Publications by authors named "Christopher A Lemmon"

27 Publications

  • Page 1 of 1

Effects of substrate stiffness and actin velocity on in silico fibronectin fibril morphometry and mechanics.

PLoS One 2021 9;16(6):e0248256. Epub 2021 Jun 9.

Department of Biomedical Engineering, Virginia Commonwealth University, Richmond, VA, United States of America.

Assembly of the extracellular matrix protein fibronectin (FN) into insoluble, viscoelastic fibrils is a critical step during embryonic development and wound healing; misregulation of FN fibril assembly has been implicated in many diseases, including fibrotic diseases and cancer. We have previously developed a computational model of FN fibril assembly that recapitulates the morphometry and mechanics of cell-derived FN fibrils. Here we use this model to probe two important questions: how is FN fibril formation affected by the contractile phenotype of the cell, and how is FN fibril formation affected by the stiffness of the surrounding tissue? We show that FN fibril formation depends strongly on the contractile phenotype of the cell, but only weakly on in vitro substrate stiffness, which is an analog for in vivo tissue stiffness. These results are consistent with previous experimental data and provide a better insight into conditions that promote FN fibril assembly. We have also investigated two distinct phenotypes of FN fibrils that we have previously identified; we show that the ratio of the two phenotypes depends on both substrate stiffness and contractile phenotype, with intermediate contractility and high substrate stiffness creating an optimal condition for stably stretched fibrils. Finally, we have investigated how re-stretch of a fibril affects cellular response. We probed how the contractile phenotype of the re-stretching cell affects the mechanics of the fibril; results indicate that the number of myosin motors only weakly affects the cellular response, but increasing actin velocity results in a decrease in the apparent stiffness of the fibril and a decrease in the stably-applied force to the fibril. Taken together, these results give novel insights into the combinatorial effects of substrate stiffness and cell contractility on FN fibril assembly.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0248256PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8189481PMC
June 2021

Immunofluorescence Image Feature Analysis and Phenotype Scoring Pipeline for Distinguishing Epithelial-Mesenchymal Transition.

Microsc Microanal 2021 May 20:1-11. Epub 2021 May 20.

Biomedical Engineering Department, The Ohio State University, Columbus, OH, USA.

Epithelial–mesenchymal transition (EMT) is an essential biological process, also implicated in pathological settings such as cancer metastasis, in which epithelial cells transdifferentiate into mesenchymal cells. We devised an image analysis pipeline to distinguish between tissues comprised of epithelial and mesenchymal cells, based on extracted features from immunofluorescence images of differing biochemical markers. Mammary epithelial cells were cultured with 0 (control), 2, 4, or 10 ng/mL TGF-β1, a well-established EMT-inducer. Cells were fixed, stained, and imaged for E-cadherin, actin, fibronectin, and nuclei via immunofluorescence microscopy. Feature selection was performed on different combinations of individual cell markers using a Bag-of-Features extraction. Control and high-dose images comprised the training data set, and the intermediate dose images comprised the testing data set. A feature distance analysis was performed to quantify differences between the treatment groups. The pipeline was successful in distinguishing between control (epithelial) and the high-dose (mesenchymal) groups, as well as demonstrating progress along the EMT process in the intermediate dose groups. Validation using quantitative PCR (qPCR) demonstrated that biomarker expression measurements were well-correlated with the feature distance analysis. Overall, we identified image pipeline characteristics for feature extraction and quantification of immunofluorescence images to distinguish progression of EMT.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1017/S1431927621000428DOI Listing
May 2021

Hydroxyapatite Particle Density Regulates Osteoblastic Differentiation Through β-Catenin Translocation.

Front Bioeng Biotechnol 2020 8;8:591084. Epub 2021 Jan 8.

Department of Biomedical Engineering and Institute for Engineering and Medicine, Virginia Commonwealth University, Richmond, VA, United States.

Substrate surface characteristics such as roughness, wettability and particle density are well-known contributors of a substrate's overall osteogenic potential. These characteristics are known to regulate cell mechanics as well as induce changes in cell stiffness, cell adhesions, and cytoskeletal structure. Pro-osteogenic particles, such as hydroxyapatite, are often incorporated into a substrate to enhance the substrates osteogenic potential. However, it is unknown which substrate characteristic is the key regulator of osteogenesis. This is partly due to the lack of understanding of how these substrate surface characteristics are transduced by cells. In this study substrates composed of polycaprolactone (PCL) and carbonated hydroxyapatite particles (HAp) were synthesized. HAp concentration was varied, and a range of surface characteristics created. The effect of each substrate characteristic on osteoblastic differentiation was then examined. We found that, of the characteristics examined, only HAp density, and indeed a specific density (85 particles/cm), significantly increased osteoblastic differentiation. Further, an increase in focal adhesion maturation and turnover was observed in cells cultured on this substrate. Moreover, β-catenin translocation from the membrane bound cell fraction to the nucleus was more rapid in cells on the 85 particle/cm substrate compared to cells on tissue culture polystyrene. Together, these data suggest that particle density is one pivotal factor in determining a substrates overall osteogenic potential. Additionally, the observed increase in osteoblastic differentiation is a at least partly the result of β-catenin translocation and transcriptional activity suggesting a β-catenin mediated mechanism by which substrate surface characteristics are transduced.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3389/fbioe.2020.591084DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7820766PMC
January 2021

Utilization of COVID-19 Treatments and Clinical Outcomes among Patients with Cancer: A COVID-19 and Cancer Consortium (CCC19) Cohort Study.

