scholarly journals Cell Force-Driven Basement Membrane Disruption Fuels EGF- and Stiffness-Induced Invasive Cell Dissemination from Benign Breast Gland Acini

2021 ◽  
Vol 22 (8) ◽  
pp. 3962
Author(s):  
Aljona Gaiko-Shcherbak ◽  
Julian Eschenbruch ◽  
Nils M. Kronenberg ◽  
Michael Teske ◽  
Benjamin Wolters ◽  
...  
Keyword(s):  
Basement Membrane ◽  
Mechanical Response ◽  
Traction Force ◽  
Initial Step ◽  
Breast Cancer Invasion ◽  
Egfr Activation ◽  
Activation Mechanisms ◽  
Cell Dissemination ◽  
Cell Force ◽  
Push And Pull

Local basement membrane (BM) disruption marks the initial step of breast cancer invasion. The activation mechanisms of force-driven BM-weakening remain elusive. We studied the mechanical response of MCF10A-derived human breast cell acini with BMs of tuneable maturation to physical and soluble tumour-like extracellular matrix (ECM) cues. Traction force microscopy (TFM) and elastic resonator interference stress microscopy (ERISM) were used to quantify pro-invasive BM stress and protrusive forces. Substrate stiffening and mechanically impaired BM scaffolds induced the invasive transition of benign acini synergistically. Robust BM scaffolds attenuated this invasive response. Additional oncogenic EGFR activation compromised the BMs’ barrier function, fuelling invasion speed and incidence. Mechanistically, EGFR-PI3-Kinase downstream signalling modulated both MMP- and force-driven BM-weakening processes. We show that breast acini form non-proteolytic and BM-piercing filopodia for continuous matrix mechanosensation, which significantly push and pull on the BM and ECM under pro-invasive conditions. Invasion-triggered acini further shear and compress their BM by contractility-based stresses that were significantly increased (3.7-fold) compared to non-invasive conditions. Overall, the highest amplitudes of protrusive and contractile forces accompanied the highest invasiveness. This work provides a mechanistic concept for tumour ECM-induced mechanically misbalanced breast glands fuelling force-driven BM disruption. Finally, this could facilitate early cell dissemination from pre-invasive lesions to metastasize eventually.

scholarly journals Structure–function analysis of oncogenic EGFR Kinase Domain Duplication reveals insights into activation and a potential approach for therapeutic targeting

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Zhenfang Du ◽  
Benjamin P. Brown ◽  
Soyeon Kim ◽  
Donna Ferguson ◽  
Dean C. Pavlick ◽  
...  
Keyword(s):  
Tandem Duplication ◽  
Function Analysis ◽  
Treatment Strategies ◽  
Kinase Domain ◽  
Time Resolved ◽  
Egfr Activation ◽  
Domain Duplication ◽  
Activation Mechanisms ◽  
Potential Therapeutic Intervention ◽  
Egfr Kinase

AbstractMechanistic understanding of oncogenic variants facilitates the development and optimization of treatment strategies. We recently identified in-frame, tandem duplication of EGFR exons 18 - 25, which causes EGFR Kinase Domain Duplication (EGFR-KDD). Here, we characterize the prevalence of ERBB family KDDs across multiple human cancers and evaluate the functional biochemistry of EGFR-KDD as it relates to pathogenesis and potential therapeutic intervention. We provide computational and experimental evidence that EGFR-KDD functions by forming asymmetric EGF-independent intra-molecular and EGF-dependent inter-molecular dimers. Time-resolved fluorescence microscopy and co-immunoprecipitation reveals EGFR-KDD can form ligand-dependent inter-molecular homo- and hetero-dimers/multimers. Furthermore, we show that inhibition of EGFR-KDD activity is maximally achieved by blocking both intra- and inter-molecular dimerization. Collectively, our findings define a previously unrecognized model of EGFR dimerization, providing important insights for the understanding of EGFR activation mechanisms and informing personalized treatment of patients with tumors harboring EGFR-KDD. Finally, we establish ERBB KDDs as recurrent oncogenic events in multiple cancers.

