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PATHOLOGY
Cells Prove to Have Strong Response to Their Physical EnvironmentForce of Extracellular Matrix Pushes Cells Toward Death or DivisionCells live in a rough and tumble world. Not only are they subject to the onslaughts of hormones and growth factors, cells are tugged, squeezed, and splayed by the dense matrix of fibers and proteins that surround them. Twenty years ago, Donald Ingber had the heretical idea that mechanical forces exerted by the extracellular matrix had as much to do with telling a cell whether to grow and divide as the commands issued by growth factors. But for years it remained an idea in search of experimental evidence. Ingber's two-decade-long quest to demonstrate his theory is finally bearing fruit. In two papers this month, Ingber and colleagues report that they got endothelial cells to divide and differentiate into blood vessels by manipulating the degree to which they were distorted by the underlying extracellular matrix. "We have discovered that mechanical distortion due to changes in the extracellular matrix can switch endothelial cells between entirely different gene programs. So we can switch between apoptosis, differentiation, and growth," says Ingber, HMS professor of pathology at Children's Hospital. Distinguishing ForcesOne stumbling block in Ingber's quest has been the problem of separating the mechanical forces exerted on the cell by the extracellular matrix from the biochemical influences. Proteins in the extracellular matrix transmit chemical signals to cells via receptors, called integrins. Using a new chip technology, Ingber and his colleagues have recently grown single cells on microscopic islands of extracellular matrix protein. By varying the size of the islands but not the density of extracellular matrix protein, they were able to control the extent to which cells changed shape while keeping the binding--and hence, the biochemical influence--constant.When exposed to growth factors, only those cells grown on larger squares, and therefore able to spread, underwent division. Cells grown on the tiniest of islands died, Ingber and his colleague Sui Huang, an HMS research fellow in surgery at Children's, report in the cover story in this month's Nature Cell Biology. "We found that with all your growth factors and integrin clustering and on switches going, if you affect the degree to which the cell spreads on the extracellular matrix, you can also turn on and off the cell cycle machinery," Ingber says. By tinkering further with the physical aspects of the extracellular matrix, the researchers found they could actually get a cluster of endothelial cells to differentiate into blood vessels. Cells grown on strands of extracellular matrix 10 microns wide rounded up and formed capillaries, but when grown on bands 30 microns wide, the cells remained flat, Ingber and his colleagues report in the September In Vitro Cell Developmental Biology. Ingber believes that his once radical focus on mechanical influences on developing cells could help solve age-old questions in biology. "As we get closer to the genome, we're getting so overwhelmed with data that we need to ask, what were the fundamental questions that triggered this field. In growth, it wasn't just which signal molecule gets turned on, but how do tissues grow so that they exhibit specific form and function--and how does that get deregulated in a process like cancer?" For example, Ingber explains, one of the hallmarks of a cancer cell is a breakdown in communication between the cell and the extracellular matrix. Normally, cells are equipped with programs that allow them to sense their topological context and to divide when they sense space is available--and to stop growing when space is limited. "Cancer is basically loss of crowd control. It's not rapid growth--a tumor of the intestine can grow more slowly than normal intestine. It's growth in the wrong places at the wrong times. And it's coupling that with a loss of structural boundaries and architecture," Ingber says. Against the WindWhen Ingber began exploring the relationship between the extracellular matrix and the cell as a doctoral student two decades ago, the dogma was that extracellular matrix acted as a passive barrier to protect tissues from invasion by cancer. "But in my reading, I was finding all this literature showing extracellular matrix--and basement membrane in particular--was thinning before you get cancer," he says. Thinking that the thinning of the basement membrane might actually be promoting the development of tumor cells, Ingber explored in his thesis an even more controversial proposition: perhaps cells, by virtue of their tensile structure (see sidebar), are responsive to physical changes in the extracellular matrix not just in cancer but in normal growth.Few people accepted it. Not only was it unorthodox--most people believed growth factors controlled growth--he had no experimental data. Working in the lab of Judah Folkman, who years earlier had observed that normal cells undergo proliferation when they spread but not when they crowd, Ingber set out to show that by manipulating the physical forces operating on integrins arrayed on the cell surface, he could change the structure and function of cells. The task turned out to be technically difficult but, in 1993, Ingber and his colleagues were finally able to show that by twisting the integrins, they could cause the cytoskeleton of the cell to get stiffer and stiffer. "That was a real breakthrough," says Ingber. By pulling even harder on the integrins, they got not only the internal cytoskeletal filaments but nucleoli in the nucleus to change their alignment, suggesting the physical force had been transmitted to the core of the cell. Even more stunning, when they tried extracting a single nucleolus using a microneedle, "we saw one nucleolus come out, then another and another, as though on a string," says Ingber. They then performed a similar harpooning experiment on a single chromosome inside a mitotic human cell and reeled in all 46 chromosomes. "We got the entire human genome on a string," Ingber says. "So the cell is literally hardwired to respond to a mechanical distortion immediately all the way from integrins to cytoskeleton to chromosomes and genes." Forceful SignalsYet until recently it was unclear whether this built-in ability to respond to mechanical distortion actually affects the cell biochemically. In an experiment reported last year, Ingber and his colleagues found that by twisting integrins, they could recruit mRNA and ribosomes to the sites. "So we can actually induce the formation of a microcompartment for protein synthesis at the site of integrins, and it is stress dependent," he says. They are currently tracing the pathways by which twists on integrins can turn on genes in the nucleus.They are also pinpointing the pathway by which cell distortion--in particular spreading or crowding--may be regulating the cell cycle machinery. In their Nature Cell Biology paper, Ingber and Huang propose that mechanical signals such as tension are conducted via the cytoskeleton to the proteins p27 and cyclin D1, which act as gatekeepers for the cell cycle. In fact, Huang has found that giving cells a drug that decreases tension in the cytoskeleton actually halts the cell cycle. Knowing how cells respond to mechanical forces exerted by their environment--and specifically, how such forces turn the cell cycle machinery on and off--has implications not only for our understanding of disease but also for new treatments, says Ingber. One possibility he is currently exploring is engineering new tissues. "How do you design and fabricate artificial environments that can get cells to self-assemble into functional structures, like capillary networks, where and how you want them?" he asks. In fact, Ingber envisions the future of medicine to be built on a mimicking of natural principles, or biomimetics. "Nature is incredible in that it has materials optimally structured for their function, and they're often created using self-assembly in an aqueous environment," he says. "If we have a handle on fundamental biological design principles, then we could really revolutionize medicine." --Misia Landau |
