Experimental toolbox for quantitative evaluation of clathrin-mediated endocytosis in the plant model Arabidopsis

A.J. Johnson, N. Gnyliukh, W. Kaufmann, M. Narasimhan, G. Vert, S. Bednarek, J. Friml, Journal of Cell Science 133 (2020).

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Abstract
Clathrin-mediated endocytosis (CME) is a crucial cellular process implicated in many aspects of plant growth, development, intra- and inter-cellular signaling, nutrient uptake and pathogen defense. Despite these significant roles, little is known about the precise molecular details of how it functions in planta. In order to facilitate the direct quantitative study of plant CME, here we review current routinely used methods and present refined, standardized quantitative imaging protocols which allow the detailed characterization of CME at multiple scales in plant tissues. These include: (i) an efficient electron microscopy protocol for the imaging of Arabidopsis CME vesicles in situ, thus providing a method for the detailed characterization of the ultra-structure of clathrin-coated vesicles; (ii) a detailed protocol and analysis for quantitative live-cell fluorescence microscopy to precisely examine the temporal interplay of endocytosis components during single CME events; (iii) a semi-automated analysis to allow the quantitative characterization of global internalization of cargos in whole plant tissues; and (iv) an overview and validation of useful genetic and pharmacological tools to interrogate the molecular mechanisms and function of CME in intact plant samples.
Publishing Year
Date Published
2020-08-06
Journal Title
Journal of Cell Science
Acknowledgement
This paper is dedicated to the memory of Christien Merrifield. He pioneered quantitative imaging approaches in mammalian CME and his mentorship inspired the development of all the analysis methods presented here. His joy in research, pure scientific curiosity and microscopy excellence remain a constant inspiration. We thank Daniel Van Damme for gifting us the CLC2-GFP x TPLATE-TagRFP plants used in this manuscript. We further thank the Scientific Service Units at IST Austria; specifically, the Electron Microscopy Facility for technical assistance (in particular Vanessa Zheden) and the BioImaging Facility BioImaging Facility for access to equipment.
Volume
133
Issue
15
Article Number
jcs248062
IST-REx-ID

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Johnson AJ, Gnyliukh N, Kaufmann W, et al. Experimental toolbox for quantitative evaluation of clathrin-mediated endocytosis in the plant model Arabidopsis. Journal of Cell Science. 2020;133(15). doi:10.1242/jcs.248062
Johnson, A. J., Gnyliukh, N., Kaufmann, W., Narasimhan, M., Vert, G., Bednarek, S., & Friml, J. (2020). Experimental toolbox for quantitative evaluation of clathrin-mediated endocytosis in the plant model Arabidopsis. Journal of Cell Science, 133(15). https://doi.org/10.1242/jcs.248062
Johnson, Alexander J, Nataliia Gnyliukh, Walter Kaufmann, Madhumitha Narasimhan, G Vert, SY Bednarek, and Jiří Friml. “Experimental Toolbox for Quantitative Evaluation of Clathrin-Mediated Endocytosis in the Plant Model Arabidopsis.” Journal of Cell Science 133, no. 15 (2020). https://doi.org/10.1242/jcs.248062.
A. J. Johnson et al., “Experimental toolbox for quantitative evaluation of clathrin-mediated endocytosis in the plant model Arabidopsis,” Journal of Cell Science, vol. 133, no. 15, 2020.
Johnson AJ, Gnyliukh N, Kaufmann W, Narasimhan M, Vert G, Bednarek S, Friml J. 2020. Experimental toolbox for quantitative evaluation of clathrin-mediated endocytosis in the plant model Arabidopsis. Journal of Cell Science. 133(15).
Johnson, Alexander J., et al. “Experimental Toolbox for Quantitative Evaluation of Clathrin-Mediated Endocytosis in the Plant Model Arabidopsis.” Journal of Cell Science, vol. 133, no. 15, jcs248062, The Company of Biologists, 2020, doi:10.1242/jcs.248062.

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