The impact of mechanical compression on cortical microtubules in Arabidopsis: a quantitative pipeline

Exogenous mechanical perturbations on living tissues are commonly used to investigate whether cell effectors can respond to mechanical cues. However, in most of these experiments, the applied mechanical stress and/or the biological response are described only qualitatively. We developed a quantitative pipeline based on microindentation and image analysis to investigate the impact of a controlled and prolonged compression on microtubule behaviour in the Arabidopsis shoot apical meristem, using microtubule fluorescent marker lines. We found that a compressive stress, in the order of magnitude of turgor pressure, induced apparent microtubule bundling. Importantly, that response could be reversed several hours after the release of compression. Next, we tested the contribution of microtubule severing to compression-induced bundling: microtubule bundling seemed less pronounced in the katanin mutant, in which microtubule severing is dramatically reduced. Conversely, some microtubule bundles could still be observed 16 hours after the release of compression in the spiral2 mutant, in which severing rate is instead increased. To quantify the impact of mechanical stress on anisotropy and orientation of microtubule arrays, we used the nematic tensor based FibrilTool ImageJ/Fiji plugin. To assess the degree of apparent bundling of the network, we developed several methods, some of which were borrowed from geostatistics. The final microtubule bundling response could notably be related to tissue growth velocity that was recorded by the indenter during compression. Because both input and output are quantified, this pipeline is an initial step towards correlating more precisely the cytoskeleton response to mechanical stress in living tissues.

Keyword(s)

mechanical stress, indenter, microtubules, compression, bundling, Arabidopsis thaliana, technical advance

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Figure S1. Examples of cells pairs from indented meristems extracted from snapshots taken at time zero, i.e. before compression (left cell), and at time 6 h 45, i.e. 15 mins after release of compressi
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Figure S2. Microtubule bundling persistence 16h after release of compression in two spr2-2 GFP-MBD meristems.
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Figure S3. Methods used to rescale green intensities values, when exposure was too low, illustrated with GFP-MBD images (see also Experimental procedures).
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Figure S4. Examples of analyses performed on compressed meristems in (a) spr2-2 GFP-MBD and (b) bot1-7 GFP-MBD.
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Table S1. Wilcoxon rank sum tests p-values.
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Supporting Information Legends
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How to cite
Louveaux Marion, Rochette Sebastien, Beauzamy Lena, Boudaoud Arezki, Hamant Olivier (2016). The impact of mechanical compression on cortical microtubules in Arabidopsis: a quantitative pipeline. Plant Journal. 88 (2). 328-342. https://doi.org/10.1111/tpj.13290, https://archimer.ifremer.fr/doc/00347/45829/

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