20091216-3

Illustrations by Andrew Meehan

 

Mechanical signaling may be just as important as chemical communication in the life of a cell.

 

The-Scientist.com, December 15, 2009, by Jef Akst  —  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.”

The wave within: Mechanical forces acting inside the cell

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.

Mechanical signaling may be just as important as chemical communication in the life of a cell.

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

“Biomechanics is becoming increasingly accepted, and people are recognizing its role in development, in disease, and in general cellular and tissue function.”
-Mohammad Mofrad

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.

An early start: Mechanical forces in development

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.)

“To try to find out what’s the mechanosensor is kind of crazy at this point. As scientists are now learning, the whole cell is the mechanosensor.”
-Donald Ingber

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.”

20091216-4

HOW MECHANICAL SIGNALS AMPLIFY IN BONE CELLS
As interstitial fluid flows through the networks of cavities and canals known as the lacuno-canalicular network, it pulls on “tethering” filaments that link osteocytes, or bone cells, and the walls of the canaliculi. These drag forces on filaments can then amplify and transmit mechanical forces to the osteocytes. Projections of the canaliculi wall, attached to the osteocyte at an integrin protein, may also participate in amplifying and transmitting the signal.

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

20091216-5

PHYSICAL FORCES IN ENDOTHELIAL CELLS
Blood flowing through the vascular system inflicts shear stress on the endothelial cells of the blood vessels. The force is transmitted through cytoskeletal elements to the cell-to-cell adhesions, where a transmembrane protein known as PECAM1 responds by activating downstream signaling pathways, including those involving a Src family kinase and integrins on the basal membrane of the cell. A dense layer of macromolecules that lines the surface, known as the glycocalyx, may participate in transmitting the force across the cell membrane.

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.

The-Scientist.com, December 15, 2009, by Jef Akst  —  The renal tubules of the kidney function to reabsorb water, ions, and organic molecules from the filtrate destined to become urine. As it passes through the sections of the tubule, the majority of the fluid and electrolytes are transported back into the plasma, leaving the waste products behind, which pass on to the collecting duct system, the urethra, and out of the body. Importantly, the quantity of fluid flowing through the tubule can vary greatly-up to ten-fold-yet the total amount of reabsorption is remarkably stable. How the tubule senses such dramatic changes in flow has been a “mystery for about 4 decades now,” says bioengineer Sheldon Weinbaum of the City College of New York.

Each endothelial cell in the first section of the renal tubules is lined with thousands of densely packed protrusions known as microvilli. For years, scientists believed that the function of these protrusions was simply to increase the surface area for reabsorption, Weinbaum explains. But with the recognition of a broader role for mechanical forces, researchers are looking to the microvilli as possible sensors of filtrate flow.

Indeed, a 2003 study found that changes in torque applied to the microvilli by fluid flow corresponded almost perfectly with changes in electrolyte reabsorption, which drives water reabsorption in the tubule (PNAS 101:13068-73, 2003). Furthermore, the microvilli are “almost uniform in height,” Weinbaum says, which is critical for their collective behavior in sensing fluid flow. “If their whole function is just for transport, then it wouldn’t matter that it looked like someone went with a lawnmower and cut them all the same height.”

Alternatively, scientists have suspected that primary cilia of the tubule also sense fluid flow. These cilia, which extend like microvilli, are much less frequent-with only one per cell at most-and are a bit longer, protruding slightly above “the forest of the microvilli,” Weinbaum says. The cilia, which are essentially extensions of the internal cytoskeleton, sense fluid flow in the cortical collecting duct (CCD)-the last section of the renal tubule, where microvilli are absent. As the filtrate flows through the CCD, the cilia bend, initiating conformational changes of proteins located at the base of the cilia that together function as mechanosensitive calcium channels.

Sound comes in the form of waves of compressed air, and detecting that sound is wholly dependent on the ear’s ability to convert variations in air pressure to chemical signals that can be interpreted by the brain. When sound enters the ear, it deflects the eardrum that lies at the junction of the outer and middle ears. This deflection is then transmitted through the middle ear to a small membrane at the opening of the inner ear, resulting in the formation of waves in the fluid of the inner ear. Those waves flow through the cochlea in a rhythmic fashion and induce parallel deflections in the sensory hair cells that are physically coupled to the cochlea, where the mechanical force translates into a chemical signal.

Stereocilia-bundles of rigid actin filaments-protrude from the ends of the sensory hair cells. They are embedded in a fixed membrane and attached to one another by tiny, molecular filaments known as tip-links, which connect the actin filaments of the adjacent stereocilia. When the hair cells are shifted as a result of sound vibrations, the stereocilia bend, stretching the tip-links and opening stretch-sensitive ion channels (the identity of which remains elusive) that depolarize the membrane. This in turn activates auditory nerve fibers, which convey the sound signals to the brain.

“The cytoskeleton is key in hair cells-that’s just sort of a given,” says sensory biologist Teresa Nicolson of the Oregon Hearing Research Center at Oregon Health and Science University. “The reason why hair cells are so sensitive is because their stereocilia are so stiff, and that’s because there’s this crystalline packing of actin filaments.” The tension in the cytoskeleton and length of the stereocilia influence both the sensitivities and frequencies to which the hair cells are tuned (PNAS 97:3183-88, 2000).

