When it comes to survival, few things are more important than being able to respond quickly to a change of circumstances. And when it comes to fast-acting indicators, it turns out that signals induced by physical forces acting in and around cells, appropriately dubbed biomechanical signals, are the champions of the cellular world.
“If you look at this mechanical signaling, it’s about 30 meters per second—that’s very fast,” says bioengineer Ning Wang of the University of Illinois at Urbana-Champaign. That’s faster than most family-owned speedboats, and second only to electrical (e.g., nerve) impulses in biological signaling. By comparison, small chemicals moving by diffusion average a mere 2 micrometers per second—a speed even the slowest row boater could easily top.
Indeed, when the two signal types were pitted against each other in a cellular race last year, the mechanical signals left chemical signals in their wake, activating proteins at distant sites in the cytoplasm in just a fraction of a second, at least 40 times faster than their growth factor opponent.1 Mechanical signals are so fast, Wang adds, they are “beyond our resolution,” meaning that current imaging techniques cannot capture the very first cellular changes that result from mechanical stress, which occur within nanoseconds.
For centuries, scientists have scrutinized the molecular inner workings of the body, with little or no regard to the physical environment in which these biological reactions take place. But the growing realization that physical forces have a pervasive presence in physiology (operating in a variety of bodily systems in the bone, blood, kidney, and ear, for instance), and act with astonishing speed, has caused many to consider the possibility that mechanical signaling may be just as important as chemical communication in the life of a cell.
“Biologists have traditionally ignored the role of mechanics in biology,” says biomechanical engineer Mohammad Mofrad of the University of California, Berkley, “[but] biomechanics is becoming increasingly accepted, and people are recognizing its role in development, in disease, and in general cellular and tissue function.”
Once believed to be little more than sacks of chemically active goop, cells didn’t seem capable of transmitting physical forces into their depths, and researchers largely limited their search for molecules or structures that respond to physical forces, or mechanosensors, to the plasma membrane.
In the late 1990s, however, closer examination revealed that the cell’s interior is in fact a highly structured environment, composed of a network of filaments.2 Pull on one side of the cell, and these filaments will transmit the force all the way to other side, tugging on and bumping into a variety of cellular structures along the way—similar to how a boat’s wake sends a series of small waves lapping up on a distant and otherwise peaceful shoreline. Scientists are now realizing the potential of such intracellular jostling to induce molecular changes throughout the cell, and the search for mechanosensing molecules has escalated dramatically in scope, including, for example, several proteins of the nucleus.
It’s a search that will likely last a while, predicts cell biologist Donald Ingber, director of the Wyss Institute for Biologically Inspired Engineering at Harvard University. “To try to find out what’s the mechanosensor is kind of crazy at this point,” he says. As scientists are now learning, “the whole cell is the mechanosensor.”
A key player, most agree, is the cytoskeleton, which is comprised of a variety of microfilaments, including rigid actin filaments and active myosin motors—the two principle components of muscle. Activation of the so-called nonmuscle myosins causes the cytoskeleton to contract, much like an arm muscle does when it lifts a heavy object.
The first intimation that the cytoskeleton could go beyond its established inner-cell duties (molecule transport and cell movement and division) came in 1997, when Ingber did the logical (in hindsight, at least) experiment of pulling on the cells to see what happened inside.2 Using a tiny glass micropipette coated in ligands, Ingber and his team gently probed the surface proteins known as integrins, which secure the cell to the extracellular matrix. When they quickly pulled the micropipette away, they saw an immediate cellular makeover: cytoskeletal elements turned 90 degrees, the nucleus distorted, and the nucleolus—a small, dense structure within the nucleus that functions primarily in ribosome assembly—aligned itself with the direction of the applied force.
“That kind of blew people away,” Ingber recalls. “It revealed that cells have incredible levels of structure not only in the cytoplasm but in the nucleus as well.”
Wang (once a postdoc in Ingber’s lab at the Harvard School of Public Health) and other collaborators combined a similar technique with fluorescent imaging technology to visualize how these forces were channeled within the cell’s interior. Upping the resolution and further refining these techniques, Wang began mapping these intracellular forces as they made their way through the cell. In 2005, the maps confirmed the physical connection between the cell-surface integrins and the nucleus, and showed that these external forces follow a nonrandom path dictated by the tension of the cytoskeletal elements.3
The end point of these mechanical pathways is likely a mechanosensitive protein, which changes shape in response to the force, thereby exposing new binding areas or otherwise changing the protein’s function. In mitochondria, for example, mechanical forces may trigger the release of reactive oxygen species and activation of signaling molecules that contribute to inflammation and atherosclerosis.
