of the NSF Workshop
II. Summary of Invited Talks
A. Monday, July 24
Session 1: Molecular Perspectives
The theme of this session was the application and measurement of forces in biological systems ranging from the single molecule to whole, living cells. Force can be used as a tool to study and even change the structural state of individual biomolecules such as DNA and proteins. At the whole cell level, the forces generated by living cells play a key role in cell motility and shape. Crucial in understanding the generation of force and the resistance of cells to external forces is a characterization of the transmission of force into the cell body from the external environment. Likewise, the characterization of the material properties of the basic constituents of the cell is important.
The recent development and use of single-molecule manipulation techniques, such as various trapping methods, has permitted the study of single biomolecules in ways not possible just a few years ago. David Ben-Simon (Ecole Normal Superior, Paris) described some of the recent efforts to study single DNA molecules and associated enzymes. Specifically, force can be used as a tool to both examine and change the structural state of single DNA: different states can be induced by a combination of twist and stretch manipulation. It has also become possible recently to study the activity of single enzymes, such as DNA-polymerase and the relaxation of torsion by topoisomerase, which are important in the packing and transcription of DNA.
Michael P. Sheetz (Columbia University) focused generally on methods to measure cell mechanics in vivo, and specifically on the role of integrins in force transduction. Cells generate and respond to forces in part via integrin-matrix contacts, which are highly dynamic and which involve enzyme processes. The mechanisms of force generation and response in living cells have been studied using sensors based on silicon chips, together with laser tweezers. The various stages (extension, adhesion, and reinforcement) of cell motility have been characterized in this way.
An understanding of the generation and transmission of force in cells requires a basic understanding of the properties of the complex materials that constitute the living cell, as well as tools for their characterization. The cytoskeleton, which consists of a complex network of filamentous proteins or biopolymers, plays a key role in this force response of eukaryotic cells. Many recent efforts have focused on the characterization of such viscoelastic materials in vitro and in vivo. Frederick M. MacKintosh (University of Michigan and Vrije University of Amsterdam) summarized some of the principles and recent techniques of such microrheology, as applied to soft and biological materials. These techniques have been developed both to characterize bulk materials at a micrometer scale, as well as to probe small samples such as whole cells.
Session 2: Cellular perspectives
Peter F. Davies (Univ. of Pennsylvania) reviewed length scales of hemodynamic forces acting on the endothelium of blood vessels. Shear stresses were shown to regulate vascular endothelium over length scales ranging from tens of centimeters-millimeters throughout the tortuous geometry of the arterial tree and at flow separations, to micron scale at the topographic surface of individual cells within the endothelial monolayer, to sub-microns-nanometers during intracellular force transmission. Examples of hemodynamic-generated force quantitation at these different length scales were linked to the biological responses (including mechanotransduction signaling, gene transcription effects) and pathological consequences of hemodynamics (eg location of atherossclerotic lesions). New studies demonstrating 4-dimensional, near-real time imaging of endothelial GFP-cytoskeleton revealed spatially-defined displacement of filaments in response to external shear stress applied at the upper cell surface.
Eliot L. Elson (Washington University) described traction forces in a mutant Dictyostelium amoeba that lacks myosin II. These organisms locomote at approximately half the speed of the wild-type form (myosin II positive). Common to both types is rearward particle transport during traction but transport patterns are different in the mutant where a (undefined) low capacity alternative motor appears to operate. Myosin II was also shown to be unnecessary for cell spreading; its contractile forces actually resist cell spreading. The contributions of actin were reviewed in this system in the context of the balance between protrusive forces (actin polymerization) and restrictive forces (myosin).
Raymond E. Keller (University of Virginia) illustrated the dynamic cellular changes during Xenopus gastrulation and neurulation, events that occur through narrowing and elongation of the tissue over several hours. Dr Keller introduced biomechanical forces as a new consideration in these fundamental developmental processes that involve massive rearrangement of cellular components. Force and uniaxial compressive stress relaxation measurements revealed dorsal axial and paraxial tissue forces in the range of 0.6 microNewtons and 3-4 fold increases in stiffness in the axis of extension. Interference with the mechanical status of the tissue resulted in inappropriate development. Studies of component cells removed from the tissue are inadvisable because their behavior is context-defined. A discussion of the connections between gene-directed aspects and the role of biomechanics in these developmental processes concluded that the physical forces play an important role.
