How Cells Move
by David Pescovitz
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Dan Fletcher with a custom Atomic Force Microscope designed and built by his research group to analyze the mechanisms of cell motility. Understanding how a cell moves could someday make it possible to engineer novel devices that take inspiration from cellular mechanisms, such as advanced drug delivery systems. (David Pescovitz photo)
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As cells move through the body, navigating through tissue and pushing against obstacles, they change shape. Understanding the dynamics of this process could someday lead to therapies that improve immune cells or fight cancer. To gain insight into cell motility, UC Berkeley bioengineering professor Daniel Fletcher and his students customized an atomic force microscope (AFM), a tool commonly used in nanoengineering. Their tricked-out instrument is helping reveal how the scaffolding that gives a cell its shape is affected by its environment.
"The question we asked is, 'what happens physically when a cell pushes out and hits something?'" says Fletcher, who is affiliated with the UC San Francisco/Berkeley bioengineering graduate group and the UC Berkeley biophysics graduate group. "We wanted to figure out a way of quantifying that directly."
The researchers focused on the cytoskeleton, the network of filaments made from proteins like actin. This cytoskeletal actin network provides the cell's structure and mechanical integrity, even as its shape changes.
"When a cell crawls, its front extends forward," Fletcher says. "Actin helps power that forward motion."
A window into the cell reveals the actin network and organelles inside a macrophage as it pursues bacterial invaders. Actin forms long filaments that lie just beneath the surface of the cell, giving it structure and stability. As the immune cell crawls and looks for invaders, such as the bacterium E. coli, new actin growth helps to push the cell forward. (Nicolle Rager Fuller, National Science Foundation) [view larger image]
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The aim was to watch such an actin network grow in vitro under a microscope and study how it responds to opposing forces. But off-the-shelf microscopes wouldn't do. Instead, Fletcher and his graduate students built a custom atomic force microscope. In a standard AFM, a micron-sized cantilever physically scans a sample much like a needle travels across a record. As the probe moves over the surface of a cell, a cantilever at the end of the tip bends in response to the sample's topography and mechanical properties. That deflection is captured by a laser and translated into a measurement with nanometer (one-billionth of a meter) resolution.
"The problem is that unwanted agitation or drift over time can compromise the integrity of measurements taken," says graduate student Sapun Parekh. "So we added a second cantilever to compensate."
In their set-up, the cantilever doesn't scan the sample but rather bends like a diving board as the actin network beneath it grows and pushes against it. Meanwhile, the other cantilever acts as a reference point on the surface of the sample chamber. Using this technique, the surface can be kept at a constant position relative to the first cantilever, enabling the microscope to accurately measure the growth of the actin network.
"There haven't been many tools available to rigorously study the dynamics of these systems," says Ovijit Chaudhuri, another bioengineering graduate student involved in the research. "It's exciting to find out the interesting and complex aspects of this system now that we have the ability to probe it."
Actin in this fish cell is stained with a red fluorescent dye. The bright areas on the top edge depict growing actin networks. (courtesy the researchers)
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To conduct their experiments, the researchers used the cantilevered tip to provide an increasingly greater force to a growing network of actin for up to a half-hour. The force acts like a barrier that the cell might encounter in vivo. As the force became stronger, the speed of the actin's growth stayed constant. After a preiod of increasing force, they then reduce the force to its initial strength. Oddly though, when the pressure of the cantilever was reduced, the network grew faster than before against the same load.
"We found that the growth of the actin is dependent on its loading history, not just the load at the moment," Fletcher says. "That means the cell has some sort of 'memory' of how it has previously interacted with its environment."
The protein network seems to remodel its architecture based on the pressure it's experienced, he explains. The experiments suggest that the growing actin network adds more filaments to push back against resistance.
"Now, we'd like to determine whether the resistance causes the network to become denser," Chaudhuri says. "And if that's the case, we hope to elucidate the factors that govern those changes in the number of filaments which will help us understand how the protein network generates force."
According to Fletcher, this basic research could someday help the fight against certain diseases. For example, immune cells and cancer cells both depend on their ability to move through the body.
"If we can gain a fundamental understanding of how cells use the actin network, we have a better chance of improving an immune cell's ability to move or, in the case of cancer, removing that ability," Fletcher says.
Fletcher Lab
"Modified Microscope Proves Critical to Uncovering Cell-growth Secret" (National Science Foundation, December 27, 2005)
"A Cell's Secret Machinations" by David Pescovitz (Lab Notes, April 2004)
Biophysics Graduate Group
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