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All the (Microscopic) World’s a Stage

March 2007--In this fast-paced world, even the busiest airports and city streets cannot compare with the goings-on within our bodies: cross-talk between nerves, immune cells constantly on patrol, intestinal cells prodded by the feedback of your last meal—they’re just a fraction of the nonstop activities of cells engaged in life. At Hopkins, the Center for Cell Dynamics (CCD) aims to visualize this microscopic action as it’s unfolding so we can better understand why and how it’s unfolding.

“My colleague Scott Fraser at CalTech describes it best,” says CCD director Denise Montell. “He likes to say that studying development or any dynamic process based on images of fixed tissue is like trying to deduce the rules of football by looking at snapshots from different games. Even though all games follow the same set of rules, every game unfolds differently. The snapshots have no relationship to each other, whereas a video shows what actually happens over time in each game.”

Watching cells in real time is not a recent advancement, of course. But watching a blurry amoeba chase down a bacterium can only reveal so much. So cell dynamics researchers need to continually improve the “big four” imaging issues. As Scot Kuo, director of the IBBS Microscope Facility, says, “It’s getting faster timescales, higher visual resolution, better molecular probes—and all without damaging living cells.”

For his part, Kuo has been exploring the power of lasers. He had previously developed optical tweezers, a device that uses laser light energy to move particles around, to help with his work on the cell’s motor proteins.

“Light photons are like ping-pong balls,” he explains. “Tossing a few won’t do much, but a whole stream of them can move something.”

To expand from single molecules to whole cells, though, he needed something more powerful. 

One result of his labors has been laser-tracking microrheology (LTM), a technique that can track molecule-size gyrations.

 “At this size scale, nothing is ever stationary,” he says. “All particles constantly do the wiggly dance of Brownian motion.”

While Brownian motion stems from the inherent thermal energy in every molecule, quantifying the range of a particle’s wiggles reveals the mechanics of its surrounding environment—constrained particles gyrate less.

By monitoring microscopic “wigglers” at different locations within a cell, Kuo use LTM to get a feel for the strength and mechanical adaptability while cells perform active motions. Kuo believes this mechanical information will unravel many mysteries of movement, such as how cells divide into daughter cells, how bacteria invade host cells, and how nerves reach out for new connections.

Another CCD member, Pharmacology’s Jin Zhang, has designed her own set of imaging toys: protein-based biosensors that highlight the spatial and temporal activity of molecules in full color. With biosensors, Zhang can follow the where, when and how long of her favorite molecules in the veritable cocktail party that is the inside of a cell.

Her original design consists of a peptide substrate for the enzyme PKA, flanked by two fluorescent proteins.

 “The sensor changes its color depending on the distance between the two fluorescent proteins,” she says. “When they’re far apart, it glows cyan; if they’re close, it switches to yellow.”

Normally, the glowing proteins remain distant; however, when an active PKA grabs and distorts the substrate, the fluorescent proteins come together.

 Since coming to Hopkins, Zhang and her lab have expanded their repertoire of biosensors to include those that report activities of lipids and small messenger chemicals like cyclic AMP.

“We chose targets like PKA and cyclic AMP because they play so many different roles in cell function,” she says, “and we want to see how a cell coordinates their various duties.”

In the future, Zhang hopes to incorporate multiple biosensors into cells to understand how these multi-purpose molecules work around each other, as well as adapt the sensors’ output to produce more quantitative information. She has also teamed with fellow pharmacologist Jun Liu to use biosensors in high-throughput drug screening.

“We don’t want to imply that static data is not useful,” Montell notes. “In fact, I’m amazed at how much we have learned from fixed tissue studies. But with real-time movies—watching cells grow, divide, move and behave—can we gain a whole new level of understanding. After all, if a picture says a thousand words, then a movie tells us a million.”

--Nick Zagorski

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