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Author: Donald E. Ingber
The hierarchical molecular structures that comprise living cells, tissues, and organs are based on tensegrity principles.
The burgeoning fields of tissue engineering and nanotechnology offer exciting new approaches to address fundamental questions in biology and improve human health. But these fields are limited because we do not understand how living cells and tissues are constructed so that they exhibit their incredible organic properties, including their ability to change shape, move, grow, and self-heal. So far, we have not been able to construct man-made materials that mimic these features or to design drugs or devices to control these behaviors selectively. To accomplish this, we must first uncover the underlying design principles that govern how cells and tissues form and function as hierarchical assemblies of nanometer-scale components.
One aspect of this challenge is to understand the “hardware”—the physical structure of the whole cell. The second is to comprehend how the “software” (cellular information-processing network) functions so cells can make discrete cell-fate decisions, such as whether to grow, differentiate, or die, even when confronted by conflicting signals. The ultimate goal of this research is to explain how structural and information networks are integrated so that cells can sense their physical and chemical environments and respond appropriately.
Because a mammalian cell has a flexible membrane surrounding its cytoplasm and nucleus, people have tended to think of cells as squishy blobs, like balloons filled with molasses. However, the sculpting of tissues and organs that occurs in the embryo is an extremely physical process. Various regions of the growing cellular aggregate independently move, stretch, and pull against one another through the action of cell-generated forces. Mechanical distortion does more than change the shape of cells; it also influences cellular biochemistry and gene expression, and thereby actively controls tissue development. Adult tissues exhibit a similar sensitivity to physical forces. Compressive forces due to gravity shape bones; tension molds muscle; and hemodynamic forces govern the form and function of the cardiovascular system.
Cells could not exhibit these behaviors if they were structured like balloons. In reality, the cell has a molecular framework or “cytoskeleton” hidden within its surface membrane that mechanically stabilizes the cell and actively generates contractile forces through an actomyosin filament-shortening mechanism similar to that of muscle. Cells apply these forces to their adhesions to other cells, as well as to extracellular matrix (ECM) scaffolds that hold cells together within living tissues. These tensional forces also promote structural rearrangements within the cytoskeleton that govern multiple cellular activities (e.g., movement, contraction, intracellular transport, mitosis) at the molecular level.
The cytoskeletal network is composed of three classes of biopolymers: microfilaments, intermediate filaments, and microtubules. The challenge is to understand how the mechanical properties of a cell emerge through collective interactions among these molecular filaments. Most work on cell mechanics focuses on the gel properties of the cytoskeletal lattice, but gels made from isolated cytoskeletal filaments do not mimic complex cell behaviors. In contrast, we have explored the possibility that cells structure their cytoskeletons using “tensegrity”—the architectural principle used in Buckminster Fuller’s geodesic domes. This idea may seem strange, but molecular geodesic domes have been observed in the microfilament cytoskeletons of living cells.
The stable shape of tensegrity structures is attributable to continuous tension, rather than continuous compression. For example, a simple tensegrity structure may be constructed from a continuous series of strings under tension pulling toward the center of the object, but balanced by other filaments or struts that resist being compressed. Thus, the stable shape of the entire structure depends on the presence of isometric tension or a tensile “prestress,” just like the stability of my arm depends on my muscle tone. The key role of prestress for shape stability is the most fundamental feature of tensegrity structures. Prestress is essentially ignored in models of the cytoskeleton that focus on gel properties.
Another fundamental property of living materials, as opposed to man-made materials, is that their structures are hierarchical. For instance, when an intact nucleus is removed from one living cell and placed in another enucleated cell (e.g., to clone an embryo), both the nucleus and cytoplasm maintain their structural integrity in isolation. Once a nucleus is in the recipient cell, however, it reconnects with the surrounding cytoskeleton and regenerates a new structurally and functionally integrated cell. Moreover, smaller structures in the cells, such as organelles, transport vesicles, and enzyme complexes, exhibit similar autonomy, even though physical coupling to surrounding structures also affects their function. Whole cells are similarly integrated within tissues, tissues within organs, and organs within a whole organism. Moreover, when stress is applied at the whole organism level (e.g., gravity), there are coordinated structural and functional changes on many other levels.