scholarly journals Filling of Mater-Bi with Nanoclays to Enhance the Biofilm Rigidity

2018 ◽  
Vol 9 (4) ◽  
pp. 60 ◽  
Author(s):  
Giuseppe Cavallaro ◽  
Giuseppe Lazzara ◽  
Lorenzo Lisuzzo ◽  
Stefana Milioto ◽  
Filippo Parisi
Keyword(s):  
Tensile Properties ◽  
Food Packaging ◽  
Mechanical Response ◽  
Materials Science ◽  
Traction Force ◽  
Tensile Tests ◽  
Mechanical Analysis ◽  
Nanocomposite Films ◽  
Preliminary Study ◽  
Mechanical Performances

We investigated the efficacy of several nanoclays (halloysite, sepiolite and laponite) as nanofillers for Mater-Bi, which is a commercial bioplastic extensively used within food packaging applications. The preparation of Mater-Bi/nanoclay nanocomposite films was easily achieved by means of the solvent casting method from dichloroethane. The prepared bio-nanocomposites were characterized by dynamic mechanical analysis (DMA) in order to explore the effect of the addition of the nanoclays on the mechanical behavior of the Mater-Bi-based films. Tensile tests found that filling Mater-Bi with halloysite induced the most significant improvement of the mechanical performances under traction force, while DMA measurements under the oscillatory regime showed that the polymer glass transition was not affected by the addition of the nanoclay. The tensile properties of the Mater-Bi/halloysite nanotube (HNT) films were competitive compared to those of traditional petroleum plastics in terms of the elastic modulus and stress at the breaking point. Both the mechanical response to the temperature and the tensile properties make the bio-nanocomposites appropriate for food packaging and smart coating purposes. Here, we report a preliminary study of the development of sustainable hybrid materials that could be employed in numerous industrial and technological applications within materials science and pharmaceutics.

scholarly journals Stress fibers are embedded in a contractile cortical network

2020 ◽  
Author(s):  
Timothée Vignaud ◽  
Calina Copos ◽  
Christophe Leterrier ◽  
Qingzong Tseng ◽  
Laurent Blanchoin ◽  
...  
Keyword(s):  
Cell Fate ◽  
Actin Filaments ◽  
Mechanical Response ◽  
Force Production ◽  
Traction Force ◽  
Cortical Network ◽  
Cortical Actin ◽  
Cell Fate Decisions ◽  
Production And Distribution ◽  
Intracellular Forces

ABSTRACTContractile actomyosin networks generate intracellular forces essential for the regulation of cell shape, migration, and cell-fate decisions, ultimately leading to the remodeling and patterning of tissues. Although actin filaments aligned in bundles represent the main source of traction-force production in adherent cells, there is increasing evidence that these bundles form interconnected and interconvertible structures with the rest of the intracellular actin network. In this study, we explored how these bundles are connected to the surrounding cortical network and the mechanical impact of these interconnected structures on the production and distribution of traction forces on the extracellular matrix and throughout the cell. By using a combination of hydrogel micropatterning, traction-force microscopy and laser photoablation, we measured the relaxation of the cellular traction field in response to local photoablations at various positions within the cell. Our experimental results and modeling of the mechanical response of the network revealed that bundles were fully embedded along their entire length in a continuous and contractile network of cortical filaments. Moreover, the propagation of the contraction of these bundles throughout the entire cell was dependent on this embedding. In addition, these bundles appeared to originate from the alignment and coalescence of thin and unattached cortical actin filaments from the surrounding mesh.

scholarly journals A conformational sensor based on genetic code expansion reveals an autocatalytic component in EGFR activation

2018 ◽  
Author(s):  
Martin Baumdick ◽  
Márton Gelléri ◽  
Chayasith Uttamapinant ◽  
Václav Beránek ◽  
Jason W Chin ◽  
...  
Keyword(s):  
Genetic Code ◽  
Kinase Activity ◽  
Conformational Transition ◽  
Growth Factor Receptor ◽  
Resonance Energy ◽  
Conformational Transitions ◽  
Allosteric Activation ◽  
Egfr Activation ◽  
Activation Mechanisms ◽  
Genetic Code Expansion