Cancer Discov 2020 10 22;10(10):1514-1527. Epub 2020 Jul 22.

Vanderbilt-Ingram Cancer Center, Nashville, Tennessee.

Among 2,186 U.S. adults with invasive cancer and laboratory-confirmed SARS-CoV-2 infection, we examined the association of COVID-19 treatments with 30-day all-cause mortality and factors associated with treatment. Logistic regression with multiple adjustments (e.g., comorbidities, cancer status, baseline COVID-19 severity) was performed. Hydroxychloroquine with any other drug was associated with increased mortality versus treatment with any COVID-19 treatment other than hydroxychloroquine or untreated controls; this association was not present with hydroxychloroquine alone. Remdesivir had numerically reduced mortality versus untreated controls that did not reach statistical significance. Baseline COVID-19 severity was strongly associated with receipt of any treatment. Black patients were approximately half as likely to receive remdesivir as white patients. Although observational studies can be limited by potential unmeasured confounding, our findings add to the emerging understanding of patterns of care for patients with cancer and COVID-19 and support evaluation of emerging treatments through inclusive prospective controlled trials. SIGNIFICANCE: Evaluating the potential role of COVID-19 treatments in patients with cancer in a large observational study, there was no statistically significant 30-day all-cause mortality benefit with hydroxychloroquine or high-dose corticosteroids alone or in combination; remdesivir showed potential benefit. Treatment receipt reflects clinical decision-making and suggests disparities in medication access..
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1158/2159-8290.CD-20-0941DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7541683PMC
October 2020

Clinical impact of COVID-19 on patients with cancer (CCC19): a cohort study.

Lancet 2020 06 28;395(10241):1907-1918. Epub 2020 May 28.

Roswell Park Comprehensive Cancer Center, Buffalo, NY, USA.

Background: Data on patients with COVID-19 who have cancer are lacking. Here we characterise the outcomes of a cohort of patients with cancer and COVID-19 and identify potential prognostic factors for mortality and severe illness.

Methods: In this cohort study, we collected de-identified data on patients with active or previous malignancy, aged 18 years and older, with confirmed severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection from the USA, Canada, and Spain from the COVID-19 and Cancer Consortium (CCC19) database for whom baseline data were added between March 17 and April 16, 2020. We collected data on baseline clinical conditions, medications, cancer diagnosis and treatment, and COVID-19 disease course. The primary endpoint was all-cause mortality within 30 days of diagnosis of COVID-19. We assessed the association between the outcome and potential prognostic variables using logistic regression analyses, partially adjusted for age, sex, smoking status, and obesity. This study is registered with ClinicalTrials.gov, NCT04354701, and is ongoing.

Findings: Of 1035 records entered into the CCC19 database during the study period, 928 patients met inclusion criteria for our analysis. Median age was 66 years (IQR 57-76), 279 (30%) were aged 75 years or older, and 468 (50%) patients were male. The most prevalent malignancies were breast (191 [21%]) and prostate (152 [16%]). 366 (39%) patients were on active anticancer treatment, and 396 (43%) had active (measurable) cancer. At analysis (May 7, 2020), 121 (13%) patients had died. In logistic regression analysis, independent factors associated with increased 30-day mortality, after partial adjustment, were: increased age (per 10 years; partially adjusted odds ratio 1·84, 95% CI 1·53-2·21), male sex (1·63, 1·07-2·48), smoking status (former smoker vs never smoked: 1·60, 1·03-2·47), number of comorbidities (two vs none: 4·50, 1·33-15·28), Eastern Cooperative Oncology Group performance status of 2 or higher (status of 2 vs 0 or 1: 3·89, 2·11-7·18), active cancer (progressing vs remission: 5·20, 2·77-9·77), and receipt of azithromycin plus hydroxychloroquine (vs treatment with neither: 2·93, 1·79-4·79; confounding by indication cannot be excluded). Compared with residence in the US-Northeast, residence in Canada (0·24, 0·07-0·84) or the US-Midwest (0·50, 0·28-0·90) were associated with decreased 30-day all-cause mortality. Race and ethnicity, obesity status, cancer type, type of anticancer therapy, and recent surgery were not associated with mortality.

Interpretation: Among patients with cancer and COVID-19, 30-day all-cause mortality was high and associated with general risk factors and risk factors unique to patients with cancer. Longer follow-up is needed to better understand the effect of COVID-19 on outcomes in patients with cancer, including the ability to continue specific cancer treatments.

Funding: American Cancer Society, National Institutes of Health, and Hope Foundation for Cancer Research.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/S0140-6736(20)31187-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7255743PMC
June 2020

A hybrid model of intercellular tension and cell-matrix mechanical interactions in a multicellular geometry.

Biomech Model Mechanobiol 2020 Dec 20;19(6):1997-2013. Epub 2020 Mar 20.

Department of Biomedical Engineering, Virginia Commonwealth University, Richmond, VA, USA.

