Danahe Mohammed

Deregulation of focal adhesion formation and cytoskeletal tension due to loss of A-type lamins

ABSTRACT The nuclear lamina mechanically integrates the nucleus with the cytoskeleton and extracellular environment and regulates gene expression. These functions are exerted through direct and indirect interactions with the laminas major constituent proteins, the A-type lamins, which are encoded by the LMNA gene. Using quantitative stable isotope labeling-based shotgun proteomics we have analyzed the proteome of human dermal fibroblasts in which we have depleted A-type lamins by means of a sustained siRNA-mediated LMNA knockdown. Gene ontology analysis revealed that the largest fraction of differentially produced proteins was involved in actin cytoskeleton organization, in particular proteins involved in focal adhesion dynamics, such as actin-related protein 2 and 3 (ACTR2/3), subunits of the ARP2/3 complex, and fascin actin-bundling protein 1 (FSCN1). Functional validation using quantitative immunofluorescence showed a significant reduction in the size of focal adhesion points in A-type lamin depleted cells, which correlated with a reduction in early cell adhesion capacity and an increased cell motility. At the same time, loss of A-type lamins led to more pronounced stress fibers and higher traction forces. This phenotype could not be mimicked or reversed by experimental modulation of the STAT3-IL6 pathway, but it was partly recapitulated by chemical inhibition of the ARP2/3 complex. Thus, our data suggest that the loss of A-type lamins perturbs the balance between focal adhesions and cytoskeletal tension. This imbalance may contribute to mechanosensing defects observed in certain laminopathies.

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.