AbstractEpidermal growth factor receptor (EGFR) activation by growth factors (GFs) relies on dimerization and allosteric activation of its intrinsic kinase activity, resulting in trans-phosphorylation of tyrosines on its C-terminal tail. While structural and biochemical studies identified this EGF-induced allosteric activation, imaging collective EGFR activation in cells and molecular dynamics simulations pointed at additional catalytic EGFR activation mechanisms. To gain more insight in EGFR activation mechanisms in living cells, we developed a Förster Resonance Energy Transfer (FRET) based conformational EGFR indicator (CONEGI) using genetic code expansion that reports on conformational transitions in the EGFR activation loop. Comparing conformational transitions, self-association and auto-phosphorylation of CONEGI and its Y845F mutant revealed that Y845 phosphorylation induces a catalytically active conformation in EGFR monomers. This conformational transition depends on EGFR kinase activity and auto-phosphorylation on its C–terminal tail, generating a looped causality that leads to autocatalytic amplification of EGFR phosphorylation at low EGF dose.

Developing a multi-functional sensor for cell traction force, matrix remodeling and biomechanical assays in self-assembled 3D tissues in vitro

2021 ◽  
Author(s):  
Bashar Emon ◽  
M Saddam H Joy ◽  
M Taher A Saif
Keyword(s):  
Traction Force ◽  
Patient Specific ◽  
Matrix Remodeling ◽  
Tissue Stiffness ◽  
Cell Traction ◽  
Primary Fibroblasts ◽  
Multiple Cell ◽  
Cell Matrix Interactions ◽  
Cell Force

Abstract Cell-matrix interactions, mediated by cellular force and matrix remodeling, result in a dynamic reciprocity that drives numerous biological processes and disease progression. Currently, there is no available method for direct quantification cell traction force and matrix remodeling in 3D matrices as a function of time. To address this long-standing need, we recently developed a high-resolution microfabricated sensor1 that measures cell force, tissue-stiffness and can apply mechanical stimulation to the tissue. Here the tissue self-assembles and self-integrates with the sensor. With primary fibroblasts, cancer cells and neurons, we demonstrated the feasibility of the sensor by measuring single/multiple cell force with a resolution of 1 nN, and tissue stiffness1 due to matrix remodeling by the cells. The sensor can be translated into a high-throughput system for clinical assays such as patient-specific drug and phenotypic screening. In this paper, we present the detailed protocol for manufacturing the sensors, preparing experimental setup, developing assays with different tissues, and for imaging and analyzing the data.

scholarly journals Rac3 regulates breast cancer invasion and metastasis by controlling adhesion and matrix degradation

2017 ◽  
Vol 216 (12) ◽  
pp. 4331-4349 ◽  
Author(s):  
Sara K. Donnelly ◽  
Ramon Cabrera ◽  
Serena P.H. Mao ◽  
John R. Christin ◽  
Bin Wu ◽  
...  
Keyword(s):  
Tumor Cells ◽  
Initial Step ◽  
Surrounding Tissue ◽  
Matrix Degradation ◽  
Nucleotide Exchange ◽  
Invasion And Metastasis ◽  
Downstream Effector ◽  
Breast Cancer Invasion ◽  
The Matrix

The initial step of metastasis is the local invasion of tumor cells into the surrounding tissue. Invadopodia are actin-based protrusions that mediate the matrix degradation necessary for invasion and metastasis of tumor cells. We demonstrate that Rac3 GTPase is critical for integrating the adhesion of invadopodia to the extracellular matrix (ECM) with their ability to degrade the ECM in breast tumor cells. We identify two pathways at invadopodia important for integrin activation and delivery of matrix metalloproteinases: through the upstream recruiter CIB1 as well as the downstream effector GIT1. Rac3 activity, at and surrounding invadopodia, is controlled by Vav2 and βPIX. These guanine nucleotide exchange factors regulate the spatiotemporal dynamics of Rac3 activity, impacting GIT1 localization. Moreover, the GTPase-activating function of GIT1 toward the vesicular trafficking regulator Arf6 GTPase is required for matrix degradation. Importantly, Rac3 regulates the ability of tumor cells to metastasize in vivo. The Rac3-dependent mechanisms we show in this study are critical for balancing proteolytic activity and adhesive activity to achieve a maximally invasive phenotype.

