Thomas Grevesse

Spatial coordination between cell and nuclear shape within micropatterned endothelial cells

Growing evidence suggests that cytoplasmic actin filaments are essential factors in the modulation of nuclear shape and function. However, the mechanistic understanding of the internal orchestration between cell and nuclear shape is still lacking. Here we show that orientation and deformation of the nucleus are regulated by lateral compressive forces driven by tension in central actomyosin fibres. By using a combination of micro-manipulation tools, our study reveals that tension in central stress fibres is gradually generated by anisotropic force contraction dipoles, which expand as the cell elongates and spreads. Our findings indicate that large-scale cell shape changes induce a drastic condensation of chromatin and dramatically affect cell proliferation. On the basis of these findings, we propose a simple mechanical model that quantitatively accounts for our experimental data and provides a conceptual framework for the mechanistic coordination between cell and nuclear shape.

Super-resolution microscopy reveals LINC complex recruitment at nuclear indentation sites

Increasing evidences show that the actin cytoskeleton is a key parameter of the nuclear remodeling process in response to the modifications of cellular morphology. However, detailed information on the interaction between the actin cytoskeleton and the nuclear lamina was still lacking. We addressed this question by constraining endothelial cells on rectangular fibronectin-coated micropatterns and then using Structured Illumination Microscopy (SIM) to observe the interactions between actin stress fibers, nuclear lamina and LINC complexes at a super-resolution scale. Our results show that tension in apical actin stress fibers leads to deep nuclear indentations that significantly deform the nuclear lamina. Interestingly, indented nuclear zones are characterized by a local enrichment of LINC complexes, which anchor apical actin fibers to the nuclear lamina. Moreover, our findings indicate that nuclear indentations induce the formation of segregated domains of condensed chromatin. However, nuclear indentations and condensed chromatin domains are not irreversible processes and both can relax in absence of tension in apical actin stress fibers.

Opposite rheological properties of neuronal microcompartments predict axonal vulnerability in brain injury

Although pathological changes in axonal morphology have emerged as important features of traumatic brain injury (TBI), the mechanical vulnerability of the axonal microcompartment relative to the cell body is not well understood. We hypothesized that soma and neurite microcompartments exhibit distinct mechanical behaviors, rendering axons more sensitive to a mechanical injury. In order to test this assumption, we combined protein micropatterns with magnetic tweezer rheology to probe the viscoelastic properties of neuronal microcompartments. Creep experiments revealed two opposite rheological behaviors within cortical neurons: the cell body was soft and characterized by a solid-like response, whereas the neurite compartment was stiffer and viscous-like. By using pharmacological agents, we demonstrated that the nucleus is responsible for the solid-like behavior and the stress-stiffening response of the soma, whereas neurofilaments have a predominant contribution in the viscous behavior of the neurite. Furthermore, we found that the neurite is a mechanosensitive compartment that becomes softer and adopts a pronounced viscous state on soft matrices. Together, these findings highlight the importance of the regionalization of mechanical and rigidity-sensing properties within neuron microcompartments in the preferential damage of axons during traumatic brain injury and into potential mechanisms of axonal outgrowth after injury.

Extracellular matrix protein expression is brain region dependent.

In the brain, extracellular matrix (ECM) components form networks that contribute to structural and functional diversity. Maladaptive remodeling of ECM networks has been reported in neurodegenerative and psychiatric disorders, suggesting that the brain microenvironment is a dynamic structure. A lack of quantitative information about ECM distribution in the brain hinders an understanding of region‐specific ECM functions and the role of ECM in health and disease. We hypothesized that each ECM protein as well as specific ECM structures, such as perineuronal nets (PNNs) and interstitial matrix, are differentially distributed throughout the brain, contributing to the unique structure and function in the various regions of the brain. To test our hypothesis, we quantitatively analyzed the distribution, colocalization, and protein expression of aggrecan, brevican, and tenascin‐R throughout the rat brain utilizing immunohistochemistry and mass spectrometry analysis and assessed the effect of aggrecan, brevican, and/or tenascin‐R on neurite outgrowth in vitro. We focused on aggrecan, brevican, and tenascin‐R as they are especially expressed in the mature brain, and have established roles in brain development, plasticity, and neurite outgrowth. The results revealed a differentiated distribution of all three proteins throughout the brain and indicated that their presence significantly reduces neurite outgrowth in a 3D in vitro environment. These results underline the importance of a unique and complex ECM distribution for brain physiology and suggest that encoding the distribution of distinct ECM proteins throughout the brain will aid in understanding their function in physiology and in turn assist in identifying their role in disease. J. Comp. Neurol. 524:1309–1336, 2016.

