Alexander Dunn

All Publications

• Regulation and dynamics of force transmission at individual cell-matrix adhesion bonds. Science advances Tan, S. J., Chang, A. C., Anderson, S. M., Miller, C. M., Prahl, L. S., Odde, D. J., Dunn, A. R. 2020; 6 (20): eaax0317

  Abstract

  Integrin-based adhesion complexes link the cytoskeleton to the extracellular matrix (ECM) and are central to the construction of multicellular animal tissues. How biological function emerges from the tens to thousands of proteins present within a single adhesion complex remains unclear. We used fluorescent molecular tension sensors to visualize force transmission by individual integrins in living cells. These measurements revealed an underlying functional modularity in which integrin class controlled adhesion size and ECM ligand specificity, while the number and type of connections between integrins and F-actin determined the force per individual integrin. In addition, we found that most integrins existed in a state of near-mechanical equilibrium, a result not predicted by existing models of cytoskeletal force transduction. A revised model that includes reversible cross-links within the F-actin network can account for this result and suggests one means by which cellular mechanical homeostasis can arise at the molecular level.

  View details for DOI 10.1126/sciadv.aax0317

  View details for PubMedID 32440534

  View details for PubMedCentralID PMC7228748

• Vinculin forms a directionally asymmetric catch bond with F-actin SCIENCE Huang, D. L., Bax, N. A., Buckley, C. D., Weis, W. I., Dunn, A. R. 2017; 357 (6352): 703–6

  Abstract

  Vinculin is an actin-binding protein thought to reinforce cell-cell and cell-matrix adhesions. However, how mechanical load affects the vinculin-F-actin bond is unclear. Using a single-molecule optical trap assay, we found that vinculin forms a force-dependent catch bond with F-actin through its tail domain, but with lifetimes that depend strongly on the direction of the applied force. Force toward the pointed (-) end of the actin filament resulted in a bond that was maximally stable at 8 piconewtons, with a mean lifetime (12 seconds) 10 times as long as the mean lifetime when force was applied toward the barbed (+) end. A computational model of lamellipodial actin dynamics suggests that the directionality of the vinculin-F-actin bond could establish long-range order in the actin cytoskeleton. The directional and force-stabilized binding of vinculin to F-actin may be a mechanism by which adhesion complexes maintain front-rear asymmetry in migrating cells.

  View details for PubMedID 28818948

• Energetics and forces in living cells PHYSICS TODAY Dunn, A. R., Price, A. 2015; 68 (2): 27-32

  View details for DOI 10.1063/PT.3.2686

  View details for Web of Science ID 000352078600014

• Cell adhesion. The minimal cadherin-catenin complex binds to actin filaments under force. Science Buckley, C. D., Tan, J., Anderson, K. L., Hanein, D., Volkmann, N., Weis, W. I., Nelson, W. J., Dunn, A. R. 2014; 346 (6209)

  Abstract

  Linkage between the adherens junction (AJ) and the actin cytoskeleton is required for tissue development and homeostasis. In vivo findings indicated that the AJ proteins E-cadherin, β-catenin, and the filamentous (F)-actin binding protein αE-catenin form a minimal cadherin-catenin complex that binds directly to F-actin. Biochemical studies challenged this model because the purified cadherin-catenin complex does not bind F-actin in solution. Here, we reconciled this difference. Using an optical trap-based assay, we showed that the minimal cadherin-catenin complex formed stable bonds with an actin filament under force. Bond dissociation kinetics can be explained by a catch-bond model in which force shifts the bond from a weakly to a strongly bound state. These results may explain how the cadherin-catenin complex transduces mechanical forces at cell-cell junctions.

  View details for DOI 10.1126/science.1254211

  View details for PubMedID 25359979

• Molecular tension sensors report forces generated by single integrin molecules in living cells. Nano letters Morimatsu, M., Mekhdjian, A. H., Adhikari, A. S., Dunn, A. R. 2013; 13 (9): 3985-3989

  Abstract

  Living cells are exquisitely responsive to mechanical cues, yet how cells produce and detect mechanical force remains poorly understood due to a lack of methods that visualize cell-generated forces at the molecular scale. Here we describe Förster resonance energy transfer (FRET)-based molecular tension sensors that allow us to directly visualize cell-generated forces with single-molecule sensitivity. We apply these sensors to determine the distribution of forces generated by individual integrins, a class of cell adhesion molecules with prominent roles throughout cell and developmental biology. We observe strikingly complex distributions of tensions within individual focal adhesions. FRET values measured for single probe molecules suggest that relatively modest tensions at the molecular level are sufficient to drive robust cellular adhesion.

  View details for DOI 10.1021/nl4005145

  View details for PubMedID 23859772

• Spatially controlled stem cell differentiation via morphogen gradients: A comparison of static and dynamic microfluidic platforms JOURNAL OF VACUUM SCIENCE & TECHNOLOGY A Cui, K. W., Engel, L., Dundes, C. E., Nguyen, T. C., Loh, K. M., Dunn, A. R. 2020; 38 (3)

  View details for DOI 10.1116/1.5142012

  View details for Web of Science ID 000522020800001

• Scaling up single-cell mechanics to multicellular tissues - the role of the intermediate filament-desmosome network. Journal of cell science Broussard, J. A., Jaiganesh, A., Zarkoob, H., Conway, D. E., Dunn, A. R., Espinosa, H. D., Janmey, P. A., Green, K. J. 2020; 133 (6)

  Abstract

  Cells and tissues sense, respond to and translate mechanical forces into biochemical signals through mechanotransduction, which governs individual cell responses that drive gene expression, metabolic pathways and cell motility, and determines how cells work together in tissues. Mechanotransduction often depends on cytoskeletal networks and their attachment sites that physically couple cells to each other and to the extracellular matrix. One way that cells associate with each other is through Ca2+-dependent adhesion molecules called cadherins, which mediate cell-cell interactions through adherens junctions, thereby anchoring and organizing the cortical actin cytoskeleton. This actin-based network confers dynamic properties to cell sheets and developing organisms. However, these contractile networks do not work alone but in concert with other cytoarchitectural elements, including a diverse network of intermediate filaments. This Review takes a close look at the intermediate filament network and its associated intercellular junctions, desmosomes. We provide evidence that this system not only ensures tissue integrity, but also cooperates with other networks to create more complex tissues with emerging properties in sensing and responding to increasingly stressful environments. We will also draw attention to how defects in intermediate filament and desmosome networks result in both chronic and acquired diseases.

  View details for DOI 10.1242/jcs.228031

  View details for PubMedID 32179593

• Tuning the Antigen Density Requirement for CAR T Cell Activity. Cancer discovery Majzner, R. G., Rietberg, S. P., Sotillo, E., Dong, R., Vachharajani, V. T., Labanieh, L., Myklebust, J. H., Kadapakkam, M., Weber, E. W., Tousley, A. M., Richards, R. M., Heitzeneder, S., Nguyen, S. M., Wiebking, V., Theruvath, J., Lynn, R. C., Xu, P., Dunn, A. R., Vale, R. D., Mackall, C. L. 2020

  Abstract

  Insufficient reactivity against cells with low antigen density has emerged as an important cause of CAR resistance. Little is known about factors that modulate the threshold for antigen recognition. We demonstrate that CD19 CAR activity is dependent upon antigen density and the CAR construct in axicabtagene-ciloleucel (CD19-CD28z) outperforms that in tisagenlecleucel (CD19-4-1BBz) against antigen low tumors. Enhancing signal strength by including additional ITAMs in the CAR enables recognition of low antigen density cells, while ITAM deletions blunt signal and increase the antigen density threshold. Further, replacement of the CD8 hinge-transmembrane (H/T) region of a 4-1BBz CAR with a CD28-H/T lowers the threshold for CAR reactivity despite identical signaling molecules. CARs incorporating a CD28-H/T demonstrate a more stable and efficient immunological synapse. Precise design of CARs can tune the threshold for antigen recognition and endow 4-1BBz-CARs with enhanced capacity to recognize antigen low targets while retaining a superior capacity for persistence.

  View details for DOI 10.1158/2159-8290.CD-19-0945

  View details for PubMedID 32193224

• Perpendicular alignment of lymphatic endothelial cells in response to spatial gradients in wall shear stress. Communications biology Michalaki, E., Surya, V. N., Fuller, G. G., Dunn, A. R. 2020; 3 (1): 57

  Abstract

  One-way valves in the lymphatic system form from lymphatic endothelial cells (LECs) during embryonic development and are required for efficient tissue drainage. Although fluid flow is thought to guide both valve formation and maintenance, how this occurs at a mechanistic level remains incompletely understood. We built microfluidic devices that reproduce critical aspects of the fluid flow patterns found at sites of valvulogenesis. Using these devices, we observed that LECs replicated aspects of the early steps in valvulogenesis: cells oriented perpendicular to flow in the region of maximum wall shear stress (WSS) and exhibited enhanced nuclear localization of FOXC2, a transcription factor required for valvulogenesis. Further experiments revealed that the cell surface protein E-selectin was required for both of these responses. Our observations suggest that spatial gradients in WSS help to demarcate the locations of valve formation, and implicate E-selectin as a component of a mechanosensory process for detecting WSS gradients.