Session 3: Organ Perspectives
Alan J. Grodzinsky (Massachusetts Institute of Technology) presented a talk entitled "Chondrocyte Mechanotransduction: Cellular, Intracellular, and Molecular Responses to Tissue Level Forces." Extracellular matrix (ECM) adaptation to biomechanical demands in dense connective tissues such as cartilage is dependent on the ability of cells to sense and respond to physical stimuli. Recent studies suggest that there are multiple regulatory pathways (e.g., upstream signaling, transcription, translation, and post-translational modifications) by which chondrocytes in cartilage respond to mechanical stimuli and thereby alter the quantity and quality of newly synthesized ECM macromolecules. In vitro model systems including cartilage explants and 3-D chondrocyte-gel culture systems have been important in the study of mechanisms of mechano-transduction. Investigators have demonstrated that tissue shear and dynamic axial compression can each stimulate increases in proteoglycan and collagen synthesis and deposition in the ECM. Both static and dynamic compression of chondrocytes in intact tissue explants and in alginate gel culture can also alter the expression of aggrecan and type II collagen mRNA. However, mechanically-induced changes in synthesis are not necessarily dependent on gene transcription. Changes in the morphology and packing of intracellular organelles (e.g., rER, Golgi apparatus, nucleus, and mitochondria) induced by static compression may also regulate the processing and structure of molecules such as aggrecan. Finally, mechanical loading associated with joint cartilage injury is also a risk factor for development of OA. Studies in vitro have shown that injurious mechanical compression of cartilage can cause an increase in the number of apoptotic cells in a dose dependent manner.
Stephen C. Cowin (City University of New York, City College) presented a talk entitled "A possible resolution of a paradox in bone mechanosensation." Living bones adapt their structure to meet the requirements of their mechanical environment. These adaptations require a cell-based mechanosensing system with a sensor cell that perceives the mechanical deformation of the mineralized matrix in which it resides. One of the most perplexing features of this mechanosensory system in bone is the very low strain levels that a whole bone experiences in vivo compared to those needed to produce a cellular response in vitro. Strains in vivo depend strongly on frequency; they mostly fall in the range 0.04 to 0.3 percent for animal locomotion and seldom exceed 0.1 percent. These strains are nearly two orders of magnitude less than those needed (1% to 10%) to elicit biochemical responses in vitro, such as an increase in intracellular Ca2+ and prostaglandin synthesis. There is a paradox in the bone mechanosensing system in that the strains that activate the bone cells are orders of magnitude larger than the stains to which the whole bone organ is subjected. A hierarchical model, ranging from the subcellular level to the whole organ level, is used to resolve this paradox. Using this model, it is possible to explain how the fluid flow through the pericellular matrix surrounding an osteocytic cell process can lead to strains in its actin cytoskeleton which are two orders of magnitude greater than the mineralized matrix in which it resides.
Janet Braam (Rice University) presented a talk entitled "Molecular and Developmental Responses of Plants to Mechanostimulation." Despite their passive appearance, plants sense and actively respond to environmental stimuli, including mechanical stimuli like touch. Wind blown or touched plants will undergo altered development such that they are more resistant to mechanical stress. In Arabidopsis, there are strong and rapid gene expression responses to touch. These genes, called the TCH genes, encode calmodulin, calmodulin-related proteins and a cell wall modifying enzyme. Investigation of the regulation and functions of the TCH genes is being used to attempt to uncover the mechanisms by which plants sense mechanical force, transduce signals into cells, and modify growth patterns.
Both plant and animal tissues adapt their shape and form to the mechanical loadings to which they are subjected. While this influence is particularly strong when the tissue is growing, it also occurs in mature tissues. The three talks in this session consider a sample of plant and animal tissues that demonstrate this stress adaptation, articular cartilage, bone and several plant tissues.
Contemporary research has as its objective the description of the cellular and molecular mechanisms that make this structural adaptation possible. Generally these mechanisms involve sensor cells, material (protein) manufacturing cells, deconstruction (phagocytic) cells and systems of inter- or intra- cellular communication. The specifics of these features vary between tissue types, but all feature mechanosensation.