Epithelial cells form continuous sheets of cells that exist in tensional homeostasis. Homeostasis is maintained through cell-to-cell junctions that distribute tension and balance forces between cells and their underlying matrix. Disruption of tensional homeostasis can lead to epithelial-mesenchymal transition (EMT), a transdifferentiation process in which epithelial cells adopt a mesenchymal phenotype, losing cell-cell adhesion and enhancing cellular motility. This process is critical during embryogenesis and wound healing, but is also dysregulated in many disease states. To further understand the role of intercellular tension in spatial patterning of epithelial cell monolayers, we developed a multicellular computational model of cell-cell and cell-substrate forces. This work builds on a hybrid cellular Potts model (CPM)-finite element model to evaluate cell-matrix mechanical feedback of an adherent multicellular cluster. Cellular movement is governed by thermodynamic constraints from cell volume, cell-cell and cell-matrix contacts, and durotaxis, which arises from cell-generated traction forces on a finite element substrate. Junction forces at cell-cell contacts balance these traction forces, thereby producing a mechanically stable epithelial monolayer. Simulations were compared to in vitro experiments using fluorescence-based junction force sensors in clusters of cells undergoing EMT. Results indicate that the multicellular CPM model can reproduce many aspects of EMT, including epithelial monolayer formation dynamics, changes in cell geometry, and spatial patterning of cell-cell forces in an epithelial tissue.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/s10237-020-01321-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7502553PMC
December 2020

Cell Fate Forecasting: A Data-Assimilation Approach to Predict Epithelial-Mesenchymal Transition.

Biophys J 2020 04 15;118(7):1749-1768. Epub 2020 Feb 15.

Department of Biomedical Engineering, The Ohio State University, Columbus, Ohio; Department of Biomedical Engineering, Virginia Commonwealth University, Richmond, Virginia; The Dorothy M. Davis Heart and Lung Research Institute, The Ohio State University Wexner Medical Center, Columbus, Ohio. Electronic address:

Epithelial-mesenchymal transition (EMT) is a fundamental biological process that plays a central role in embryonic development, tissue regeneration, and cancer metastasis. Transforming growth factor-β (TGFβ) is a potent inducer of this cellular transition, which is composed of transitions from an epithelial state to intermediate or partial EMT state(s) to a mesenchymal state. Using computational models to predict cell state transitions in a specific experiment is inherently difficult for reasons including model parameter uncertainty and error associated with experimental observations. In this study, we demonstrate that a data-assimilation approach using an ensemble Kalman filter, which combines limited noisy observations with predictions from a computational model of TGFβ-induced EMT, can reconstruct the cell state and predict the timing of state transitions. We used our approach in proof-of-concept "synthetic" in silico experiments, in which experimental observations were produced from a known computational model with the addition of noise. We mimic parameter uncertainty in in vitro experiments by incorporating model error that shifts the TGFβ doses associated with the state transitions and reproduces experimentally observed variability in cell state by either shifting a single parameter or generating "populations" of model parameters. We performed synthetic experiments for a wide range of TGFβ doses, investigating different cell steady-state conditions, and conducted parameter studies varying properties of the data-assimilation approach including the time interval between observations and incorporating multiplicative inflation, a technique to compensate for underestimation of the model uncertainty and mitigate the influence of model error. We find that cell state can be successfully reconstructed and the future cell state predicted in synthetic experiments, even in the setting of model error, when experimental observations are performed at a sufficiently short time interval and incorporate multiplicative inflation. Our study demonstrates the feasibility and utility of a data-assimilation approach to forecasting the fate of cells undergoing EMT.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.bpj.2020.02.011DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7136288PMC
April 2020

A novel method for quantifying traction forces on hexagonal micropatterned protein features on deformable poly-dimethyl siloxane sheets.

MethodsX 2019 31;6:1343-1352. Epub 2019 May 31.

Department of Biomedical Engineering, Virginia Commonwealth University, United States.

Many methods exist for quantifying cellular traction forces, including traction force microscopy and microfabricated post arrays. However, these methodologies have limitations, including a requirement to remove cells to determine undeflected particle locations and the inability to quantify forces of cells with low cytoskeletal stiffness, respectively. Here we present a novel method of traction force quantification that eliminates both of these limitations. Through the use of a hexagonal pattern of microcontact-printed protein spots, a novel computational algorithm, and thin surfaces of polydimethyl siloxane (PDMS) blends, we demonstrate a system that: •quantifies cellular forces on a homogeneous surface that is stable and easily manufactured.•utilizes hexagonal patterns of protein spots and computational geometry to quantify cellular forces without need for cell removal.•quantifies cellular forces in cells with low cytoskeletal rigidity.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.mex.2019.05.011DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6690417PMC
May 2019

Mechanochemical Signaling of the Extracellular Matrix in Epithelial-Mesenchymal Transition.

Front Cell Dev Biol 2019 19;7:135. Epub 2019 Jul 19.

Department of Biomedical Engineering, Virginia Commonwealth University, Richmond, VA, United States.

Epithelial-Mesenchymal Transition (EMT) is a critical process in embryonic development in which epithelial cells undergo a transdifferentiation into mesenchymal cells. This process is essential for tissue patterning and organization, and it has also been implicated in a wide array of pathologies. While the intracellular signaling pathways that regulate EMT are well-understood, there is increasing evidence that the mechanical properties and composition of the extracellular matrix (ECM) also play a key role in regulating EMT. In turn, EMT drives changes in the mechanics and composition of the ECM, creating a feedback loop that is tightly regulated in healthy tissues, but is often dysregulated in disease. Here we present a review that summarizes our understanding of how ECM mechanics and composition regulate EMT, and how in turn EMT alters ECM mechanics and composition.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3389/fcell.2019.00135DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6658819PMC
July 2019

Multiple Cryptic Binding Sites are Necessary for Robust Fibronectin Assembly: An In Silico Study.

Sci Rep 2017 12 22;7(1):18061. Epub 2017 Dec 22.

