Mechanotransduction at the nucleus in plants: from chromatin organization to cell fate decision – Mecha-NUC

Various arabidopsis transgenic lines co-expressing fluorescent markers of the cytoskeleton (microtubules or actin filaments, green markers) and the nuclear envelope (red marker) in wild-type or mutated plants were established. Different microfluidic and micromechanical tools were developed.

At the tissue level:

- Proteomic and transcriptomic analyses under experimental mechanical stress conditions (hyperosmotic stress)

- Modeling tests of chromatin organization at the periphery of the nuclear envelope

At the cellular level:

- Analysis of cytoskeletal and nuclear dynamics by high-resolution microscopy of root hairs using microfluidic chips

- Mathematical modeling of root hair development by integrating the forces acting on the nuclear envelope

- AFM measurement of root hairs in the sheath regions and near the root hair tip

- Compression of root hairs or protoplasts isolated from roots

- Modeling tests of chromatin organization at the periphery of the nuclear envelope.

Our major findings concern the impact of dynamic cytoskeletal and nuclear changes on root hair growth in Arabidopsis using microfluidic chips. We monitored cytoskeletal (microtubules, actin filaments) and nuclear dynamics in plants expressing fluorescent cytoskeletal and nuclear markers using high-resolution real-time imaging. We identified three distinct stages—rapid growth, slow growth, and early maturation—and quantified growth kinetics with high temporal resolution. The transition from rapid to slow growth is associated with dynamic cytoskeletal changes, leading to reduced apical growth and a decreased nucleus-tip distance. Based on these observations, we developed a mathematical model linking cytoskeletal and nuclear dynamics to apical growth by integrating the forces acting on the nuclear envelope. Using genetic and pharmacological approaches, we were able to disrupt or trigger this transition, thus confirming the model and revealing a crucial interaction between actin filaments and microtubules at the nuclear envelope.

Vacuole dynamics, as well as cell diameter and rigidity, vary in the subapex region during the transition from rapid to slow growth, indicating coordinated regulation of several subcellular systems.

Furthermore, root hair growth on different agar concentrations can be correlated with mechanical stress induced by changes in substrate rigidity. An increase in substrate rigidity leads to slowed root hair growth and a reduction in nuclear displacement velocity within the root hair. Finally, to correlate dynamic changes in nuclear shape with the 3D organization of chromatin, statistical analysis tools were developed (BIP program).

- Mecha-NUC represents a stepping stone towards modeling apical growth by integrating various parameters such as cytoskeletal dynamics, nuclear dynamics (shape and movement), and cellular mechanics (viscoelastic model).

- This project will be continued within the framework of a CNRS-MITI program between P1 and P2, and in collaboration with P3 to address in more detail the dynamics of changes in the spatial organization of chromatin, its modeling as well as the epigenetic and transcriptional responses after a mechanical stress applied to a single cell in microfluidic chips.

- This should also lead to a better understanding of root growth and improve the agronomic characteristics of plant growth under environmental stress conditions ( osmotic, mechanical stresses).

All living organisms are able to sense mechanical forces such as compression, tension or shear. The transduction of such mechanical signals contributes to changes in cell shape and fate. Relevant cortical mechanosensing pathways start to be deciphered, but the nuclear mechanotransduction linking nuclear mechanics to specific gene expression regulation and cell fate decisions remains unclear. Our work, recently published in Current Biology, indicates that physiological mechanical perturbations applied on root meristematic cells in the plant model Arabidopsis, impacts the shape, stiffness and chromatin organization of nuclei together with the expression of mechanosensitive genes. GIP proteins are present at the nuclear envelope. They control nuclear shape and rigidity, microtubule nucleation and centromere architecture. GIP-dependent changes in rheological properties of the nucleus may affect cell fate too. Based on our data, we hypothesize that, in plants, the nuclear envelope integrates peripheral mechanical cues to control nuclear mechanics, chromatin state and gene expression. In the Mecha-NUC project, we will address two main questions in plants: What is the identity of the nuclear mechanotransduction pathway? and to what extent it affects nuclear mechanics, resulting in changes in chromatin organization, gene expression and cell fate decisions? The questions we address are novel in plants and open new avenues in nuclear mechanotransduction in walled cells at different scales, from nuclei in isolated single cells to nuclei in living tissues. Focusing on the microtubule-based GIP-dependent signaling pathway in relationship with the plant specific nucleoskeleton, we will decipher a new mechanotransduction pathway in plants. Our project is at the interface of cell biology and physics, and includes approaches of micromechanics, molecular biology, omics, and high-resolution in vivo imaging as well as computational 3D modelling.