Session 1: Molecular Perspectives
Julio Fernandez (Mayo Clinic) discussed the mechanical stretching in vivo, which is thought to regulate the function of many proteins. The application of mechanical force to biological polymers produces conformations that are different than those that have been investigated by chemical or thermal denaturation, and are inaccessible to conventional methods of measurement such as NMR spectroscopy and X-ray crystallography. Force-induced conformational transitions may therefore be physiologically relevant, and may offer novel perspectives on the structure of biomolecules. Recent developments in single molecule force spectroscopy have enabled study of the mechanical properties of single biological polymers. For example, the force-measuring mode of the atomic force microscope (AFM) is capable of measuring force-induced domain unfolding in proteins. Furthermore, through the use of protein engineering, we have examined the mechanical stability and topology of immunoglobulin and fibronectin protein modules which are common muscle and cell adhesion proteins. These experiments have demonstrated a number of mechanical phenotypes that are readily captured by the single molecule AFM technique. We recently demonstrated that point mutations can have large effects on the mechanical stability of an immunoglobulin module. Hence, the AFM may help to elucidate the molecular determinants of mechanical stability in proteins and the role of force-induced conformational changes in the regulation of their physiological function.
Klaus Schulten (University of Illinois, Urbana-Champaign) discussed the structure, dynamics, and function of biopolymer aggregates, including lipids and water forming membrane bilayers, proteins complexing with DNA and regulating gene expression, and proteins involved in complexes with other proteins. Schulten uses very-large-scale computer simulations to study their behavior.
John Frangos (University of California, San Diego) discussed fluid shear stress (FSS) which has been shown to be an ubiquitous stimulator of mammalian cell metabolism. While many of the biochemical transduction pathways have been characterized, the primary mechanoreceptor for FSS remains unknown. His hypothesis is that the cytoplasmic membrane acts as the receptor for FSS. He proposes that FSS increases membrane fluidity, a change that leads to the activation of heterotrimetric G proteins (Gudi et al, PNAS 90: 2515-2519, 1998). 9-(dicyanovinyl)-julolidine (DCVJ) is a fluorescent probe that integrates into the cell membrane and changes quantum yield with the viscosity of the environment. In a parallel-plate flow chamber, a confluent layer of DCVJ-labeled human umbilical cord venous endothelial cells were exposed to different levels of FSS. With increased FSS, a reduced fluorescence intensity was observed, indicating an increase of membrane fluidity. Step changes of FSS caused an approximately linear drop of fluorescence within 5 seconds, showing fast and almost full recovery after shear stopped. A linear relationship between shear stress and membrane fluidity changes was observed. This study clearly shows the direct link between fluid shear stress and membrane fluidity, and suggests that the membrane may be the primary flow mechanosensor of the cell.
Session 2: Cellular Perspectives
Gabor Forgacs (University of Missouri) discussed a general network model for information transmission by diffusion along cytoskeletal elements. This model was contrasted to the current simple diffusional models for soluble signals. He outlined a method of magnetic bead rheology with which he hopes to test the model, although some listeners were unclear about what specific rheological predictions the model makes other than some evidence of network structure. He also introduced a novel magnetic tweezer apparatus, capable of producing forces of orders of magnitude stronger than existing tweezers. He is planning to use this apparatus to investigate the proposed interconnected nature of the cytoskeleton. In connection with his talk Michael Sheetz reminded that he had earlier demonstrated the possibility for microtubule associated proteins to indeed diffuse along these filaments, thus giving support to the suggested mechanism of signaling. Alan Hunt noted that there must exist a lower size cutoff for molecules diffusing along cytoskeletal filaments. Below this cutoff he expects free diffusion to be the principal mechanism for intracellular protein translocation.
Steven Heidemann (Michigan State University) argued that the tensegrity model of intracellular architecture is too specific to explain a number of observations. In particular he argued that "tensegrity lacks time scale aspects", cortical tension is not the primary determinant of cell shape and stress hardening (being an important feature of tensegrity) characterizes also the cell models of Hiramoto (rubber model) an of Yonegida (liquid drop model). He cited Fuller's statement that tensegrity in no way mimics living structures. He described experiments in which GFP labeled cytoskeletal proteins had been used to follow the consequences of pulling on cytoplasmic processes. Since the applied forces produced only local responses, he concluded that the results of these experiments, performed on fibroblasts, are inconsistent with the predictions of the tensegrity model. He noted that tensegrity still may be a useful representation for other cell types (i. e. neurons).
Donald Ingber (Harvard University) defended the tensegrity model. He disputed the arguments of Heidemann and reasoned that tensegrity is the only structure which has built in prestress necessary to understand a number of cellular phenomena. He presented experimental results in favor of the model. In particular, he has shown that disrupting the actin cytoskeleton leads to the same effect as changing cell shape (which he and his collaborators can do in a controlled manner using special "moulds"). He argued, this finding is consistent with the tensegrity model. Furthermore, he showed that when the cell spreads, so does its nucleus, which (according to him) can be understood only if a prestressed tensegrity structure extends in the interior of the cell including the nucleus.