Department of Biomedical Engineering, Virginia Commonwealth University, Richmond, VA, 23298, USA.

The mechanism of assembly of the extracellular matrix protein fibronectin (FN) into elastic, insoluble fibrils is still poorly understood. FN fibrillogenesis requires cell-generated forces, which expose cryptic FN-FN binding sites buried in FN Type III domains. The number and location of cryptic binding sites have been debated, but experimental evidence suggests multiple domains may contain FN-FN binding sites. The requirement of cell-dependent forces to generate FN fibrils restricts investigation of the mechanism of assembly. To address this, we use a recently developed biophysical model of fibrillogenesis to test competing hypotheses for the location and number of cryptic FN-FN binding sites and quantify the effect of these molecular alterations on assembled FN fibril properties. Simulations predict that a single FN-FN binding site facilitates either negligible fibrillogenesis or produces FN fibrils that are neither robust nor physiological. However, inclusion of multiple FN-FN binding sites predicts robust fibrillogenesis, which minimally depends on individual domain properties. Multiple FN-FN binding site models predict a heterogeneous fibril population that contains two distinct phenotypes with unique viscoelastic properties, which we speculate may play a key role in generating heterogeneous mechanical signaling in the extracellular matrix of developing and regenerating tissues.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41598-017-18328-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5741729PMC
December 2017

Fibronectin Conformation and Assembly: Analysis of Fibronectin Deletion Mutants and Fibronectin Glomerulopathy (GFND) Mutants.

Biochemistry 2017 08 11;56(34):4584-4591. Epub 2017 Aug 11.

Department of Cell Biology, Duke University Medical Center , Durham, North Carolina 27710, United States.

To study fibronectin (FN) conformation and assembly, we generated several deletion mutants: FNΔ1-5, FNΔ1-3, FNΔ4-8, and FNΔ11-14. A monomeric form, FNmono, which lacked the C-terminal dimerization region, was also created. FNtnA-D was generated by swapping FNIII domains 1-8 in FNΔ11-14 with seven FNIII domains from tenascin-C. The conformations of these mutants were analyzed by glycerol gradient sedimentation under low-salt (20 mM NaCl) and high-salt (200 mM NaCl) conditions. Surprisingly, most of the mutants showed a compact conformation under low-salt conditions, except for FNtnA-D. When we tested these mutants in cell culture, FNΔ1-5, FNΔ1-3, and FNtnA-D were unable to form a matrix. Interestingly, FNΔ1-3 and FNtnA-D were capable of co-assembly with full-length FN, while FNΔ1-5 was not. This indicates that the segment 1-5 is crucial for matrix assembly and segment 1-3 is also important. Mutations in FN are associated with glomerulopathy, but when we studied mutant proteins, the single-nucleotide mutations had only minor effects on conformation and matrix assembly. The mutations may destabilize their FNIII domains or generate dimers of dimers by disulfide cross-linking.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1021/acs.biochem.7b00589DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5729893PMC
August 2017

Mechanotransduction Dynamics at the Cell-Matrix Interface.

Biophys J 2017 May;112(9):1962-1974

Department of Biomedical Engineering, Virginia Commonwealth University, Richmond, Virginia. Electronic address:

The ability of cells to sense and respond to mechanical cues from the surrounding environment has been implicated as a key regulator of cell differentiation, migration, and proliferation. The extracellular matrix (ECM) is an oft-overlooked component of the interface between cells and their surroundings. Cells assemble soluble ECM proteins into insoluble fibrils with unique mechanical properties that can alter the mechanical cues a cell receives. In this study, we construct a model that predicts the dynamics of cellular traction force generation and subsequent assembly of fibrils of the ECM protein fibronectin (FN). FN fibrils are the primary component in primordial ECM and, as such, FN assembly is a critical component in the cellular mechanical response. The model consists of a network of Hookean springs, each representing an extensible domain within an assembling FN fibril. As actomyosin forces stretch the spring network, simulations predict the resulting traction force and FN fibril formation. The model accurately predicts FN fibril morphometry and demonstrates a mechanism by which FN fibril assembly regulates traction force dynamics in response to mechanical stimuli and varying surrounding substrate stiffness.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.bpj.2017.02.027DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5425358PMC
May 2017

Fibronectin fibrils regulate TGF-β1-induced Epithelial-Mesenchymal Transition.

Matrix Biol 2017 07 19;60-61:157-175. Epub 2017 Jan 19.

Department of Biomedical Engineering, Virginia Commonwealth University, 800 E. Leigh St., Richmond, VA 23298, United States; Massey Cancer Center, Virginia Commonwealth University, 101 W Franklin St., Richmond, VA 23220, United States. Electronic address:

Epithelial-Mesenchymal Transition (EMT) is a dynamic process through which epithelial cells transdifferentiate from an epithelial phenotype into a mesenchymal phenotype. Previous studies have demonstrated that both mechanical signaling and soluble growth factor signaling facilitate this process. One possible point of integration for mechanical and growth factor signaling is the extracellular matrix. Here we investigate the role of the extracellular matrix (ECM) protein fibronectin (FN) in this process. We demonstrate that inhibition of FN fibrillogenesis blocks activation of the Transforming Growth Factor-Beta (TGF-β) signaling pathway via Smad2 signaling, decreases cell migration and ultimately leads to inhibition of EMT. Results show that soluble FN, FN fibrils, or increased contractile forces are insufficient to independently induce EMT. We further demonstrate that inhibition of latent TGF-β1 binding to FN fibrils via either a monoclonal blocking antibody against the growth factor binding domain of FN or through use of a FN deletion mutant that lacks the growth factor binding domains of FN blocks EMT progression, indicating a novel role for FN in EMT in which the assembly of FN fibrils serves to localize TGF-β1 signaling to drive EMT.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.matbio.2017.01.001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5438896PMC
July 2017

Nesprin-2G, a Component of the Nuclear LINC Complex, Is Subject to Myosin-Dependent Tension.

