Navid Bonakdar

3D Traction forces in cancer cell invasion.

Cell invasion through a dense three-dimensional (3D) matrix is believed to depend on the ability of cells to generate traction forces. To quantify the role of cell tractions during invasion in 3D, we present a technique to measure the elastic strain energy stored in the matrix due to traction-induced deformations. The matrix deformations around a cell were measured by tracking the 3D positions of fluorescent beads tightly embedded in the matrix. The bead positions served as nodes for a finite element tessellation. From the strain in each element and the known matrix elasticity, we computed the local strain energy in the matrix surrounding the cell. We applied the technique to measure the strain energy of highly invasive MDA-MB-231 breast carcinoma and A-125 lung carcinoma cells in collagen gels. The results were compared to the strain energy generated by non-invasive MCF-7 breast and A-549 lung carcinoma cells. In all cases, cells locally contracted the matrix. Invasive breast and lung carcinoma cells showed a significantly higher contractility compared to non-invasive cells. Higher contractility, however, was not universally associated with higher invasiveness. For instance, non-invasive A-431 vulva carcinoma cells were the most contractile cells among all cell lines tested. As a universal feature, however, we found that invasive cells assumed an elongated spindle-like morphology as opposed to a more spherical shape of non-invasive cells. Accordingly, the distribution of strain energy density around invasive cells followed patterns of increased complexity and anisotropy. These results suggest that not so much the magnitude of traction generation but their directionality is important for cancer cell invasion.

Filamin A Is Essential for Active Cell Stiffening but not Passive Stiffening under External Force

The material properties of a cell determine how mechanical forces are transmitted through and sensed by that cell. Some types of cells stiffen passively under large external forces, but they can also alter their own stiffness in response to the local mechanical environment or biochemical cues. Here we show that the actin-binding protein filamin A is essential for the active stiffening of cells plated on collagen-coated substrates. This appears to be due to a diminished capability to build up large internal contractile stresses in the absence of filamin A. To show this, we compare the material properties and contractility of two human melanoma cell lines that differ in filamin A expression. The filamin A-deficient M2 cells are softer than the filamin A-replete A7 cells, and exert much smaller contractile stresses on the substratum, even though the M2 cells have similar levels of phosphorylated myosin II light chain and only somewhat diminished adhesion strength. In contrast to A7 cells, the stiffness and contractility of M2 cells are insensitive to either myosin-inhibiting drugs or the stiffness of the substratum. Surprisingly, however, filamin A is not required for passive stiffening under large external forces.

Integrin adhesion and force coupling are independently regulated by localized PtdIns(4,5)2 synthesis

The 90‐kDa isoform of the lipid kinase PIP kinase Type I γ (PIPKIγ) localizes to focal adhesions (FAs), where it provides a local source of phosphatidylinositol 4,5‐bisphosphate (PtdIns(4,5)P2). Although PtdIns(4,5)P2 regulates the function of several FA‐associated molecules, the role of the FA‐specific pool of PtdIns(4,5)P2 is not known. We report that the genetic ablation of PIPKIγ specifically from FAs results in defective integrin‐mediated adhesion and force coupling. Adhesion defects in cells deficient in FA‐PtdIns(4,5)P2 synthesis are corrected within minutes while integrin–actin force coupling remains defective over a longer period. Talin and vinculin, but not kindlin, are less efficiently recruited to new adhesions in these cells. These data demonstrate that the specific depletion of PtdIns(4,5)P2 from FAs temporally separates integrin–ligand binding from integrin–actin force coupling by regulating talin and vinculin recruitment. Furthermore, it suggests that force coupling relies heavily on locally generated PtdIns(4,5)P2 rather than bulk membrane PtdIns(4,5)P2.

Biomechanical characterization of a desminopathy in primary human myoblasts.

