Assessing the hydromechanical control of plant growth.

Multicellular organisms grow and acquire their shapes through the differential expansion and deformation of their cells. Recent research has addressed the role of cell and tissue mechanical properties in these processes. In plants, it is believed that growth rate is a function of the mechanical stress exerted on the cell wall, the thin polymeric layer surrounding cells, involving an effective viscosity. Nevertheless, recent studies have questioned this view, suggesting that cell wall elasticity sets the growth rate or that uptake of water is limiting for plant growth. To assess these issues, we developed a microfluidic device to quantify the growth rates, elastic properties and hydraulic conductivity of individual Marchantia polymorpha plants in a controlled environment with a high throughput. We characterized the effect of osmotic treatment and abscisic acid on growth and hydromechanical properties. Overall, the instantaneous growth rate of individuals is correlated with both bulk elastic modulus and hydraulic conductivity. Our results are consistent with a framework in which the growth rate is determined primarily by the elasticity of the wall and its remodelling, and secondarily by hydraulic conductivity. Accordingly, the coupling between the chemistry of the cell wall and the hydromechanics of the cell appears as key to set growth patterns during morphogenesis.

A Magnetic Pincher for the Dynamic Measurement of the Actin Cortex Thickness in Live Cells.

The actin cortex is an essential element of the cytoskeleton allowing cells to control and modify their shape. It is involved in cell division and migration. However, probing precisely the physical properties of the actin cortex has proved to be challenging: it is a thin and dynamic material, and its location in the cell-directly under the plasma membrane-makes it difficult to study with standard light microscopy and cell mechanics techniques. In this chapter, we present a novel protocol to probe dynamically the thickness of the cortex and its fluctuations using superparamagnetic microbeads in a uniform magnetic field. A bead ingested by the cell and another outside the cell attract each other due to dipolar forces. By tracking their position with nanometer precision, one can measure the thickness of the cortex pinched between two beads and monitor its evolution in time. We first present the set of elements necessary to realize this protocol: a magnetic field generator adapted to a specific imaging setup and the aforementioned superparamagnetic microbeads. Then we detail the different steps of a protocol that can be used on diverse cell types, adherent or not.

Pinching the cortex of live cells reveals thickness instabilities caused by myosin II motors.

The cell cortex is a contractile actin meshwork, which determines cell shape and is essential for cell mechanics, migration, and division. Because its thickness is below optical resolution, there is a tendency to consider the cortex as a thin uniform two-dimensional layer. Using two mutually attracted magnetic beads, one inside the cell and the other in the extracellular medium, we pinch the cortex of dendritic cells and provide an accurate and time-resolved measure of its thickness. Our observations draw a new picture of the cell cortex as a highly dynamic layer, harboring large fluctuations in its third dimension because of actomyosin contractility. We propose that the cortex dynamics might be responsible for the fast shape-changing capacity of highly contractile cells that use amoeboid-like migration.

Mechanosensitivity of Cancer Cells in Contact with Soft Substrates Using AFM.

Cancer cells are usually found to be softer than normal cells, but their stiffness changes when they are in contact with different environments because of mechanosensitivity. For example, they adhere to a given substrate by tuning their cytoskeleton, thus affecting their rheological properties. This mechanism could become efficient when cancer cells invade the surrounding tissues, and they have to remodel their cytoskeleton in order to achieve particular deformations. Here we use an atomic force microscope in force modulation mode to study how local rheological properties of cancer cells are affected by a change of the environment. Cancer cells were plated on functionalized polyacrylamide substrates of different stiffnesses as well as on an endothelium substrate. A new correction of the Hertz model was developed because measurements require one to account for the precise properties of the thin, layered viscoelastic substrates. The main results show the influence of local cell rheology (the nucleus, perinuclear region, and edge locations) and the role of invasiveness. A general mechanosensitive trend is found by which the cell elastic modulus and transition frequency increase with substrate elasticity, but this tendency breaks down with a real endothelium substrate. These effects are investigated further during cell transmigration, when the actin cytoskeleton undergoes a rapid reorganization process necessary to push through the endothelial gap, in agreement with the local viscoelastic changes measured by atomic force microscopy. Taken together, these results introduce a paradigm for a new-to our knowledge-possible extravasation mechanism.