Biophys J 2016 Jan;110(1):34-43

Department of Biomedical Engineering, Virginia Commonwealth University, Richmond, Virginia. Electronic address:

The nucleus of a cell has long been considered to be subject to mechanical force. Despite the observation that mechanical forces affect nuclear geometry and movement, how forces are applied onto the nucleus is not well understood. The nuclear LINC (linker of nucleoskeleton and cytoskeleton) complex has been hypothesized to be the critical structure that mediates the transfer of mechanical forces from the cytoskeleton onto the nucleus. Previously used techniques for studying nuclear forces have been unable to resolve forces across individual proteins, making it difficult to clearly establish if the LINC complex experiences mechanical load. To directly measure forces across the LINC complex, we generated a fluorescence resonance energy transfer-based tension biosensor for nesprin-2G, a key structural protein in the LINC complex, which physically links this complex to the actin cytoskeleton. Using this sensor we show that nesprin-2G is subject to mechanical tension in adherent fibroblasts, with highest levels of force on the apical and equatorial planes of the nucleus. We also show that the forces across nesprin-2G are dependent on actomyosin contractility and cell elongation. Additionally, nesprin-2G tension is reduced in fibroblasts from Hutchinson-Gilford progeria syndrome patients. This report provides the first, to our knowledge, direct evidence that nesprin-2G, and by extension the LINC complex, is subject to mechanical force. We also present evidence that nesprin-2G localization to the nuclear membrane is altered under high-force conditions. Because forces across the LINC complex are altered by a variety of different conditions, mechanical forces across the LINC complex, as well as the nucleus in general, may represent an important mechanism for mediating mechanotransduction.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.bpj.2015.11.014DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4805861PMC
January 2016

Bioengineered silk scaffolds in 3D tissue modeling with focus on mammary tissues.

Mater Sci Eng C Mater Biol Appl 2016 Feb 9;59:1168-1180. Epub 2015 Oct 9.

Department of Biological Sciences, University of North Carolina at Charlotte, Charlotte, NC 28223, USA. Electronic address:

In vitro generation of three-dimensional (3D) biological tissues and organ-like structures is a promising strategy to study and closely model complex aspects of the molecular, cellular, and physiological interactions of tissue. In particular, in vitro 3D tissue modeling holds promises to further our understanding of breast development. Indeed, biologically relevant 3D structures that combine mammary cells and engineered matrices have improved our knowledge of mammary tissue growth, organization, and differentiation. Several polymeric biomaterials have been used as scaffolds to engineer 3D mammary tissues. Among those, silk fibroin-based biomaterials have many biologically relevant properties and have been successfully used in multiple medical applications. Here, we review the recent advances in engineered scaffolds with an emphasis on breast-like tissue generation and the benefits of modified silk-based scaffolds.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.msec.2015.10.007DOI Listing
February 2016

Fibronectin fibrillogenesis facilitates mechano-dependent cell spreading, force generation, and nuclear size in human embryonic fibroblasts.

Integr Biol (Camb) 2015 Nov;7(11):1454-65

Department of Biomedical Engineering, Virginia Commonwealth University, 401 West Main Street, Richmond, VA 23284-3067, USA.

Cells respond to mechanical cues from the substrate to which they are attached. These mechanical cues drive cell migration, proliferation, differentiation, and survival. Previous studies have highlighted three specific mechanisms through which substrate stiffness directly alters cell function: increasing stiffness drives (1) larger contractile forces; (2) increased cell spreading and size; and (3) altered nuclear deformation. While studies have shown that substrate mechanics are an important cue, the role of the extracellular matrix (ECM) has largely been ignored. The ECM is a crucial component of the mechanosensing system for two reasons: (1) many ECM fibrils are assembled by application of cell-generated forces, and (2) ECM proteins have unique mechanical properties that will undoubtedly alter the local stiffness sensed by a cell. We specifically focused on the role of the ECM protein fibronectin (FN), which plays a critical role in de novo tissue production. In this study, we first measured the effects of substrate stiffness on human embryonic fibroblasts by plating cells onto microfabricated pillar arrays (MPAs) of varying stiffness. Cells responded to increasing substrate stiffness by generating larger forces, spreading to larger sizes, and altering nuclear geometry. These cells also assembled FN fibrils across all stiffnesses, with optimal assembly occurring at approximately 6 kPa. We then inhibited FN assembly, which resulted in dramatic reductions in contractile force generation, cell spreading, and nuclear geometry across all stiffnesses. These findings suggest that FN fibrils play a critical role in facilitating cellular responses to substrate stiffness.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1039/c5ib00217fDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4630078PMC
November 2015

Mammary epithelial cell adhesion, viability, and infiltration on blended or coated silk fibroin-collagen type I electrospun scaffolds.

Mater Sci Eng C Mater Biol Appl 2014 Oct 8;43:37-44. Epub 2014 Jul 8.

Department of Biological Sciences, University of North Carolina at Charlotte, Charlotte, NC 28223 USA.

