Correlative light-environmental scanning electron microscopy of plasma membrane efflux carriers of plant hormone auxin

0546239 - ÚPT 2022 RIV CH eng J - Journal Article
Stelate, A. - Tihlaříková, Eva - Schwarzerová, K. - Neděla, Vilém - Petrášek, J.
Correlative light-environmental scanning electron microscopy of plasma membrane efflux carriers of plant hormone auxin.
Biomolecules. Roč. 11, č. 10 (2021), č. článku 1407. E-ISSN 2218-273X
R&D Projects: GA ČR(CZ) GA19-03909S
Institutional support: RVO:68081731
Keywords : correlative microscopy * plasma membrane * nanodomains * auxin carriers
OECD category: Plant sciences, botany
Impact factor: 6.064, year: 2021
Method of publishing: Open access
https://www.mdpi.com/2218-273X/11/10/1407

Fluorescence light microscopy provided convincing evidence for the domain organization of plant plasma membrane (PM) proteins. Both peripheral and integral PM proteins show an inhomo-geneous 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.
Permanent Link: http://hdl.handle.net/11104/0325294