Similarly, proteins on the nuclear membrane may pass mechanical signals into the nucleus by way of a specialized structure known as LINC (linker of nucleoskeleton and cytoskeleton), which physically links the actin cytoskeleton to proteins important in nuclear organization and gene function. To determine if mechanical forces directly affect gene expression, last year scientists began exploiting the increasingly popular fluorescence resonance energy transfer (FRET) technology,1 in which energy emitted by one fluorescent molecule can stimulate another, resulting in a visible energy transfer that can track enzymatic activities in live cells. By combining FRET technology with the techniques that apply physical forces to specific cell membrane proteins, scientists can visualize entire mechanochemical transduction pathways, Wang says.
“The big issue right now in the field of mechanotransduction is whether the genes in the nucleus can be directly activated by forces applied to the cell surface,” Wang explains. While the physical maps of the cytoskeleton tentatively sketch out a path that supports this possibility, confirmatory data is lacking. This combination of new technologies will be “tremendously” helpful in answering that question, he says, and “push the field” towards a more complete understanding of how mechanical forces can influence cellular life.
In the world of developmental biology, the cytoskeleton’s role in biomechanics really comes into its own. As the embryo develops, the cells themselves are the force generators, and by contracting at critical times, the cytoskeleton can initiate many key developmental steps, from invagination and gastrulation to proliferation and differentiation, and overall cellular organization.
The idea that physical forces play a role in development is not a new one. In the early 20th century, back when Albert Einstein was first developing the molecular basis of viscosity and scientists were realizing molecules are distinct particles, biologist and mathematician D’Arcy Thompson of the University of Dundee in Scotland suggested that mechanical strain is a key player in morphogenesis. Now, nearly a century later, biologists are finally beginning to agree.
Because Thompson “couldn’t measure [the forces] at that time, that kind of thinking got pushed to the wayside as genetic thinking took over biology,” says bioengineer Christopher Chen of the University of Pennsylvania. That is, until 2003, when Emmanuel Farge of the Curie Institute in France squeezed Drosophila embryos to mimic the compression experienced during early development and activated twist—a critical gene in the formation of the digestive tract.4 These results gave weight to Thompson’s idea that stress in the embryo stimulates development and growth, and inspired developmental scientists to begin considering mechanical effects, Chen says. “Now we’re at the stage where there’s a lot of interest and willingness to consider the fact that mechanical forces are not only shaping the embryo, but are linked to the differentiation programs that are going on.”
Again, the cytoskeleton is a key player in this process. In fruit flies and frogs, for example, nonmuscle myosins contract the actin filaments to generate the compressive forces necessary for successful gastrulation—the first major shape-changing event of development. Myosins similarly influence proliferation in the development of the Drosophila egg chamber, with increased myosin activity resulting in increased cell division.
Cytoskeleton contractility also appears to direct stem cell differentiation. In 2006, Dennis Discher of the University of Pennsylvania demonstrated that the tension of the substrate on which cells are grown in culture is important for determining what type of tissue the cells will form.5 Cells grown on soft matrices that mimic brain tissue tended to grow into neural cells, while cells grown on stiffer matrices grew into muscle cell precursors, and hard matrices yielded bone. In this case, it seems that stiffer substrates increased the expression of nonmuscle myosin, generating greater tension in the actin cytoskeleton and affecting differentiation. (Altering or inhibiting myosin contraction can also affect differentiation.)
More recently, in October, Wang induced changes in mouse embryonic stem (mES) cells by simply probing the cell surface.6 Almost immediately after applying a small force to a surface integrin, each cell began spreading across the substrate—a key process in morphogenesis and germ layer formation. Tugging on the cells also down-regulated oct3/4 expression—a sign of cell differentiation—further supporting a role for external forces in embryogenesis.
Developing specific cell types for clinical uses hinges on a more complete understanding of how cell fate is shaped in vivo, and the recognition that the physical environment plays a role in this process has “had a big effect on extending the importance of mechanics,” Chen says. “There’s always a good mechanical aspect of these biological problems,” Mofrad adds. “[As] this is becoming increasingly evident, mechanics is taking a more prominent role.”