Many of the properties of living systems are mimicked by simple tensegrity structures. For example, hier-archical tensegrity models of a cell containing a nucleus can be constructed by linking larger and smaller tensegrity structures composed of elastic sticks and strings, with additional tensile connections. Because they are prestressed, when these tensegrity models are not anchored, they take on a round shape. However, both the cell and nucleus spontaneously flatten out and spread in a coordinated way when they are attached to a rigid substrate. Furthermore, when their anchors are clipped, both the cell and the nucleus spontaneously retract into a round shape. This is exactly what is observed when cells adhere to and detach from a culture substrate. Analysis of these structural models also reveals that applying stress locally on the surface of a hierarchical tensegrity results in global structural rearrangements in various locations and on several levels.
Experimental studies from various laboratories support the possibility that cells use tensegrity to structure themselves. These experiments confirm that cell shape is stabilized through a balance of mechanical forces. Cytoskeletal contractile forces are resisted and balanced by internal cytoskeletal struts and by external adhesions to ECM and to other cells, thereby generating prestress that stabilizes the cell. The cytoskeletal lattice connects to the ECM and neighboring cells via transmembrane adhesion receptors, known as “cadherins” and “integrins,” that form spot weld-like adhesion sites on the cell surface. The tensed strings of the tensegrity model mimic the contractile microfilaments of the cytoskeleton; the struts represent other cytoskeletal elements that resist compression, such as microtubules and stiffened (e.g., cross-linked) bundles of actin microfilaments. Intermediate filaments act like molecular guy wires to help individual microtubules resist buckling under compression and link the nucleus to the surface membrane, thereby ensuring hierarchical coordination. Finally, the surface membrane and underlying cortical cytoskeleton (a thin shell composed of actin, ankryin, and spectrin molecules) form a third level in the structural hierarchy of the cell. This submembranous cytoskeleton is also a prestressed molecular lattice that is highly flexible, except where the membrane connects with the microfilament-microtubule-intermediate filament lattice at sites of cell-cell and cell-ECM adhesion.
Tensegrity also appears on both smaller and larger scales in the hierarchy of life. Viruses, enzyme complexes, transport vesicles, actin geodomes, the submembranous cytoskeleton, transport vesicles, enzyme complexes, and viruses all exhibit geodesic forms. On a larger scale, specialized ECM compo-nents, including elastic (elastin) fibers, stiffened (cross-linked) collagen bundles, and compression-resistant (hydroscopically swollen) polysaccharide gels, interplay with contractile cells to maintain a stabilizing tensile prestress at the tissue and organ levels. Bones, muscles, tendons, and ligaments organized in a similar way stabilize the shapes of our bodies; tensegrity even has been invoked to explain structural stability in insects and plants.
Cellular Mechanotransduction and Tissue Morphogenesis
One prediction based on the cellular tensegrity model is that adhesion receptors linked to the deep cytoskeleton, such as integrins and cadherins, provide preferred paths for mechanical signals to enter the cell. For instance, if one were to pull on a transmembrane protein that only connects to the flexible submembranous cytoskeleton, stress would dissipate locally. In contrast, if one were to tweak a receptor linked to the internal microfilament-intermediate filament-microtubule lattice, the entire cytoskeletal network would bear the load and become stronger as a result of structural rearrangements at multiple levels.
To test this prediction, we developed micro-engineering approaches to apply mechanical stresses to specific receptors on the surface of living cells. Magnetic fields were applied to cells bound to micrometer-sized magnetic beads precoated with receptor ligands, and bead displacements were measured simultaneously. With Ning Wang (Harvard School of Public Health) and Dimitrije Stamenovic (Boston University), we have used this approach to demonstrate that the mechanical behavior of mammalian cells is like the behavior of tensegrity structures. A theoretical formulation of the tensegrity model starting from first mechanical principles also yields accurate qualitative and quantitative predictions of many static and dynamic mechanical behaviors.
But the cytoskeleton is more than a structural scaffold; it also orients much of the biochemical machinery of the cell, including many of the enzymes and substrates that mediate signal transduction. This type of “solid-state” biochemistry has important implications for the way cells sense mechanical signals and transduce them into a biochemical response, a process known as mechanotransduction. For example, when molecular (e.g., enzymatic) components of cytoskeletal filaments that bear mechanical loads are deformed, their thermodynamic and kinetic properties change. In this way, tensegrity provides a way for cells to channel mechanical forces in distinct patterns and focus them on specific sites where mechanochemical conversion may take place.
