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In JoVE (1)
- A Protocol for Using Förster Resonance Energy Transfer (FRET)-force Biosensors to Measure Mechanical Forces across the Nuclear LINC Complex
Other Publications (14)
- Annals of Biomedical Engineering
- Cardiovascular Research
- American Journal of Physiology. Heart and Circulatory Physiology
- American Journal of Physiology. Cell Physiology
- Current Biology : CB
- Journal of Cell Science
- Cell Adhesion & Migration
- The Journal of Cell Biology
- The Journal of Cell Biology
- The Journal of Cell Biology
- Biophysical Journal
- Nano Letters
Articles by Daniel E. Conway in JoVE
A Protocol for Using Förster Resonance Energy Transfer (FRET)-force Biosensors to Measure Mechanical Forces across the Nuclear LINC Complex
Paul T. Arsenovic1, Kranthidhar Bathula1, Daniel E. Conway1
1Department of Biomedical Engineering, Virginia Commonwealth University
Other articles by Daniel E. Conway on PubMed
In Vitro Cytotoxicity of Redox Radical Initiators for Cross-linking of Oligo(poly(ethylene Glycol) Fumarate) Macromers
Biomacromolecules. Nov-Dec, 2003 | Pubmed ID: 14606886
A novel hydrogel system based on oligo(poly(ethylene glycol) fumarate) (OPF) is currently being investigated as an injectable carrier for marrow stromal cells (MSCs) for orthopedic tissue engineering applications. This hydrogel is cross-linked using the redox radical initiators ammonium persulfate (APS) and ascorbic acid (AA). In this study, two different persulfate oxidizing agents (APS and sodium persulfate (NaPS)) with three reducing agents derived from ascorbic acid (AA, sodium ascorbate (Asc), and magnesium ascorbate-2-phosphate (Asc-2)) and their combinations were examined to determine the relationship between pH, exposure time, and cytotoxicity for rat MSCs. In addition, gelation times for specific combinations were determined using rheometry. pH and cell viability data after 2 h for combinations ranging from 10 to 500 mM in each reagent showed that there was a smaller pH change and a corresponding higher viability at lower concentrations, regardless of the reagents used. At 10 mM, there was less than a 1.5 unit drop in pH and greater than 90% viability for all initiator combinations examined. However, MSC viability was significantly reduced with concentrations of 100 mM and higher of the initiator combinations. At 100 mM, exposure to NaPS/Asc-2 resulted in significantly more live cells than exposure to APS/AA or NaPS/Asc, but at this concentration, NaPS/Asc-2 exhibited significantly longer OPF gelation onset times than APS/AA. At all combination concentrations, exposure time (10 min vs 2 h) did not significantly affect MSC viability. These data indicate that final pH and/or radical formation have a large impact on MSC viability and that multiple, intertwined testing procedures are required for identification of appropriate initiators for cell encapsulation applications.
Thermally Cross-linked Oligo(poly(ethylene Glycol) Fumarate) Hydrogels Support Osteogenic Differentiation of Encapsulated Marrow Stromal Cells in Vitro
Biomacromolecules. Jan-Feb, 2004 | Pubmed ID: 14715001
A novel polymer, oligo(poly(ethylene glycol) fumarate) (OPF), cross-linked with a thermal radical initiation system has recently been developed in our laboratory as an injectable, biodegradable cell carrier for regeneration of orthopaedic tissues. The cross-linking, swelling, and degradative properties of hydrogels prepared from OPF with poly(ethylene glycol) of two different chain lengths were assessed. The two OPF types had similar gelation onset times ( approximately 3.6 min) but, when cross-linked for 8 min at 37 degrees C, exhibited significantly different swelling characteristics (fold swelling: 17.5 +/- 0.2 vs 13.4 +/- 0.4). Rat marrow stromal cells (MSCs) were then directly combined with the hydrogel precursors and encapsulated in a model OPF formulation at approximately 14 million cells/mL, cultured in vitro in the presence of osteogenic supplements (dexamethasone), and monitored over 28 days via histology. MSC differentiation in these samples (6 mm diameter x 0.5 mm thick before swelling), as determined by Von Kossa staining for calcified matrix, was apparent by day 21. At day 28, mineralized matrix could be seen throughout the samples, many microns away from the cells. These experiments strongly support the usefulness of thermally cross-linked OPF hydrogels as injectable cell carriers for bone regeneration.
