A PLA-iRoCS Pipeline for the Localization of Protein–Protein Interactions In Situ

Part of the Methods in Molecular Biology book series (MIMB, volume 1787)


In plants as well as other organisms, protein localization alone is insufficient to provide a mechanistic link between stimulus and process regulation. This is because protein–protein interactions are central to the regulation of biological processes. However, they remain very difficult to detect in situ, with the choice of tools for the detection of protein–protein interaction in situ still in need of expansion. Here, we provide a protocol for the detection and accurate localization of protein interactions based on the combination of a whole-mount proximity ligation assay and iRoCS, a coordinate system able to standardize subtle differences between the architecture of individual Arabidopsis roots.

Key words

Proximity ligation assay iRoCS Protein complex Protein–protein interactions In situ In planta 3D imaging Root apical meristem Arabidopsis thaliana 


  1. 1.
    Uhlen M, Ponten F (2005) Antibody-based proteomics for human tissue profiling. Mol Cell Proteomics 4:384–393CrossRefPubMedCentralGoogle Scholar
  2. 2.
    Sprinzak E, Sattath S, Margalit H (2003) How reliable are experimental protein–protein interaction data? J Mol Biol 327:919–923CrossRefPubMedCentralGoogle Scholar
  3. 3.
    Hu CD, Chinenov Y, Kerppola TK (2002) Visualization of interactions among bZip and Rel family proteins in living cells using bimolecular fluorescence complementation. Mol Cell 9:789–798CrossRefPubMedCentralGoogle Scholar
  4. 4.
    Bartel PL, Fields S (1995) Analyzing protein–protein initeractions using yeast 2-hybrid system. Oncogene Tech 254:241–263CrossRefGoogle Scholar
  5. 5.
    Soderberg O, Gullberg M, Jarvius M, Ridderstrale K, Leuchowius KJ, Jarvius J et al (2006) Direct observation of individual endogenous protein complexes in situ by proximity ligation. Nat Methods 3:995–1000CrossRefPubMedCentralGoogle Scholar
  6. 6.
    Soderberg O, Leuchowius KJ, Gullberg M, Jarvius M, Weibrecht I, Larsson LG et al (2008) Characterizing proteins and their interactions in cells and tissues using the in situ proximity ligation assay. Methods 45:227–232CrossRefPubMedCentralGoogle Scholar
  7. 7.
    Apelt F, Breuer D, Nikoloski Z, Stitt M, Kragler F (2015) Phytotyping(4D): a light-field imaging system for non-invasive and accurate monitoring of spatio-temporal plant growth. Plant J 82:693–706CrossRefPubMedCentralGoogle Scholar
  8. 8.
    Fernandez R, Das P, Mirabet V, Moscardi E, Traas J, Verdeil JL et al (2010) Imaging plant growth in 4D: robust tissue reconstruction and lineaging at cell resolution. Nat Methods 7:547–U594CrossRefPubMedCentralGoogle Scholar
  9. 9.
    Montenegro-Johnson TD, Stamm P, Strauss S, Topham AT, Tsagris M, Wood ATA et al (2015) Digital single-cell analysis of plant organ development using 3DCellAtlas. Plant Cell 27:1018–1033CrossRefPubMedCentralGoogle Scholar
  10. 10.
    de Reuille PB, Routier-Kierzkowska AL, Kierzkowski D, Bassel GW, Schupbach T, Tauriello G et al (2015) MorphoGraphX: a platform for quantifying morphogenesis in 4D. Elife 4:05864Google Scholar
  11. 11.
    Schmidt T, Pasternak T, Liu K, Blein T, Aubry-Hivet D, Dovzhenko A et al (2014) The iRoCS toolbox: 3D analysis of the plant root apical meristem at cellular resolution. Plant J 77:806–814CrossRefPubMedCentralGoogle Scholar

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© Springer Science+Business Media, LLC, part of Springer Nature 2018

Authors and Affiliations

  1. 1.Faculty of BiologyInstitute of Biology II/Molecular Plant Physiology, University of FreiburgFreiburgGermany
  2. 2.BIOSS Centre for Biological Signaling Studies, University of FreiburgFreiburgGermany
  3. 3.Department of Computer Science, Technical FacultyUniversity of FreiburgFreiburgGermany
  4. 4.Department of Agronomy, Food, Natural Resources, Animals and EnvironmentUniversity of Padova, AgripolisLegnaro (Padova)Italy
  5. 5.Center for Biological Systems AnalysisUniversity of FreiburgFreiburgGermany

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