Shedding light on protein interaction networks in a developing organism
Researchers succeeded for the first time in mapping protein-protein interactions in living developing plant roots. The findings of the international team of the department of Plant Developmental Biology at Wageningen University and Research, the section of Molecular Cytology at the University of Amsterdam and the University of Düsseldorf were published in Nature .
This work is considered as a breakthrough in the field of cell and developmental biology because it is now possible to visualize interactions between proteins under physiological conditions within intact living tissue in a non-invasive manner. This approach will allow better understanding of protein-binding events, which are essential for cellular function as well as their dysfunction in diseases.
The published work in Nature entitled 'In vivo FRET-FLIM reveals cell type-specific protein interactions in Arabidopsis roots' combined FRET-FLIM (Förster Resonance Energy Transfer) –Fluorescence Lifetime imaging Microscopy) technology with genetics and gene expression analysis. The work unravelled that in living tissues, protein complexes can change their conformation in a cell type dependent manner to regulate specific gene expression programs leading to precise specification and maintenance of particular cell fates within the Arabidopsis root meristem.
Understanding root growth
At the tip of a developing and growing root a population of 'stem cells' reside. These cells divide and differentiate into different cell types generating different tissues in the plant root. The fate of each cell, hence what kind of root tissue it will become, is determined by special proteins, among which transcription factors play a crucial role. In order to better understand how these proteins regulate root growth the team of Ikram Blilou, together with Yuchn Long, set out to determine the interactions between a set of key transcription factors. In particular they focused on how these interactions change during growth and susbequent impact on cell fate and root development.
It is technically very challenging to elucidate protein interactions in living organisms at the cellular level because the expression level of these proteins is low, making it difficult to pick up these interactions in living tissue. In this study the researchers used state-of-the-art functional imaging and molecular biology techniques to visualize specific interactions using FRET-FLIM (Förster Resonance Energy Transfer –fluorescence lifetime imaging) technique in combination with genetics and gene expression analysis. Proteins interact by direct binding, this means they come very close to each other and in particular FRET techniques can be used to detect these interactions by tagging the proteins of interest with genetically encoded fluorescent proteins.
This study is the result of a successful collaboration between three international teams, in addition to Plant Developmental Biology group at Wageningen University headed by Prof Ben Scheres, the work involved Prof. Theodorus Gadella from the Van Leeuwenhoek Centre for Advanced Microscopy in Amsterdam. The team of Gadella, including Joachim Goedhart and Marten Postma, has a long standing expertise in optimization of fluorescent proteins, quantitative microscopy and advanced FRET-FLIM analysis. Together with the team of Rüdiger Simon from Düsseldorf University, including Yvonne Stahl and Stephanie Stefanie-Weidtkamp-Peters with expertise on high resolution life time imaging, the FRET-FLIM technology was implemented overcoming the current limitations for studying protein complex dynamics at cellular resolution.
Yuchen Long, Yvonne Stahl, Stefanie Weidtkamp-Peters, Marten Postma, Wenkun Zhou, Joachim Goedhart, María-Isabel Sánchez-Pérez, Theodorus W. J. Gadella, Rüdiger Simon, Ben Scheres & Ikram Blilou: ‘In vivo FRET–FLIM reveals cell-type-specific protein interactions in Arabidopsis roots’, in Nature (26 July 2017). http://dx.doi.org/ 10.1038/nature23317