Gene Expression of Endothelial Cells Under Pulsatile Non-reversing Vs. Steady Shear Stress; Comparison of Nitric Oxide Production
Annals of Biomedical Engineering. Apr, 2008 | Pubmed ID: 18256937
Pulsations in arterial blood flow expose the endothelium to diverse mechanical forces that may differentially regulate endothelial cell (EC) phenotype. We postulated that pulsatile non-reversing shear stress (typical of the common carotid artery), would produce a more "athero-protective" gene expression pattern compared with steady shear stress of the same mean value. Transcriptional analysis of human umbilical vein endothelial cells (HUVEC) subjected to 24 h of pulsatile shear stress (average = 13 dyne/cm(2), range = 7-25 dyne/cm(2); 1 Hz) or steady shear stress (13 dyne/cm(2)) identified approximately 200 differentially expressed genes. Hierarchical cluster analysis indicated that HUVEC respond similarly to both types of shear stress (Pearson correlation coefficient = 0.785). However, categorization of the differentially expressed genes with Ingenuity Pathways Analysis and with Expression Analysis Systematic Explorer revealed possible differences in nitric oxide (NO) production and signaling. Consistent with gene expression analysis, pulsatile shear stress significantly attenuated NO production relative to steady shear stress (0.77 +/- 0.08, p < 0.01) in HUVEC without significantly altering the levels of intracellular reactive oxygen species (0.95 +/- 0.14, p = 0.65). These results demonstrate that the common carotid flow waveform elicits subtle changes in HUVEC responses to arterial levels of shear stress, which lead to differences in NO production.
Cardiovascular Research. Mar, 2009 | Pubmed ID: 19126602
CYP1A1 and CYP1B1, members of the cytochrome P450 protein family, are regulated by fluid shear stress. This study describes the effects of duration, magnitude and pattern of shear stress on CYP1A1 and CYP1B1 expressions in human endothelial cells, towards the goal of understanding the role(s) of these genes in pro-atherogenic or anti-atherogenic endothelial cell functions.
Endothelial Cell Responses to Atheroprone Flow Are Driven by Two Separate Flow Components: Low Time-average Shear Stress and Fluid Flow Reversal
American Journal of Physiology. Heart and Circulatory Physiology. Feb, 2010 | Pubmed ID: 19915176
To simulate the effects of shear stress in regions of the vasculature prone to developing atherosclerosis, we subjected human umbilical vein endothelial cells to reversing shear stress to mimic the hemodynamic conditions at the wall of the carotid sinus, a site of complex, reversing blood flow and commonly observed atherosclerosis. We compared the effects of reversing shear stress (time-average: 1 dyn/cm(2), maximum: +11 dyn/cm(2), minimum: -11 dyn/cm(2), 1 Hz), arterial steady shear stress (15 dyn/cm(2)), and low steady shear stress (1 dyn/cm(2)) on gene expression, cell proliferation, and monocyte adhesiveness. Microarray analysis revealed that most differentially expressed genes were similarly regulated by all three shear stress regimens compared with static culture. Comparisons of the three shear stress regimens to each other identified 138 genes regulated by low average shear stress and 22 genes regulated by fluid reversal. Low average shear stress induced increased cell proliferation compared with high shear stress. Only reversing shear stress exposure induced monocyte adhesion. The adhesion of monocytes was partially inhibited by the incubation of endothelial cells with ICAM-1 blocking antibody. Increased heparan sulfate proteoglycan expression was observed on the surface of cells exposed to reversing shear stress. Heparinase III treatment significantly reduced monocyte adhesion. Our results suggest that low steady shear stress is the major impetus for differential gene expression and cell proliferation, whereas reversing flow regulates monocyte adhesion.
