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Single-molecule fluorescence imaging to quantify membrane protein dynamics and oligomerization in living plant cells

Nature Protocols volume 10, pages 2054–2063 (2015)Cite this article

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Abstract

Measuring the mobility and interactions of proteins is key to understanding cellular signaling mechanisms; however, quantitative analysis of protein dynamics in living plant cells remains a major challenge. Here we describe an automated, single-molecule protocol based on total internal reflection fluorescence microscopy (TIRFM) imaging that allows protein tracking and subunit counting in living plant cells. This protocol uses TIRFM to image transgenic plant tissues expressing fluorescently tagged proteins that are localized to the plasma membrane. Next, a tracking algorithm quantifies dynamic changes in fluorescent protein motion types, temporary particle displacement and protein photobleaching steps. This protocol allows researchers to study the kinetic characteristics of heterogeneously distributed proteins. The approach has potential applications for studies of protein dynamics and subunit stoichiometry for a wide variety of plasma membrane and intracellular proteins in living plant cells and other biological specimens visualized by TIRFM or other fluorescence imaging techniques. The whole protocol can be completed in 5–6 h.

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Figure 1: The detection and tracking interface.
Figure 2: Automated tracking of GFP-PIP2;1 and GFP-AMT1;3 particles on the plasma membrane.
Figure 3: Photobleaching traces of mGFP, GFP-AMT1;3 and GFP-PIP2;1 single particles on the plasma membrane.

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Acknowledgements

We thank K. Jaqaman and R. Parthasarathy for providing the original algorithm source code. This work is supported by the Program of Introducing Talents of Discipline to Universities (111 project, B13007), the Major Science Foundation of the Ministry of Education of China (no. 313008), the National Basic Research Program of China (973 Program 2011CB809103) and the National Nature Science Foundation of China Project (grant nos. 31270412 and 31270224).

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Author notes

  1. Xiaohua Wang and Xiaojuan Li: These authors contributed equally to this work.

Authors and Affiliations

  1. Key Laboratory of Plant Resources, Institute of Botany, Chinese Academy of Sciences, Beijing, China

    Xiaohua Wang & Xin Deng

  2. Key Laboratory for Genetics and Breeding of Forest Trees and Ornamental Plants, Ministry of Education, College of Biological Sciences and Technology, Beijing Forestry University, Beijing, China

    Xiaojuan Li & Jinxing Lin

  3. Biochimie et Physiologie Moléculaire des Plantes, Institut de Biologie Intégrative des Plantes, Unité Mixte de Recherche (UMR) 5004 Centre National de la Recherche Scientifique (CNRS) UMR 0386 Institut National de la Recherche Agronomique,

    Doan-Trung Luu & Christophe Maurel

  4. /Montpellier SupAgro/Université Montpellier 2, Montpellier, France

    Doan-Trung Luu & Christophe Maurel

Authors
  1. Xiaohua Wang
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Contributions

X.W. and X.L. combined the MATLAB scripts and wrote the paper. X.D. and D.-T.L. performed the computational analysis. C.M. edited and revised the paper. J.L. designed the experiment and revised the paper.

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Correspondence to Jinxing Lin.

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Wang, X., Li, X., Deng, X. et al. Single-molecule fluorescence imaging to quantify membrane protein dynamics and oligomerization in living plant cells. Nat Protoc 10, 2054–2063 (2015). https://doi.org/10.1038/nprot.2015.132

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