Cell stretching is amplified by active actin remodelling to deform and recruit proteins in mechanosensitive structures.

Detection and conversion of mechanical forces into biochemical signals controls cell functions during physiological and pathological processes. Mechanosensing is based on protein deformations and reorganizations, yet the molecular mechanisms are still unclear. Using a cell-stretching device compatible with super-resolution microscopy and single-protein tracking, we explored the nanoscale deformations and reorganizations of individual proteins inside mechanosensitive structures. We achieved super-resolution microscopy after live stretching on intermediate filaments, microtubules and integrin adhesions. Simultaneous single-protein tracking and stretching showed that while integrins followed the elastic deformation of the substrate, actin filaments and talin also displayed lagged and transient inelastic responses associated with active acto-myosin remodelling and talin deformations. Capturing acute reorganizations of single molecules during stretching showed that force-dependent vinculin recruitment is delayed and depends on the maturation of integrin adhesions. Thus, cells respond to external forces by amplifying transiently and locally cytoskeleton displacements, enabling protein deformation and recruitment in mechanosensitive structures.

Using Single-Protein Tracking to Study Cell Migration.

To get a complete understanding of cell migration, it is critical to study its orchestration at the molecular level. Since the recent developments in single-molecule imaging, it is now possible to study molecular phenomena at the single-molecule level inside living cells. In this chapter, we describe how such approaches have been and can be used to decipher molecular mechanisms involved in cell migration.

Fluorescent nanodiamond tracking reveals intraneuronal transport abnormalities induced by brain-disease-related genetic risk factors.

Brain diseases such as autism and Alzheimer's disease (each inflicting >1% of the world population) involve a large network of genes displaying subtle changes in their expression. Abnormalities in intraneuronal transport have been linked to genetic risk factors found in patients, suggesting the relevance of measuring this key biological process. However, current techniques are not sensitive enough to detect minor abnormalities. Here we report a sensitive method to measure the changes in intraneuronal transport induced by brain-disease-related genetic risk factors using fluorescent nanodiamonds (FNDs). We show that the high brightness, photostability and absence of cytotoxicity allow FNDs to be tracked inside the branches of dissociated neurons with a spatial resolution of 12 nm and a temporal resolution of 50 ms. As proof of principle, we applied the FND tracking assay on two transgenic mouse lines that mimic the slight changes in protein concentration (∼30%) found in the brains of patients. In both cases, we show that the FND assay is sufficiently sensitive to detect these changes.

Exposure to high static or pulsed magnetic fields does not affect cellular processes in the yeast Saccharomyces cerevisiae.

We report results of a study of the effects of strong static (up to 16 T for 8 h) and pulsed (up to 55 T single-shot and 4 x 20 T repeated shots) magnetic fields on Saccharomyces cerevisiae cultures in the exponential phase of growth. In contrast to previous reports restricted to only a limited number of cellular parameters, we have examined a wide variety of cellular processes: genome-scale gene expression, proteome profile, cell viability, morphology, and growth, metabolic and fermentation activity after magnetic field exposure. None of these cellular activities were impaired in response to static or pulsed magnetic field exposure. Our results confirm and extend previous reports on the absence of magnetic field effects on yeast and support the hypothesis that magnetic fields have no impact on the transcriptional machinery and on the integrity of unicellular biological systems.

Energetics and metastability of the adenine dication observed in proton-adenine collisions.

We present here a study that deals with the correlated fragmentation of a doubly charged adenine molecular target induced by a 100 keV proton beam. We have elucidated part of the dissociation dynamics for several channels and have obtained the corresponding kinetic energy released values. We have extracted activation energies by combining our experimental data with computations using the ab initio GAMESS code. We have observed metastability patterns against fragmentation, for which we have extracted the temporal mechanism (one or two steps). Subsequently, we have obtained lifetimes in the 100-200 ns range. In the simplest case of two-body fragmentation with the emission of mass 28, the determination of transition states and reaction paths has showed that emission of the H-C-N-H fragment is preferred to that of C-N-H(2). From the calculated activation barriers and lifetimes, we have deduced an equivalent temperature of the dication that we have compared with the existing models.