期刊
BIOMOLECULES
卷 11, 期 10, 页码 -出版社
MDPI
DOI: 10.3390/biom11101407
关键词
correlative microscopy; plasma membrane; nanodomains; auxin carriers
资金
- Czech Science Foundation [19-03909S, CZ.01.1.02/0.0/0.0/17_176/0015020]
- MEYS of the Czech Republic
- OPPK project [CZ.2.16/3.1.00/21515]
- large RI project National Infrastructure for Biological and Medical Imaging Czech-BioImaging [LM2018129]
A novel correlative light electron microscopy method has been developed for accurate study of domain organization of plant plasma membrane proteins, allowing quantification of the number of auxin efflux carriers within nanodomains.
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.
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