Alan Hunt asked whether the tensegrity model can be used to understand structure from the atomic scale all the way to cosmic scales, to which Ingber responded that indeed it can. Christian Oddou noted that numerous experimental results obtained in his lab, using stick and string representation of cytoskeletal filaments are consistent with the predictions of the tensegrity model and as long the model does not fail, it should not be abandoned. Several participants stressed that tensegrity structures as conceived by Buckminster Fuller are passive engineering constructions and they are not necessarily correct representations of the rapidly varying cytoskeleton, with these variations being controlled by gene activity.
These talks and following discussions indicated a consensus on the role of the cytoskeleton in intracellular force transduction. Although a number of experimental observations can be explained by assuming the cytoskeleton to be an interconnected network of specific filaments (either via a percolation or a tensegrity structure), other observations seem to inconsistent with this hypothesis (at least with the model based on tensegrity). Thus, the topic remains contentious and further studies are needed to clarify the precise mechanism through which the cytoskeleton may participate in intracellular signal and force transmission.
Session 3: Tissue/Organ Perspective
The topics in this session included asthma, muscle implants and hearing, all three are relevant to human health. The speakers presented tissue/organ perspectives based on molecular and cellular mechanisms.
Roger D. Kamm (Massachusetts Institute of Technology) began the session by describing asthmatic tissue remodeling that decreases the dimensions of the airway. His central hypothesis is that airway remodeling is a response to a mechanical stimulus rather than generalized inflammation. He went on to present results based on in vitro culture models showing the mechanical stimulus (most likely shear stress) is transduced by epithelial cells into a biochemical signal that acts on co-cultured fibroblasts.
Herman H. Vandenburgh (Brown University) followed with a description of bioartifical muscles (BAMs). BAMs are fabricated from mammalian skeletal muscle stem cells. A variety of strategies involving both the intensity and temporal properties (including quiescence) of applied stress were described for guiding the modeling of this tissue. The goal was to enhance its ability of generate mechanical force. BAMs are less efficient than native muscle vis a vis force transduction but they have potential for therapeutic protein delivery. Genetic induction of protein expression reveals they are able secrete therapeutic proteins (growth factors, kinases, etc.) at high levels.
William E. Brownell (Baylor Medical School) then described how electromechanical force transduction by outer hair cells enhances mammalian hearing. Outer hair cells provide a positive feedback of mechanical force that counteracts viscous damping forces. The cells convert electrical energy directly to mechanical energy at frequencies >100 kHz. Experimental evidence locates this piezoelectric-like force generator in the plasma membrane of the cell's lateral wall. Electromechanical force transduction has not previously been associated with membranes. The potential for membranes to provide useful work is a novel biological and physical concept.
Session 1: Molecular Perspectives
Fred Sachs (State University of New York, Buffalo) presented a talk entitled "A blocker for cationic SACs, from channels to animals." He discussed how a 4 kD peptide isolated from tarantula venom blocks cationic SACs with an affinity of about 500 nM. The peptide noted GsMTx-4 is specific for SACs. It doesn't affect steady state I/V curves of heart cells or of astrocytes. It does, however block stretch induced effects. It reduces volume activated currents in astrocytes and can block atrial fibrillation induced by dilatation in the rabbit heart with affecting the action potential.
Evan Evans (Boston University, University of British Columbia) presented a talk entitled "Exploring the Complex Relation between Force - Time - Chemistry in Single Biomolecular Bonds." He discussed how noncovalent-macromolecular bonds are the fundament of nanoscale chemistry in recognition, adhesion, signaling, activation, regulation, and a host of other processes from outside to inside cells. But not well-appreciated is that energy landscapes of these biomolecular bonds are rugged terrains with more than one prominent activation barrier. Near-equilibrium kinetics in conventional test tube assays only reveal a single-outer barrier, which is the classical paradigm of biological chemistry. However, when bonds are detached under a large range of loading rates (force/time), the measurements of single bond strength on a scale of Log(loading rate) provide a spectroscopic image of prominent energy barriers traversed along the force-driven reaction coordinate. In this way, dynamic force spectroscopy DFS exposes barriers - especially inner barriers - that are difficult or impossible to detect in solution assays. Because of the inherent logarithmic dependence of rupture force on speed of loading, the DFS method is most revealing when applied over many orders of magnitude in loading rate. Examining biomolecular bonds with dynamic force spectroscopy is leading to a new perspective of the important connection between force - time - chemistry in biology.