Some of the major cellular sites for solid-state signaling are “focal adhesions” where integrin receptors mediate the transfer of mechanical force between the cytoskeleton and the ECM. When mechanical forces are applied directly to integrin receptors (e.g., using magnetic forces), cellular biochemistry and gene expression are altered in a stress-dependent way. Forces applied to integrins activate many signaling pathways in these sites, including protein tyrosine phosphorylation, ion fluxes, cAMP production, and G protein signaling. In contrast, if the same stress is applied to a peripheral membrane receptor, there is no effect. Thus, cells use specific transmembrane receptors that link to the deep cytoskeleton—in this case, integrins—to mediate mechanochemical transduction.
However, the tensegrity model suggests that a local stress may also produce global structural responses. In fact, when tension is applied to surface integrins (e.g., with a micropipette), this results in stress-dependent displacements of mitochondria, focal adhesions, and even molecular realignment of nucleoli inside the nucleus. Moreover, as predicted by tensegrity, this type of force transduction is mediated by cytoskeletal filaments and modulated by the level of cytoskeletal prestress. Thus, a mechanical force applied on one point at the surface may alter cell behavior by influencing biochemical activities at multiple sites.
These actions at a distance are important physiologically. For example, although cells may sense mechanical forces locally within focal adhesions, the whole cell must process this information before orchestrating a concerted functional response. This was demonstrated by controlling cell distortion independently of other factors (e.g., soluble hormones), by plating cells on different sized adhesive islands created with a microcontact printing technique originally developed by George Whitesides’ laboratory (Harvard Univer-sity) as an inexpensive way to fabricate microchips for the computer industry. The islands, made adhesive for cells by coating them with ECM molecules, were surrounded by nonadhesive regions covered with polyethylene glycol. The cells spread to take the shape of the island to which they adhered as a result of pulling themselves flat against the ECM substrate. Cells appeared round on circular islands and literally displayed 90-degree corners when cultured on square islands. Thus, if we hold the shape of the island constant and vary its size, we can control the degree of cell distortion.
The cytoskeleton orients much of the biomechnical
machinery of the cell.
Spread and round cells cultured on different sized islands produce similar intracellular signals (e.g., cAMP production) when their integrin receptors are magnetically stressed. A flattened cell takes in this signal, integrates it with other cues conveyed by its overall structural state, and switches on a growth (proliferation) program, whereas a round cell shuts off growth and activates a suicide program, known as “apoptosis.” Furthermore, when spreading is only partially restricted on an intermediate-sized island, the cell neither grows nor dies; instead, the cell differentiates and expresses tissue-specific features (e.g., capillary endothelial cells form hollow capillary tubes, liver cells increase production of specialized blood proteins).
Cell distortion also impacts cell movement. When cells on square islands are stimulated with motility factors, the cells preferentially extend new motile processes from their corners, whereas they extend from all points along the edge of round cells. These methods have led to new approaches to tissue engineering using microfabricated substrates, in which it is possible to direct cell migration, growth, and differentiation in specific locations by modifying the surface chemistry and topography of artificial materials, instead of adding soluble stimulants.
Regional variations in cell distortion may similarly drive tissue patterning in the embryo. For a capillary network to form, for example, only a subset of cells must respond to soluble growth factors by proliferating locally and sprouting outward relative to neighboring non-growing cells. This process is repeated along the sides of the newly formed sprouts, and then is repeated over time; this is how the fractal-like patterns of all tissues develop. This process is mediated by regional changes in ECM structure; the ECM thins in regions where new sprouts will form due to local enzymatic degradation. Because tissues are prestressed, a local region of the tensed ECM may thin out more than the rest, like a “run” in a nylon stocking. Cells anchored to this region will also stretch, whereas neighboring cells on intact ECM remain unchanged. If cell stretching promotes growth, then this would generate local cell growth differentials. In short, these studies suggest that tissue morphogenesis may be controlled mechanically, and recent experimental studies in embryonic systems support this possibility.
Biologists commonly speak of a “growth pathway” versus “differentiation pathway” and assume that cell-fate switching is controlled through activation of a specific series of regulatory events that “instruct” the cell to express one distinct phenotype or another. Work on controlling cell shape suggests that this model does not take into account the larger frame of reference that is critical for understanding cell structure and function—the framework of the whole cell. Sui Huang in my group has noted that when a single control parameter—cell shape—is varied continuously, abrupt all-or-none changes in cell fate are produced reminiscent of a “phase transition” in physical systems (e.g., water going from solid to liquid to gas when temperature is varied). Macroscopic (system-level) features of simple inorganic materials are emergent properties of the network of interactions among multiple components (e.g., a single water molecule has no boiling point). Given that different stable cell fates similarly emerge out of a network of gene and protein regulatory interactions, we began to ask if this could work in a similar manner.