  View details for DOI 10.1038/s42003-019-0732-8

  View details for PubMedID 32029852

• Limited Dishevelled/Axin oligomerization determines efficiency of Wnt/β-catenin signal transduction. eLife Kan, W., Enos, M. D., Korkmazhan, E., Muennich, S., Chen, D. H., Gammons, M. V., Vasishtha, M., Bienz, M., Dunn, A. R., Skiniotis, G., Weis, W. I. 2020; 9

  Abstract

  In Wnt/β-catenin signaling, the transcriptional coactivator β-catenin is regulated by its phosphorylation in a complex that includes the scaffold protein Axin and associated kinases. Wnt binding to its coreceptors activates the cytosolic effector Dishevelled (Dvl), leading to the recruitment of Axin and the inhibition of β-catenin phosphorylation. This process requires interaction of homologous DIX domains present in Dvl and Axin, but is mechanistically undefined. We show that Dvl DIX forms antiparallel, double-stranded oligomers in vitro, and that Dvl in cells forms oligomers typically <10 molecules at endogenous expression levels. Axin DIX (DAX) forms small single-stranded oligomers, but its self-association is stronger than that of DIX. DAX caps the ends of DIX oligomers, such that a DIX oligomer has at most four DAX binding sites. The relative affinities and stoichiometry of the DIX-DAX interaction provide a mechanism for efficient inhibition of β-catenin phosphorylation upon Axin recruitment to the Wnt receptor complex.

  View details for DOI 10.7554/eLife.55015

  View details for PubMedID 32297861

• Binding partner- and force-promoted changes in alphaE-catenin conformation probed by native cysteine labeling. Scientific reports Terekhova, K., Pokutta, S., Kee, Y. S., Li, J., Tajkhorshid, E., Fuller, G., Dunn, A. R., Weis, W. I. 2019; 9 (1): 15375

  Abstract

  Adherens Junctions (AJs) are cell-cell adhesion complexes that sense and propagate mechanical forces by coupling cadherins to the actin cytoskeleton via beta-catenin and the F-actin binding protein alphaE-catenin. When subjected to mechanical force, the cadherincatenin complex can tightly link to F-actin through alphaE-catenin, and also recruits the F-actin-binding protein vinculin. In this study, labeling of native cysteines combined with mass spectrometry revealed conformational changes in alphaE-catenin upon binding to the E-cadherinbeta-catenin complex, vinculin and F-actin. A method to apply physiologically meaningful forces in solution revealed force-induced conformational changes in alphaE-catenin when bound to F-actin. Comparisons of wild-type alphaE-catenin and a mutant with enhanced vinculin affinity using cysteine labeling and isothermal titration calorimetry provide evidence for allosteric coupling of the N-terminal beta-catenin-binding and the middle (M) vinculin-binding domain of alphaE-catenin. Cysteine labeling also revealed possible crosstalk between the actin-binding domain and the rest of the protein. The data provide insight into how binding partners and mechanical stress can regulate the conformation of full-length alphaE-catenin, and identify the M domain as a key transmitter of conformational changes.

  View details for DOI 10.1038/s41598-019-51816-3

  View details for PubMedID 31653927

• Oscillatory cortical forces promote three dimensional cell intercalations that shape the murine mandibular arch. Nature communications Tao, H., Zhu, M., Lau, K., Whitley, O. K., Samani, M., Xiao, X., Chen, X. X., Hahn, N. A., Liu, W., Valencia, M., Wu, M., Wang, X., Fenelon, K. D., Pasiliao, C. C., Hu, D., Wu, J., Spring, S., Ferguson, J., Karuna, E. P., Henkelman, R. M., Dunn, A., Huang, H., Ho, H. H., Atit, R., Goyal, S., Sun, Y., Hopyan, S. 2019; 10 (1): 1703

  Abstract

  Multiple vertebrate embryonic structures such as organ primordia are composed of confluent cells. Although mechanisms that shape tissue sheets are increasingly understood, those which shape a volume of cells remain obscure. Here we show that 3D mesenchymal cell intercalations are essential to shape the mandibular arch of the mouse embryo. Using a genetically encoded vinculin tension sensor that we knock-in to the mouse genome, we show that cortical force oscillations promote these intercalations. Genetic loss- and gain-of-function approaches show that Wnt5a functions as a spatial cue to coordinate cell polarityand cytoskeletal oscillation. Theseprocessesdiminish tissue rigidity and help cells to overcome the energy barrier to intercalation. YAP/TAZ and PIEZO1 serve as downstream effectors of Wnt5a-mediated actomyosin polarity and cytosolic calcium transients that orient and drive mesenchymal cell intercalations. These findings advance our understanding of how developmental pathways regulate biophysical properties and forces to shape a solid organ primordium.

  View details for PubMedID 30979871

• Lymphatic endothelial cell calcium pulses are sensitive to spatial gradients in wall shear stress MOLECULAR BIOLOGY OF THE CELL Surya, V. N., Michalaki, E., Fuller, G. G., Dunn, A. R. 2019; 30 (7): 923–31

  View details for DOI 10.1091/mbc.E18-10-0618

  View details for Web of Science ID 000461869400011

• Lymphatic endothelial cell calcium pulses are sensitive to spatial gradients in wall shear stress. Molecular biology of the cell Surya, V. N., Michalaki, E., Fuller, G. G., Dunn, A. R. 2019: mbcE18100618

  Abstract

  Cytosolic calcium (Ca2+) is a ubiquitous second messenger that influences numerous aspects of cellular function. In many cell types cytosolic Ca2+ concentrations are characterized by periodic pulses whose dynamics can influence downstream signal transduction. Here, we examined the general question of how cells use Ca2+ pulses to encode input stimuli in the context of the response of lymphatic endothelial cells (LECs) to fluid flow. Previous work shows that fluid flow regulates Ca2+ dynamics in LECs, and that Ca2+-dependent signaling plays a key role in regulating lymphatic valve formation during embryonic development. However, how fluid flow might influence the Ca2+ pulse dynamics of individual LECs remained, to our knowledge, little explored. We used live-cell imaging to characterize Ca2+ pulse dynamics in LECs exposed to fluid flow in an in vitro flow device that generates spatial gradients in wall shear stress (WSS) such as are found at sites of valve formation. We found that the frequency of Ca2+ pulses was sensitive to the magnitude of WSS, while the duration of individual Ca2+ pulses increased in the presence of spatial gradients in WSS. These observations reveal an example of how cells can separately modulate Ca2+ pulse frequency and duration to encode distinct forms of information, a phenomenon that could extend to other cell types. Movie S1 Movie S1 HLMVEC Ca2+ dynamics in the IFC, recorded for 30 minutes starting from the onset of flow at t = 50 s. Regions corresponding to Rings 1 and 2 are shown, which have average WSSs of 32 and 65 dyn/cm2, respectively. The flow direction is radially outward and symmetric about the jet center at the center of Ring 1. Frames were recorded every 5 seconds. Scale bar, 100 mum. Movie S2 Movie S2 HLMVEC Ca2+ dynamics as in Movie 1 for Rings 2 - 6, which have average WSSs of 65, 53, 30, 17 and 11 dyn/cm2. The flow direction is radially outward and here is roughly from left to right. Frames were recorded every 5 seconds. Scale bar, 100 mum. Movie S3 Movie S3 HLMVEC Ca2+ dynamics for cells exposed to uniform WSS (parallel plate flow), recorded for 30 minutes from the onset of flow at t = 50 s. Here, all HLMVECs experience a WSS of 50 dyn/cm2. The flow direction is from the bottom of the video to the top. Frames were recorded every 5 seconds. Scale bar, 100 mum. Movie S4 Movie S4 HLMVEC Ca2+ dynamics under no flow conditions, recorded for 30 minutes. Frames were recorded every 5 seconds. Scale bar, 100 mum.

  View details for PubMedID 30811261

• Myosin-II mediated traction forces evoke localized Piezo1-dependent Ca2+ flickers. Communications biology Ellefsen, K. L., Holt, J. R., Chang, A. C., Nourse, J. L., Arulmoli, J., Mekhdjian, A. H., Abuwarda, H., Tombola, F., Flanagan, L. A., Dunn, A. R., Parker, I., Pathak, M. M. 2019; 2 (1): 298

  Abstract

  Piezo channels transduce mechanical stimuli into electrical and chemical signals to powerfully influence development, tissue homeostasis, and regeneration. Studies on Piezo1 have largely focused on transduction of "outside-in" mechanical forces, and its response to internal, cell-generated forces remains poorly understood. Here, using measurements of endogenous Piezo1 activity and traction forces in native cellular conditions, we show that cellular traction forces generate spatially-restricted Piezo1-mediated Ca2+ flickers in the absence of externally-applied mechanical forces. Although Piezo1 channels diffuse readily in the plasma membrane and are widely distributed across the cell, their flicker activity is enriched near force-producing adhesions. The mechanical force that activates Piezo1 arises from Myosin II phosphorylation by Myosin Light Chain Kinase. We propose that Piezo1 Ca2+ flickers allow spatial segregation of mechanotransduction events, and that mobility allows Piezo1 channels to explore a large number of mechanical microdomains and thus respond to a greater diversity of mechanical cues.

