Molecular tension microscopy of the LINC complex in live cells.

We present a protocol to measure the effect of pharmacological treatments on the mechanical tension experienced by nesprins at the cytoplasmic surface of the nuclear envelope of mammalian cells in culture. We apply this protocol to MDCK epithelial cells exposed to the actin depolymerization agent cytochalasin D. To do so, we perform confocal spectral imaging of transiently expressed molecular tension sensors of mini-nesprin 2G and analyze the FRET signal from the sensors with a custom-made Fiji script. For complete details on the use and execution of this protocol, please refer to Déjardin et al. (2020).

[Light-sheet based fluorescence microscopy: the dark side of the sample finally revealed]. / Microscopie de fluorescence à feuille de lumière - La face cachée de l'échantillon enfin dévoilée.

Light-sheet based fluorescence microscopy (LSM) is an optical technique that becomes more and more popular for multi-view imaging of in vivo sample in its physiological environment. LSM combines the advantages of the direct optical sectioning to the ones of optical tomography by angular scanning. In fact, a thin light-sheet illuminates laterally a section of the sample, thus limiting the effects of photobleaching and phototoxicity only to the plane of interest. The spatial resolution can be improved by combining multiple views obtained along different angle into a single data, leading to a 3D isotropic rendering of the sample. Such an approach provides several advantages in comparison to conventional 3D microscopic techniques: confocal and multiphoton microscopies. It makes LSM an optical tool suited for imaging specimens with a subcellular resolution even inside an embryo and with temporal resolution adapted for real-time monitoring of biological processes.

Molecular Tension Microscopy of E-Cadherin During Epithelial-Mesenchymal Transition.

Molecular Tension Microscopy has been increasingly used in the last years to investigate mechanical forces acting in cells at the molecular scale. Here, we describe a protocol to image the tension of the junctional protein E-cadherin in cultured epithelial cells undergoing Epithelial-Mesenchymal Transition (EMT). We report how to prepare cells and induce EMT, and how to acquire, analyze, and quantitatively interpret FRET data.

Cellular chemomechanics at interfaces: sensing, integration and response.

Living cells are complex entities whose remarkable, emergent capacity to sense, integrate, and respond to environmental cues relies on an intricate series of interactions among the cell's macromolecular components. Defects in mechanosensing, transduction,or responses underlie many diseases such as cancers, immune disorders, cardiac hypertrophy, genetic malformations, and neuropathies. Here, we highlight micro- and nanotechnology-based tools that have been used to study how chemical and mechanical cues modulate the responses of single cells in contact with the extracellular environment. Understanding the physical aspects of these complex processes at the micro- and nanometer scale could produce profound and fundamental new insights into how the processes of cell migration, metastasis, immune function and other areas which are regulated by mechanical forces.