Endothelial Metallothionein Expression and Intracellular Free Zinc Levels Are Regulated by Shear Stress
American Journal of Physiology. Cell Physiology. Dec, 2010 | Pubmed ID: 20861469
We examined the effects of fluid shear stress on metallothionein (MT) gene and protein expression and intracellular free zinc in mouse aorta and in human umbilical vein endothelial cells (HUVECs). Immunostaining of the endothelial surface of mouse aorta revealed increased expression of MT protein in the lesser curvature of the aorta relative to the descending thoracic aorta. HUVECs were exposed to high steady shear stress (15 dyn/cm(2)), low steady shear stress (1 dyn/cm(2)), or reversing shear stress (mean of 1 dyn/cm(2), 1 Hz) for 24 h. Gene expression of three MT-1 isoforms, MT-2A, and zinc transporter-1 was upregulated by low steady shear stress and reversing shear stress. HUVECs exposed to 15 dyn/cm(2) had increased levels of free zinc compared with cells under other shear stress regimes and static conditions. The increase in free zinc was partially blocked with an inhibitor of nitric oxide synthesis, suggesting a role for shear stress-induced endothelial nitric oxide synthase activity. Cells subjected to reversing shear stress in zinc-supplemented media (50 Î¼M ZnSO(4)) had increased intracellular free zinc, reduced surface intercellular adhesion molecule-1 expression, and reduced monocyte adhesion compared with cells exposed to reversing shear stress in normal media. The sensitivity of intracellular free zinc to differences in shear stress suggests that intracellular zinc levels are important in the regulation of the endothelium and in the progression of vascular disease.
Current Biology : CB. Jun, 2013 | Pubmed ID: 23684974
Fluid shear stress (FSS) from blood flow acting on the endothelium critically regulates vascular morphogenesis, blood pressure, and atherosclerosis. FSS applied to endothelial cells (ECs) triggers signaling events including opening of ion channels, activation of signaling pathways, and changes in gene expression. Elucidating how ECs sense flow is important for understanding both normal vascular function and disease. EC responses to FSS are mediated in part by a junctional mechanosensory complex consisting of VE-cadherin, PECAM-1, and VEGFR2. Previous work suggested that flow increases force on PECAM-1, which initiates signaling. Deletion of PECAM-1 blocks responses to flow in vitro and flow-dependent vascular remodeling in vivo. To understand this process, we developed and validated FRET-based tension sensors for VE-cadherin and PECAM-1 using our previously developed FRET tension biosensor. FRET measurements showed that in static culture, VE-cadherin in cell-cell junctions bears significant myosin-dependent tension, whereas there was no detectable tension on VE-cadherin outside of junctions. Onset of shear stress triggered a rapid (<30 s) decrease in tension across VE-cadherin, which paralleled a decrease in total cell-cell junctional tension. Flow triggered a simultaneous increase in tension across junctional PECAM-1, while nonjunctional PECAM-1 was unaffected. Tension on PECAM-1 was mediated by flow-stimulated association with vimentin. These data confirm the prediction that shear increases force on PECAM-1. However, they also argue against the current model of passive transfer of force through the cytoskeleton to the junctions, showing instead that flow triggers cytoskeletal remodeling, which alters forces across the junctional receptors.
Journal of Cell Science. Nov, 2013 | Pubmed ID: 24190880
Atherosclerosis depends on risk factors such as hyperlipidemia, smoking, hypertension and diabetes. Although these risk factors are relatively constant throughout the arterial circulation, atherosclerotic plaques occur at specific sites where flow patterns are disturbed, with lower overall magnitude and complex changes in speed and direction. Research over the past few decades has provided new insights into the cellular mechanisms of force transduction and how mechanical effects act in concert with conventional risk factors to mediate plaque formation and progression. This Commentary summarizes our current understanding of how mechanotransduction pathways synergize with conventional risk factors in atherosclerosis. We attempt to integrate cellular studies with animal and clinical data, and highlight major questions that need to be answered to develop more effective therapies.
Mechanotransduction of Shear Stress Occurs Through Changes in VE-cadherin and PECAM-1 Tension: Implications for Cell Migration
Cell Adhesion & Migration. 2015 | Pubmed ID: 25482618
Recent work has shown that cadherins at cell-cell junctions bear tensile forces. Using novel FRET-based tension sensors, we showed first that in response to shear stress, endothelial cells rapidly reduce mechanical tension on vascular endothelial (VE)-cadherin. Second, we observed a simultaneous increase in tension on platelet endothelial cell adhesion molecule (PECAM)-1, induced by an interaction with vimentin. In this commentary, we discuss how our results fit with existing data on cadherins as important mediators of mechanotransduction, in particular, in cell migration where mechanical tension across cadherins may communicate the direction of movement. The ability of PECAM-1 to bear mechanical tension may also be important in other PECAM-1 functions, such as leukocyte transmigration through the endothelium. Additionally, our observation that vimentin expression was required for PECAM-1 tension and mechanotransduction of fluid flow suggests that intermediate filaments are capable of transmitting tension. Overall, our results argue against models where an external force is passively transferred across the cytoskeleton, and instead suggest that cells actively respond to extracellular forces by modulating tension across junctional proteins.