  View details for DOI 10.1038/s42003-019-0514-3

  View details for PubMedID 31925072

• Mechanical loading of desmosomes depends on themagnitude and orientation of external stress. Nature communications Price, A. J., Cost, A., UngewiSS, H., Waschke, J., Dunn, A. R., Grashoff, C. 2018; 9 (1): 5284

  Abstract

  Desmosomes are intercellular adhesion complexes that connect the intermediate filament cytoskeletons of neighboring cells, and are essential for the mechanical integrity of mammalian tissues. Mutations in desmosomal proteins cause severe human pathologies including epithelial blistering and heart muscle dysfunction. However, direct evidence for their load-bearing nature is lacking. Here we develop Forster resonance energy transfer (FRET)-based tension sensors to measure the forces experienced by desmoplakin, an obligate desmosomal protein that links the desmosomal plaque to intermediate filaments. Our experiments reveal that desmoplakin does not experience significant tension under most conditions, but instead becomes mechanically loaded when cells are exposed to external mechanical stresses. Stress-induced loading of desmoplakin is transient and sensitive to the magnitude and orientation of the applied tissue deformation, consistent with a stress absorbing function for desmosomes that is distinct from previously analyzed cell adhesion complexes.

  View details for PubMedID 30538252

• Mechanobiology: ubiquitous and useful. Molecular biology of the cell Dunn, A. R. 2018; 29 (16): 1917–18

  View details for PubMedID 30088797

• DACH1 stimulates shear stress-guided endothelial cell migration and coronary artery growth through the CXCL12-CXCR4 signaling axis GENES & DEVELOPMENT Chang, A. H., Raftrey, B. C., D'Amato, G., Surya, V. N., Poduri, A., Chen, H. I., Goldstone, A. B., Woo, J., Fuller, G. G., Dunn, A. R., Red-Horse, K. 2017; 31 (13): 1308–24

  Abstract

  Sufficient blood flow to tissues relies on arterial blood vessels, but the mechanisms regulating their development are poorly understood. Many arteries, including coronary arteries of the heart, form through remodeling of an immature vascular plexus in a process triggered and shaped by blood flow. However, little is known about how cues from fluid shear stress are translated into responses that pattern artery development. Here, we show that mice lacking endothelial Dach1 had small coronary arteries, decreased endothelial cell polarization, and reduced expression of the chemokine Cxcl12 Under shear stress in culture, Dach1 overexpression stimulated endothelial cell polarization and migration against flow, which was reversed upon CXCL12/CXCR4 inhibition. In vivo, DACH1 was expressed during early arteriogenesis but was down in mature arteries. Mature artery-type shear stress (high, uniform laminar) specifically down-regulated DACH1, while the remodeling artery-type flow (low, variable) maintained DACH1 expression. Together, our data support a model in which DACH1 stimulates coronary artery growth by activating Cxcl12 expression and endothelial cell migration against blood flow into developing arteries. This activity is suppressed once arteries reach a mature morphology and acquire high, laminar flow that down-regulates DACH1. Thus, we identified a mechanism by which blood flow quality balances artery growth and maturation.

  View details for PubMedID 28779009

• A cytoskeletal clutch mediates cellular force transmission in a soft, 3D extracellular matrix. Molecular biology of the cell Owen, L. M., Adhikari, A. S., Patel, M., Grimmer, P., Leijnse, N., Kim, M. C., Notbohm, J., Franck, C., Dunn, A. R. 2017

  Abstract

  The ability of cells to impart forces and deformations on their surroundings underlies cell migration and extracellular matrix (ECM) remodeling, and is thus an essential aspect of complex, metazoan life. Previous work has resulted in a refined understanding, commonly termed the molecular clutch model, of how cells adhering to flat surfaces such as a microscope coverslip transmit cytoskeletally generated forces to their surroundings. Comparatively less is known about how cells adhere to and exert forces in soft, three-dimensional, and structurally heterogeneous ECM environments such as occur in vivo We used timelapse 3D imaging and quantitative image analysis to determine how the actin cytoskeleton was mechanically coupled to the surrounding matrix for primary dermal fibroblasts embedded in a 3D fibrin matrix. Under these circumstances the cytoskeletal architecture was dominated by contractile actin bundles attached at their ends to large, stable integrin-based adhesions. Time-lapse imaging revealed that α-actinin-1 puncta within actomyosin bundles moved more quickly than the paxillin-rich adhesion plaques, which in turn moved more quickly than the local matrix, an observation reminiscent of the molecular clutch model. However, closer examination did not reveal a continuous rearward flow of the actin cytoskeleton over slower moving adhesions. Instead, we found that a subset of stress fibers continuously elongated at their attachment points to integrin adhesions, providing stable yet structurally dynamic coupling to the ECM. Analytical modeling and numerical simulation provide a plausible physical explanation for this result, and support a picture in which cells respond to the effective stiffness of the local matrix attachment points. The resulting dynamic equilibrium can explain how cells maintain stable, contractile connections to discrete points within ECM during cell migration, and provides a plausible means by which fibroblasts contract provisional matrices during wound healing.

  View details for DOI 10.1091/mbc.E17-02-0102

  View details for PubMedID 28592635

• Integrin-mediated traction force enhances paxillin molecular associations and adhesion dynamics that increase the invasiveness of tumor cells into a three-dimensional extracellular matrix. Molecular biology of the cell Mekhdjian, A. H., Kai, F., Rubashkin, M. G., Prahl, L. S., Przybyla, L. M., McGregor, A. L., Bell, E. S., Barnes, J. M., DuFort, C. C., Ou, G., Chang, A. C., Cassereau, L., Tan, S. J., Pickup, M. W., Lakins, J. N., Ye, X., Davidson, M. W., Lammerding, J., Odde, D. J., Dunn, A. R., Weaver, V. M. 2017; 28 (11): 1467-1488

  Abstract

  Metastasis requires tumor cells to navigate through a stiff stroma and squeeze through confined microenvironments. Whether tumors exploit unique biophysical properties to metastasize remains unclear. Data show that invading mammary tumor cells, when cultured in a stiffened three-dimensional extracellular matrix that recapitulates the primary tumor stroma, adopt a basal-like phenotype. Metastatic tumor cells and basal-like tumor cells exert higher integrin-mediated traction forces at the bulk and molecular levels, consistent with a motor-clutch model in which motors and clutches are both increased. Basal-like nonmalignant mammary epithelial cells also display an altered integrin adhesion molecular organization at the nanoscale and recruit a suite of paxillin-associated proteins implicated in invasion and metastasis. Phosphorylation of paxillin by Src family kinases, which regulates adhesion turnover, is similarly enhanced in the metastatic and basal-like tumor cells, fostered by a stiff matrix, and critical for tumor cell invasion in our assays. Bioinformatics reveals an unappreciated relationship between Src kinases, paxillin, and survival of breast cancer patients. Thus adoption of the basal-like adhesion phenotype may favor the recruitment of molecules that facilitate tumor metastasis to integrin-based adhesions. Analysis of the physical properties of tumor cells and integrin adhesion composition in biopsies may be predictive of patient outcome.

  View details for DOI 10.1091/mbc.E16-09-0654

  View details for PubMedID 28381423

• A semi-interpenetrating network of polyacrylamide and recombinant basement membrane allows pluripotent cell culture in a soft, ligand-rich microenvironment. Biomaterials Price, A. J., Huang, E. Y., Sebastiano, V., Dunn, A. R. 2017; 121: 179-192

  Abstract

  The physical properties of the extracellular matrix play an essential role in guiding stem cell differentiation and tissue morphogenesis both in vivo and in vitro. Existing work to investigate the role of matrix mechanics in directing stem cell proliferation, self-renewal, and differentiation has been limited by the poor attachment and survival of human pluripotent cells cultured on soft matrices (Young's modulus E ≲ 1000 Pa). To address this limitation we developed a protocol for generating semi-interpenetrating networks of polyacrylamide and recombinant basement membrane. Using these materials, we found that human embryonic stem cells (hESCs) remained proliferative and pluripotent even when grown in small colonies and on surfaces ranging in stiffness from 150 to 12000 Pa, spanning the range of tissue stiffnesses likely to be encountered in the embryo. Considerable recent attention has focused on the role of the transcriptional coactivator and Hippo effector YAP in regulating differentiation and cell proliferation both in the early embryo and in vitro. We found that while YAP localized to the nucleus on substrates of E ≳ 1000 Pa, its localization was heterogeneous on substrates of moduli ≲ 450 Pa, with predominantly nuclear localization at the colony periphery and mixed cytoplasmic and nuclear localization for cells in the colony interior, a pattern reminiscent of YAP subcellular localization in the inner cell mass (ICM) of the early embryo. In addition, hESC colony dynamics were highly responsive to substrate stiffness, with cells assembling into monolayers, multilayer structures, and transient, hollow rosettes in response to decreasing substrate stiffnesses in the range of 12000 to 150 Pa. We suggest that soft, ligand-rich substrates such as are described here provide a promising means of recapitulating aspects of early mammalian development that are otherwise inaccessible, and more broadly may be useful in the derivation of complex tissues from pluripotent cells in an in vitro setting.

