OPTICALLY TRACKING THE MOTION OF MICROBEADS TO STUDY PHYSICAL BEHAVIORS OF THE LIVING CELL IN RESPONSE TO TRANSIENT STRETCH OR COMPRESSION

2011 ◽  
Vol 04 (02) ◽  
pp. 143-150
Author(s):  
LINHONG DENG ◽  
XUEMEI JIANG ◽  
CHENG CHEN ◽  
AIJING SONG ◽  
FENG LIN
Keyword(s):  
Cell Mechanics ◽  
Living Cell ◽  
Actin Polymerization ◽  
Traction Force ◽  
Optical Methods ◽  
Cell Stiffness ◽  
Immunofluorescent Staining ◽  
Adherent Cells ◽  
Force Dynamics ◽  
Research Tools

Optical magnetic twisting cytometry and traction force microscopy are two advanced cell mechanics research tools that employ optical methods to track the motion of microbeads that are either bound to the surface or embedded in the substrate underneath the cell. The former measures rheological properties of the cell such as cell stiffness, and the latter measures cell traction force dynamics. Here we describe the principles of these two cell mechanics research tools and an example of using them to study physical behaviors of the living cell in response to transient stretch or compression. We demonstrate that, when subjected to a stretch–unstretch manipulation, both the stiffness and traction force of adherent cells promptly reduced, and then gradually recover up to the level prior to the stretch. Immunofluorescent staining and Western blotting results indicate that the actin cytoskeleton of the cells underwent a corresponding disruption and reassembly process almost in step with the changes of cell mechanics. Interestingly, when subjected to compression, the cells did not show such particular behaviors. Taken together, we conclude that adherent cells are very sensitive to the transient stretch but not transient compression, and the stretch-induced cell response is due to the dynamics of actin polymerization.

scholarly journals Finite Element Analysis of Traction Force Microscopy: Influence of Cell Mechanics, Adhesion, and Morphology

2013 ◽  
Vol 135 (7) ◽  
Author(s):  
Rachel Zielinski ◽  
Cosmin Mihai ◽  
Douglas Kniss ◽  
Samir N. Ghadiali
Keyword(s):  
Sensitivity Analysis ◽  
Energy Density ◽  
Traction Force ◽  
Biomechanical Properties ◽  
Contractile Force ◽  
Adhesion Energy ◽  
Cell Stiffness ◽  
Adherent Cells ◽  
Force Microscopy ◽  
Contractile Forces

The interactions between adherent cells and their extracellular matrix (ECM) have been shown to play an important role in many biological processes, such as wound healing, morphogenesis, differentiation, and cell migration. Cells attach to the ECM at focal adhesion sites and transmit contractile forces to the substrate via cytoskeletal actin stress fibers. This contraction results in traction stresses within the substrate/ECM. Traction force microscopy (TFM) is an experimental technique used to quantify the contractile forces generated by adherent cells. In TFM, cells are seeded on a flexible substrate and displacements of the substrate caused by cell contraction are tracked and converted to a traction stress field. The magnitude of these traction stresses are normally used as a surrogate measure of internal cell contractile force or contractility. We hypothesize that in addition to contractile force, other biomechanical properties including cell stiffness, adhesion energy density, and cell morphology may affect the traction stresses measured by TFM. In this study, we developed finite element models of the 2D and 3D TFM techniques to investigate how changes in several biomechanical properties alter the traction stresses measured by TFM. We independently varied cell stiffness, cell-ECM adhesion energy density, cell aspect ratio, and contractility and performed a sensitivity analysis to determine which parameters significantly contribute to the measured maximum traction stress and net contractile moment. Results suggest that changes in cell stiffness and adhesion energy density can significantly alter measured tractions, independent of contractility. Based on a sensitivity analysis, we developed a correction factor to account for changes in cell stiffness and adhesion and successfully applied this correction factor algorithm to experimental TFM measurements in invasive and noninvasive cancer cells. Therefore, application of these types of corrections to TFM measurements can yield more accurate estimates of cell contractility.

scholarly journals Actin cytoskeleton deregulation confers midostaurin resistance in FLT3-mutant acute myeloid leukemia

