Alexander R. Dunn, Ph.D. - US grants

DESCRIPTION (Provided by the applicant) Abstract: Mechanical forces exerted by cells control processes of central importance in modern biology and medicine, for example cancer metastasis, stem cell differentiation, and embryonic development. However, the mechanisms by which cells exert and detect force remain poorly understood. Our understanding of how mechanical signaling modulates the behavior of whole tissues or organs is likewise in its infancy. We will use the tools of single-molecule biophysics to test transformative hypotheses about the roles of mechanical force in biology. Current techniques do not provide quantitative measurements of forces within and between cells. We will use novel molecular force sensors to directly observe myosin-based tension generation in living cells. These measurements will provide an unprecedented look at cells as mechanical entities-we will watch cells exert and respond to force in real time. Our measurements thus constitute a radical departure from the traditional forms of microscopy that characterize cellular structure, but that are blind to the underlying mechanical forces that shape and maintain both cells and tissues. Our work will clarify long-standing controversies about how the cytoskeleton is constructed, with important implications for our understanding of stem cell differentiation and cancer metastasis. In a separate set of measurements we will test the hypothesis that mechanical forces directly modulate the remodeling of the extracellular matrix by matrix metalloproteinases (MMPs). Confirmation of this model will open up new avenues in the investigation of heart disease. Further, our measurements will provide crucial insight into the mechanism by which MMPs differentially recognize substrates, thus contributing to the development of improved treatments for cancer. Finally, we will integrate these two strands of inquiry by measuring both myosin force generation and extracellular matrix remodeling in whole Drosophila embryos. Our experiments in Drosophila represent a first step toward a quantitative understanding of molecular force generation and mechanical signaling in living organisms. We feel that this transition to in vivo measurement represents a necessary progression both in our own research and for the field of biophysics as a whole. Public Health Relevance: Cells use nanometer-sized molecular motors to move, grow, and divide. Cells inside the human body also pull and tug on each other. This mechanical signaling is a crucial component of normal growth and development, but failures in mechanical communication can result in to the development of multiple diseases. We will watch cells in living organisms create and respond to force in real time. By learning more about how cellular mechanical signaling works we will be better able to understand and treat cancer, heart disease, and other important illnesses.

This award by the Office of Emerging Frontiers in Research and Innovation supports work to study mechanical interactions between cells that govern the basic processes of life and underpin many unresolved questions in multicellular biology. Mechanical stresses can modulate healthy and diseased cell responses such as renewal, growth, cell death or disease progression. Such mechanical signals can also directly regulate signaling pathways controlling cancer metastasis, cardiovascular remodeling or stem cell differentiation. The mechano-response of cell-cell adhesive structures to applied force is only recently documented, yet remains poorly characterized. Our inter-disciplinary team is addressing critical measurement challenges in biology to understand the cellular responses of dynamic mechanical load at cell-cell junctions. In this program, we will: 1) Develop novel engineering devices to image cell-cell junctions in living cells under dynamic applied load. These measurements will test models of cell adhesion remodeling in response to external mechanical load in multicellular tissue-like assemblies; 2) Apply innovative single-molecule assays to characterize force-dependent protein-protein interactions that are hypothesized to underlie cell adhesion remodeling; 3) Demonstrate a new class of molecular force sensors that can directly visualize the transmission of molecular-scale mechanical force through cell-cell adhesions. Our team unites an unusual combination of expertise in cell biology, structural biology, engineering, and biophysics and is well-positioned to tackle fundamental questions in mechanobiology that would be impossible for each individual research group to address alone. This work has transformative potential to revolutionize quantitative biology and unites unique views and skills in the growing, interdisciplinary field of mechanobiology.
The intellectual merit of the work lies in the development of basic knowledge and new models for cell response to environmental cues. Inter- and intra-cellular responses to mechanical stimuli offer a test bed for characterizing the thresholds and mechanisms of environmental adaptation and remodeling of multicellular assemblies. The outcomes, methods, devices and probes developed for our experiments will be made available through publications, detailed specifications, and databases for other researchers. Models and results will be disseminated through our webpage, publications and seminars for researchers in the field, and public seminar forums.
The broad impact of our work lies in enhancing knowledge of multicellular mechanical signaling, the role of the mechanical environment in cell behaviors, fundamental mechanisms for force and displacement sensing at the molecular scale, and the development of enhanced protocols, probes, and technologies to study the mechanobiology of multicellular systems in vivo and in vitro. Topics of our research will be incorporated in modules for teaching basic Engineering and Biology courses, and the development of undergraduate research experiences within our laboratories. The PIs actively participate in community outreach, undergraduate research opportunities, and research experience for teachers and under-represented student research programs. This award allows us to expand these efforts and include more participation to this work, as well as to showcase the work through public outreach via booths at community fairs and public talks.