  View details for DOI 10.1016/j.biomaterials.2016.12.005

  View details for PubMedID 28088685

• Genetic defects in beta-spectrin and tau sensitize C.elegans axons to movement-induced damage via torque-tension coupling ELIFE Krieg, M., Stuehmer, J., Cueva, J. G., Fetter, R., Spilker, K., Cremers, D., Shen, K., Dunn, A. R., Goodman, M. B. 2017; 6

  View details for DOI 10.7554/eLife.20172

  View details for Web of Science ID 000394255800001

• Sphingosine 1-phosphate receptor 1 regulates the directional migration of lymphatic endothelial cells in response to fluid shear stress JOURNAL OF THE ROYAL SOCIETY INTERFACE Surya, V. N., Michalaki, E., Huang, E. Y., Fuller, G. G., Dunn, A. R. 2016; 13 (125)

  Abstract

  The endothelial cells that line blood and lymphatic vessels undergo complex, collective migration and rearrangement processes during embryonic development, and are known to be exquisitely responsive to fluid flow. At present, the molecular mechanisms by which endothelial cells sense fluid flow remain incompletely understood. Here, we report that both the G-protein-coupled receptor sphingosine 1-phosphate receptor 1 (S1PR1) and its ligand sphingosine 1-phosphate (S1P) are required for collective upstream migration of human lymphatic microvascular endothelial cells in an in vitro setting. These findings are consistent with a model in which signalling via S1P and S1PR1 are integral components in the response of lymphatic endothelial cells to the stimulus provided by fluid flow.

  View details for DOI 10.1098/rsif.2016.0823

  View details for Web of Science ID 000391108100013

  View details for PubMedID 27974574

  View details for PubMedCentralID PMC5221531

• The tubulin repertoire of Caenorhabditis elegans sensory neurons and its context-dependent role in process outgrowth MOLECULAR BIOLOGY OF THE CELL Lockhead, D., Schwarz, E. M., O'Hagan, R., Bellotti, S., Krieg, M., Barr, M. M., Dunn, A. R., Sternberg, P. W., Goodman, M. B. 2016; 27 (23): 3717-3728

  View details for DOI 10.1091/mbc.E16-06-0473

  View details for Web of Science ID 000389601600004

• Single Molecule Force Measurements in Living Cells Reveal a Minimally Tensioned Integrin State. ACS nano Chang, A. C., Mekhdjian, A. H., Morimatsu, M., Denisin, A. K., Pruitt, B. L., Dunn, A. R. 2016: -?

  Abstract

  Integrins mediate cell adhesion to the extracellular matrix and enable the construction of complex, multicellular organisms, yet fundamental aspects of integrin-based adhesion remain poorly understood. Notably, the magnitude of the mechanical load experienced by individual integrins within living cells is unclear, due principally to limitations inherent to existing techniques. Here we use Förster resonance energy transfer-based molecular tension sensors to directly measure the distribution of loads experienced by individual integrins in living cells. We find that a large fraction of integrins bear modest loads of 1-3 pN, while subpopulations bearing higher loads are enriched within adhesions. Further, our data indicate that integrin engagement with the fibronectin synergy site, a secondary binding site specifically for α5β1 integrin, leads to increased levels of α5β1 integrin recruitment to adhesions but not to an increase in overall cellular traction generation. The presence of the synergy site does, however, increase cells' resistance to detachment by externally applied loads. We suggest that a substantial population of integrins experiencing loads well below their peak capacities can provide cells and tissues with mechanical integrity in the presence of widely varying mechanical loads.

  View details for PubMedID 27779848

• Kank2 activates talin, reduces force transduction across integrins and induces central adhesion formation NATURE CELL BIOLOGY Sun, Z., Tseng, H., Tan, S., Senger, F., Kurzawa, L., Dedden, D., Mizuno, N., Wasik, A. A., Thery, M., Dunn, A. R., Faessler, R. 2016; 18 (9): 941-953

  Abstract

  Integrin-based adhesions play critical roles in cell migration. Talin activates integrins and flexibly connects integrins to the actomyosin cytoskeleton, thereby serving as a 'molecular clutch' that transmits forces to the extracellular matrix to drive cell migration. Here we identify the evolutionarily conserved Kank protein family as novel components of focal adhesions (FAs). Kank proteins accumulate at the lateral border of FAs, which we term the FA belt, and in central sliding adhesions, where they directly bind the talin rod domain through the Kank amino-terminal (KN) motif and induce talin and integrin activation. In addition, Kank proteins diminish the talin-actomyosin linkage, which curbs force transmission across integrins, leading to reduced integrin-ligand bond strength, slippage between integrin and ligand, central adhesion formation and sliding, and reduced cell migration speed. Our data identify Kank proteins as talin activators that decrease the grip between the integrin-talin complex and actomyosin to regulate cell migration velocity.

  View details for DOI 10.1038/ncb3402

  View details for Web of Science ID 000382416800005

• Multiplexed Fluid Flow Device to Study Cellular Response to Tunable Shear Stress Gradients ANNALS OF BIOMEDICAL ENGINEERING Ostrowski, M. A., Huang, E. Y., Surya, V. N., Poplawski, C., Barakat, J. M., Lin, G. L., Fuller, G. G., Dunn, A. R. 2016; 44 (7): 2261-2272

  Abstract

  Endothelial cells (ECs) line the interior of blood and lymphatic vessels and experience spatially varying wall shear stress (WSS) as an intrinsic part of their physiological function. How ECs, and mammalian cells generally, sense spatially varying WSS remains poorly understood, due in part to a lack of convenient tools for exposing cells to spatially varying flow patterns. We built a multiplexed device, termed a 6-well impinging flow chamber, that imparts controlled WSS gradients to a six-well tissue culture plate. Using this device, we investigated the migratory response of lymphatic microvascular ECs, umbilical vein ECs, primary fibroblasts, and epithelial cells to WSS gradients on hours to days timescales. We observed that lymphatic microvascular ECs migrate upstream, against the direction of flow, a response that was unique among all the cells types investigated here. Time-lapse, live cell imaging revealed that the microtubule organizing center relocated to the upstream side of the nucleus in response to the applied WSS gradient. To further demonstrate the utility of our device, we screened for the involvement of canonical signaling pathways in mediating this upstream migratory response. These data highlight the importance of WSS magnitude and WSS spatial gradients in dictating the cellular response to fluid flow.

  View details for DOI 10.1007/s10439-015-1500-7

  View details for Web of Science ID 000377437600015

  View details for PubMedID 26589597

  View details for PubMedCentralID PMC4874920

• How Hydra Eats. Biophysical journal Dunn, A. R. 2016; 110 (7): 1467-1468

  View details for DOI 10.1016/j.bpj.2016.01.036

  View details for PubMedID 27074672

  View details for PubMedCentralID PMC4833776

• Nanoscale Patterning of Extracellular Matrix Alters Endothelial Function under Shear Stress NANO LETTERS Nakayama, K. H., Surya, V. N., Gole, M., Walker, T. W., Yang, W., Lai, E. S., Ostrowski, M. A., Fuller, G. G., Dunn, A. R., Huang, N. F. 2016; 16 (1): 410-419

  Abstract

  The role of nanotopographical extracellular matrix (ECM) cues in vascular endothelial cell (EC) organization and function is not well-understood, despite the composition of nano- to microscale fibrillar ECMs within blood vessels. Instead, the predominant modulator of EC organization and function is traditionally thought to be hemodynamic shear stress, in which uniform shear stress induces parallel-alignment of ECs with anti-inflammatory function, whereas disturbed flow induces a disorganized configuration with pro-inflammatory function. Since shear stress acts on ECs by applying a mechanical force concomitant with inducing spatial patterning of the cells, we sought to decouple the effects of shear stress using parallel-aligned nanofibrillar collagen films that induce parallel EC alignment prior to stimulation with disturbed flow resulting from spatial wall shear stress gradients. Using real time live-cell imaging, we tracked the alignment, migration trajectories, proliferation, and anti-inflammatory behavior of ECs when they were cultured on parallel-aligned or randomly oriented nanofibrillar films. Intriguingly, ECs cultured on aligned nanofibrillar films remained well-aligned and migrated predominantly along the direction of aligned nanofibrils, despite exposure to shear stress orthogonal to the direction of the aligned nanofibrils. Furthermore, in stark contrast to ECs cultured on randomly oriented films, ECs on aligned nanofibrillar films exposed to disturbed flow had significantly reduced inflammation and proliferation, while maintaining intact intercellular junctions. This work reveals fundamental insights into the importance of nanoscale ECM interactions in the maintenance of endothelial function. Importantly, it provides new insight into how ECs respond to opposing cues derived from nanotopography and mechanical shear force and has strong implications in the design of polymeric conduits and bioengineered tissues.