George Oster (University of California Berkeley) presented a talk entitled "How F1 ATPase uses nucleotide hydrolysis to generate a rotary torque." He discussed how the experimentally measured mechanical efficiency of the F1 ATPase under viscous loading is nearly 100%, far higher than any other hydrolysis driven molecular motor. A structural and bioenergetic analysis provides a molecular explanation for this remarkable property.
Session 2: Cellular Perspectives
These three talks [Yale Goldman (University of Pennsylvania), Joyce Wang (Boston University), and, Charles Lindemann (Oakland University)] and those by Sheetz, and Elson earlier in the meeting, have the common theme that the motor proteins and the cytoskeletal polymers exhibit bi-directional communication and energy transduction. The conventional energy transduction pathway is from metabolic energy into motion. Many cellular machines use energy liberated by splitting ATP or GTP to perform useful functions, such as motility, ion pumping, untwisting of tangled DNA or proof-reading of the genetic code during translation. It can easily be shown thermodynamically that this energy transduction implies an influence of the work output or mechanical properties of the load, such as its mass, stiffness or viscosity, on the rates of some of the accompanying biochemical reactions. In muscle, non-muscle myosin-based intracellular motility, locomoting cells and the flagellar axoneme the mechanical conditions, forces on the motors and properties of the substrate, strongly control the kinetics of the energy transduction process. Decoding the details of this 'reverse communication' and understanding the mechanisms at the molecular and atomic levels remain crucial tasks in most examples of cell motility.
Yale Goldman (University of Pennsylvania) showed several examples of the feedback of the loading conditions on actomyosin kinetics and some new methods for detecting the relevant mechanical and structural signals. This feedback is essential to minimize energy consumption. Non-muscle myosins participate in myriad cell biological roles, including development of the cell morphology, maintenance of native ultrastructure and signaling. Members of the myosin superfamily transduce force signals and move crucial cargoes to specific sub-cellular target. Wang used a new manipulatable substrate (cross-linked polyacrylamide) to detect traction forces of locomoting cells. He addressed the production of such forces, their magnitudes and mechanisms. The results are compatible with an engine-cargo model. How the cells detect and respond to mechanical properties of the substrate is just beginning to be understood. The functions of such detection may be probing the environment or long range signaling between cells or from the environment. Lindemann presented a model of the eukaryotic flagellum in which the transverse-force acting on the outer microtubule doublets regulates the dynein motors. A simulation based on this model replicates the behavior of cilia and flagella including mechanical sensitivity.
Force transduction by the force generators themselves controls their output and may also influence many other cellular processes. Another thread in these talks is that development of new methods is essential to obtain discriminating experimental data. Using the widest possible armamentarium, including physical and engineering approaches, toward solving biological problems is the most fruitful avenue.
Session 3: Tissue/Organ Perspectives
The session on Biological Forces, Tissue-Organ Perspectives focused on the mechanisms of mechanotransduction in biological systems.
Shu Chien (University of California, San Diego) reported that the shear stress can activate integrins and a vascular endothelial growth factor receptor. The activation of these membrane proteins triggers intracellular signaling pathways to modulate gene expression and cellular functions. The temporal and spatial natures of the mechanochemical transduction in relation to flow dynamics may explain the preferential localization of atherosclerosis in branch points of the arterial tree.
Elisabeth Burger (Vrije University of Amsterdam) presented data showing that the flow of interstitial fluid in the canaliculi in strained bones induces significant shear stresses which are sensed by the osteocytes to induce bone remodeling. High bone strain and interstitial flow causes osteoblast recruitment and bone growth, whereas reduced bone strain and interstitial flow leads to osteoclast attack and bone loss. In addition to the modulation of cellular functions such as proliferation, motility, and secretion, mechanical forces also cause structural remodeling, e.g., the reorganization of cytoskeletal fibers and the alignment of endothelial cells and bone trabeculae and osteons with the direction of force application.
Stephan Levin (private practice) presented the tensegrity model of spine mechanics. In the tensegrity model, the bones act as compression elements enmeshed in soft tissues. In contrast to the traditional "stack of block" models, tensigrity structures are omni-directional, hierarchical, nonlinear, and independent of gravity, and local load distributing. The tensegrity model allows the synegistic linkage of structure and function for the creation of an integrated hierarchical system.
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