Alex Dunn is an Assistant Professor in the Department of Chemical Engineering at Stanford University. His research focuses on understanding how living cells sense mechanical stimuli, with particular interests in stem cell biology and tissue engineering. Dr. Dunn worked as a postdoctoral scholar with James Spudich in the Department of Biochemistry at the Stanford University School of Medicine. He received his Ph.D. at the California Institute of Technology under the direction of Harry Gray, where his work focused on understanding the catalytic mechanism selective C-H bond oxidation by cytochrome P450 enzymes. His work has been recognized with numerous awards, including the Hertz Fellowship, Jane Coffin Childs Postdoctoral Fellowship, the Burroughs Wellcome Career Award at the Scientific Interface, and NIH Director’s New Innovator Award.

Academic Appointments

Honors & Awards

  • New Innovator Award, National Institutes of Health (2010)
  • Burroughs Wellcome Career Award, Scientific Interface (2008)
  • Postdoctoral Fellowship, American Heart Association (2007)
  • Jane Coffin Childs Fellowship, Jane Coffin Childs Memorial Fund for Medical Research (2003)
  • Herbert Newby McCoy Award, McCoy family (2003)
  • Fannie and John Hertz Fellowship, Fannie and John Hertz Foundation (1998)

Professional Education

  • PhD, Caltech (2003)

Current Research and Scholarly Interests

The knowledge that the mechanical properties of living tissue reflect health or disease dates at least to Hippocrates. However, the underlying cellular and molecular mechanisms have only just begun to be discovered. We now know that mechanical forces at the cellular level guide stem cell differentiation, convey the health benefits of exercise, and underlie our ability to sense touch, sound, and pain. Conversely, faulty cellular responses to mechanical signals contribute to atherosclerosis, aneurism, and cancer metastasis. Understanding how cells sense and respond to their mechanical environment is thus a problem of deep intellectual and practical significance.

Below I describe three projects that together add up to a concerted effort to understand how cells detect and process mechanical signals. I’m happy to discuss any of the following ideas in greater detail, so please feel free to email me with your questions or comments.

Project 1: Understand how motor proteins use chemical energy to generate force and motion. The most familiar motor protein is myosin, which generates the force responsible for muscle contraction. Additional motor proteins are found in every cell in the human body, where they allow the cell to move and divide, transport cargo from one part of the cell to another, and participate in the detection of external mechanical stimuli. We are using sophisticated biophysical techniques to directly observe the motion of single motor proteins in order to better understand the physical principles that allow proteins to convert chemical energy into useful motion. More broadly, recent work suggests that enzymes in general may derive their incredible catalytic ability by coupling protein motion to bond making and breaking. Single-molecule biophysical measurements offer a potentially powerful way to test this idea.

Project 2: Uncovering previously unrecognized functions of mechanical force in extracellular matrix remodeling. The material properties of our own bodies are governed largely by the extracellular matrix, a complex protein and carbohydrate network that gives shape to tissues and organs. Previously, the ECM was dismissed as passive glue that simply held cells together. We now know that failure to maintain the ECM leads not only to aching knees and wrinkles, but also contributes to heart disease and cancer metastasis. We are using techniques ranging from single-molecule assays to live cell imaging to test the hypothesis that mechanical force is an unrecognized regulator of ECM remodeling. We are particularly interested in the possibility that mechanical force may increase the susceptibility of ECM proteins to proteolysis by matrix metalloproteinases, enzymes that are the subject of intense medical interest due to their prominence in cancer biology.

Project 3: Role of intercellular forces in cell and developmental biology. Our goal in this project is to determine how cells generate, detect, and respond to tension at the molecular level. To do so, we are using new microscopy techniques that allow us to measure mechanical forces inside living cells, and even in whole organisms. Questions we hope to answer in the next five years are: 1) Do cells communicate by pulling on each other, and if so, what are the biological consequences? 2) How do cells coordinate their actions over long distances in order to shape organs and tissues? 3) How do stem cells sense the mechanical properties of their environment in order to properly differentiate? Results from this project will be highly relevant to multiple aspects of human disease, to the development of stem-cell-based therapies, and to engineering complex, three-dimensional tissues in the lab.

2015-16 Courses

Stanford Advisees

Graduate and Fellowship Programs

All Publications

  • 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


    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

    View details for PubMedID 26200877

  • 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


    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

  • 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)


    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

  • 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


    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

  • 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


    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 Web of Science ID 000330158900004

  • 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


    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


    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 ?E-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

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


    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 Web of Science ID 000349954100016

  • Mechano-Transduction: From Molecules to Tissues PLOS BIOLOGY Pruitt, B. L., Dunn, A. R., Weis, W. I., Nelson, W. J. 2014; 12 (11)
  • 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
  • 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

    View details for DOI 10.1038/ncomms4613

    View details for Web of Science ID 000335220700018

  • Mechanical control of the sense of touch by beta-spectrin NATURE CELL BIOLOGY Krieg, M., Dunn, A. R., Goodman, M. B. 2014; 16 (3): 224-?