  View details for DOI 10.1021/acs.nanolett.5b04028

  View details for Web of Science ID 000368322700064

  View details for PubMedCentralID PMC4758680

• A Force Balance Can Explain Local and Global Cell Movements during Early Zebrafish Development BIOPHYSICAL JOURNAL Chai, J., Hamilton, A. L., Krieg, M., Buckley, C. D., Riedel-Kruse, I. H., Dunn, A. R. 2015; 109 (2): 407-414

  Abstract

  Embryonic morphogenesis takes place via a series of dramatic collective cell movements. The mechanisms that coordinate these intricate structural transformations across an entire organism are not well understood. In this study, we used gentle mechanical deformation of developing zebrafish embryos to probe the role of physical forces in generating long-range intercellular coordination during epiboly, the process in which the blastoderm spreads over the yolk cell. Geometric distortion of the embryo resulted in nonuniform blastoderm migration and realignment of the anterior-posterior (AP) axis, as defined by the locations at which the head and tail form, toward the new long axis of the embryo and away from the initial animal-vegetal axis defined by the starting location of the blastoderm. We found that local alterations in the rate of blastoderm migration correlated with the local geometry of the embryo. Chemical disruption of the contractile ring of actin and myosin immediately vegetal to the blastoderm margin via Ca(2+) reduction or treatment with blebbistatin restored uniform migration and eliminated AP axis reorientation in mechanically deformed embryos; it also resulted in cellular disorganization at the blastoderm margin. Our results support a model in which tension generated by the contractile actomyosin ring coordinates epiboly on both the organismal and cellular scales. Our observations likewise suggest that the AP axis is distinct from the initial animal-vegetal axis in zebrafish.

  View details for DOI 10.1016/j.bpj.2015.04.029

  View details for Web of Science ID 000358312800025

• Visualizing the Interior Architecture of Focal Adhesions with High-Resolution Traction Maps NANO LETTERS Morimatsu, M., Mekhdjian, A. H., Chang, A. C., Tan, S. J., Dunn, A. R. 2015; 15 (4): 2220-2228

  Abstract

  Focal adhesions (FAs) are micron-sized protein assemblies that coordinate cell adhesion, migration, and mechanotransduction. How the many proteins within FAs are organized into force sensing and transmitting structures is poorly understood. We combined fluorescent molecular tension sensors with super-resolution light microscopy to visualize traction forces within FAs with <100 nm spatial resolution. We find that αvβ3 integrin selectively localizes to high force regions. Paxillin, which is not generally considered to play a direct role in force transmission, shows a higher degree of spatial correlation with force than vinculin, talin, or α-actinin, proteins with hypothesized roles as force transducers. These observations suggest that αvβ3 integrin and paxillin may play important roles in mechanotransduction.

  View details for DOI 10.1021/nl5047335

  View details for Web of Science ID 000352750200002

  View details for PubMedID 25730141

• Mechanical systems biology of C. elegans touch sensation. BioEssays Krieg, M., Dunn, A. R., Goodman, M. B. 2015; 37 (3): 335-344

  Abstract

  The sense of touch informs us of the physical properties of our surroundings and is a critical aspect of communication. Before touches are perceived, mechanical signals are transmitted quickly and reliably from the skin's surface to mechano-electrical transduction channels embedded within specialized sensory neurons. We are just beginning to understand how soft tissues participate in force transmission and how they are deformed. Here, we review empirical and theoretical studies of single molecules and molecular ensembles thought to be involved in mechanotransmission and apply the concepts emerging from this work to the sense of touch. We focus on the nematode Caenorhabditis elegans as a well-studied model for touch sensation in which mechanics can be studied on the molecular, cellular, and systems level. Finally, we conclude that force transmission is an emergent property of macromolecular cellular structures that mutually stabilize one another.

  View details for DOI 10.1002/bies.201400154

  View details for PubMedID 25597279

• The CDC42-Interacting Protein 4 Controls Epithelial Cell Cohesion and Tumor Dissemination DEVELOPMENTAL CELL Rolland, Y., Marighetti, P., Malinverno, C., Confalonieri, S., Luise, C., Ducano, N., Palamidessi, A., Bisi, S., Kajiho, H., Troglio, F., Shcherbakova, O. G., Dunn, A. R., Oldani, A., Lanzetti, L., Di Fiore, P. P., Disanza, A., Scita, G. 2014; 30 (5): 553-568

  Abstract

  The role of endocytic proteins and the molecular mechanisms underlying epithelial cell cohesion and tumor dissemination are not well understood. Here, we report that the endocytic F-BAR-containing CDC42-interacting protein 4 (CIP4) is required for ERBB2- and TGF-β1-induced cell scattering, breast cancer (BC) cell motility and invasion into 3D matrices, and conversion from ductal breast carcinoma in situ to invasive carcinoma in mouse xenograft models. CIP4 promotes the formation of an E-cadherin-CIP4-SRC complex that controls SRC activation, E-cadherin endocytosis, and localized phosphorylation of the myosin light chain kinase, thereby impinging on the actomyosin contractility required to generate tangential forces to break cell-cell junctions. CIP4 is upregulated in ERBB2-positive human BC, correlates with increased distant metastasis, and is an independent predictor of poor disease outcome in subsets of BC patients. Thus, it critically controls cell-cell cohesion and is required for the acquisition of an invasive phenotype in breast tumors.

  View details for DOI 10.1016/j.devcel.2014.08.006

  View details for Web of Science ID 000341296100010

• Mechanical control of the sense of touch by ß-spectrin. Nature cell biology Krieg, M., Dunn, A. R., Goodman, M. B. 2014; 16 (3): 224-233

  Abstract

  The ability to sense and respond to mechanical stimuli emanates from sensory neurons and is shared by most, if not all, animals. Exactly how such neurons receive and distribute mechanical signals during touch sensation remains mysterious. Here, we show that sensation of mechanical forces depends on a continuous, pre-stressed spectrin cytoskeleton inside neurons. Mutations in the tetramerization domain of Caenorhabditis elegans β-spectrin (UNC-70), an actin-membrane crosslinker, cause defects in sensory neuron morphology under compressive stress in moving animals. Through atomic force spectroscopy experiments on isolated neurons, in vivo laser axotomy and fluorescence resonance energy transfer imaging to measure force across single cells and molecules, we show that spectrin is held under constitutive tension in living animals, which contributes to elevated pre-stress in touch receptor neurons. Genetic manipulations that decrease such spectrin-dependent tension also selectively impair touch sensation, suggesting that such pre-tension is essential for efficient responses to external mechanical stimuli.

  View details for DOI 10.1038/ncb2915

  View details for PubMedID 24561618

• Mechanical control of the sense of touch by ß-spectrin. Nature cell biology Krieg, M., Dunn, A. R., Goodman, M. B. 2014; 16 (3): 224-233

  Abstract

  The ability to sense and respond to mechanical stimuli emanates from sensory neurons and is shared by most, if not all, animals. Exactly how such neurons receive and distribute mechanical signals during touch sensation remains mysterious. Here, we show that sensation of mechanical forces depends on a continuous, pre-stressed spectrin cytoskeleton inside neurons. Mutations in the tetramerization domain of Caenorhabditis elegans β-spectrin (UNC-70), an actin-membrane crosslinker, cause defects in sensory neuron morphology under compressive stress in moving animals. Through atomic force spectroscopy experiments on isolated neurons, in vivo laser axotomy and fluorescence resonance energy transfer imaging to measure force across single cells and molecules, we show that spectrin is held under constitutive tension in living animals, which contributes to elevated pre-stress in touch receptor neurons. Genetic manipulations that decrease such spectrin-dependent tension also selectively impair touch sensation, suggesting that such pre-tension is essential for efficient responses to external mechanical stimuli.

  View details for DOI 10.1038/ncb2915

  View details for PubMedID 24561618

• Microvascular Endothelial Cells Migrate Upstream and Align Against the Shear Stress Field Created by Impinging Flow BIOPHYSICAL JOURNAL Ostrowski, M. A., Huang, N. F., Walker, T. W., Verwijlen, T., Poplawski, C., Khoo, A. S., Cooke, J. P., Fuller, G. G., Dunn, A. R. 2014; 106 (2): 366-374

  Abstract

  At present, little is known about how endothelial cells respond to spatial variations in fluid shear stress such as those that occur locally during embryonic development, at heart valve leaflets, and at sites of aneurysm formation. We built an impinging flow device that exposes endothelial cells to gradients of shear stress. Using this device, we investigated the response of microvascular endothelial cells to shear-stress gradients that ranged from 0 to a peak shear stress of 9-210 dyn/cm(2). We observe that at high confluency, these cells migrate against the direction of fluid flow and concentrate in the region of maximum wall shear stress, whereas low-density microvascular endothelial cells that lack cell-cell contacts migrate in the flow direction. In addition, the cells align parallel to the flow at low wall shear stresses but orient perpendicularly to the flow direction above a critical threshold in local wall shear stress. Our observations suggest that endothelial cells are exquisitely sensitive to both magnitude and spatial gradients in wall shear stress. The impinging flow device provides a, to our knowledge, novel means to study endothelial cell migration and polarization in response to gradients in physical forces such as wall shear stress.