DESCRIPTION (provided by applicant): FRET-based tension sensors to study zebrafish development forces and other mechanical variables play a significant role in animal development, as they shape tissue morphology, are part of signal transduction pathways, and drive cellular differentiation. Quantitative in-vivo tools for measuring forces and other biophysicl properties inside developing embryos are key for further understanding of these processes and their deregulation in disease, but these tools are largely lacking. We propose to develop genetically encoded, FRET-based tension sensors that harness a fluorescent signal to report force. Zebrafish is an ideal model organism for these studies due to the transparency and fast development of the embryo. We will build and screen multiple tension sensors based on native zebrafish genes, characterize the functionality of these sensors in the simpler and established cell-culture context, and test and fully characterize the most promising sensor constructs in the more challenging in-vivo context. Our preliminary data demonstrate that our imaging and data analysis modalities are sensitive enough for the proposed in-vivo measurements in zebrafish. We have already built multiple tension sensors based on native zebrafish mechanoproteins (ezrin, EpCAM) that properly localize in MDCK cells and in zebrafish. Further, we have established chemical and mechanical methods to characterize these sensors in-vitro and in- vivo. The main goal of this project is to translate the established in-vitro cell culture measurements into the in- vivo context of the zebrafish and to expand our repertoire of zebrafish-native force reporter constructs. The risk of this project is appropriate to the FOA and will be mitigated by screening a large number of probes. Our major intended deliverable is the construction and validation of one or more sensors that report in- vivo tension, with the successful demonstration of at least one force measurement at subcellular resolution within one developmentally relevant context. Depending on our progress, we hope to use our sensor(s) to significantly advance our understanding of how subcellular or inter-cellular tension drives a morphogenic process such as epiboly. Our team has all of the necessary expertise and an active collaboration that to date has generated a joint publication and the preliminary results tha predict the success of the proposed project. All resources and technologies are therefore at hand to revolutionize developmental biology research by developing robust, validated tools for measuring mechanical properties such as intercellular tension with subcellular resolution inside intact, living animals during development. We anticipate that empowering the developmental biology community to see forces inside living organisms will impact the field of mechano-biology much as seeing gene expression via GFP-tagging and seeing neuronal activity via Ca2+ imaging opened tremendous opportunities in developmental biology and neurobiology, respectively.

DESCRIPTION (provided by applicant): Our goal is to discover the molecular mechanisms by which integrins sense and transduce mechanical cues. Integrins are heterodimeric transmembrane proteins that link the cell's cytoskeleton to the extracellular matrix (ECM). Cells use integrins to migrate, exert force on their surroundings, and to sense the physical properties of the ECM. This latter property, termed mechanotransduction, is particularly important in human health and disease. Physical tension transmitted through integrins activates intracellular signaling that in turn exerts profound effects on processes as diverse as immune function, stem cell differentiation, and cancer cell metastasis. Despite this great physiological and medical importance, the physical mechanisms by which integrins sense mechanical force are not known. We aim to close this fundamental gap in our understanding of cell biology. In published work, we have developed F?rster resonance energy transfer (FRET) based molecular tension sensors (MTSs) that report on the mechanical tensions experienced by individual integrins in living cells. We have since combined MTSs and superresolution light microscopy to, for the first time, map force transmission within integrin adhesions with nanometer spatial resolution. The qualitatively new capabilities of MTS-based imaging allow us to tackle two fundamental questions in integrin biology that until now could not be directly addressed. In Aim 1, we will determine the physical mechanisms by which integrins sense mechanical tension. In particular, we will examine the overarching hypothesis that different integrin classes sense tension via fundamentally different mechanisms, and that these differences allow the cell to sense mechanical stimuli over a wide range of forces and timescales. In Aim 2, we will characterize the force transducing and sensing machinery in micron-sized integrin assemblies, termed focal adhesions (FAs), for the first time. Specifically, we will test the hypothesis that FAs contain highly coordinated, force-sensing microdomains, a prediction that cannot be tested using conventional techniques. This work will transform our understanding of cellular mechanotransduction by uncovering the molecular assemblies and biophysical mechanisms by which cells sense and transduce mechanical signals. More broadly, the mechano-responsiveness and compositional complexity that characterize FAs are also present in many other cellular structures. The conceptual and technical approaches developed in this project have the capacity to transform multiple fields of research by introducing powerful new single-molecule biophysical measurements in the context of intact, living cells.