2021 ◽  
Vol 4 (1) ◽  
Author(s):  
Andoni Garitano-Trojaola ◽  
Ana Sancho ◽  
Ralph Götz ◽  
Patrick Eiring ◽  
Susanne Walz ◽  
...  
Keyword(s):  
Acute Myeloid Leukemia ◽  
Actin Cytoskeleton ◽  
Myeloid Leukemia ◽  
Actin Polymerization ◽  
Cell Stiffness ◽  
Adhesion Forces ◽  
Frequent Mutations ◽  
New Perspective ◽  
The Impact ◽  
Acute Myeloid

AbstractThe presence of FMS-like tyrosine kinase 3-internal tandem duplication (FLT3-ITD) is one of the most frequent mutations in acute myeloid leukemia (AML) and is associated with an unfavorable prognosis. FLT3 inhibitors, such as midostaurin, are used clinically but fail to entirely eradicate FLT3-ITD + AML. This study introduces a new perspective and highlights the impact of RAC1-dependent actin cytoskeleton remodeling on resistance to midostaurin in AML. RAC1 hyperactivation leads resistance via hyperphosphorylation of the positive regulator of actin polymerization N-WASP and antiapoptotic BCL-2. RAC1/N-WASP, through ARP2/3 complex activation, increases the number of actin filaments, cell stiffness and adhesion forces to mesenchymal stromal cells (MSCs) being identified as a biomarker of resistance. Midostaurin resistance can be overcome by a combination of midostaruin, the BCL-2 inhibitor venetoclax and the RAC1 inhibitor Eht1864 in midostaurin-resistant AML cell lines and primary samples, providing the first evidence of a potential new treatment approach to eradicate FLT3-ITD + AML.

scholarly journals Dissipation of contractile forces: the missing piece in cell mechanics

2017 ◽  
Vol 28 (14) ◽  
pp. 1825-1832 ◽  
Author(s):  
Laetitia Kurzawa ◽  
Benoit Vianay ◽  
Fabrice Senger ◽  
Timothée Vignaud ◽  
Laurent Blanchoin ◽  
...  
Keyword(s):  
Cell Mechanics ◽  
Force Production ◽  
Traction Force ◽  
Structural Rearrangements ◽  
Force Transmission ◽  
Traction Forces ◽  
Cell Traction Forces ◽  
Cell Traction ◽  
Contractile Forces ◽  
Fiber Contraction

Mechanical forces are key regulators of cell and tissue physiology. The basic molecular mechanism of fiber contraction by the sliding of actin filament upon myosin leading to conformational change has been known for decades. The regulation of force generation at the level of the cell, however, is still far from elucidated. Indeed, the magnitude of cell traction forces on the underlying extracellular matrix in culture is almost impossible to predict or experimentally control. The considerable variability in measurements of cell-traction forces indicates that they may not be the optimal readout to properly characterize cell contractile state and that a significant part of the contractile energy is not transferred to cell anchorage but instead is involved in actin network dynamics. Here we discuss the experimental, numerical, and biological parameters that may be responsible for the variability in traction force production. We argue that limiting these sources of variability and investigating the dissipation of mechanical work that occurs with structural rearrangements and the disengagement of force transmission is key for further understanding of cell mechanics.

scholarly journals Force localization modes in dynamic epithelial colonies

2018 ◽  
Vol 29 (23) ◽  
pp. 2835-2847 ◽  
Author(s):  
Erik N. Schaumann ◽  
Michael F. Staddon ◽  
Margaret L. Gardel ◽  
Shiladitya Banerjee
Keyword(s):  
Cancer Metastasis ◽  
Wide Spectrum ◽  
Traction Force ◽  
Cellular Motility ◽  
Force Transmission ◽  
Mechanical Coupling ◽  
Cell Behaviors ◽  
Force Dynamics ◽  
Epithelial Tissues ◽  
Cell Matrix Interactions

Collective cell behaviors, including tissue remodeling, morphogenesis, and cancer metastasis, rely on dynamics among cells, their neighbors, and the extracellular matrix. The lack of quantitative models precludes understanding of how cell–cell and cell–matrix interactions regulate tissue-scale force transmission to guide morphogenic processes. We integrate biophysical measurements on model epithelial tissues and computational modeling to explore how cell-level dynamics alter mechanical stress organization at multicellular scales. We show that traction stress distribution in epithelial colonies can vary widely for identical geometries. For colonies with peripheral localization of traction stresses, we recapitulate previously described mechanical behavior of cohesive tissues with a continuum model. By contrast, highly motile cells within colonies produce traction stresses that fluctuate in space and time. To predict the traction force dynamics, we introduce an active adherent vertex model (AAVM) for epithelial monolayers. AAVM predicts that increased cellular motility and reduced intercellular mechanical coupling localize traction stresses in the colony interior, in agreement with our experimental data. Furthermore, the model captures a wide spectrum of localized stress production modes that arise from individual cell activities including cell division, rotation, and polarized migration. This approach provides a robust quantitative framework to study how cell-scale dynamics influence force transmission in epithelial tissues.

