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Single-particle tracking photoactivated localization microscopy of membrane proteins in living plant tissues

Nature Protocols volume 16, pages 1600–1628 (2021)Cite this article

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Abstract

Super-resolution microscopy techniques have pushed the limit of optical imaging to unprecedented spatial resolutions. However, one of the frontiers in nanoscopy is its application to intact living organisms. Here we describe the implementation and application of super-resolution single-particle tracking photoactivated localization microscopy (sptPALM) to probe single-molecule dynamics of membrane proteins in live roots of the model plant Arabidopsis thaliana. We first discuss the advantages and limitations of sptPALM for studying the diffusion properties of membrane proteins and compare this to fluorescence recovery after photobleaching (FRAP) and fluorescence correlation spectroscopy (FCS). We describe the technical details for handling and imaging the samples for sptPALM, with a particular emphasis on the specificity of imaging plant cells, such as their thick cell walls or high degree of autofluorescence. We then provide a practical guide from data collection to image analyses. In particular, we introduce our sptPALM_viewer software and describe how to install and use it for analyzing sptPALM experiments. Finally, we report an R statistical analysis pipeline to analyze and compare sptPALM experiments. Altogether, this protocol should enable plant researchers to perform sptPALM using a benchmarked reproducible protocol. Routinely, the procedure takes 3–4 h of imaging followed by 3–4 d of image processing and data analysis.

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Fig. 1: Schematic representation of sptPALM.
Fig. 2: Comparison of the typical dynamic range of FRAP, FCS, sptPALM and SMdM.
Fig. 3: Coverslip cleaning and sample preparation.
Fig. 4: sptPALM track acquisition.
Fig. 5: Track reconstruction using MTT.
Fig. 6: MSD and diffusion analysis using the sptPALM_viewer software.
Fig. 7: MSD and diffusion results from the sptPALM_viewer.
Fig. 8: Mixture modeling reveals two latent subpopulations of mEos2-PHEVECT2 molecules.

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Data availability

All the raw data used to generate Figs. 4–8 and Extended Data Figs. 1–3 are available at http://bioserv.cbs.cnrs.fr/DOWNLOAD/sptPALM_data/.

Code availability

The code used for the analysis of TrackMate or MTT data have been uploaded to https://github.com/jbfiche/sptPALM/, and explanations on how to run an analysis can be found in the README.md file. A standalone application and a test set are available from http://bioserv.cbs.cnrs.fr/DOWNLOAD/sptPALM_data/. Additional codes (including FIJI plugins and R code for the statistics) as well as data used for the figures are accessible from http://bioserv.cbs.cnrs.fr/DOWNLOAD/sptPALM_data/. Additional advice on how to use them can be obtained from the authors upon reasonable request.

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Acknowledgements

We acknowledge the contribution of SFR Biosciences (UMS3444/CNRS, US8/Inserm, ENS de Lyon, UCBL) facilities: C. Lionnet, E. Chattre and J. Brocard. We thank A. Johnson and G. Vert for their initial input and advice in setting up TIRF microscopy. Y.J. is funded by ERC no. 3363360-APPL under FP/2007-2013 and ANR caLIPSO (ANR18-CE13-0025). Y.J. and A.M. are funded by the innovative project iRhobot from the department of Biologie et Amélioration des Plantes of INRAE. A.M. is funded by the French National Agency ANR CellOsmo (ANR-19-CE20-0008-01). C.B. is funded by the Austrian Science Fund (FWF W1225). We acknowledge support by France-BioImaging (ANR-10-INBS-04, ‘Investments for the future’).

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Author notes

  1. These authors contributed equally: Vincent Bayle, Jean-Bernard Fiche.

Authors and Affiliations

  1. Laboratoire Reproduction et Développement des Plantes, Université de Lyon, ENS de Lyon, UCB Lyon 1, CNRS, INRAE, Lyon, France

    Vincent Bayle, Matthieu Pierre Platre & Yvon Jaillais

  2. Centre de Biochimie Structurale, Centre National de la Recherche Scientifique Unité Mixte de Recherche 5048, Institut National de la Santé et de la Recherche Médicale U1054, Université de Montpellier, Montpellier, France

    Jean-Bernard Fiche & Marcelo Nollmann

  3. Institut für Populationsgenetik, Vetmeduni Vienna, Vienna, Austria

    Claire Burny

  4. Vienna Graduate School of Population Genetics, Vetmeduni Vienna, Vienna, Austria

    Claire Burny

  5. BPMP, Univ Montpellier, CNRS, INRAE, Montpellier SupAgro, Montpellier, France

    Alexandre Martinière

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Contributions

V.B., M.P.P. and A.M. set up the imaging conditions and performed imaging. J.B.F. established the sptPALM analysis pipeline and coded the sptPALM_viewer software. V.B., A.M. and J.B.F. performed image analyses. C.B. established the statistical analysis pipeline and performed statistics. V.B., J.B.F., C.B., M.P.P., M.N., A.M. and Y.J. wrote the manuscript.

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Correspondence to Alexandre Martinière or Yvon Jaillais.

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Peer review information Nature Protocols thanks Guido Grossmann, Jinxing Lin and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Key references using this protocol:

Platre, M. P. et al. Science 364, 57–62 (2019): https://doi.org/10.1126/science.aav9959

Martinière, A. et al. Plant Physiol. 179, 1581–1593 (2019): https://doi.org/10.1104/pp.18.01065

Smokvarska, M. et al. Curr. Biol. 30, 4654–4664 (2020): https://doi.org/10.1016/j.cub.2020.09.013

Extended data

Extended Data Fig. 1 Fluorescence intensity of a typical mEOS2 sub-diffractive spot along time.

a, Pictures showing a single molecule through time (20 ms between each picture) and (b) corresponding trace of fluorescent intensity. Note that the signal intensity observed is not continuous, and the OFF state varies in duration between seconds and milliseconds. This blinking behavior is typical of single-molecule observation. Scale bar, 1 µm.

Extended Data Fig. 2 Example of false tracks identification.

a, Example of mis-reconnected tracks (white arrow). b, Example of track with a very long duration (white arrowhead), indicating that it is background fluorescence rather than true signal. The color gradient from blue, green to red indicates the early versus late recorded positions. Scale bars, 1 µm.

Extended Data Fig. 3 Mixture modeling reveals two latent subpopulations of Lti6B-mEos2 and PIP2;1-mEos2 molecules.

GMM (a and c) and FMSMSN (b and d) fits. Either a GMM-2V (a) or a FMSMSN-2 (d) model is retained for Lti6b and PIP2;1, respectively, using the BIC criterion. See the legend in Fig. 8c for more details.

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Bayle, V., Fiche, JB., Burny, C. et al. Single-particle tracking photoactivated localization microscopy of membrane proteins in living plant tissues. Nat Protoc 16, 1600–1628 (2021). https://doi.org/10.1038/s41596-020-00471-4

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  • DOI: https://doi.org/10.1038/s41596-020-00471-4

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