Given the ostensible inflexibility of bone, it may seem counterintuitive to imagine mechanical force playing a significant role in the skeletal system. But as every astronaut knows, bones are actually quite dynamic, and physical force (or lack thereof) can trigger changes that affect bone growth and strength. Astronauts, for example, experience significant bone degeneration after long stints in space, where their bodies are not exposed to the constant pull of gravity, and paraplegic patients lose between 25 and 30% of their bone mass within the first month of being paralyzed.
Despite the well-established response of bone to mechanical loading, however, the mechanism by which it senses such forces has been “an age-long mystery,” says bioengineer Sheldon Weinbaum of the City College of New York. Because bone is so stiff, normal physiological stress rarely induces more than a 0.1% strain, meaning that bone is compressed just 1/10 of 1% of its length. Yet in vitro experiments on bone required strains of 1–3% to produce a cellular response—a force that would likely cause bone damage.
The answer came in the mid-1990s in the form of fluid flow. The calcified matrix of bone consists of cavities known as lacunae that are connected via a network of canals known as canaliculi, which carries interstitial fluid through the skeletal system. Originally proposed as a system for delivering nutrients and removing waste products from bone cells called osteocytes, scientists now recognize fluid flow through this lacuno-canalicular network as providing bone tissue with important mechanical loading information.
In 2001, Weinbaum and his colleagues suggested that “tethering” filaments strung between bone cells and the walls of the lacuno-canalicular network may act as a sensor—and amplifier—of physical forces.10 Indeed, the drag forces inflicted on these tethers as the result of fluid flow can amplify a mechanical signal 10 to 100 times greater than a signal imposed directly on the bone matrix, but how this signal elicits a biochemical response is unclear. An alternative hypothesis arose in 2007, when Weinbaum and his colleagues identified integrin attachments on the canalicular wall. Their work suggested that these integrins—which transmit and receive mechanical forces via the cytoskeleton in other systems—may be the primary mechanotransducer in bone, resulting in intracellular signals two orders of magnitude greater than the strains of the bone itself.11
Bioengineer John Tarbell of the City College of New York points to a small device that holds a matrix of dancing ink spots, lengthening and warping with the tug of the machine. “The stretch-and-shear device,” he explains. “[In here], the cells get exposed to flow and to stretch.” The spots, placed on an artificial membrane within the device’s plastic walls, illustrate the effect of the machine’s mechanical forces, to which Tarbell will eventually subject cell cultures and record the effects. It’s like a drug-testing experiment, only instead of a drug, he and his team are exposing the cells to friction and stretching, two of the many mechanical forces cells lining blood vessels experience every day.
Recently, scientists have been gathering information showing how physical forces direct the development and restructuring of the cardiovascular system. Forces from blood flow can trigger blood vessels to dilate or contract. In particular, shear stress—the frictional force resulting from blood flow, which can range from just 1 pascal when an individual is resting to 10 pascals during heavy exercise—may initiate biochemical responses inside the cell that can affect such changes.
In 2005, researchers identified a transmembrane protein at cell-cell adhesions that connect endothelial cells to one another called PECAM1, which responds to stress by rapidly activating a Src family kinase.7 This kinase appears to initiate downstream signaling pathways, including those involving integrins on the basal membrane of the cell. This activation is likely triggered by a conformational change in PECAM1 or other proteins, but “the understanding of those physical mechanisms isn’t very good,” says cell biologist Martin Schwartz of the University of Virginia.
To reach this lateral site of mechanotransduction, the shear forces are transmitted through cytoskeletal elements that link the membrane exposed to the flow to the cell-to-cell adhesions. Recently, work by Tarbell and others has suggested that the forces are propagated across the membrane through a dense layer of macromolecules that lines the surface, known as the glycocalyx. Compromising the glycocalyx, however, does not completely abolish the cell’s response to physical force, suggesting that other membrane proteins play key roles, as well.
Most recently, scientists have recognized a role for shear stress in early development. Two studies published this past summer demonstrated that the initiation of the heartbeat and the first pulses of blood flowing through the young aorta spur the development of hematopoietic stem cell (HSC) production.8,9 These findings suggest that the physical forces exerted by blood play a lifelong role in the physiology of the vascular system.