Marie Versaevel

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.

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.

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.

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