  View details for DOI 10.1016/j.bpj.2013.11.4502

  View details for Web of Science ID 000330132500005

  View details for PubMedID 24461011

  View details for PubMedCentralID PMC3907231

• Quantification of nanowire penetration into living cells. Nature communications Xu, A. M., Aalipour, A., Leal-Ortiz, S., Mekhdjian, A. H., Xie, X., Dunn, A. R., Garner, C. C., Melosh, N. A. 2014; 5: 3613-?

  Abstract

  High-aspect ratio nanostructures such as nanowires and nanotubes are a powerful new tool for accessing the cell interior for delivery and sensing. Controlling and optimizing cellular access is a critical challenge for this new technology, yet even the most basic aspect of this process, whether these structures directly penetrate the cell membrane, is still unknown. Here we report the first quantification of hollow nanowires-nanostraws-that directly penetrate the membrane by observing dynamic ion delivery from each 100-nm diameter nanostraw. We discover that penetration is a rare event: 7.1±2.7% of the nanostraws penetrate the cell to provide cytosolic access for an extended period for an average of 10.7±5.8 penetrations per cell. Using time-resolved delivery, the kinetics of the first penetration event are shown to be adhesion dependent and coincident with recruitment of focal adhesion-associated proteins. These measurements provide a quantitative basis for understanding nanowire-cell interactions, and a means for rapidly assessing membrane penetration.

  View details for DOI 10.1038/ncomms4613

  View details for PubMedID 24710350

• Conformational Dynamics Accompanying the Proteolytic Degradation of Trimeric Collagen I by Collagenases JOURNAL OF THE AMERICAN CHEMICAL SOCIETY Adhikari, A. S., Glassey, E., Dunn, A. R. 2012; 134 (32): 13259-13265

  Abstract

  Collagenases are the principal enzymes responsible for the degradation of collagens during embryonic development, wound healing, and cancer metastasis. However, the mechanism by which these enzymes disrupt the highly chemically and structurally stable collagen triple helix remains incompletely understood. We used a single-molecule magnetic tweezers assay to characterize the cleavage of heterotrimeric collagen I by both the human collagenase matrix metalloproteinase-1 (MMP-1) and collagenase from Clostridium histolyticum. We observe that the application of 16 pN of force causes an 8-fold increase in collagen proteolysis rates by MMP-1 but does not affect cleavage rates by Clostridium collagenase. Quantitative analysis of these data allows us to infer the structural changes in collagen associated with proteolytic cleavage by both enzymes. Our data support a model in which MMP-1 cuts a transient, stretched conformation of its recognition site. In contrast, our findings suggest that Clostridium collagenase is able to cleave the fully wound collagen triple helix, accounting for its lack of force sensitivity and low sequence specificity. We observe that the cleavage of heterotrimeric collagen is less force sensitive than the proteolysis of a homotrimeric collagen model peptide, consistent with studies suggesting that the MMP-1 recognition site in heterotrimeric collagen I is partially unwound at equilibrium.

  View details for DOI 10.1021/ja212170b

  View details for Web of Science ID 000307487200030

  View details for PubMedID 22720833

• E-cadherin is under constitutive actomyosin-generated tension that is increased at cell-cell contacts upon externally applied stretch PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Borghi, N., Sorokina, M., Shcherbakova, O. G., Weis, W. I., Pruitt, B. L., Nelson, W. J., Dunn, A. R. 2012; 109 (31): 12568-12573

  Abstract

  Classical cadherins are transmembrane proteins at the core of intercellular adhesion complexes in cohesive metazoan tissues. The extracellular domain of classical cadherins forms intercellular bonds with cadherins on neighboring cells, whereas the cytoplasmic domain recruits catenins, which in turn associate with additional cytoskeleton binding and regulatory proteins. Cadherin/catenin complexes are hypothesized to play a role in the transduction of mechanical forces that shape cells and tissues during development, regeneration, and disease. Whether mechanical forces are transduced directly through cadherins is unknown. To address this question, we used a Förster resonance energy transfer (FRET)-based molecular tension sensor to test the origin and magnitude of tensile forces transmitted through the cytoplasmic domain of E-cadherin in epithelial cells. We show that the actomyosin cytoskeleton exerts pN-tensile force on E-cadherin, and that this tension requires the catenin-binding domain of E-cadherin and αE-catenin. Surprisingly, the actomyosin cytoskeleton constitutively exerts tension on E-cadherin at the plasma membrane regardless of whether or not E-cadherin is recruited to cell-cell contacts, although tension is further increased at cell-cell contacts when adhering cells are stretched. Our findings thus point to a constitutive role of E-cadherin in transducing mechanical forces between the actomyosin cytoskeleton and the plasma membrane, not only at cell-cell junctions but throughout the cell surface.

  View details for DOI 10.1073/pnas.1204390109

  View details for Web of Science ID 000307538200062

  View details for PubMedID 22802638

  View details for PubMedCentralID PMC3411997

• Multiplexed Single-molecule Force Proteolysis Measurements Using Magnetic Tweezers JOVE-JOURNAL OF VISUALIZED EXPERIMENTS Adhikari, A. S., Chai, J., Dunn, A. R. 2012

  Abstract

  The generation and detection of mechanical forces is a ubiquitous aspect of cell physiology, with direct relevance to cancer metastasis(1), atherogenesis(2) and wound healing(3). In each of these examples, cells both exert force on their surroundings and simultaneously enzymatically remodel the extracellular matrix (ECM). The effect of forces on ECM has thus become an area of considerable interest due to its likely biological and medical importance(4-7). Single molecule techniques such as optical trapping(8), atomic force microscopy(9), and magnetic tweezers(10,11) allow researchers to probe the function of enzymes at a molecular level by exerting forces on individual proteins. Of these techniques, magnetic tweezers (MT) are notable for their low cost and high throughput. MT exert forces in the range of ~1-100 pN and can provide millisecond temporal resolution, qualities that are well matched to the study of enzyme mechanism at the single-molecule level(12). Here we report a highly parallelizable MT assay to study the effect of force on the proteolysis of single protein molecules. We present the specific example of the proteolysis of a trimeric collagen peptide by matrix metalloproteinase 1 (MMP-1); however, this assay can be easily adapted to study other substrates and proteases.

  View details for DOI 10.3791/3520

  View details for Web of Science ID 000209223200005

• Strain Tunes Proteolytic Degradation and Diffusive Transport in Fibrin Networks BIOMACROMOLECULES Adhikari, A. S., Mekhdjian, A. H., Dunn, A. R. 2012; 13 (2): 499-506

  Abstract

  Proteolytic degradation of fibrin, the major structural component in blood clots, is critical both during normal wound healing and in the treatment of ischemic stroke and myocardial infarction. Fibrin-containing clots experience substantial strain due to platelet contraction, fluid shear, and mechanical stress at the wound site. However, little is understood about how mechanical forces may influence fibrin dissolution. We used video microscopy to image strained fibrin clots as they were degraded by plasmin, a major fibrinolytic enzyme. Applied strain causes up to 10-fold reduction in the rate of fibrin degradation. Analysis of our data supports a quantitative model in which the decrease in fibrin proteolysis rates with strain stems from slower transport of plasmin into the clot. We performed fluorescence recovery after photobleaching (FRAP) measurements to further probe the effect of strain on diffusive transport. We find that diffusivity perpendicular to the strain axis decreases with increasing strain, while diffusivity along the strain axis remains unchanged. Our results suggest that the properties of the fibrin network have evolved to protect mechanically loaded fibrin from degradation, consistent with its function in wound healing. The pronounced effect of strain upon diffusivity and proteolytic susceptibility within fibrin networks offers a potentially useful means of guiding cell growth and morphology in fibrin-based biomaterials.

  View details for DOI 10.1021/bm2015619

  View details for Web of Science ID 000300115900025

  View details for PubMedID 22185486

• Nucleotide Pocket Thermodynamics Measured by EPR Reveal How Energy Partitioning Relates Myosin Speed to Efficiency JOURNAL OF MOLECULAR BIOLOGY Purcell, T. J., Naber, N., Franks-Skiba, K., Dunn, A. R., Eldred, C. C., Berger, C. L., Malnasi-Csizmadia, A., Spudich, J. A., Swank, D. M., Pate, E., Cooke, R. 2011; 407 (1): 79-91

  Abstract

  We have used spin-labeled ADP to investigate the dynamics of the nucleotide-binding pocket in a series of myosins, which have a range of velocities. Electron paramagnetic resonance spectroscopy reveals that the pocket is in equilibrium between open and closed conformations. In the absence of actin, the closed conformation is favored. When myosin binds actin, the open conformation becomes more favored, facilitating nucleotide release. We found that faster myosins favor a more closed pocket in the actomyosin•ADP state, with smaller values of ΔH(0) and ΔS(0), even though these myosins release ADP at a faster rate. A model involving a partitioning of free energy between work-generating steps prior to rate-limiting ADP release explains both the unexpected correlation between velocity and opening of the pocket and the observation that fast myosins are less efficient than slow myosins.