Cell confinement: putting the squeeze on the nucleus

The nucleus has long been considered as a passive compartment containing the genetic information. However, recent attention to its structure, mechanical properties and physical connections with other cellular compartments has shown that the nucleus changes dynamically its morphology and internal organization for important cellular processes, especially those associated with cellular confinement. In this paper, we review some recent progress in experimental investigations of nuclear squeezing that lead to a better understanding of the nuclear remodeling in response to various situations of cellular confinement. We will discuss compelling examples of original experiments performed with microsystems that have recently brought new insights into the close relationship between nuclear mechanics and cellular organization. We will show that the study of nuclear confinement with microsystems has opened up new experimental avenues that already offer promising clues for understanding diseases that are associated with defective nuclear mechanics.

Soy Protein/Cellulose Nanofiber Scaffolds Mimicking Skin Extracellular Matrix for Enhanced Wound Healing

Historically, soy protein and extracts have been used extensively in foods due to their high protein and mineral content. More recently, soy protein has received attention for a variety of its potential health benefits, including enhanced skin regeneration. It has been reported that soy protein possesses bioactive molecules similar to extracellular matrix (ECM) proteins and estrogen. In wound healing, oral and topical soy has been heralded as a safe and cost-effective alternative to animal protein and endogenous estrogen. However, engineering soy protein-based fibrous dressings, while recapitulating ECM microenvironment and maintaining a moist environment, remains a challenge. Here, the development of an entirely plant-based nanofibrous dressing comprised of cellulose acetate (CA) and soy protein hydrolysate (SPH) using rotary jet spinning is described. The spun nanofibers successfully mimic physicochemical properties of the native skin ECM and exhibit a high water retaining capability. In vitro, CA/SPH nanofibers promote fibroblast proliferation, migration, infiltration, and integrin β1 expression. In vivo, CA/SPH scaffolds accelerate re-epithelialization and epidermal thinning as well as reduce scar formation and collagen anisotropy in a similar fashion to other fibrous scaffolds, but without the use of animal proteins or synthetic polymers. These results affirm the potential of CA/SPH nanofibers as a novel wound dressing.

Probing cytoskeletal pre-stress and nuclear mechanics in endothelial cells with spatiotemporally controlled (de-)adhesion kinetics on micropatterned substrates

ABSTRACT The mechanical properties of living cells reflect their propensity to migrate and respond to external forces. Both cellular and nuclear stiffnesses are strongly influenced by the rigidity of the extracellular matrix (ECM) through reorganization of the cyto- and nucleoskeletal protein connections. Changes in this architectural continuum affect cell mechanics and underlie many pathological conditions. In this context, an accurate and combined quantification of the mechanical properties of both cells and nuclei can contribute to a better understanding of cellular (dys-)function. To address this challenge, we have established a robust method for probing cellular and nuclear deformation during spreading and detachment from micropatterned substrates. We show that (de-)adhesion kinetics of endothelial cells are modulated by substrate stiffness and rely on the actomyosin network. We combined this approach with measurements of cell stiffness by magnetic tweezers to show that relaxation dynamics can be considered as a reliable parameter of cellular pre-stress in adherent cells. During the adhesion stage, large cellular and nuclear deformations occur over a long time span (>60 min). Conversely, nuclear deformation and condensed chromatin are relaxed in a few seconds after detachment. Finally, our results show that accumulation of farnesylated prelamin leads to modifications of the nuclear viscoelastic properties, as reflected by increased nuclear relaxation times. Our method offers an original and non-intrusive way of simultaneously gauging cellular and nuclear mechanics, which can be extended to high-throughput screens of pathological conditions and potential countermeasures.