Heterozygous mutations of the human desmin gene on chromosome 2q35 cause hereditary and sporadic myopathies and cardiomyopathies. The expression of mutant desmin brings about partial disruption of the extra sarcomeric desmin cytoskeleton and abnormal protein aggregation in the sarcoplasm of striated muscle cells. The precise molecular pathways and sequential steps that lead from a desmin gene defect to progressive muscle damage are still unclear. We tested whether mutant desmin changes the biomechanical properties and the intrinsic mechanical stress response of primary cultured myoblasts derived from a patient carrying a heterozygous R350P desmin mutation. Compared to wildtype controls, undifferentiated mutant desmin myoblasts revealed increased cell death and substrate detachment in response to cyclic stretch on flexible membranes. Moreover, magnetic tweezer microrheometry of myoblasts using fibronectin-coated beads showed increased stiffness of diseased cells. Our findings provide the first evidence that altered mechanical properties may contribute to the progressive striated muscle pathology in desminopathies. We postulate that the expression of mutant desmin leads to increased mechanical stiffness, which results in excessive mechanical stress in response to strain and consecutively to increased mechanical vulnerability and damage of muscle cells.

NEDD9 stabilizes focal adhesions, increases binding to the extra-cellular matrix and differentially effects 2D versus 3D cell migration.

The speed of cell migration on 2-dimensional (2D) surfaces is determined by the rate of assembly and disassembly of clustered integrin receptors known as focal adhesions. Different modes of cell migration that have been described in 3D environments are distinguished by their dependence on integrin-mediated interactions with the extra-cellular matrix. In particular, the mesenchymal invasion mode is the most dependent on focal adhesion dynamics. The focal adhesion protein NEDD9 is a key signalling intermediary in mesenchymal cell migration, however whether NEDD9 plays a role in regulating focal adhesion dynamics has not previously been reported. As NEDD9 effects on 2D migration speed appear to depend on the cell type examined, in the present study we have used mouse embryo fibroblasts (MEFs) from mice in which the NEDD9 gene has been depleted (NEDD9 −/− MEFs). This allows comparison with effects of other focal adhesion proteins that have previously been demonstrated using MEFs. We show that focal adhesion disassembly rates are increased in the absence of NEDD9 expression and this is correlated with increased paxillin phosphorylation at focal adhesions. NEDD9−/− MEFs have increased rates of migration on 2D surfaces, but conversely, migration of these cells is significantly reduced in 3D collagen gels. Importantly we show that myosin light chain kinase is activated in 3D in the absence of NEDD9 and is conversely inhibited in 2D cultures. Measurement of adhesion strength reveals that NEDD9−/− MEFs have decreased adhesion to fibronectin, despite upregulated α5β1 fibronectin receptor expression. We find that β1 integrin activation is significantly suppressed in the NEDD9−/−, suggesting that in the absence of NEDD9 there is decreased integrin receptor activation. Collectively our data suggest that NEDD9 may promote 3D cell migration by slowing focal adhesion disassembly, promoting integrin receptor activation and increasing adhesion force to the ECM.

Glomerular podocytes: a study of mechanical properties and mechano-chemical signaling.

Kidney glomeruli function as filters, allowing the passage of small solutes and waste products into the urinary tract, while retaining essential proteins and macromolecules in the blood stream. These structures are under constant mechanical stress due to fluid pressure, driving filtration across the barrier. We mechanically stimulated adherent wildtype podocytes using the methods of magnetic tweezer and twisting as well as cell stretching. Attaching collagen IV-coated or poly-l-lysine-coated magnetic beads to cell receptors allowed for the determination of cellular stiffness. Angiotensin II-treated podocytes showed slightly higher stiffness than untreated cells, the cell fluidity (i.e. internal dynamics) remained similar, and showed an increase with force. The bead detachment (a measure of the binding strength) was higher in angiotensin II-treated compared to untreated podocytes. Magnetic twisting confirmed that angiotensin II treatment of podocytes increases and CDTA treatment decreases cell stiffness. However, treatment with both angiotensin II and CDTA increased the cell stiffness only slightly compared to solely CDTA-treated cells. Exposing podocytes to cyclic, uniaxial stretch showed an earlier onset of ERK(1/2) phosphorylation compared to MEF (control) cells. These results indicate that angiotensin II might free intracellularly stored calcium and affects actomyosin contraction, and that mechanical stimulation influences cell signaling.

Plectin reinforces vascular integrity by mediating crosstalk between the vimentin and the actin networks.