    View details for DOI 10.1038/ncb2915

    View details for Web of Science ID 000332124000008

  • 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


    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

  • 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-?


    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

  • Cellular biomechanics at the molecular scale Dunn, A., R. 2013
  • Mechanotransduction at cell-cell and cell-matrix adhesions Dunn, A., R. 2013
  • Biomechanics at the molecular scale Dunn, A., R. 2013
  • Multiplexed Single-molecule Force Proteolysis Measurements Using Magnetic Tweezers JOVE-JOURNAL OF VISUALIZED EXPERIMENTS Adhikari, A. S., Chai, J., Dunn, A. R. 2012

    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


    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

  • Multiplexed single-molecule force proteolysis measurements using magnetic tweezers. Journal of visualized experiments : JoVE Adhikari, A. S., Chai, J., Dunn, A. R. 2012


    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 PubMedID 22871786

  • Force Dunn, A., R. 2012
  • Metalloproteinase conformational dynamics accompanying the proteolytic degradation of trimeric collagen I Dunn, A., R. 2012
  • Roles of Mechanical Force in Extracellular Matrix Remodeling Dunn, A., R. 2012
  • E-cadherin experiences constitutive mechanical tension in living cells Dunn, A., R. 2012
  • 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


    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 ?H(0) and ?S(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

  • 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


    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

  • 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


    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

  • 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
  • Molecular force probes for measuring cellular mechanotransduction Dunn, A., R. 2011
  • Mechanical forces in developmental biology Dunn, A., R. 2011
  • Mechanical force induces a 100-fold increase in the rate of collagen proteolysis by MMP-1 Dunn, A., R., Adhikari, A., S., Chai, J. 2011
  • Using single molecule measurements to study cellular force sensors Dunn, A., R., Adhikari, A., S., Chai, J., Chuan, P., Y., Spudich, J., A. 2011
  • Using single molecule measurements to study cellular force sensors Dunn, A., R. 2011
  • Measurement of cytoskeletal forces in living epithelial cells Dunn, A., R. 2011
  • Exploring the role of mechanical forces in cell biology Dunn, A., R. 2011
  • Effect of mechanical load on extracellular matrix remodeling from single molecules to millimeters Dunn, A., R., Adhikari, A., S., Mekhdjian, A., Chai, J. 2011
  • Building biology Dunn, A., R. 2011
  • 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


    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

  • 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


    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

  • Contribution of the myosin VI medial tail domain to processive stepping and intramolecular tension sensing. Dunn, A., R., Chuan, P., Y., Spudich, J., A. 2010
  • 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


    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

  • Velocity, Processivity, and Individual Steps of Single Myosin V Molecules in Live Cells BIOPHYSICAL JOURNAL Pierobon, P., Achouri, S., Courty, S., Dunn, A. R., Spudich, J. A., Dahan, M., Cappello, G. 2009; 96 (10): 4268-4275


    We report the tracking of single myosin V molecules in their natural environment, the cell. Myosin V molecules, labeled with quantum dots, are introduced into the cytoplasm of living HeLa cells and their motion is recorded at the single molecule level with high spatial and temporal resolution. We perform an intracellular measurement of key parameters of this molecular transporter: velocity, processivity, step size, and dwell time. Our experiments bridge the gap between in vitro single molecule assays and the indirect measurements of the motor features deduced from the tracking of organelles in live cells.

    View details for DOI 10.1016/j.bpj.2009.02.045

    View details for Web of Science ID 000266312900038

    View details for PubMedID 19450497

  • Contribution of the myosin VI medial tail domain to processive stepping and intramolecular tension sensing. Dunn, A., R., Chuan, P., Y., Spudich, J., A. 2009
  • The mechanism of load detection in the molecular motor myosin VI. Dunn, A., R., Spudich, J., A. 2009
  • Myosin VI as a transporter and an anchor: A model for kinetics of the motor under load. Dunn, A., R., Chuan, P., Y., Spudich, J., A. 2009
  • 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


    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 to share this tool with the wider community (

    View details for DOI 10.1016/j.jmb.2007.08.059

    View details for Web of Science ID 000250712600021

    View details for PubMedID 17900617

  • 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


    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

  • Structural dynamics of myosin V: characterization of the one-head bound intermediate. Dunn, A., R., Spudich, J., A. 2007
  • Structural dynamics of single molecular motors. Dunn, A., R., Spudich, J., A. 2007
  • Single molecule measurements link myosin V biophysics and cellular function. Dunn, A., R., Spudich, J., A. 2007
  • Regulation of the cell’s dynamic city plan and the myosin family of molecular motors. Dunn, A., R., Spudich, J., A. 2007
  • 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
  • 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


    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


    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
  • 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
  • Luminescent probes for cytochrome P450 Dunn, A., R., Hays, A.-M., A., Goodin, D., G., Stout, C., D., Chiu, R., Winkler, J., A. 2003
  • 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
  • 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