Nejvíce citovaný článek - PubMed ID 31610051
Cell wall contributes to the stability of plasma membrane nanodomain organization of Arabidopsis thaliana FLOTILLIN2 and HYPERSENSITIVE INDUCED REACTION1 proteins
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.
- Klíčová slova
- auxin carriers, correlative microscopy, nanodomains, plasma membrane,
- MeSH
- Arabidopsis genetika růst a vývoj MeSH
- buněčná membrána genetika metabolismus ultrastruktura MeSH
- konfokální mikroskopie MeSH
- kovové nanočástice chemie MeSH
- kyseliny indoloctové metabolismus MeSH
- mikroskopie elektronová rastrovací * MeSH
- počítačové zpracování obrazu MeSH
- protoplasty metabolismus ultrastruktura MeSH
- regulátory růstu rostlin genetika metabolismus MeSH
- tabák genetika metabolismus ultrastruktura MeSH
- zlato chemie MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- kyseliny indoloctové MeSH
- regulátory růstu rostlin MeSH
- zlato MeSH