More recently, scientists began to suspect that nonmuscle myosins in hair cells also participate in this process as mutations to these proteins are associated with hearing loss in humans and other animals. Myosins may serve a structural role, linking the actin of the stereocilia to the plasma membrane or tacking down the plasma membrane to the actin meshwork in which the stereocilia are embedded. Maintaining the tension in these structures is essential to the propagation of sound signals. Additionally, myosins may participate in adaptation to continuous noises, which involves the closing of transduction channels after periods of sustained stimuli (Phil Trans R Soc Lond B, 359:1895-905, 2004).

20091216-2

The synthetic red blood cells that Mitragotri and his team developed
Image: Nishit Doshi 

 

The-Scientist.com, December 15, 2009, by Bob Grant  —  Newly created synthetic particles that mimic red blood cells may one day carry drug molecules and/or oxygen through bloodstreams, according to researchers writing in this week’s issue of the Proceedings of the National Academy of Sciences (PNAS).

What’s more, the team of scientists in Michigan and California say the particles could also be used to improve the resolution of magnetic resonance imaging.

 

“It’s a very nice paper and very exciting work,” Krishnendo Roy, a biomedical engineer at the University of Texas at Austin who wasn’t involved with the study, told The Scientist. “The beauty of their method is its simplicity.”

University of California, Santa Barbara, chemical engineer Samir Mitragotri led the team of scientists and told The Scientist that the blood cell-like particles could evolve into useful tools in the clinic. “What we got very excited about was making a structure with synthetic materials that begins to mimic a natural object,” said Mitragotri. “If we can bridge the gap [between synthetic materials and living cells] it will open up tremendous opportunities for synthetic materials.”

Mitragotri said that he and his team tested the ability of the particles to carry oxygen, finding that they had a “comparable” oxygen-carrying capacity to actual red blood cells. He added that it may be possible in the future to link therapeutic agents destined for the vascular system, such as heparin, to the particles so that they can be easily distributed throughout the blood. The artificial blood cells, with attached iron oxide nanoparticles, could also one day improve MRI resolution by serving as contrast agents that provide a different imaging signal compared to the surrounding tissue, Mitragotri said.

Mitragotri and his colleagues created the artificial red blood cells by first making tiny spheres out of a biodegradable polymer called poly(lactic acid-co-glycolide) (PLGA). They then exposed the spheres to isopropanol, which collapsed them into the discoid shape characteristic of red blood cells. The researchers then layered proteins — either albumin or hemoglobin — onto the doughnut-shaped disks, cross-linked the proteins to give them extra strength and stability, and finally dissolved away the PLGA template to leave only a strong but flexible shell of proteins in the shape and size (about 7 microns in diameter) of a red blood cell.

Mitragotri and his team then tested the ability of the artificial cells to behave like real blood cells, passing them through glass capillary tubes that were narrower than the diameter of the particles and testing their oxygen-carrying capacity. They showed that the particles could carry about 90 percent of the oxygen real red blood cells can carry. They also showed that a drug-mimicking molecule could easily be loaded into and off of the artificial blood cells.

“They conclusively demonstrated some stuff concerning oxygen-carrying capacity and the potential for drug release,” Patrick Doyle, a chemical engineer at the Massachusetts Institute of Technology who was not involved with the study, told The Scientist.

But years of continued testing lie between Mitragotri’s synthetic red blood cells and clinical application. Several questions, including how long the particles will remain in circulation, how the immune system will react to the synthetic blood cells, and how efficiently they transport oxygen, remain to be answered. Mitragotri said that his lab plans on answering these questions by studying the particles in model organisms, research that is set to begin soon.

“Whether this is applicable in an in vivo setting,” said Roy, “we won’t know that for 3, 4, or 5 years.”

“I don’t think these [clinical applications] are far off ideas, but you have to go through all the usual regulatory hurdles,” said Doyle, noting that the synthetic cells might also be used to study how cellular aberrations, such as tumor cells, behave in the body. “Ultimately they can also be model systems, by which you can understand disease states of cells,” he added.

20091216-1

Image by Anselm Levskaya

There’s an ever-growing armament of tools for tagging proteins to watch cellular events unfold, but until recently, researchers lacked ways to experimentally manipulate those events with the same molecular-level precision. A handful of genetically encoded light-sensitive systems have now been reported that do just that, but most require a heavy dose of protein engineering (see this issue’s Lab Tools).

Wendell Lim and his colleagues at the University of California, San Francisco, may have found a solution. Normally, the light-sensitive plant protein phytochrome and its binding partner, phytochrome interaction factor (PIF), link up and translocate to the nucleus in response to red light; infrared light breaks the bond. The researchers modified the genes so that the pair, when activated, instead moved to the cell membrane. They then linked PIF to a cytoskeletal protein. Spatially targeted pulses of red light flipped on PIF, which in turn activated the cytoskeletal protein, reshaping the cell (Nature, 461:997-1001, 2009).

Phytochrome “converts light into a protein-protein interaction,” says Lim. Researchers can link PIF to any number of proteins, potentially making the system applicable to a broader array of cell processes than other light-controlled systems, he adds.

The group submitted the mutant phytochrome and PIF plasmids to Addgene, a nonprofit plasmid repository that facilitates distribution of plasmids among the scientific community. Researchers can request the plasmids for about $65 each.

WILEY: Because the system is genetically encoded, modular (works with any pair of proteins), reversible, and uses nontoxic wavelengths of light, it is likely to have an extremely high impact.

LEVY: This data may offer an unprecedented ability to control protein interaction and localization in the cell.