ABSTRACT Mutations in the cytoskeletal linker protein plectin result in multisystemic diseases affecting skin and muscle with indications of additional vascular system involvement. To study the mechanisms underlying vascular disorders, we established plectin-deficient endothelial cell and mouse models. We show that apart from perturbing the vimentin cytoskeleton of endothelial cells, plectin deficiency leads to severe distortions of adherens junctions (AJs), as well as tight junctions, accompanied by an upregulation of actin stress fibres and increased cellular contractility. Plectin-deficient endothelial cell layers were more leaky and showed reduced mechanical resilience in fluid-shear stress and mechanical stretch experiments. We suggest that the distorted AJs and upregulated actin stress fibres in plectin-deficient cells are rooted in perturbations of the vimentin cytoskeleton, as similar phenotypes could be mimicked in wild-type cells by disruption of vimentin filaments. In vivo studies in endothelium-restricted conditional plectin-knockout mice revealed significant distortions of AJs in stress-prone aortic arch regions and increased pulmonary vascular leakage. Our study opens a new perspective on cytoskeleton-controlled vascular permeability, where a plectin-organized vimentin scaffold keeps actomyosin contractility ‘in-check’ and maintains AJ homeostasis. Summary: Plectin-arranged vimentin scaffolds keep actomyosin contractility ‘in-check’ and maintain cell–cell junction homeostasis, providing a new perspective on cytoskeleton-controlled vascular permeability.

Determining the mechanical properties of plectin in mouse myoblasts and keratinocytes

Plectin is the prototype of an intermediate filament (IF)-based cytolinker protein. It affects cells mechanically by interlinking and anchoring cytoskeletal filaments and acts as scaffolding and docking platform for signaling proteins to control cytoskeleton dynamics. The most common disease caused by mutations in the human plectin gene, epidermolysis bullosa simplex with muscular dystrophy (EBS-MD), is characterized by severe skin blistering and progressive muscular dystrophy. Therefore, we compared the biomechanical properties and the response to mechanical stress of murine plectin-deficient myoblasts and keratinocytes with wild-type cells. Using a cell stretching device, plectin-deficient myoblasts exhibited lower mechanical vulnerability upon external stress compared to wild-type cells, which we attributed to lower cellular pre-stress. Contrary to myoblasts, wild-type and plectin-deficient keratinocytes showed no significant differences. In magnetic tweezer measurements using fibronectin-coated paramagnetic beads, the stiffness of keratinocytes was higher than of myoblasts. Interestingly, cell stiffness, adhesion strength, and cytoskeletal dynamics were strikingly altered in plectin-deficient compared to wild-type myoblasts, whereas smaller differences were observed between plectin-deficient and wild-type keratinocytes, indicating that plectin might be more important for stabilizing cytoskeletal structures in myoblasts than in keratinocytes. Traction forces strongly correlated with the stiffness of plectin-deficient and wild-type myoblasts and keratinocytes. Contrary to that cell motility was comparable in plectin-deficient and wild-type myoblasts, but was significantly increased in plectin-deficient compared to wild-type keratinocytes. Thus, we postulate that the lack of plectin has divergent implications on biomechanical properties depending on the respective cell type.

Measuring mechanical properties in cells: three easy methods for biologists

The mechanism by which cells sense stresses and transmit them throughout the cytoplasm and the cytoskeleton (CSK) and by which these mechanical signals are converted into biochemical signaling responses is not clear. Specifically, there is little direct experimental evidence on how intracellular CSK structural elements in living cells deform and transmit stresses in response to external mechanical forces. Existing theories have invoked various biophysical and biochemical mechanisms to explain how cells spread, deform, divide, move, and change shape in response to mechanical inputs, but rigorous tests in cells are lacking. The lack of data and understanding is preventing the identification of mechanisms and sites of mechano‐regulation in cells. Here, we introduce and describe three unique and easy methods for biologists to determine mechanical properties and signaling events in cells.

Innovations in Measuring Cellular Mechanics

This article describes several novel mechanical methods for elucidating cellular responses to different types of mechanical loading (adhesive, pulling, pushing, shearing, and stretching forces). Understanding how cells deform and transmit stresses into the cell is important for gene expression, cytoskeletal remodeling, and focal adhesion reorganization and crucial for a variety of higher fundamental cell functions including cell division, motility, and differentiation. Introducing these unique methods of measuring and understanding cellular mechanics, therefore, provides a valuable platform for cell biology research.