Interactions between cells and the extracellular matrix (ECM) play a crucial role in regulating biological tissue function. Silk biomaterials from Bombyx mori (B. mori) silkworm silk are widely used in tissue engineering. As this silk fibroin (SF) contains no strong adhesion sites, we assessed whether the blending or coating of SF with collagen would further improve SF biocompatibility, in part through the addition of the specific integrin recognition sequences. In the present study, electrospun scaffolds were developed by blending 7% SF and 7% type I collagen solutions at ratios of 100:0 (pure SF), 95:5, 90:10, and 85:15 (SF:collagen, v/v) prior to electrospinning. Pure SF scaffolds were further coated with collagen type I. The physical and mechanical properties of these scaffolds and MCF10A mammary epithelial cell adhesion, viability, and infiltration into these blended or coated SF-collagen (SF-C) scaffolds were determined. The blending of SF with collagen decreased average pore sizes and fiber diameters of the electrospun scaffolds regardless of the ratio (p<0.01). The mechanical strength of these scaffolds did not change in their hydrated state (ns), but was decreased for 85:15 SF-C blended scaffolds in the dry state (p<0.05). The adhesion of MCF10A cells was significantly increased in SF-C blended or coated scaffolds compared to pure SF scaffolds (p<0.01). MCF10A cell viability and infiltration on SF-C coated scaffolds were significantly higher compared to all other conditions tested (p<0.01).
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.msec.2014.06.037DOI Listing
October 2014

Endothelial matrix assembly during capillary morphogenesis: insights from chimeric TagRFP-fibronectin matrix.

J Histochem Cytochem 2014 Nov 25;62(11):774-90. Epub 2014 Jul 25.

Anesthesiology and Critical Care Medicine (FC, LR), Johns Hopkins Medical Institutions, Baltimore, MDDepartment of Biomedical Engineering, Virginia Commonwealth University, Richmond, VA (CAL)Cell Biology (LR), Johns Hopkins Medical Institutions, Baltimore, MDBiomedical Engineering (LR), Johns Hopkins Medical Institutions, Baltimore, MDPediatrics (LR), Johns Hopkins Medical Institutions, Baltimore, MDCenter for Cell Dynamics (LR), Johns Hopkins Medical Institutions, Baltimore, MDGraduate Program in Cellular and Molecular Medicine (VN), Johns Hopkins Medical Institutions, Baltimore, MDDepartment of Molecular and Cell Biology, The Weizmann Institute of Science, Rehovot, Israel (VR)

Biologically relevant, three-dimensional extracellular matrix is an essential component of in vitro vasculogenesis models. WI-38 fibroblasts assemble a 3D matrix that induces endothelial tubulogenesis, but this model is challenged by fibroblast senescence and the inability to distinguish endothelial cell-derived matrix from matrix made by WI-38 fibroblasts. Matrices produced by hTERT-immortalized WI-38 recapitulated those produced by wild type fibroblasts. ECM fibrils were heavily populated by tenascin-C, fibronectin, and type VI collagen. Nearly half of the total type I collagen, but only a small fraction of the type IV collagen, were incorporated into ECM. Stable hTERT-WI-38 transfectants expressing TagRFP-fibronectin incorporated TagRFP into ~90% of the fibronectin in 3D matrices. TagRFP-fibronectin colocalized with tenascin-C and with type I collagen in a pattern that was similar to that seen in matrices from wild type WI-38. Human Umbilical Vein Endothelial Cells (HUVEC) formed 3D adhesions and tubes on WI38-hTERT-TagRFP-FN-derived matrices, and the TagRFP-fibronectin component of this new 3D human fibroblast matrix model facilitated the demonstration of concentrated membrane type 1 metalloprotease and new HUVEC FN and collagen type IV fibrils during EC tubulogenesis. These findings indicate that WI-38-hTERT- and WI-38-hTERT-TagRFP-FN-derived matrices provide platforms for the definition of new matrix assembly and remodeling events during vasculogenesis.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1369/0022155414547419DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4209295PMC
November 2014

Probing the folded state of fibronectin type III domains in stretched fibrils by measuring buried cysteine accessibility.

J Biol Chem 2011 Jul 7;286(30):26375-82. Epub 2011 Jun 7.

Department of Cell Biology, Duke University Medical Center, Durham, North Carolina 27710, USA.

Fibronectin (FN) is an extracellular matrix protein that is assembled into fibrils by cells during tissue morphogenesis and wound healing. FN matrix fibrils are highly elastic, but the mechanism of elasticity has been debated: it may be achieved by mechanical unfolding of FN-III domains or by a conformational change of the molecule without domain unfolding. Here, we investigate the folded state of FN-III domains in FN fibrils by measuring the accessibility of buried cysteines. Four of the 15 FN-III domains (III-2, -3, -9, and -11) appear to unfold in both stretched fibrils and in solution, suggesting that these domains spontaneously open and close even in the absence of tension. Two FN-III domains (III-6 and -12) appear to unfold only in fibrils and not in solution. These results suggest that domain unfolding can at best contribute partially to the 4-fold extensibility of fibronectin fibrils.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1074/jbc.M111.240028DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3143600PMC
July 2011

A predictive model of cell traction forces based on cell geometry.

Biophys J 2010 Nov;99(9):L78-80

Department of Cell Biology, Duke University Medical Center, Durham, North Carolina, USA.