? DESCRIPTION (provided by applicant): Our OBJECTIVE is to determine the molecular mechanisms by which fluid flow guides the formation and growth of lymphatic valves. Valves ensure one-way fluid flow in the lymphatic system, and are essential to physiological function. Relatively little is known about how lymphatic valves form. This knowledge gap, coupled with the limited regenerative ability of the lymphatic system, represents a central roadblock in the development of effective treatments for lymphedema, a debilitating condition for which there is no cure. Understanding the molecular basis of lymphatic valvulogenesis would thus have a transformative impact on the treatment of lymphedema and other diseases of the lymphatic system. RATIONALE: Lymphatic valves form preferentially near vessel junctions. These regions feature complex, recirculating flow that is not present in straight portions of the lymphatic vasculature. We hypothesized that these flow patterns might provide a critical cue that triggers valve formation specifically in these locations. To test this hypothesis, we developed a unique in vitro assay that exposes lymphatic endothelial cells (LECs) to spatial gradients in wall shear stress (WSS) that mimic those found at the sites of valve formation. Remarkably, LECs exposed to this flow pattern recapitulate the migratory, morphogenetic, and signaling events that occur during the initial stages of valve formation in vivo. Further, we find that these responses depend on activation of sphingosine-1-phosphate receptor 1 (S1PR1) a GPCR that is activated by fluid flow in blood endothelial cells, and that is known to play an important role in lymphangiogenesis. These and other preliminary data strongly suggest that spatial patterns in WSS play a central role in sculpting lymphatic valve development. STRATEGY: We have created and characterized in vitro culture systems that expose LECs to key attributes of the flow environment found at sites of valve formation. These devices provide quantitative control of the flow stimuli experienced by the LECs, allow time-lapse, multi-day imaging, and provide high experimental throughput, capabilities that are difficult to attain in an in vivo setting. This combination of attributes is nique in its ability to uncover the molecular mechanisms by which LECs sense and respond to fluid flow. The GOALS of our research are: Aim 1. Elucidate the role of fluid flow in guiding lymphatic valve formation. We will determine the role of WSS gradients and oscillating flow in shaping lymphatic valve development (Aim 1a), and discover how signaling pathways known to be required for valvulogenesis are coupled to LEC flow sensing (Aim 1b). Further, we will create 3D cell culture systems that fully recapitulate the flow environment found at sites of valve growth, and use this powerful technology to recreate key attributes of valvulogenesis in vitro. Aim 2. Determine the molecular mechanism by which S1PR1 mediates flow sensing in LECs. We will elucidate the role of flow-activated S1PR1 signaling in valve formation (Aim 2a), and use cell biological and biophysical approaches to determine the molecular mechanism by which flow activates S1PR1 (Aims 2b and 2c).

