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Using Steady-State Fluorescence Anisotropy to Study Protein Clustering

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Plasmodesmata

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2457))

Abstract

Signaling pathways rely on the precise control of protein-protein interactions. Therefore, it is essential to be able to investigate such interactions with spatiotemporal resolution and in live cells. Here we describe a microscope-based fluorescence spectrometry technique to investigate homotypic interactions between GFP-labeled fusion proteins in a rapid and reproducible fashion using fluorescence anisotropy. This method is of great value for the study of protein complexes in live tissue with subcellular resolution.

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References

  1. Harter K, Meixner AJ, Schleifenbaum F (2012) Spectro-microscopy of living plant cells. Mol Plant 5:14–26

    Article  CAS  Google Scholar 

  2. Bücherl CA, Bader A, Westphal AH, Laptenok SP, Borst JW (2014) FRET-FLIM applications in plant systems. Protoplasma 251:383–394

    Article  Google Scholar 

  3. Long Y, Stahl Y, Weidtkamp-Peters S, Postma M, Zhou W, Goedhart J et al (2017) In vivo FRET-FLIM reveals cell-type-specific protein interactions in Arabidopsis roots. Nature 548:97–102

    Article  CAS  Google Scholar 

  4. Weidtkamp-Peters S, Stahl Y (2017) The use of FRET/FLIM to study proteins interacting with plant receptor kinases. Methods Mol Biol 1621:163–175

    Article  CAS  Google Scholar 

  5. Shah K, Gadella TW, van Erp H, Hecht V, de Vries SC (2001) Subcellular localization and oligomerization of the Arabidopsis thaliana somatic embryogenesis receptor kinase 1 protein. J Mol Biol 309:641–655

    Article  CAS  Google Scholar 

  6. Somssich M, Ma Q, Weidtkamp-Peters S, Stahl Y, Felekyan S, Bleckmann A et al (2015) Real-time dynamics of peptide ligand-dependent receptor complex formation in planta. Sci Signal 8:ra76

    Article  Google Scholar 

  7. Stahl Y, Grabowski S, Bleckmann A, Kuhnemuth R, Weidtkamp-Peters S, Pinto KG et al (2013) Moderation of Arabidopsis root stemness by CLAVATA1 and ARABIDOPSIS CRINKLY4 receptor kinase complexes. Curr Biol 23:362–371

    Article  CAS  Google Scholar 

  8. Vaddepalli P, Herrmann A, Fulton L, Oelschner M, Hillmer S, Stratil TF et al (2014) The C2-domain protein QUIRKY and the receptor-like kinase STRUBBELIG localize to plasmodesmata and mediate tissue morphogenesis in Arabidopsis thaliana. Development 141:4139–4148

    Article  CAS  Google Scholar 

  9. Rosas-Diaz T, Zhang D, Fan P, Wang L, Ding X, Jiang Y et al (2018) A virus-targeted plant receptor-like kinase promotes cell-to-cell spread of RNAi. Proc Natl Acad Sci U S A 115:1388–1393

    Article  CAS  Google Scholar 

  10. Cheval C, Samwald S, Johnston MG, de Keijzer J, Breakspear A, Liu X et al (2020) Chitin perception in plasmodesmata characterizes submembrane immune-signaling specificity in plants. Proc Natl Acad Sci U S A 117:9621–9629

    Article  CAS  Google Scholar 

  11. Denay G, Schultz P, Hänsch S, Weidtkamp-Peters S, Simon R (2019) Over the rainbow: a practical guide for fluorescent protein selection in plant FRET experiments. Plant Direct 3:e00189

    Article  Google Scholar 

  12. Bader AN, Hoetzl S, Hofman EG, Voortman J et al (2011) Homo-FRET imaging as a tool to quantify protein and lipid clustering. ChemPhysChem 12:475–483

    Article  CAS  Google Scholar 

  13. Jares-Erijman EA, Jovin TM (2003) FRET imaging. Nat Biotechnol 21:1387–1395

    Article  CAS  Google Scholar 

  14. JR L (2006) Principles of fluorescence spectroscopy. Springer Science and Business Media, New York

    Google Scholar 

  15. Chevalier D, Batoux M, Fulton L, Pfister K, Yadav RK, Schellenberg M et al (2005) STRUBBELIG defines a receptor kinase-mediated signaling pathway regulating organ development in Arabidopsis. Proc Natl Acad Sci U S A 102:9074–9079

    Article  CAS  Google Scholar 

  16. Kwak SH, Shen R, Schiefelbein J (2005) Positional signaling mediated by a receptor-like kinase in Arabidopsis. Science 307:1111–1113

    Article  CAS  Google Scholar 

  17. Vaddepalli P, Fulton L, Batoux M, Yadav RK, Schneitz K (2011) Structure-function analysis of STRUBBELIG, an Arabidopsis atypical receptor-like kinase involved in tissue morphogenesis. PLoS One 6:e19730

    Article  CAS  Google Scholar 

  18. Chaudhary A, Chen X, Leśniewska B, Gao J, Wolf S, Schneitz K (2020) Cell wall damage impairs root hair cell patterning and tissue morphogenesis mediated by the Arabidopsis receptor kinase STRUBBELIG bioRxiv 2020.11.04.368241

    Google Scholar 

  19. Imlau A, Truernit E, Sauer N (1999) Cell-to-cell and long-distance trafficking of the green fluorescent protein in the phloem and symplastic unloading of the protein into sink tissues. Plant Cell 11:309–322

    Article  CAS  Google Scholar 

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Acknowledgments

Our work is currently supported by grants SFB 924 (TPA2) and FOR2581 (TP7) from the German Research Council (DFG) to KS. We thank Ramón A. Torres-Ruiz for help with FA imaging and comments on the manuscript. We also thank Wanney Walja and Sebastian Scholz for sharing data. We further acknowledge support by the Center for Advanced Light Microscopy (CALM) of the TUM School of Life Sciences.

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Author notes

  1. Ajeet Chaudhary

    Present address: Department of Plant Biology, Carnegie Institution for Science, Stanford, CA, USA

Authors and Affiliations

  1. Plant Developmental Biology, TUM School of Life Sciences, Technical University of Munich, Freising, Germany

    Ajeet Chaudhary & Kay Schneitz

Authors
  1. Ajeet Chaudhary
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  2. Kay Schneitz
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Corresponding author

Correspondence to Kay Schneitz .

Editor information

Editors and Affiliations

  1. Centre for Plant Sciences, School of Biology, University of Leeds, Leeds, UK

    Yoselin Benitez-Alfonso

  2. Institut de Biologie Moléculaire des Plantes, CNRS, Université de Strasbourg, Strasbourg, France

    Manfred Heinlein

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Chaudhary, A., Schneitz, K. (2022). Using Steady-State Fluorescence Anisotropy to Study Protein Clustering. In: Benitez-Alfonso, Y., Heinlein, M. (eds) Plasmodesmata. Methods in Molecular Biology, vol 2457. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-2132-5_16

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  • DOI: https://doi.org/10.1007/978-1-0716-2132-5_16

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-2131-8

  • Online ISBN: 978-1-0716-2132-5

  • eBook Packages: Springer Protocols

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