protocols

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