? DESCRIPTION (provided by applicant): Cell-cell adhesion defines solid tissues, and dysregulation of adhesion is an essential step in cancer cell metastasis. The protein aE-catenin has critical roles in cell and tissue development by transducing mechanical tension between cadherin cell adhesion molecules and the actin cytoskeleton into biochemical signals. We will investigate the molecular basis of how aE-catenin structure changes in response to force, and how its molecular behavior contributes to the formation and dissociation of cell-cell contacts. Our approach is to use a combination of rigorous biochemical characterization (Weis) and innovative single-molecule optical trapping assays (Dunn) to discover how the protein a-catenin both reinforces cell-cell junctions and triggers downstream signal transduction in response to mechanical stress. This question has deep biomedical significance, since a-catenin is known to be required for the formation of multicellular tissues and is a central player in both organogenesis and cancer metastasis. Cell biological data show that a-catenin and its binding partner ß-catenin are required to link the intracellular adhesion protein E-cadherin (epithelial cadherin) to the actin cytoskeleton. However, the a- catenin/ß-catenin/E-cadherin does not bind actin in bulk biochemical assays. In preliminary work, we used a novel single-molecule optical trap assay to show that the cadherin/catenin ternary complex can indeed bind actin, but only in the presence of mechanical load. Further, we find that the strength of the a-catenin-actin bond increases with mechanical load, and that binding of the cadherin/catenin complex to the actin filament is highly cooperative. The implication of these findings is that a-catenin acts as a force sensitive linker that can reinforce cell-cell contacts in response to mechanical load. This mechanism provides an elegant means to maintain tissue integrity in the presence of mechanical strain, and provides an explanation for how cells may sense tension at cell-cell junctions, a topic of intense current interest. However, how exactly a-catenin senses mechanical tension is not known. We will use a combination of biochemical and single-molecule biophysical approaches to: 1) determine the molecular mechanism by which a-catenin forms a force-sensitive linkage between cadherins and the actin cytoskeleton; and 2) discover how cooperative structural transformations in a-catenin, actin, or both regulate binding between the cadherin/catenin complex and filamentous actin. These measurements will reveal the molecular mechanism by which a-catenin senses force at cell-cell junctions. In addition, this work will provide a mechanistic basis for understanding how groups of cadherin-catenin complexes work in concert to remodel cell-cell junctions in response to changes in mechanical load, with potentially broad implications for our understanding of epithelial remodeling and morphogenesis.

? DESCRIPTION (provided by applicant): We propose to acquire a combined atomic force and optical microscope that will be equally useful for dynamic biological sample and soft/wet material research (Bio-AFM). The BioScope Resolve-Bio AFM (Bruker, Inc.) head and AxioObserver Z1 epifluorescence microscope (Zeiss, Inc.) base we are requesting has a versatile, modular design that combines the sub-molecular resolution imaging and precision force measurements of AFM with a broad range of optical microscopy techniques for simultaneous, correlative AFM and optical microscopy. This is a high performance Bio-AFM consisting of the following components: 1) Bio-AFM head equipped with an extended z-range head, Petri dish holder and stage heater, CO2 control, active vibration control and acoustic enclosure. 2) Inverted epifluorescence optical microscope on which to mount the AFM head; provides automated, diffraction limited resolution, wide-field epifluorescence and brightfield /phase imaging capabilities with piezo z-drive, sCMOS camera, and splitting optics, needed for correlative biological imaging studies utilizing AFM. The Bio- AFM and its requested accessories will serve first the specific needs of the NIH funded research projects described in this proposal and then the anticipated needs of the greater Stanford University and surrounding research community. This instrument will enable innovative experiments that will allow high resolution force measurements and mapping over the surface of soft materials, cells and other biological material. These force measurements will be correlated with macromolecules, proteins and subcellular structures as cells sense and respond to mechanical cues and environmental changes via epifluorescence, brightfield and phase contrast optical imaging. These otherwise not possible combinations of imaging experiments will result in dramatic and rapid progress in important health-related research topics in a variety of disciplines and interdisciplinary areas including the molecular and biophysical mechanisms underlying sensation of mechanical stimuli, engineering organoid cultures for regenerative medicine research, analysis of stem cell derived cardiomyocyte contractility and modeling familial dilated cardiomyopathy, correlating proteins involved in cell division with force distribution in dividing microbes and the manipulatio of T cell signaling pathways to control immunologically-mediated diseases. Stanford researchers, students, and staff are well-qualified - and eager - to carry out the research described here, to use the Bio-AFM to further these and other research efforts, to develop and publish novel methodologies for Bio-AFM, and to maintain the instrument within a shared imaging facility that will enable additional research.