The Journal of Cell Biology. 01, 2015 | Pubmed ID: 25559184
The role of the RhoGTPase Rac1 in stabilizing mature endothelial adherens junctions (AJs) is not well understood. In this paper, using a photoactivatable probe to control Rac1 activity at AJs, we addressed the relationship between Rac1 and the dynamics of vascular endothelial cadherin (VE-cadherin). We demonstrated that Rac1 activation reduced the rate of VE-cadherin dissociation, leading to increased density of VE-cadherin at AJs. This response was coupled to a reduction in actomyosin-dependent tension across VE-cadherin adhesion sites. We observed that inhibiting myosin II directly or through photo-release of the caged Rho kinase inhibitor also reduced the rate of VE-cadherin dissociation. Thus, Rac1 functions by stabilizing VE-cadherin trans-dimers in mature AJs by counteracting the actomyosin tension. The results suggest a new model of VE-cadherin adhesive interaction mediated by Rac1-induced reduction of mechanical tension at AJs, resulting in the stabilization of VE-cadherin adhesions.
ZO-1 Controls Endothelial Adherens Junctions, Cell-cell Tension, Angiogenesis, and Barrier Formation
The Journal of Cell Biology. Mar, 2015 | Pubmed ID: 25753039
Intercellular junctions are crucial for mechanotransduction, but whether tight junctions contribute to the regulation of cell-cell tension and adherens junctions is unknown. Here, we demonstrate that the tight junction protein ZO-1 regulates tension acting on VE-cadherin-based adherens junctions, cell migration, and barrier formation of primary endothelial cells, as well as angiogenesis in vitro and in vivo. ZO-1 depletion led to tight junction disruption, redistribution of active myosin II from junctions to stress fibers, reduced tension on VE-cadherin and loss of junctional mechanotransducers such as vinculin and PAK2, and induced vinculin dissociation from the α-catenin-VE-cadherin complex. Claudin-5 depletion only mimicked ZO-1 effects on barrier formation, whereas the effects on mechanotransducers were rescued by inhibition of ROCK and phenocopied by JAM-A, JACOP, or p114RhoGEF down-regulation. ZO-1 was required for junctional recruitment of JACOP, which, in turn, recruited p114RhoGEF. ZO-1 is thus a central regulator of VE-cadherin-dependent endothelial junctions that orchestrates the spatial actomyosin organization, tuning cell-cell tension, migration, angiogenesis, and barrier formation.
The Journal of Cell Biology. Apr, 2015 | Pubmed ID: 25847538
Biophysical Journal. Jan, 2016 | Pubmed ID: 26745407
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.
Nano Letters. Mar, 2016 | Pubmed ID: 26824190
Recent development and applications of calibrated, fluorescence resonance energy transfer (FRET)-based tension sensors have led to a new understanding of single molecule mechanotransduction in a number of biological systems. To expand the range of accessible forces, we systematically measured FRET versus force trajectories for 25, 40, and 50 amino acid peptide repeats derived from spider silk. Single molecule fluorescence-force spectroscopy showed that the peptides behaved as linear springs instead of the nonlinear behavior expected for a disordered polymer. Our data are consistent with a compact, rodlike structure that measures 0.26 nm per 5 amino acid repeat that can stretch by 500% while maintaining linearity, suggesting that the remarkable elasticity of spider silk proteins may in part derive from the properties of individual chains. We found the shortest peptide to have the widest range of force sensitivity: between 2 pN and 11 pN. Live cell imaging of the three tension sensor constructs inserted into vinculin showed similar force values around 2.4 pN. We also provide a lookup table for force versus intracellular FRET for all three constructs.