  View details for DOI 10.1016/j.jmb.2010.11.053

  View details for Web of Science ID 000288725500007

  View details for PubMedID 21185304

  View details for PubMedCentralID PMC3347976

• Mechanical Load Induces a 100-Fold Increase in the Rate of Collagen Proteolysis by MMP-1 JOURNAL OF THE AMERICAN CHEMICAL SOCIETY Adhikari, A. S., Chai, J., Dunn, A. R. 2011; 133 (6): 1686-1689

  Abstract

  Although mechanical stress is known to profoundly influence the composition and structure of the extracellular matrix (ECM), the mechanisms by which this regulation occurs remain poorly understood. We used a single-molecule magnetic tweezers assay to study the effect of force on collagen proteolysis by matrix metalloproteinase-1 (MMP-1). Here we show that the application of ∼10 pN in extensional force causes an ∼100-fold increase in proteolysis rates. Our results support a mechanistic model in which the collagen triple helix unwinds prior to proteolysis. The data and resulting model predict that biologically relevant forces may increase localized ECM proteolysis, suggesting a possible role for mechanical force in the regulation of ECM remodeling.

  View details for DOI 10.1021/ja109972p

  View details for Web of Science ID 000287831800020

  View details for PubMedID 21247159

  View details for PubMedCentralID PMC3320677

• Robust Mechanosensing and Tension Generation by Myosin VI JOURNAL OF MOLECULAR BIOLOGY Chuan, P., Spudich, J. A., Dunn, A. R. 2011; 405 (1): 105-112

  Abstract

  Myosin VI is a molecular motor that is thought to function both as a transporter and as a cytoskeletal anchor in vivo. Here we use optical tweezers to examine force generation by single molecules of myosin VI under physiological nucleotide concentrations. We find that myosin VI is an efficient transporter at loads of up to ∼2 pN but acts as a cytoskeletal anchor at higher loads. Our data and the resulting model are consistent with an indirect coupling of global structural motions to nucleotide binding and release. The model provides a mechanism by which load may regulate the dual functions of myosin VI in vivo. Our results suggest that myosin VI kinetics are tuned such that the motor maintains a consistent level of mechanical tension within the cell, a property potentially shared by other mechanosensitive proteins.

  View details for DOI 10.1016/j.jmb.2010.10.010

  View details for Web of Science ID 000286700800011

  View details for PubMedID 20970430

  View details for PubMedCentralID PMC3200311

• Mechanical force induces a 100-fold increase in the rate of collagen proteolysis by MMP-1 J. Am. Chem. Soc. Adhikari, A., S., Chai, J., Dunn, A., R. 2011; 133: 1686-1689
• Contribution of the myosin VI tail domain to processive stepping and intramolecular tension sensing PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Dunn, A. R., Chuan, P., Bryant, Z., Spudich, J. A. 2010; 107 (17): 7746-7750

  Abstract

  Myosin VI is proposed to act as both a molecular transporter and as an anchor in vivo. A portion of the molecule C-terminal to the canonical lever arm, termed the medial tail (MT), has been proposed to act as either a lever arm extension or as a dimerization motif. We describe constructs in which the MT is interrupted by a glycine-rich molecular swivel. Disruption of the MT results in decreased processive run lengths measured using single-molecule fluorescence microscopy and a decreased step size under applied load as measured in an optical trap. We used single-molecule gold nanoparticle tracking and optical trapping to examine the mechanism of coordination between the heads of dimeric myosin VI. We detect two rate-limiting kinetic processes at low (< 200 micromolar) ATP concentrations. Our data can be explained by a model in which intramolecular tension greatly increases the affinity of the lead head for ADP, likely by slowing ADP release from the lead head. This mechanism likely increases both the motor's processivity and its ability to act as an anchor under physiological conditions.

  View details for DOI 10.1073/pnas.1002430107

  View details for Web of Science ID 000277088700028

  View details for PubMedID 20385849

  View details for PubMedCentralID PMC2867888

• Electron tunneling through sensitizer wires bound to proteins COORDINATION CHEMISTRY REVIEWS Hartings, M. R., Kurnikov, I. V., Dunn, A. R., Winkler, J. R., Gray, H. B., Ratner, M. A. 2010; 254 (3-4): 248-253

  Abstract

  We report a quantitative theoretical analysis of long-range electron transfer through sensitizer wires bound in the active-site channel of cytochrome P450cam. Each sensitizer wire consists of a substrate group with high binding affinity for the enzyme active site connected to a ruthenium-diimine through a bridging aliphatic or aromatic chain. Experiments have revealed a dramatic dependence of electron transfer rates on the chemical composition of both the bridging group and the substrate. Using combined molecular dynamics simulations and electronic coupling calculations, we show that electron tunneling through perfluorinated aromatic bridges is promoted by enhanced superexchange coupling through virtual reduced states. In contrast, electron flow through aliphatic bridges occurs by hole-mediated superexchange. We have found that a small number of wire conformations with strong donor-acceptor couplings can account for the observed electron tunneling rates for sensitizer wires terminated with either ethylbenzene or adamantane. In these instances, the rate is dependent not only on electronic coupling of the donor and acceptor but also on the nuclear motion of the sensitizer wire, necessitating the calculation of average rates over the course of a molecular dynamics simulation. These calculations along with related recent findings have made it possible to analyze the results of many other sensitizer-wire experiments that in turn point to new directions in our attempts to observe reactive intermediates in the catalytic cycles of P450 and other heme enzymes.

  View details for DOI 10.1016/j.ccr.2009.08.008

  View details for Web of Science ID 000273933300005

  View details for PubMedCentralID PMC2797321

• SINGLE-MOLECULE DUAL-BEAM OPTICAL TRAP ANALYSIS OF PROTEIN STRUCTURE AND FUNCTION METHODS IN ENZYMOLOGY, VOL 475: SINGLE MOLECULE TOOLS, PT B Sung, J., Sivaramakrishnan, S., Dunn, A. R., Spudich, J. A. 2010; 475: 321-375

  Abstract

  Optical trapping is one of the most powerful single-molecule techniques. We provide a practical guide to set up and use an optical trap, applied to the molecular motor myosin as an example. We focus primarily on studies of myosin function using a dual-beam optical trap, a protocol to build such a trap, and the experimental and data analysis protocols to utilize it.

  View details for DOI 10.1016/S0076-6879(10)75014-X

  View details for Web of Science ID 000280733800014

  View details for PubMedID 20627164

• Force dependence of myosin VI nucleotide binding kinetics J. Mol. Biol. Chuan, P., Y., Spudich, J., A., Dunn, A., R. 2010; 405: 105-112
• Nanosecond photoreduction of inducible nitric oxide synthase by a Ru-diimine electron tunneling wire bound distant from the active site JOURNAL OF INORGANIC BIOCHEMISTRY Whited, C. A., Belliston-Bittner, W., Dunn, A. R., Winkler, J. R., Gray, H. B. 2009; 103 (6): 906-911

  Abstract

  A Ru-diimine wire, [(4,4',5,5'-tetramethylbipyridine)2Ru(F9bp)]2+ (tmRu-F9bp, where F9bp is 4-methyl-4'-methylperfluorobiphenylbipyridine), binds tightly to the oxidase domain of inducible nitric oxide synthase (iNOSoxy). The binding of tmRu-F9bp is independent of tetrahydrobiopterin, arginine, and imidazole, indicating that the wire resides on the surface of the enzyme, distant from the active-site heme. Photoreduction of an imidazole-bound active-site heme iron in the enzyme-wire conjugate (k(ET) = 2(1) x 10(7) s(-1)) is fully seven orders of magnitude faster than the in vivo process.

  View details for DOI 10.1016/j.jinorgbio.2009.04.001

  View details for Web of Science ID 000266646100006

  View details for PubMedID 19427703

  View details for PubMedCentralID PMC2700734

• Probing the heme-thiolate oxygenase domain of inducible nitric oxide synthase with Ru(II) and Re(I) electron tunneling wires. J. Porphyrins Phthalocyanines Whited, C., A., Belliston-Bittner, W., Dunn, A., R., Winkler, J., R., Gray, H., B. 2008; 12: 971-978
• Predicting allosteric communication in myosin via a pathway of conserved residues JOURNAL OF MOLECULAR BIOLOGY Tang, S., Liao, J., Dunn, A. R., Altman, R. B., Spudich, J. A., Schmidt, J. P. 2007; 373 (5): 1361-1373

  Abstract

  We present a computational method that predicts a pathway of residues that mediate protein allosteric communication. The pathway is predicted using only a combination of distance constraints between contiguous residues and evolutionary data. We applied this analysis to find pathways of conserved residues connecting the myosin ATP binding site to the lever arm. These pathway residues may mediate the allosteric communication that couples ATP hydrolysis to the lever arm recovery stroke. Having examined pre-stroke conformations of Dictyostelium, scallop, and chicken myosin II as well as Dictyostelium myosin I, we observed a conserved pathway traversing switch II and the relay helix, which is consistent with the understood need for allosteric communication in this conformation. We also examined post-rigor and rigor conformations across several myosin species. Although initial residues of these paths are more heterogeneous, all but one of these paths traverse a consistent set of relay helix residues to reach the beginning of the lever arm. We discuss our results in the context of structural elements and reported mutational experiments, which substantiate the significance of the pre-stroke pathways. Our method provides a simple, computationally efficient means of predicting a set of residues that mediate allosteric communication. We provide a refined, downloadable application and source code (on https://simtk.org) to share this tool with the wider community (https://simtk.org/home/allopathfinder).

