Microscopy of Live Cells in Motion


Seest thou her locks, whose sunny glow
Half shows, half shades, her neck of snow?

———Sir Walter Scott, Ivanhoe


By Jason Socrates Bardi

Cells are in motion on the computer monitor in the office of Clare Waterman-Storer, who is assistant professor in the Department of Cell Biology and the Institute for Childhood and Neglected Diseases.

Big speckled cells—awash with activity. She is showing a video of the cells she recorded earlier and is busy pointing out some of their more subtle features: These are probably actin bundles moving around organelles, here is where the nucleus should be (off-screen above the monitor), this is how large the cell really is (about four times larger than the screen), and this is cell’s leading edge, she says, pointing to a speckling mass in the lower left hand of the screen.

Actin filaments polymerizing along the leading edge and moving backwards to the cell center. They look like a waterfall but act more like a treadmill, she tells me.

“I love seeing these movies,” she says.

Waterman-Storer came to The Scripps Research Institute (TSRI) last year to start up the Laboratory for Cell Motility, an area that has important implications for many fields.

She studies the molecules that are involved with cell motility—particularly microtubules, actin, and all the proteins responsible for regulating them. In particular, she is interested in the structural and regulatory interactions between actin and microtubules—how they touch and move each other and how they affect each other.

Her laboratory is specifically interested in how these molecules interact during the control of motility and how those interactions impact such diverse areas as cancer, wound healing, and early embryonic development.

In order to address these questions, Waterman-Storer uses a microscopy method with which she can image both the microtubules and actin directly. She can also quantitatively assess what happens to these structural proteins in living cells when she makes changes to the signaling and regulatory proteins that bind to them.

This method, called fluorescence speckle microscopy (FSM), was developed by Waterman-Storer and her former boss E.D. Salmon while she was doing a post doctoral fellowship is his laboratory at the University of North Carolina at Chapel Hill in the late 1990s.

"We discovered it by accident," she says.

Like Looking at a Brick Wall

Waterman-Storer and Salmon had been using another technique, called fluorescence analog cytochemistry, to study actin and microtubule motion in the cell.

In this technique, fluorophores are covalently attached to actin or microtubule subunits and then microinjected back in the cell. Fluorophores are simply small molecules that absorb and reemit photons of a particular wavelength.

One can then illuminate the cells with a monochromatic light source and train a microscope camera to capture the reemitted photons. For instance, one can attach green florescent protein to the tubulin subunits from which microtubules, which are shaped somewhat like brick chimneys, are constructed.

But this technique always had problems imaging the cytoskeleton in living cells because of the high concentration of fluorophores.

An overabundance of labeled subunits would be injected into a cell so that the microtubules assembled with a high concentration of these subunits. But the microtubules could never assemble all the fluorescing subunits. This would cause too much background florescence to see individual filaments in certain parts of the cell.

Another, related, problem was that fluorescence analog cytochemistry could not resolve microtubule motion because the microtubules would be too evenly labeled with fluorescing molecules.

"It’s like looking at a brick wall from a distance, where you cannot see the individual bricks and they are all red," she says. "That wall could be moving in front of you, but if you can’t resolve the individual bricks, all you would see is a red wall, and you wouldn’t know if it were moving or stationary."

FSM seeks to resolve the movement of the wall by painting only certain bricks white. Then the movement of the wall could be followed by tracking the position of the white bricks. In fact, FSM uses about 100 times less fluorescent material than fluorescence analog cytochemistry—only about one tenth of one percent of the subunits are labeled—but the few that are show how the whole wall moves.

"FSM," says Waterman-Storer, "Is basically fluorescence analog cytochemistry with less florescence.

This may sound simple, but people had been using the technique of fluorescence analog cytochemistry for over 20 years, and for years they had occasionally injected too little fluorescing material into the cells. Over and over, researchers wound up with spotty, or speckled microtubules, and started over.

"We realized that this was something that could give us information," says Waterman-Storer. This realization was enough to turn what were formerly regarded as anomalous mistakes into a new technology.

Using FSM, the growing actin or microtubule molecules appear speckled, and the movement of these speckles stands out to the eye, making it apparent. One can watch the growth of the protein bundles and their retrograde flow, which is thought to pull the cells along.

Next Page | Wound Healing, Nerve Cell Development, and Cancer Metastasis

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Assistant Professor Clare Waterman-Storer came to TSRI last year to start the Laboratory for Cell Motility.