Cortical tension overrides geometrical cues to orient microtubules in confined protoplasts

Abstract

In plant cells, cortical microtubules (CMTs) generally control morphogenesis by guiding cellulose synthesis. CMT alignment has been proposed to depend on geometrical cues, with microtubules aligning with the cell long axis in silico and in vitro. Yet, CMTs are usually transverse in vivo, i.e., along predicted maximal tension, which is transverse for cylindrical pressurized vessels. Here, we adapted a microwell setup to test these predictions in a single-cell system. We confined protoplasts laterally to impose a curvature ratio and modulated pressurization through osmotic changes. We find that CMTs can be longitudinal or transverse in wallless protoplasts and that the switch in CMT orientation depends on pressurization. In particular, longitudinal CMTs become transverse when cortical tension increases. This explains the dual behavior of CMTs in planta: CMTs become longitudinal when stress levels become low, while stable transverse CMT alignments in tissues result from their autonomous response to tensile stress fluctuations.

Imaging the living plant cell: From probes to quantification

“…Such an approach has been successfully used to study the anisotropy of the microtubule network after genetic perturbation or pharmacological treatment in different systems (Robinson and Kuhlemeier, 2018;Riglet et al, 2020;Zhao et al, 2020). Similar approaches were recently used to quantify how geometry affects cytoskeletal organization by confining single cells (or protoplasts) within microfabricated microwells of various geometries (see (Colin et al, 2020;Durand-Smet et al, 2020) and the last paragraph of this review). This plugin has been integrated into the MorphographX platform (de Reuille et al, 2015), thus allowing microtubule organization on computer-assisted cell segmentations to be analyzed (see next paragraph).…”

“…In developmental mechanobiology, single-cell systems represent a simpler model to study the role of mechanical forces in cellulo, avoiding the additional complexity brought about by the tissue context (e.g., chemical signals, impact of neighboring cells, complex mechanical stress patterns). Recent studies have used such single cell approaches to assess the relative contributions of both cell geometry and cortex tension to cortical microtubule behavior (Colin et al, 2020;Durand-Smet et al, 2020). In these studies, wall-less plant cells, also called protoplasts, were confined in microfabricated wells of various shapes and sizes (Figure 4A).…”

“…The protoplasts were either placed under hyperosmotic conditions (i.e., with a reduced cortex tension) or under hypoosmotic conditions (i.e., with an increased cortex tension), and cortical microtubule orientation was then analyzed (Figure 4B and C). In cells confined in rectangular microwells and exhibiting a reduced cortex tension, cortical microtubules tended to align with the long axis of the cell (Colin et al, 2020;Durand-Smet et al, 2020). By contrast, using microwells of a similar shape, in cells with an increased cortex tension, cortical microtubules mainly aligned with the shortest axis of the cell, which also corresponds to the principal stress direction in these cells (Colin et al, 2020).…”

Mechanobiology of cell division in plant growth

“…1e). However, when turgor pressure is high enough to induce surface tension the microtubules align with the direction of maximal tensile stress, which is transverse to the long axis in elongated cells (Colin et al, 2020;Fig. 1f).…”

“…(e) At low turgor the microtubules align along the long axis of the cell (Durand‐Smet et al ., 2020). (f) At high turgor the microtubules align with the direction of maximal tensile stress (Colin et al ., 2020). (g) The polarised protein BASL (green) may align with mechanical stress or global polarity factors, BASL displaces the nucleus away from the centre of mass to produce an asymmetrical division (Bringmann & Bergmann, 2017; Mansfield et al ., 2018; Muroyama et al ., 2020).…”