Correlative Light-Environmental Scanning Electron Microscopy of Plasma Membrane Efflux Carriers of Plant Hormone Auxin.

Ayoub Stelate,Kateřina Schwarzerová,Vilém Neděla,Jan Petrášek,Eva Tihlaříková

doi:10.3390/biom11101407

Ayoub Stelate, Kateřina Schwarzerová + Show 3 more

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https://doi.org/10.3390/biom11101407

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Abstract

Fluorescence light microscopy provided convincing evidence for the domain organization of plant plasma membrane (PM) proteins. Both peripheral and integral PM proteins show an inhomogeneous distribution within the PM. However, the size of PM nanodomains and protein clusters is too small to accurately determine their dimensions and nano-organization using routine confocal fluorescence microscopy and super-resolution methods. To overcome this limitation, we have developed a novel correlative light electron microscopy method (CLEM) using total internal reflection fluorescence microscopy (TIRFM) and advanced environmental scanning electron microscopy (A-ESEM). Using this technique, we determined the number of auxin efflux carriers from the PINFORMED (PIN) family (NtPIN3b-GFP) within PM nanodomains of tobacco cell PM ghosts. Protoplasts were attached to coverslips and immunostained with anti-GFP primary antibody and secondary antibody conjugated to fluorochrome and gold nanoparticles. After imaging the nanodomains within the PM with TIRFM, the samples were imaged with A-ESEM without further processing, and quantification of the average number of molecules within the nanodomain was performed. Without requiring any post-fixation and coating procedures, this method allows to study details of the organization of auxin carriers and other plant PM proteins.

Highlights

Plant plasma membrane (PM) is a dynamic structure composed of lipids and proteins that forms a boundary between intracellular and cell wall space
The heterogeneity in the distribution of this protein within the PM of protoplasts, which we show here by total internal reflection fluorescence microscopy (TIRFM), is in good agreement with several recently published reports on the distribution of Arabidopsis thaliana PIN proteins within PM, including nanodomains of PIN3 in the hypocotyl epidermal cells shown by Airyscan confocal laser scanning microscopy [17]
Clusters of PIN2-GFP in the root epidermal cells shown by transmission electron microscopy on immunostained platinum replicas [35]

Summary

Introduction

Plant PM is a dynamic structure composed of lipids and proteins that forms a boundary between intracellular and cell wall space. PM is a highly dynamic structure, it contains functionally and structurally defined domains [2]. Domain organization of the PM allows correct progression of transport and signaling processes, which coordinate plant development and their reactions to abiotic and biotic stimuli from the environment [7,8]. Auxin efflux carriers from PIN family are one of the most extensively studied plant-integral PM proteins with numerous developmental roles [9]. Their heterogeneity in various polar PM domains is collectively supported by several mechanisms, including targeted secretion, constitutive recycling, and lateral diffusion within the PM [10,11]

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Conclusion