Preparation of Hydroxy-PAAm Hydrogels for Decoupling the Effects of Mechanotransduction Cues

It is now well established that many cellular functions are regulated by interactions of cells with physicochemical and mechanical cues of their extracellular matrix (ECM) environment. Eukaryotic cells constantly sense their local microenvironment through surface mechanosensors to transduce physical changes of ECM into biochemical signals, and integrate these signals to achieve specific changes in gene expression. Interestingly, physicochemical and mechanical parameters of the ECM can couple with each other to regulate cell fate. Therefore, a key to understanding mechanotransduction is to decouple the relative contribution of ECM cues on cellular functions. Here we present a detailed experimental protocol to rapidly and easily generate biologically relevant hydrogels for the independent tuning of mechanotransduction cues in vitro. We chemically modified polyacrylamide hydrogels (PAAm) to surmount their intrinsically non-adhesive properties by incorporating hydroxyl-functionalized acrylamide monomers during the polymerization. We obtained a novel PAAm hydrogel, called hydroxy-PAAm, which permits immobilization of any desired nature of ECM proteins. The combination of hydroxy-PAAm hydrogels with microcontact printing allows to independently control the morphology of single-cells, the matrix stiffness, the nature and the density of ECM proteins. We provide a simple and rapid method that can be set up in every biology lab to study in vitro cell mechanotransduction processes. We validate this novel two-dimensional platform by conducting experiments on endothelial cells that demonstrate a mechanical coupling between ECM stiffness and the nucleus.

A linked organ-on-chip model of the human neurovascular unit reveals the metabolic coupling of endothelial and neuronal cells

The neurovascular unit (NVU) regulates metabolic homeostasis as well as drug pharmacokinetics and pharmacodynamics in the central nervous system. Metabolic fluxes and conversions over the NVU rely on interactions between brain microvascular endothelium, perivascular pericytes, astrocytes and neurons, making it difficult to identify the contributions of each cell type. Here we model the human NVU using microfluidic organ chips, allowing analysis of the roles of individual cell types in NVU functions. Three coupled chips model influx across the blood–brain barrier (BBB), the brain parenchymal compartment and efflux across the BBB. We used this linked system to mimic the effect of intravascular administration of the psychoactive drug methamphetamine and to identify previously unknown metabolic coupling between the BBB and neurons. Thus, the NVU system offers an in vitro approach for probing transport, efficacy, mechanism of action and toxicity of neuroactive drugs.

Altering the Cellular Morphology Results in the Mechanical Regulation of Nuclear Shape and Functions by Central Actin Filaments

Growing evidence suggests that cytoplasmic actin filaments are essential players in the modulation of nuclear shape and functions. However, the mechanistic understanding of the internal orchestration between cell and nuclear shape is still lacking. In this communication, we shape-engineered single endothelial cells to quantitatively and non-invasively assess the nuclear morphology and the intracellular force balance in response to large-scale cell elongations. Our study reveals for the first time that nuclear orientation and deformation are regulated by lateral compressive forces driven by tension in central actomyosin stress fibers. We show that tension in central stress fibers is gradually generated by anisotropic force contraction dipoles as the cell elongates and strongly dependent on the cell spreading area. Our findings indicate that large-scale cell shape changes induce a chromatin condensation and dramatically affect cell proliferation. On the basis of these findings, we propose a simple mechanical model that quantitatively accounts for our experimental data and provides a conceptual framework for the mechanistic coordination between cell and nuclear shape.View Large Image | View Hi-Res Image | Download PowerPoint Slide