Nesprins are mechanotransducers that discriminate epithelial-mesenchymal transition programs.

LINC complexes are transmembrane protein assemblies that physically connect the nucleoskeleton and cytoskeleton through the nuclear envelope. Dysfunctions of LINC complexes are associated with pathologies such as cancer and muscular disorders. The mechanical roles of LINC complexes are poorly understood. To address this, we used genetically encoded FRET biosensors of molecular tension in a nesprin protein of the LINC complex of fibroblastic and epithelial cells in culture. We exposed cells to mechanical, genetic, and pharmacological perturbations, mimicking a range of physiological and pathological situations. We show that nesprin experiences tension generated by the cytoskeleton and acts as a mechanical sensor of cell packing. Moreover, nesprin discriminates between inductions of partial and complete epithelial-mesenchymal transitions. We identify the implicated mechanisms, which involve α-catenin capture at the nuclear envelope by nesprin upon its relaxation, thereby regulating ß-catenin transcription. Our data thus implicate LINC complex proteins as mechanotransducers that fine-tune ß-catenin signaling in a manner dependent on the epithelial-mesenchymal transition program.

Nesprin-2 accumulates at the front of the nucleus during confined cell migration.

The mechanisms by which cells exert forces on their nuclei to migrate through openings smaller than the nuclear diameter remain unclear. We use CRISPR/Cas9 to fluorescently label nesprin-2 giant, which links the cytoskeleton to the nuclear interior. We demonstrate that nesprin-2 accumulates at the front of the nucleus during nuclear deformation through narrow constrictions, independently of the nuclear lamina. We find that nesprins are mobile at time scales similar to the accumulation. Using artificial constructs, we show that the actin-binding domain of nesprin-2 is necessary and sufficient for this accumulation. Actin filaments are organized in a barrel structure around the nucleus in the direction of movement. Using two-photon ablation and cytoskeleton-inhibiting drugs, we demonstrate an actomyosin-dependent pulling force on the nucleus from the front of the cell. The elastic recoil upon ablation is dampened when nesprins are reduced at the nuclear envelope. We thus show that actin redistributes nesprin-2 giant toward the front of the nucleus and contributes to pulling the nucleus through narrow constrictions, in concert with myosin.

Src- and confinement-dependent FAK activation causes E-cadherin relaxation and ß-catenin activity.

In epithelia, E-cadherin cytoplasmic tail is under cytoskeleton-generated tension via a link that contains ß-catenin. A cotranscription factor, ß-catenin, is also active in morphogenetic processes associated with epithelial-to-mesenchymal transition. ß-Catenin signaling appears mechanically inducible and was proposed to follow phosphorylation-induced ß-catenin release from E-cadherin. Evidence for this mechanism is lacking, and whether E-cadherin tension is involved is unknown. To test this, we combined quantitative fluorescence microscopies with genetic and pharmacological perturbations of epithelial-to-mesenchymal transition-induced cells in culture. We showed that ß-catenin nuclear activity follows a substantial release from the membrane specific to migrating cells and requires multicellular deconfinement and Src activity. Selective nuclear translocation occurs downstream of focal adhesion kinase activation, which targets E-cadherin tension relaxation through actomyosin remodeling. In contrast, phosphorylations of the cadherin/catenin complex are not substantially required. These data demonstrate that E-cadherin acts as a sensor of intracellular mechanics in a crosstalk with cell-substrate adhesions that target ß-catenin signaling.

Microtopographical cues promote peripheral nerve regeneration via transient mTORC2 activation.

Despite microsurgical repair, recovery of function following peripheral nerve injury is slow and often incomplete. Outcomes could be improved by an increased understanding of the molecular biology of regeneration and by translation of experimental bioengineering strategies. Topographical cues have been shown to be powerful regulators of the rate and directionality of neurite regeneration, and in this study we investigated the downstream molecular effects of linear micropatterned structures in an organotypic explant model. Linear topographical cues enhanced neurite outgrowth and our results demonstrated that the mTOR pathway is important in regulating these responses. mTOR gene expression peaked between 48 and 72h, coincident with the onset of rapid neurite outgrowth and glial migration, and correlated with neurite length at 48h. mTOR protein was located to glia and in a punctate distribution along neurites. mTOR levels peaked at 72h and were significantly increased by patterned topography (p<0.05). Furthermore, the topographical cues could override pharmacological inhibition. Downstream phosphorylation assays and inhibition of mTORC1 using rapamycin highlighted mTORC2 as an important mediator, and more specific therapeutic target. Quantitative immunohistochemistry confirmed the presence of the mTORC2 component rictor at the regenerating front where it co-localised with F-actin and vinculin. Collectively, these results provide a deeper understanding of the mechanism of action of topography on neural regeneration, and support the incorporation of topographical patterning in combination with pharmacological mTORC2 potentiation within biomaterial constructs used to repair peripheral nerves. STATEMENT OF SIGNIFICANCE: Peripheral nerve injury is common and functionally devastating. Despite microsurgical repair, healing is slow and incomplete, with lasting functional deficit. There is a clear need to translate bioengineering approaches and increase our knowledge of the molecular processes controlling nerve regeneration to improve the rate and success of healing. Topographical cues are powerful determinants of neurite outgrowth and represent a highly translatable engineering strategy. Here we demonstrate, for the first time, that microtopography potentiates neurite outgrowth via the mTOR pathway, with the mTORC2 subtype being of particular importance. These results give further evidence for the incorporation of microtopographical cues into peripheral nerve regeneration conduits and indicate that mTORC2 may be a suitable therapeutic target to potentiate nerve regeneration.

Directed nerve regeneration enabled by wirelessly powered electrodes printed on a biodegradable polymer.

Wirelessly directed nerve regeneration: inductively powered electrical stimulation circuits on the biodegradable polymer polycaprolactone demonstrate directed regeneration of sensory neurons from a dorsal root ganglion. These circuits, produced using a unique transfer printing process, illustrate progress towards the use of electrical stimulation systems on biodegradable materials to improve peripheral nerve repair functional outcomes.

Influence of both a static magnetic field and penetratin on magnetic nanoparticle delivery into fibroblasts.

AIM: With regards to nanoparticles, all biomedical applications require cellular uptake, which to date remains a hurdle to further progress. This study aims to compare both the attractive force of a static magnetic field and the cell penetrating capability of penetratin; two techniques currently employed to enhance cell uptake. MATERIALS & METHODS: Fluorescent magnetic nanoparticles were functionalized with penetratin and cells were challenged with or without the particles in the presence/absence of a static magnetic field (350 mT). Following analysis of the magnetic field applied, cellular uptake and behavior was assessed in terms of fluorescence microscopy, clathrin and caveolin levels, scanning electron microscopy and transmission electron microscopy. RESULTS: Modeling of the field applied demonstrated varying field patterns across the cell culture area, reflected by higher particle uptake at higher field strengths. Both penetratin and the magnetic field increased cell uptake with penetratin proving more efficient. Interestingly, the magnetic field stimulated clathrin-mediated endocytosis and subsequent particle uptake.