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

Abstract

SummaryExogenous 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 h 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.

External mechanical cues reveal core molecular pathway behind tissue bending in plants

“…At first glance, our 407 observations are in conflict with the role of katanin in mediating the response of CMTs to 408 external mechanical cues (Sampathkumar, et al, 2014). However, our data are consistent 409 with slower but pronounced reorganization of CMTs in meristems of katanin mutants 410 subjected to prolonged mechanical cues (Louveaux, et al, 2016; Uyttewaal, et al, 2012). 411 Katanin-independent CMT reorganization can happen by motor protein mediated 412 reorientation of CMTs (Inoue, et al, 2016).…”

An Automated Confocal Micro-Extensometer Enables in Vivo Quantification of Mechanical Properties with Cellular Resolution

“…A range of methods have been used to demonstrate that plants respond to mechanical stress. These include making cuts or ablations (Hamant et al, 2008;Sampathkumar et al, 2014), osmotic stress , compressing the tissue Sampathkumar et al, 2014;Louveaux et al, 2016), or large-scale deformations (Bringmann and Bergmann, 2017). These methods also induce other types of stress to the tissue, for example, wounding or drought.…”

“…Extensometers are naturally easier to couple to a confocal microscope compared with indentation devices, as they measure perpendicular to the imaging axis. Indentation methods by contrast obscure the image and must be used sequentially Louveaux et al, 2016).…”

An automated confocal micro-extensometer enables in vivo quantification of mechanical properties with cellular resolution

“…Extensometers are naturally easier to couple to a confocal microscope as they measure perpendicular to the imaging axis. Indentation methods by contrast obscure the image, and must be used sequentially, Louveaux et al, 2016).…”