Microscopy of Live Cells in Motion
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 cellsawash 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 cells 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 motilityparticularly microtubules, actin, and all the proteins responsible for regulating them. In particular, she is interested in the structural and regulatory interactions between actin and microtubuleshow 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.
"Its 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 cant resolve the individual bricks, all you would see is a red wall, and you wouldnt 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 cytochemistryonly about one tenth of one percent of the subunits are labeledbut 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.
Cell Crawling and Wound Healing
Understanding the signals and interactions driving the molecules that move the cells should help to elucidate the mechanisms that are common to a number of specific health-related problems studied in the Laboratory for Cell Motility.
Any time the skin is cut, the body will work to heal the wound. Wounds will over time close and the skin will grow back, connecting the two sides of the cut.
What drives this process is the action of the cells, which crawl forward to close and heal the wound. Once the two sides meet, the cells stop crawling and simply adhere to each other to make a solid tissue again, in a process known as contact inhibition of cell motility.
One area that interests Waterman-Storer is how the cells stop crawling once they contact each other. Presumably when the leading edge contacts another cell, a signal cascade occurs that ultimately shuts down the polymerization of the actin and halts cell motility.
"But what is happening to the cytoskeleton when that happens?" she asks. "What is regulating it and what are the kinetics when that occurs?"
Another area of interest is that of embryonic development. Cell motility is of huge importance to this area, because stem cells that will develop into nerve cells have to move to the correct location in the embryo before they can form nerve tissue and extend their axons so they can communicate with other cells.
The problem of motility is also closely related to cancer studies, since metastasis of cancerous cells is caused by the loss of this contact inhibition in cell motility. In fact, metastatic cells can be identified on plates by their ability to form colonies that continue to crawl and divide.
"If there were a way to selectively control tumor cell motility," says Waterman-Storer, "It could be used as an anti-metastatic therapeutic agent." Then cancers would remain local, forming tumors that could be easily excised.
And in the Laboratory for Cell Motility, this understanding starts with microscopy of the live cells in migration.
Waterman-Storer uses a spinning disk confocal microscope with excellent optics to produce the images with low background florescence. Images are captured with a high-resolution charge coupled device (CCD), which was originally designed for astronomers and is electronically cooled to improve the signal-to-noise. "These have improved greatly over the last five years," says Waterman-Storer.
The cameras have gotten so good, in fact, that they can now image cells at the optical limit of diffraction, capturing images with a rich dynamic range where each pixel represents an area of about six microns. And the cameras are so sensitive that they can detect groups of only two or three fluorescently labeled molecules above the noise.
To image these cells over time, Waterman-Storer captures a frame every 10 seconds and uses a mechanical filter wheel to take nearly simultaneous exposures at several wavelengths in order to capture the overall dynamics on the cell. One image will be of microtubules that fluoresce at one wavelength, and another will be of the actin, which fluoresce at another.
"One of the specialties of the laboratory is multimode imaging," says Waterman-Storer, "the ability to look at multiple probes simultaneously."
Multimodal imaging can be used to look at whether the microtubules track along the actin bundles or are pushed in the same direction as actin moves backwards in the cell. Viewing this sort of relationship is evidence that the two cytoskeletal structures contact each other and stick together.
Then this data can be correlated with in vitro assays that characterize which particular proteins are mediating these interactions.
Between Two Worlds
The interaction of different types of cytoskeletal proteins puts Waterman-Storer between two worlds, so to speak.
These cytoskeletal proteins are polar, and cells use their polarity to generate polarized cell morphology. Everything from the formation of a microtubule spindle for mitotic cell division to adhesion and movement of the cells comes from this polarity.
And just as these proteins polarize the cells so have they polarized cell biologists, who have traditionally fallen into separate camps dedicated to studying the distinct proteins, either microtubules or actin.
Actin research has benefited from years of biochemical studies of the molecule and the myriad proteins that bind to and regulate it. But while many of these regulatory molecules have been characterized biochemically, nobody has ever looked at their effect directly in cells. FSM has changed that completely.
"FSM has provided a way to quantitatively analyze actin dynamics in vivo," says Waterman-Storer. "And you can study all the actin in a cell at one time."
Actin filaments form bundles and cross-linked meshes, making it difficult to see individual filaments with traditional florescence. Another technique, photobleaching, had previously been used, but it was time-consuming and technically demanding. Also, this technique was difficult to use for studying actin in the whole cell since only a small part of the cell could be photobleached.
Microtubules, for their part, have had a long history under the microscope. They have the ability to rotate polarized light, so they have been studied in vivo for many years. But what people havent done is to look at how proteins that bind to actin affect microtubule dynamics and vice versa.
"There are some old observations that tell us their interactions are important," says Waterman-Storer. "But nobody has really followed up to figure out the kinetics of these interactions, what mediates them, and how they are regulated."
Years ago, a Russian biologist observed that when microtubules are destroyed in a cell, the actin depolarizes and cell motility stops, leading many to believe that microtubules provide instructions to the actin in a cell. Microtubules are known to bind regulator molecules of the actin-mediating GTPase signaling molecule, but the mechanism is not yet clear.
"What is clear," says Waterman-Storer, "is that we can image them simultaneously to look for evidence if interactions between the two."
Some little motion in the cell catches my eye and Waterman-Storer follows my gaze to the computer screen. She is in mid-sentence, saying, "Every genesis involves huge migrations of cells. You have these neural crest cells..." and her words trail off.
"I love watching these movies." She says.
Assistant Professor Clare Waterman-Storer came to TSRI last year to start the Laboratory for Cell Motility.
A schematic diagram of how microtubules obtain fluorescent speckles by co-assembling very few fluorescently labeled tubulin subunits with many unlabeled subunits