? DESCRIPTION (provided by applicant): The purpose of this project is to elucidate the molecular mechanisms by which intercellular adhesion complexes form and remodel in response to mechanical load. Recent evidence demonstrates that mechanically initiated signaling at cell-cell junctions is a fundamental aspect of cell and developmental biology. Aberrant assembly and remodeling of intercellular junctions has likewise emerged as a defining feature of diseases including metastatic cancers, cardiomyopathies, and skin barrier defects. However, at present very little is known about how the complex protein assemblies present at cell-cell contacts convert molecule-scale forces into biochemical signals, or how mechanical cues govern the complex junctional dynamics that typify multicellular tissues. Previous work from our collaboration showed that a complex of E-cadherin, ?-catenin, and ?E-catenin forms a minimal force-sensing unit at adherens junctions (AJs). Separate work suggests that ?E-catenin additionally plays a central role in organizing epithelial tissues based on its interactions with vinculin, Epithelial Protein Lost in Neoplasm (EPLIN), Zonula Occludens (ZO)-1, and afadin, all of which bind actin and recruit other scaffolding and signaling proteins. In Aim 1 we will test the hypothesis that force-sensitive, cooperative actin binding by ?E-catenin and vinculin leads to dramatic increases in actin affinity over a very small range in force. This idea, if correct, would explain how a four-protein system amplifies small changes in force into dramatic alterations in adhesion stability and downstream signal transduction. Further, we will perform the first detailed biochemical and biophysical characterization of the interaction of the cadherin-catenin complex with EPLIN, ZO-1, and afadin. These studies lay the foundation for a quantitative understanding for how the AJ functions as an integrated, multifunctional force-sensing assembly. In Aim 2 we will examine force sensitivity in desmosomes. These junctions link desmosomal cadherins to the intermediate filament (IF) cytoskeleton, and are essential for tissue integrity. However, while cel biological data suggest a role of desmosomes in transmitting force between cells, there is currently no direct evidence for when, where, and even whether desmosomal cadherins transmit tension between neighboring cells in the absence of externally applied force. To address this gap, we will use genetically encoded molecular tension sensors to determine when and where desmosomal cadherins transduce force between neighboring cells. We will then critically evaluate the role of desmoplakin in transmitting force at desmosomes, analogous to the role established for ?E-catenin at AJs. Finally, we will use a single-molecule magnetic tweezers assay to test the innovative hypothesis that recruitment of plakoglobin, plakophilin, or both to desmoplakin is inherently force sensitive. These experiments will dramatically enhance our basic understanding of how desmosomes function as a mechanical linkage between cells.

Our objective is to elucidate the molecular mechanisms by which cellular adhesion complexes form and remodel in response to mechanical load. Cell-cell and cell-matrix adhesions are a defining feature of metazoan life and are essential to the physiological function of virtually every tissue in the human body. Despite this central importance, only a few of the protein-protein interactions that make up adhesion complexes have been characterized biochemically, and even less is known about the underlying mechanisms by which these structures respond to mechanical load. This lack of quantitative data presents an unavoidable roadblock in the collective effort to understand how cells build and remodel multicellular tissues. We will use single-molecule biophysical approaches to develop a detailed understanding of how adhesion complexes templated by E-cadherin sense and transduce mechanical cues. Previously, we demonstrated that a complex of E-cadherin, ?-catenin, and ?E-catenin forms a minimal force-sensing unit at intercellular adhesions. Here, we build on this result to test the hypothesis that this complex lies at the heart of a mechanosensory assembly that converts small changes in input forces into dramatic alterations in adhesion architecture, size, and stability. In parallel work, we will use biophysical techniques unique to our laboratory to determine how directional interactions between proteins within adhesion complexes and filamentous (F)-actin may give rise to long-range organization in the cytoskeleton. Recently, we found that the protein vinculin, which is recruited to both cell- matrix and intercellular adhesions, forms a directionally asymmetric interaction with F-actin that is stabilized ~10- fold when load is oriented toward the pointed (-) vs. barbed (+) end of the actin filament. Preliminary data suggest that force-dependent, asymmetric binding interactions with F-actin are not unique to vinculin, and likely extend to other adhesion proteins. These observations suggest that asymmetric interactions between F-actin and proteins within adhesion complexes may play a central and previously unsuspected role in organizing cells and tissues, a hypothesis that we will test during the next funding period. Cell and developmental biological data indicate that ?E-catenin plays a central role in organizing epithelial tissues through its interactions with zonula occludens-1 (ZO-1) and afadin, both of which bind F-actin and recruit other scaffolding and signaling proteins. We will perform the first detailed biochemical and biophysical characterization of the interaction of the cadherin-catenin complex with ZO-1 and afadin, and use cutting-edge imaging techniques to determine how these proteins interact in living cells. These studies will lay the foundation for a quantitative understanding of how intercellular adhesion complexes function as integrated, multifunctional force-sensing assemblies.