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 prestress 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.

Role of the Nucleus as a Sensor of Cell Environment Topography

“…Combining this approach with other micromanipulation tools revealed that tension in lateral stress fibers is gradually generated by anisotropic force contraction dipoles as the cell elongates and is strongly dependent on the cell spreading area . Similarly, the combination of the analysis of adhesion and de‐adhesion of endothelial cells on 2D patterns with different stiffness allowed the probing of nuclear mechanics without applying external forces and without any alteration of the nucleo‐cytoskeletal interactions such as those observed in micropipette aspiration experiments on isolated nuclei . Interestingly, differences in terms of regulation of the nuclear shapes on various rectangular 2D patterns were observed between fibroblasts and a model cancerous cell .…”

Nanoparticle–Cell Interaction: A Cell Mechanics Perspective

“…MBs on the cellular membrane in the presence of magnetic field will stretch and mechanically activate the targeted membrane receptors. This technique is useful to study mechanotransduction as well as the rheological properties of cells . Different types of forces can be applied on membrane‐conjugated MBs ( Figure ): unidirectional tension delivered by the use of a permanent magnet (magnetic drag) or magnetic tweezers; torsional or rotational forces are applied by magnetic twisting/rotation.…”

“…Aside from studying internal biochemical pathways, MB‐assisted mechanical stimulation has been used to study the elastic and viscoelastic behavior of cytoplasm and nucleus for example, using spherical beads and microrods . When magnetic rods and wires are embedded in the cells and are subjected to a rotational magnetic field they behave as microrheometers.…”

FAM83G/PAWS1 controls cytoskeletal dynamics and cell migration through association with the SH3 adaptor CD2AP

“…Bearing in mind the role of PAWS1 in cell morphology, migration, cytoskeletal organization and focal adhesion distribution, we decided to examine its contribution to the architecture of cortactin and actin fibres. To this end, PAWS1 -/and control U2OS cells were plated onto fibronectin-coated crossbow and H-shaped (double-crossbow) micropatterns (Versaevel et al, 2016). We first noted that the lamellipodia of PAWS1 -/cells plated on the 'crossbow' fibronectin micropattern had a disorganized actin pattern ( Figure 3A; Supplementary Figure 4).…”