scholarly journals Astigmatic traction force microscopy (aTFM)

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Di Li ◽  
Huw Colin-York ◽  
Liliana Barbieri ◽  
Yousef Javanmardi ◽  
Yuting Guo ◽  
...  
Keyword(s):  
Total Internal Reflection ◽  
Three Dimensional ◽  
Traction Force ◽  
Super Resolution ◽  
Traction Force Microscopy ◽  
Force Measurements ◽  
Complete Understanding ◽  
Force Microscopy ◽  
Cell Force ◽  
Limited Sensitivity

AbstractQuantifying small, rapidly progressing three-dimensional forces generated by cells remains a major challenge towards a more complete understanding of mechanobiology. Traction force microscopy is one of the most broadly applied force probing technologies but ascertaining three-dimensional information typically necessitates slow, multi-frame z-stack acquisition with limited sensitivity. Here, by performing traction force microscopy using fast single-frame astigmatic imaging coupled with total internal reflection fluorescence microscopy we improve the temporal resolution of three-dimensional mechanical force quantification up to 10-fold compared to its related super-resolution modalities. 2.5D astigmatic traction force microscopy (aTFM) thus enables live-cell force measurements approaching physiological sensitivity.

scholarly journals The biochemical composition of the actomyosin network sets the magnitude of cellular traction forces

2021 ◽  
Vol 32 (18) ◽  
pp. 1737-1748
Author(s):  
Somanna Kollimada ◽  
Fabrice Senger ◽  
Timothée Vignaud ◽  
Manuel Théry ◽  
Laurent Blanchoin ◽  
...  
Keyword(s):  
Focal Adhesion ◽  
Biochemical Composition ◽  
Force Production ◽  
Traction Force ◽  
Traction Force Microscopy ◽  
Traction Forces ◽  
Force Microscopy ◽  
Cell Force

The endogenous content of proteins associated with force production and the resultant traction forces were quantified in the same cells using a new traction force-microscopy assay. Focal adhesion size correlated with force in stationary cells. Relative numbers of motors and cross-linkers per actin required an optimum to maximize cell force production.

scholarly journals Fiber Network Models Predict Enhanced Cell Mechanosensing on Fibrous Gels

2016 ◽  
Vol 138 (10) ◽  
Author(s):  
Maziar Aghvami ◽  
Kristen L. Billiar ◽  
Edward A. Sander
Keyword(s):  
Mechanical Response ◽  
Traction Force ◽  
Network Models ◽  
Fiber Network ◽  
High Fiber ◽  
Fibrous Network ◽  
Cell Traction ◽  
Cell Niche ◽  
Fibrous Tissues ◽  
Fiber Connectivity

The propagation of mechanical signals through nonlinear fibrous tissues is much more extensive than through continuous synthetic hydrogels. Results from recent studies indicate that increased mechanical propagation arises from the fibrous nature of the material rather than the strain-stiffening property. The relative importance of different parameters of the fibrous network structure to this propagation, however, remains unclear. In this work, we directly compared the mechanical response of substrates of varying thickness subjected to a constant cell traction force using either a nonfibrous strain-stiffening continuum-based model or a volume-averaged fiber network model consisting of two different types of fiber network structures: one with low fiber connectivity (growth networks) and one with high fiber connectivity (Delaunay networks). The growth network fiber models predicted a greater propagation of substrate displacements through the model and a greater sensitivity to gel thickness compared to the more connected Delaunay networks and the nonlinear continuum model. Detailed analysis of the results indicates that rotational freedom of the fibers in a network with low fiber connectivity is critically important for enhanced, long-range mechanosensing. Our findings demonstrate the utility of multiscale models in predicting cells mechanosensing on fibrous gels, and they provide a more complete understanding of how cell traction forces propagate through fibrous tissues, which has implications for the design of engineered tissues and the stem cell niche.

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