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Biomechanics

Biomechanics - Poster Session C

Poster E5 - Integrating a mechanosensitive reporter and a plant cell-on-a-chip system for quantitative mechanobiology studies in plants.

Friday, October 25, 2024
10:00 AM - 11:00 AM EST
Location: Exhibit Hall E, F & G

Introduction: The ability to respond to mechanical forces is a core function of all cells. In plants, thigmomorphogenesis, touch-responsive growth, is a long-known but poorly understood phenomenon. We aim to push this field forward by developing two complimentary reagents: a mechanosensitive live-cell reporter system and scaffolds compatible with plant cell adherence.
Group I bZIP transcription factors in several plant species 1–3 translocate from the cytoplasm to the nucleus following mechanical stimulation, analogous to YAP/TAZ movement in animal cells4. Using GFP-tagged bZIPs as live-cell reporters, we can actively monitor plant cell mechanosensation.
Cell-on-a chip systems are common in animal mechanobiology, with cells mechanically stimulated through deformation of the cell-bound scaffolds. Previously, plant cells were not amenable to such studies, as they do not adhere to most substrates. However, this is now possible using electro-spun PVDF-TrFE as biomimetic scaffolds for plant culture5.
We have united these tools in the model moss Physcomitrium patens, which is easily cultivated and imaged, and has highly tractable genetics. We have generated a stable bZIP-GFP reporter line in P. patens and validated that P. patens binds to the PVDF-TrFE scaffold6. Furthermore, we have defined the force threshold for initiating bZIP translocation and shown that physical manipulation of scaffolds can transduce mechanical forces that trigger translocation of the bZIP reporter. Together, these tools create a system for quantitatively studying mechanobiology in plant cells.

Materials and

Methods: The moss homolog of AtbZIP18 was identified by amino acid BLAST search as Pp13770. An overexpression construct was made using the maize ubiquitin promoter driving a Pp13770-GFP fusion (UBQ:Pp13770-GFP) and was transformed into the moss genome at the Pp108B locus.
For osmotic shock assays, moss samples were mounted in a flow cell setup on microscope slides in BCDAT media containing 400mM mannitol. Hypotonic treatments were conducted by flowing mannitol solutions ranging from -50 to -200 mM (Fig. 1A). Samples were imaged under a fluorescent dissecting microscope for two hours to visualize bZIP nuclear translocation. ImageJ was used to quantify the number of nuclei and the level of bZIP-GFP fluorescence in each nucleus.
To test mechanotransduction on scaffolds, moss cells were seeded on the PVDF-TrFE scaffolds, allowed to grow for four to six days, and then washed in liquid media for 30 minutes to remove unbound cells. Scaffolds were mounted in flow cells as described above and tested with osmotic downshocks. To test the ability of the scaffolds to transduce mechanical force to the cells, the seeded scaffolds were fixed to two aligned glass coverslips. The scaffolds were gently folded along the hinged axis between coverslips to perturb the bound cells (Fig. 1B). bZIP translocation was then observed as described above.

Results, Conclusions, and Discussions: The degree of nuclear translocation of bZIP-GFP correlated positively with the intensity of osmotic shock. Nuclear translocation was detectable beginning at a -100 mM mannitol down shock, with no significant nuclear signal apparent at -50mM mannitol down shock or with isotonic controls. Under conditions that induce nuclear translocation, the nuclear signal rises steadily until peaking at between 50 and 60 minutes post-shock, then decreasing back to initial levels (Fig. 2). There is a similar response to osmotic shock from cells seeded on scaffolds, with the same trend of bZIP nuclear translocation at the same time scale. This shows that adhesion of moss cells to a scaffold does not affect their sensitivity to osmotic stimulation.
In our scaffold folding experiments, bZIP nuclear translocation occurred at much earlier time points compared to the osmotic shock treatment, with a peak of nuclear signal at around 10 minutes post scaffold folding and a decrease to baseline signal by 30-40 minutes.
Together, our results demonstrate the utility of the bZIP reporter system to monitor the effects of osmotic and mechanical perturbations in living plant cells. This tool opens the door for further research in understanding the mechanisms underlying the mechanobiology of plant cells.

Acknowledgements (Optional):