Recent work has indicated that the shape and size of a cell can influence how a cell spreads, develops focal adhesions, and exerts forces on the substrate. However, it is unclear how cell shape regulates these events. Here we present a computational model that uses cell shape to predict the magnitude and direction of forces generated by cells. The predicted results are compared to experimentally measured traction forces, and show that the model can predict traction force direction, relative magnitude, and force distribution within the cell using only cell shape as an input. Analysis of the model shows that the magnitude and direction of the traction force at a given point is proportional to the first moment of area about that point in the cell, suggesting that contractile forces within the cell act on the entire cytoskeletal network as a single cohesive unit. Through this model, we demonstrate that intrinsic properties of cell shape can facilitate changes in traction force patterns, independently of heterogeneous mechanical properties or signaling events within the cell.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.bpj.2010.09.024DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2966001PMC
November 2010

Cell traction forces direct fibronectin matrix assembly.

Biophys J 2009 Jan;96(2):729-38

Department of Biomedical Engineering, The Johns Hopkins University School of Medicine, Baltimore, Maryland, USA.

Interactions between cells and the surrounding matrix are critical to the development and engineering of tissues. We have investigated the role of cell-derived traction forces in the assembly of extracellular matrix using what we believe is a novel assay that allows for simultaneous measurement of traction forces and fibronectin fibril growth at discrete cell-matrix attachment sites. NIH3T3 cells were plated onto arrays of deformable cantilever posts for 2-24 h. Data indicate that developing fibril orientation is guided by the direction of the traction force applied to that fibril. In addition, cells initially establish a spatial distribution of traction forces that is largest at the cell edge and decreases toward the cell center. This distribution progressively shifts from a predominantly peripheral pattern to a more uniform pattern as compressive strain at the cell perimeter decreases with time. The impact of these changes on fibrillogenesis was tested by treating cells with blebbistatin or calyculin A to tonically block or augment, respectively, myosin II activity. Both treatments blocked the inward translation of traction forces, the dissipation of compressive strain, and fibronectin fibrillogenesis over time. These data indicate that dynamic spatial and temporal changes in traction force and local strain may contribute to successful matrix assembly.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.bpj.2008.10.009DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2716473PMC
January 2009

Selective fluorescent labeling of S-nitrosothiols (S-FLOS): a novel method for studying S-nitrosation.

Nitric Oxide 2008 Nov 30;19(3):295-302. Epub 2008 Jul 30.

The Johns Hopkins School of Medicine, Anesthesiology and CCM, 720 Rutland Avenue, Traylor 621, Baltimore, MD 21205, USA.

Protein S-nitrosation is a reversible post-translation modification critical for redox-sensitive cell signaling that is typically studied using the Biotin Switch method. This method and subsequent modifications usually require avidin binding or Western blot analysis to detect biotin labeled proteins. We describe here a modification of the Biotin Switch assay that eliminates the need for Western blot or avidin enrichment protocols and allows direct comparison of the S-nitrosation state proteins from two different samples in the same gel lane or on the same 2D gel. This S-FLOS method offers detection, identification and quantification of S-nitrosated proteins, with the potential for site-specific identification of nitrosation events.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.niox.2008.07.007DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3705760PMC
November 2008

FAK potentiates Rac1 activation and localization to matrix adhesion sites: a role for betaPIX.

Mol Biol Cell 2007 Jan 8;18(1):253-64. Epub 2006 Nov 8.

Department of Anesthesiology and Critical Care Medicine, Johns Hopkins University School of Medicine, Baltimore, MD 21287, USA.

FAK, a cytoplasmic protein tyrosine kinase, is activated and localized to focal adhesions upon cell attachment to extracellular matrix. FAK null cells spread poorly and exhibit altered focal adhesion turnover. Rac1 is a member of the Rho-family GTPases that promotes membrane ruffling, leading edge extension, and cell spreading. We investigated the activation and subcellular location of Rac1 in FAK null and FAK reexpressing fibroblasts. FAK reexpressers had a more robust pattern of Rac1 activation after cell adhesion to fibronectin than the FAK null cells. Translocation of Rac1 to focal adhesions was observed in FAK reexpressers, but seldom in FAK null cells. Experiments with constitutively active L61Rac1 and dominant negative N17Rac1 indicated that the activation state of Rac1 regulated its localization to focal adhesions. We demonstrated that FAK tyrosine-phosphorylated betaPIX and thereby increased its binding to Rac1. In addition, betaPIX facilitated the targeting of activated Rac1 to focal adhesions and the efficiency of cell spreading. These data indicate that FAK has a role in the activation and focal adhesion translocation of Rac1 through the tyrosine phosphorylation of betaPIX.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1091/mbc.e06-03-0207DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1751318PMC
January 2007

Oxidized low-density lipoprotein-dependent endothelial arginase II activation contributes to impaired nitric oxide signaling.

Circ Res 2006 Oct 28;99(9):951-60. Epub 2006 Sep 28.

Department of Anesthesiology, The Johns Hopkins Medical Institutions, Baltimore, MD, USA.