scholarly journals Cholesterol-Dependent Modulation of Stem Cell Biomechanics: Application to Adipogenesis

2019 ◽  
Vol 141 (8) ◽  
Author(s):  
Shan Sun ◽  
Djanybek Adyshev ◽  
Steven Dudek ◽  
Amit Paul ◽  
Andrew McColloch ◽  
...  
Keyword(s):  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Stem Cell ◽  
Young’S Modulus ◽  
Cell Mechanics ◽  
Young's Modulus ◽  
Adipogenic Differentiation ◽  
Stem Cell Differentiation ◽  
Cell Stiffness ◽  
Cholesterol Depletion

Cell mechanics has been shown to regulate stem cell differentiation. We have previously reported that altered cell stiffness of mesenchymal stem cells can delay or facilitate biochemically directed differentiation. One of the factors that can affect the cell stiffness is cholesterol. However, the effect of cholesterol on differentiation of human mesenchymal stem cells remains elusive. In this paper, we demonstrate that cholesterol is involved in the modulation of the cell stiffness and subsequent adipogenic differentiation. Rapid cytoskeletal actin reorganization was evident and correlated with the cell's Young's modulus measured using atomic force microscopy. In addition, the level of membrane-bound cholesterol was found to increase during adipogenic differentiation and inversely varied with the cell stiffness. Furthermore, cholesterol played a key role in the regulation of the cell morphology and biomechanics, suggesting its crucial involvement in mechanotransduction. To better understand the underlying mechanisms, we investigated the effect of cholesterol on the membrane–cytoskeleton linker proteins (ezrin and moesin). Cholesterol depletion was found to upregulate the ezrin expression which promoted cell spreading, increased Young's modulus, and hindered adipogenesis. In contrast, cholesterol enrichment increased the moesin expression, decreased Young's modulus, and induced cell rounding and facilitated adipogenesis. Taken together, cholesterol appears to regulate the stem cell mechanics and adipogenesis through the membrane-associated linker proteins.

Hypoxia alters biophysical properties of endothelial cells via p38 MAPK- and Rho kinase-dependent pathways

2005 ◽  
Vol 289 (3) ◽  
pp. C521-C530 ◽  
Author(s):  
Steven S. An ◽  
Corin M. Pennella ◽  
Achuta Gonnabathula ◽  
Jianxin Chen ◽  
Ning Wang ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Actin Polymerization ◽  
Rho Kinase ◽  
Small Heat Shock Protein ◽  
Cell Stiffness ◽  
Microvascular Endothelial Cells ◽  
Pulmonary Vasculature ◽  
Biophysical Properties ◽  
Traction Forces ◽  
Downstream Effector

Hypoxia alters the barrier function of the endothelial cells that line the pulmonary vasculature, but underlying biophysical mechanisms remain unclear. Using rat pulmonary microvascular endothelial cells (RPMEC) in culture, we report herein changes in biophysical properties, both in space and in time, that occur in response to hypoxia. We address also the molecular basis of these changes. At the level of the single cell, we measured cell stiffness, the distribution of traction forces exerted by the cell on its substrate, and spontaneous nanoscale motions of microbeads tightly bound to the cytoskeleton (CSK). Hypoxia increased cell stiffness and traction forces by a mechanism that was dependent on the activation of Rho kinase. These changes were followed by p38-mediated decreases in spontaneous bead motions, indicating stabilization of local cellular-extracellular matrix (ECM) tethering interactions. Cells overexpressing phospho-mimicking small heat shock protein (HSP27-PM), a downstream effector of p38, exhibited decreases in spontaneous bead motions that correlated with increases in actin polymerization in these cells. Together, these findings suggest that hypoxia differentially regulates endothelial cell contraction and cellular-ECM adhesion.