  View details for DOI 10.1016/j.jmb.2007.08.059

  View details for Web of Science ID 000250712600021

  View details for PubMedID 17900617

  View details for PubMedCentralID PMC2128046

• Dynamics of the unbound head during myosin V processive translocation NATURE STRUCTURAL & MOLECULAR BIOLOGY Dunn, A. R., Spudich, J. A. 2007; 14 (3): 246-248

  Abstract

  Myosin V moves cargoes along actin filaments by walking hand over hand. Although numerous studies support the basic hand-over-hand model, little is known about the fleeting intermediate that occurs when the rear head detaches from the filament. Here we use submillisecond dark-field imaging of gold nanoparticle-labeled myosin V to directly observe the free head as it releases from the actin filament, diffuses forward and rebinds. We find that the unbound head rotates freely about the lever-arm junction, a trait that likely facilitates travel through crowded actin meshworks.

  View details for DOI 10.1038/nsmb1206

  View details for Web of Science ID 000244715200016

  View details for PubMedID 17293871

• Single-molecule gold-nanoparticle tracking with high temporal and spatial resolution and without an applied load. Laboratory Manual for Single Molecule Studies Dunn, A., R., Spudich, J., A. Cold Spring Harbor Laboratory Press, Woodbury, NY. 2007
• Tracking single gold nanoparticle-myosin V conjugates using darkfield imaging Dunn, A., R., Churchman, L., S., Bryant, Z., Spudich, J., A. 2006
• Picosecond photoreduction of inducible nitric oxide synthase by rhenium(I)-diimine wires JOURNAL OF THE AMERICAN CHEMICAL SOCIETY Belliston-Bittner, W., Dunn, A. R., Nguyen, Y. H., Stuehr, D. J., Winkler, J. R., Gray, H. B. 2005; 127 (45): 15907-15915

  Abstract

  In a continuing effort to unravel mechanistic questions associated with metalloenzymes, we are developing methods for rapid delivery of electrons to deeply buried active sites. Herein, we report picosecond reduction of the heme active site of inducible nitric oxide synthase bound to a series of rhenium-diimine electron-tunneling wires, [Re(CO)3LL']+, where L is 4,7-dimethylphenanthroline and L' is a perfluorinated biphenyl bridge connecting a rhenium-ligated imidazole or aminopropylimidazole to a distal imidazole (F8bp-im (1) and C3-F8bp-im (2)) or F (F9bp (3) and C3-F9bp (4)). All four wires bind tightly (Kd in the micromolar to nanomolar range) to the tetrahydrobiopterin-free oxidase domain of inducible nitric oxide synthase (iNOSoxy). The two fluorine-terminated wires displace water from the active site, and the two imidazole-terminated wires ligate the heme iron. Upon 355-nm excitation of iNOSoxy conjugates with 1 and 2, the active site Fe(III) is reduced to Fe(II) within 300 ps, almost 10 orders of magnitude faster than the naturally occurring reduction.

  View details for DOI 10.1021/ja0543088

  View details for Web of Science ID 000233535400053

  View details for PubMedID 16277534

• A flexible domain is essential for the large step size and processivity of myosin VI MOLECULAR CELL Rock, R. S., Ramamurthy, B., Dunn, A. R., Beccafico, S., Rami, B. R., Morris, C., Spink, B. J., Franzini-Armstrong, C., Spudich, J. A., Sweeney, H. L. 2005; 17 (4): 603-609

  Abstract

  Myosin VI moves processively along actin with a larger step size than expected from the size of the motor. Here, we show that the proximal tail (the approximately 80-residue segment following the IQ domain) is not a rigid structure but, rather, a flexible domain that permits the heads to separate. With a GCN4 coiled coil inserted in the proximal tail, the heads are closer together in electron microscopy (EM) images, and the motor takes shorter processive steps. Single-headed myosin VI S1 constructs take nonprocessive 12 nm steps, suggesting that most of the processive step is covered by a diffusive search for an actin binding site. Based on these results, we present a mechanical model that describes stepping under an applied load.

  View details for DOI 10.1016/j.molcel.2005.01.015

  View details for Web of Science ID 000227143400016

  View details for PubMedID 15721263

• Luminescent ruthenium(II)- and rhenium(I)-diimine wires bind nitric oxide synthase. J. Am. Chem. Soc. Dunn, A., R., Belliston-Bittner, W., Winkler, J., R., Getzoff, E., D., Stuehr, D., J., Gray, H., B. 2005; 127: 5169-5173
• Reversible inhibition of copper amine oxidase activity by channel-blocking ruthenium(II) and rhenium(I) molecular wires. Contakes, S., M., Juda, G., A., Langley, D., B., Halpern-Manners, N., W., Duff, A., P., Dunn, A., R. 2005
• Conformational states of cytochrome P450cam revealed by trapping of synthetic molecular wires. J. Mol. Biol. Hays, A.-M., A., Dunn, A., R., Chiu, R., Gray, H., B., Stout, C., D., Goodin, D., B. 2004; 2: 455-469
• Mechanism of sequence-specific fluorescent detection of DNA by N-methyl-imidazole, N-methyl-pyrrole, and β-alanine linked polyamides. J. Phys. Chem. B Rucker, V., C., Dunn, A., R., Sharma, S., Dervan, P., B., Gray, H., B. 2004; 108: 7490-7494
• Nanosecond photoreduction of cytochrome P450cam by channel-specific electron tunneling Ru-diimine wires. J. Am. Chem. Soc. Dunn, A., R., Dmochowski, I., J., Winkler, J., R., Gray, H., B. 2003; 41: 12450-12456
• Luminescent probes for cytochrome P450 Dunn, A., R., Hays, A.-M., A., Goodin, D., G., Stout, C., D., Chiu, R., Winkler, J., A. 2003
• Dark-to-light luminescent probes for metalloenzymes Dunn, A., R., Belliston, W., Chiu, R., Hays, A.-M., A., Goodin, D., B., Stout, C., D. 2003
• Ruthenium probes of P450 structure and mechanism. Meth. Enzymol. Dmochowski, I., J., Dunn, A., R., Wilker, J., J., Crane, B., R., Green, M., Dawson, J., H. 2002; 357: 120-133
• Sensitizer-linked substrates for cytochrome P450: Photoinduced electron transfer and structural insights Dunn, A., R., Crane, B., R., Dmochowski, I., J., Winkler, J., R., Gray, H., B. 2002
• Fluorescent probes for cytochrome P450 structural characterization and inhibitor screening. J. Am. Chem. Soc. Dunn, A., R., Hays, A.-M., A., Goodin, D., B., Stout, C., D., Chiu, R., Winkler, J., R. 2002; 124: 10254-10255
• Probing the open state of cytochrome P450cam with ruthenium-linker substrates. Dunn, A., R., Dmochowski, I., J., Bilwes, A., M., Gray, H., B., Crane, B., R. 2001
• Sensitizer-linked substrates for cytochrome P450: Photoinduced electron transfer and structural insights Dunn, A., R., Crane, B., R., Dmochowski, I., J., Winkler, J., R., Gray, H., B. 2001
• Influence of perfluoroarene-arene interactions on the phase behavior of liquid crystalline and polymeric materials. Angew. Chem. Int. Ed. Engl. Weck, M., Dunn, A., R., Matsumoto, K., Coates, G., W., Lobkovsky, E., B., Grubbs, R., H. 1999; 38: 2741-2745
• Comparison of the allosteric properties of the Co(II)- and Zn(II)-substituted insulin hexamers. Biochemistry Bloom, C., R., Wu, N., Dunn, A., Kaarsholm, N., C., Dunn, M., F. 1998; 37: 10937-10944
• Phenyl-perfluorophenyl stacking interactions: Topochemical[2+2] photodimerization and photopolymerization of olefinic compounds. J. Am. Chem. Soc. Coates, G., W., Dunn, A., R., Henling, L., M., Ziller, J., W., Lobkovsky, E., B., Grubbs, R., H. 1998; 120: 3641-3649
• Phenyl-perfluorophenyl stacking interactions: A new strategy for supermolecule construction. Angew. Chem. Int. Ed. Engl. Coates, G., W., Dunn, A., R., Henling, L., M., Dougherty, D., A., Grubbs, R., H. 1997; 36: 248-251