Oxidized low-density lipoprotein (OxLDL) impairs NO signaling and endothelial function, and contributes to the pathogenesis of atherosclerosis. Arginase reciprocally regulates NO levels in endothelial cells by competing with NO synthase for the substrate l-arginine. In human aortic endothelial cells, OxLDL stimulation increased arginase enzyme activity in a time- and dose-dependent manner. Arginase activity reached its maximum as early as 5 minutes, was maintained for a period of more than 48 hours, and was associated with a reciprocal decrease in NO metabolite (NOx [nitrite and nitrate]) production. Furthermore, OxLDL induced arginase II mRNA expression after 4 hours. Small interfering RNA targeted to arginase II decreased both the quantity and the activity of arginase from baseline, prevented OxLDL-dependent increases in arginase activity, and induced an increase in NOx production. Immunofluorescence analysis revealed an association of arginase II with the microtubule cytoskeleton. Microtubule disruption with nocodazole caused a dramatic redistribution of arginase II to a diffuse cytosolic pattern, increased arginase activity, and decreased NOx production, which was restored in the presence of the specific arginase inhibitor (S)-(2-boronoethyl)-l-cysteine (BEC). On the other hand, epothilone B prevented microtubule disruption and inhibited OxLDL-dependent increases in arginase activity and attenuated OxLDL-dependent decreases in NOx. Preincubation of rat aortic rings with OxLDL resulted in an increase in arginase activity and a decrease in NOx production. This was reversed by arginase inhibition with the BEC. Thus, OxLDLs increase arginase activity by a sequence of regulatory events that involve early activation through decreased association with microtubules and a later increase in transcription. Furthermore, increased arginase activity contributes to OxLDL-dependent impairment of NOx production. Arginase, therefore, represents a novel target for therapy in atherosclerosis.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1161/01.RES.0000247034.24662.b4DOI Listing
October 2006

An inhibitory role for FAK in regulating proliferation: a link between limited adhesion and RhoA-ROCK signaling.

J Cell Biol 2006 Jul;174(2):277-88

Department of Biomedical Engineering, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA.

Focal adhesion kinase (FAK) transduces cell adhesion to the extracellular matrix into proliferative signals. We show that FAK overexpression induced proliferation in endothelial cells, which are normally growth arrested by limited adhesion. Interestingly, displacement of FAK from adhesions by using a FAK-/- cell line or by expressing the C-terminal fragment FRNK also caused an escape of adhesion-regulated growth arrest, suggesting dual positive and negative roles for FAK in growth regulation. Expressing kinase-dead FAK-Y397F in FAK-/- cells prevented uncontrolled growth, demonstrating the antiproliferative function of inactive FAK. Unlike FAK overexpression-induced growth, loss of growth control in FAK-/- or FRNK-expressing cells increased RhoA activity, cytoskeletal tension, and focal adhesion formation. ROCK inhibition rescued adhesion-dependent growth control in these cells, and expression of constitutively active RhoA or ROCK dysregulated growth. These findings demonstrate the ability of FAK to suppress and promote growth, and underscore the importance of multiple mechanisms, even from one molecule, to control cell proliferation.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1083/jcb.200510062DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2064187PMC
July 2006

Shear force at the cell-matrix interface: enhanced analysis for microfabricated post array detectors.

Mech Chem Biosyst 2005 ;2(1):1-16

Dept. of Biomedical Engineering, Johns Hopkins University, Baltimore, MD 21205, USA.

The interplay of mechanical forces between the extracellular environment and the cytoskeleton drives development, repair, and senescence in many tissues. Quantitative definition of these forces is a vital step in understanding cellular mechanosensing. Microfabricated post array detectors (mPADs) provide direct measurements of cell-generated forces during cell adhesion to extracellular matrix. A new approach to mPAD post labeling, volumetric imaging, and an analysis of post bending mechanics determined that cells apply shear forces and not point moments at the matrix interface. In addition, these forces could be accurately resolved from post deflections by using images of post tops and bases. Image analysis tools were then developed to increase the precision and throughput of post centroid location. These studies resulted in an improved method of force measurement with broad applicability and concise execution using a fully automated force analysis system. The new method measures cell-generated forces with less than 5% error and less than 90 seconds of computational time. Using this approach, we demonstrated direct and distinct relationships between cellular traction force and spread cell surface area for fibroblasts, endothelial cells, epithelial cells and smooth muscle cells.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1480360PMC
June 2006

Nitric oxide regulates the heart by spatial confinement of nitric oxide synthase isoforms.

Nature 2002 Mar;416(6878):337-9

Department of Medicine (Cardiology Division), The Johns Hopkins Medical Institutions, Baltimore, Maryland 21287, USA.

Subcellular localization of nitric oxide (NO) synthases with effector molecules is an important regulatory mechanism for NO signalling. In the heart, NO inhibits L-type Ca2+ channels but stimulates sarcoplasmic reticulum (SR) Ca2+ release, leading to variable effects on myocardial contractility. Here we show that spatial confinement of specific NO synthase isoforms regulates this process. Endothelial NO synthase (NOS3) localizes to caveolae, where compartmentalization with beta-adrenergic receptors and L-type Ca2+ channels allows NO to inhibit beta-adrenergic-induced inotropy. Neuronal NO synthase (NOS1), however, is targeted to cardiac SR. NO stimulation of SR Ca2+ release via the ryanodine receptor (RyR) in vitro, suggests that NOS1 has an opposite, facilitative effect on contractility. We demonstrate that NOS1-deficient mice have suppressed inotropic response, whereas NOS3-deficient mice have enhanced contractility, owing to corresponding changes in SR Ca2+ release. Both NOS1-/- and NOS3-/- mice develop age-related hypertrophy, although only NOS3-/- mice are hypertensive. NOS1/3-/- double knockout mice have suppressed beta-adrenergic responses and an additive phenotype of marked ventricular remodelling. Thus, NOS1 and NOS3 mediate independent, and in some cases opposite, effects on cardiac structure and function.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/416337aDOI Listing
March 2002
-->