scholarly journals Isolation and culture of endothelial cells from embryonic rat yolk sac

2017 ◽  
Vol 1 (2) ◽  
pp. 149-154
Author(s):  
Harun Ülger ◽  
Ahmet K. Karabulut ◽  
Margaret K. Pratten
Keyword(s):  
Endothelial Cells ◽  
Bovine Serum ◽  
Cultured Cells ◽  
Fetal Bovine Serum ◽  
Wistar Rat ◽  
Yolk Sac ◽  
Immunofluorescent Staining ◽  
Adherent Cells ◽  
Growth Supplement ◽  
Fetal Bovine

Abstract Yolk sac blood islands are the first morphologic evidence of hematopoietic development during mammalian embryogenesis, and visseral yolk sac mesoderm gives rise to the first embryonic blood cells within a rich endothelial network. Present study reports the isolation and culture of endothelial cells from 11.5 days old embryonic rat yolk sac. The embryos were dissected from 11.5 days pregnant Wistar rat (Rattus norvegicus) and the external yolk sac membrane and embryos were removed under aseptic condition. After washing three times with Calcium-Magnesium free Hank’s balanced salt solution (CMF-HBSS), the tissue was minced, and fragments were incubated in CMF-HBSS containing 2mg/ml Trypsin, 100mg/ml collagenase I and 40mg/ml DNAse at 37°C until the tissue was completely dispersed. The digestion effect was then neutralized by fetal bovine serum at 1:3 (v/v). The cell suspension was centrifuged at 1000 rpm for 10 min., the supernatants were discarded and the cell pellets resuspended in Dulbecco modified Eagle medium containing 15% fetal bovine serum, 1.25mg/ml amphotericin B, 25mg/ml gentamycin sulphate and 100mg/ml endothelial cell growth supplement. The resuspended cells were plated in two diverse 25cm2 culture flasks for overnight differential adherence at 37°C. The non-adherent cells were removed by gentle aspiration and adherent cells refed with fresh medium. The cells were transferred using 1ml of 0.2% Trypsin when cultures reached near-confluence. The cultured yolk sac endothelial cells had characteristic cobblestone appearence and positive immunofluorescent staining for von Willebrand Factor (vWF). Weibel-Palade bodies, the major ultrastructural marker for endothelium, were also detected in cultured cells by electron microscopy.

scholarly journals Force-dependent activation of actin elongation factor mDia1 protects the cytoskeleton from mechanical damage and facilitates stress fiber repair

2021 ◽  
Author(s):  
Fernando R. Valencia ◽  
Eduardo Sandoval ◽  
Jian Liu ◽  
Sergey V. Plotnikov
Keyword(s):  
Cell Mechanics ◽  
Actin Polymerization ◽  
Safety Valve ◽  
Mechanical Damage ◽  
Elongation Factor ◽  
Focal Adhesions ◽  
Stress Fiber ◽  
Stress Fibers ◽  
Mechanical Tension ◽  
Contractile Forces

ABSTRACTPlasticity of cell mechanics, which relies heavily on the spatiotemporal regulation of the actomyosin cytoskeleton, safeguards cells against mechanical damage. Yet, mechanisms of adaptive change in cell mechanics remain elusive. Here, we report a new mechanism whereby mechanically activated actin elongation factor mDia1 controls the dynamics of actin polymerization at focal adhesions, force bearing linkages between the actin cytoskeleton and extracellular matrix. By combining live-cell imaging with mathematical modelling, we show that actin polymerization at focal adhesions exhibits pulsatile dynamics where the spikes of mDia1 activity are triggered by cell-generated contractile forces. We show that suppression of mDia1-mediated actin polymerization at focal adhesions results in two-fold increase in mechanical tension on the stress fibers. This elevated tension leads to an increased frequency of spontaneous stress fiber damage and decreased efficiency of zyxin-mediated stress fiber repair. We conclude that tension-controlled actin polymerization at focal adhesions acts as a safety valve dampening excessive mechanical tension on the actin cytoskeleton and safeguarding stress fibers against mechanical damage.SUMMARYValencia et al. reports that stress fiber elongation at focal adhesion requires mDia1 activity, furthermore contractile forces trigger mDia1-dependent actin polymerization. mDia1-mediated actin polymerization acts as a safety valve to dampen mechanical stress and protect the cell from damage.

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