Probing the self-organization of plant cells by micropattern and microfluidics
Final Report Abstract
Whereas cell movement is the central mechanism for animal morphogenesis, plant cell development rather relies on flexible alignment of cell axiality adjusting cellular differentiation to directional cues. As central input, vectorial fields of mechanical stress and gradients of the phytohormone auxin have been discussed. In tissue contexts, mechanical and chemical signals will always act in concert; experimentally it is difficult to dissect their individual roles. We therefore established an experimental system that allows to follow the generation of cellular directionality "de novo" based on regenerating protoplasts of the tobacco cell line BY-2, where An experimental system was worked out to follow the generation of cellular directionality "de novo". Transgenic cell lines expressing fusions of cytoskeletal markers with the green fluorescent protein were integrated into this system. This allowed to develop a working model on the function of actin filaments and microtubules for axis formation. We could show that the response of actin filaments was crucial for polarity induction. To identify the molecular target for auxin mediating this actin response, a panel of transgenic cell lines was generated overexpressing different actin-binding proteins. From a screen of this cell-line collection, actin-depolymerization factor 2 (ADF2) was demonstrated as central player. A functional analysis revealed that auxin regulates the interaction of actin filaments with the membrane through binding of ADF2 to PIP2 under control of G-protein controlled activity of phospholipase D. This biological system for polarity induction "de novo" was then integrated into the microfluidic devices fabricated by the Chinese partner. This step was re-iterated numerous times until a reliable model was achieved that preserved the physiology of the cells and allowed for microscopic observation. Using this system, chemical gradients (using specific auxins with differing transport properties as well as specific inhibitors of auxin efflux) were administered in a direction perpendicular to the physical cues provided by the geometry of micro-chambers and the effect on de-novo axiality of the regenerating cells was studied on a quantitative level. We show that the regenerating protoplast form a new axis in harmony with the geometry of their environment. To explore geometry, they use auxin as chemical cue, before facing constraints from physical contact with the wall of the vessel (or, in real tissues, the counterforce from a cell wall). This chemical sensing of geometry requires auxin efflux with input from local auxin concentration.
Publications
- Dynamic actin controls polarity induction de novo in protoplasts.
Journal of Integrative Plant Biology, Vol. 55. 2013, Issue 2, pp. 142–159.
Zaban B., Maisch J., Nick P.
(See online at https://doi.org/10.1111/jipb.12001) - Nicotiana tabacum actin-depolymerizing factor 2 is involved in actin-driven, auxin-dependent patterning. Journal of Plant Physiology, Vol. 170. 2013, Issue 12, pp. 1057-1066.
Durst S., Nick P., Maisch J.
(See online at https://doi.org/10.1016/j.jplph.2013.03.002) - Plant Cells Use Auxin Fluxes to Explore Geometry. Nature - Scientific Reports, Vol. 4. 2014, Article number: 5852.
Zaban B., Liu W.W., Jiang X., Nick P.
(See online at https://doi.org/10.1038/srep05852)