Systems biologists interested in nonlinear dynamics have begun to address the question of stable cell states by modeling isolated regulatory circuits consisting of a few mutually regulating genes. These low-dimensional models explain bistable, switch-like decisions (bifurcations) between stable network states that arise because of nonlinear relationships between the circuit components. However, they do not explain the coordinated changes of thousands of genes across the entire genome, which occur during a phenotypic switch in mammalian cells.
Genomic and proteomic studies also suggest that molecular pathways in the cell form a single large connected network (“giant component”) that spans almost the entire genome. Yet, cells are able to reliably integrate multiple, simultaneous, often conflicting signals that perturb genes across the entire genome and respond by selecting one of just a few possible stable cell fates. Moreover, the very same cell-fate transition can be triggered by a broad variety of unrelated signals (e.g., different hormones and adhesive molecules), including those that apparently lack molecular specificity, such as distortion of cell shape.
Theoretical models of generic networks have revealed that stable states known as high dimensional “attractors” self-organize in large interconnected networks containing thousands of elements, if they exhibit a particular class of network architecture. Virtually all biomolecular networks analyzed to date have this architecture. Stable, high-dimensional attractor states arise at the whole system level as a consequence of the particular regulatory interactions between the network components (e.g., genes) that impose constraints on the global dynamics of the network; thus, the cell cannot occupy any arbitrary network state. Stuart Kauffman (Sante Fe Institute) has proposed on these theoretical grounds that different cell types (e.g., lung vs. liver) represent different attractor states in the gene regulatory network.
Based on these observations, we proposed that the different stable cell phenotypes (e.g., growth, differentiation, motility, apoptosis, etc.) similarly represent high-dimensional attractor states, or “default” states, in the regulatory network of mammalian cells. To pursue this idea, we developed new bioinformatics software that could simultaneously visualize and compare multiple time series composed of high-dimensional, genome-wide gene profiles. Using this tool and novel nonlinear dynamics approaches to analyze the process of cell-fate switching in human blood cell precursor cells induced to differentiate into neutrophils by two different stimuli (all trans-retinoic acid and DMSO), we have obtained experimental evidence that directly supports the attractor hypothesis.
The existence of attractors in the genome-wide regulatory network that confer stability with respect to thousands of dimensions (e.g., gene expression levels) is important because it explains how cells can simultaneously sense multiple chemical, adhesive, and mechanical inputs and yet only switch on one of a limited number of specific, reproducible behavioral responses. A key feature of the attractor model is that multiple regulatory elements (e.g., ensembles of genes and signaling proteins) must change at the same time to produce an attractor switch. Given that mechanical forces and cell shape distortion probably impact many cytoskeletal-associated signaling molecules simultaneously, this may explain how global changes in shape are able to control cell-fate switching.
The cytoskeleton is a mechano-chemical
scaffold that is both structure and catalyst.
The riddle of how cells form specialized tissues and organs is more a problem in structural design, systems engineering, and architecture, than a question of chemistry. Because the hierarchical molecular structures that comprise living cells, tissues, and organs are stabilized based on tensegrity principles, cells are perfectly poised to sense physical signals, to respond mechanically, and to orchestrate a spatially coordinated biochemical response at the molecular level. For this reason, structure dictates function in living cells—cells can be switched between growth, differentiation, and death solely by varying the degree to which the cell physically distorts its shape. Thus, although a cell may be able to sense mechanical signals locally through adhesion receptors, such as integrins, the overall response of the cell is governed at the whole cell level where the mechanical status of the entire cytoskeleton is also taken into account.
The cytoskeleton can integrate these diverse signals because it is a mechanochemical scaffold that is both structure and catalyst. This structural design principle conveys mechanosensitivity to the cell because stress-dependent changes in the shape of molecules and enzymes that bear loads in these cytoskeletal structures alter their thermodynamic and kinetic parameters, thereby converting mechanical signals into a biochemical response. Cells also change their shape and move by changing their level of internal prestress, shifting forces back and forth between internal struts and external tethers, and by using these localized forces to drive biochemical remodeling events. By integrating structural networks with biochemical assemblies and information processing networks, the cell can function simultaneously as sensor, processor, and actuator, while at the same time moving, growing, and producing the energy required for these processes. The future challenge in “living materials science” is, therefore, to define the principles that govern how molecules self-assemble to form the multifunctional structural hierarchies we call living cells and tissues. If we could incorporate these principles into artificial nanomaterials, biomedical devices, and engineered tissues, we could revolutionize the way medicine is practiced in the future.
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