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Playing Up the Single Life

By Amy Maxmen Playing Up the Single Life Single-cell applications to help you explore the tiniest great unknown As researchers probe deeper into cell physiology, they are increasingly bumping into cells’ individual personalities. Identical genetic material and location, it seems, doesn’t prevent two cells from behaving differently, and in some cases this intercellular variation changes cell function and fate. Fluctuations within individual cells may

Amy Maxmen

Playing Up the Single Life

Single-cell applications to help you explore the tiniest great unknown

As researchers probe deeper into cell physiology, they are increasingly bumping into cells’ individual personalities. Identical genetic material and location, it seems, doesn’t prevent two cells from behaving differently, and in some cases this intercellular variation changes cell function and fate. Fluctuations within individual cells may even underlie cancer or neurological disease.

DNA microarrays, mainstays in assessing gene expression, aren’t sensitive enough to detect these individual-level differences, says Sunney Xie, a professor of chemistry and chemical biology at Harvard University and an early pioneer of single-cell techniques. But picking out RNAs or proteins present in low numbers within a single cell is easier said than done. And exploring forces such as tension and movement at a single-cell scale can be a Herculean task requiring collaborations between physicists, engineers, and biologists.

Here, The Scientist profiles researchers...

Sampling several single cells

DEVELOPER

Stephen Quake, Professor of Bioengineering, Stanford University, California

PROJECT

Quake and his colleagues wanted to examine the response of similar cells to the same signal. Researchers have studied this question using quantitative PCR in bulk assays, but Quake’s team focused on single cells in tissue culture and their response to the signaling molecule tumor-necrosis factor (TNF-α) (Nature, 466:267-71, 2010).

TECHNIQUE

The team used high-throughput microfluidics to measure how thousands of individual cells responded to a wide range of TNF-α concentrations by relaying information through the transcription factor nuclear factor (NF)-κB. First, they loaded cells, with their nuclei and NF-κB fluorescently labeled, onto a microfluidic cell-culture device consisting of silicon layers and microscopic gates to control fluid flow. As cells reacted to TNF-α, a programmed camera attached to the chips and connected to a microscope captured images of NF-κB proteins shuttling between the cytoplasm and the nucleus of each cell.

PROS

The technique is extremely efficient—using it, the team was able to treat several thousand cells simultaneously and observe their reaction over a period of four days.

CONS

Microfluidic experiments aren’t trivial to set up, so it’s important to use them for the right reason. If you’re looking at the reaction of cells to a single stimulus, a 96-well plate might work just as well. But if you want to expose cells to a series of conditions with precise control, microfluidics is the way to go, says Stanford systems biologist Markus Covert, who collaborated with Quake’s team on the paper.

CONSIDERATIONS

Microfluidic devices can be finicky, so expect some trial and error early on. Also, like other high-throughput tools, the device yields an enormous amount of data, so you’ll need image analysis tools to avoid spending months on analysis, says Covert. He and his colleagues are open to providing the software they built, which tracks the movement of the proteins and the cells, upon request.

SPECIAL EQUIPMENT

Fluidigm, a South San Francisco-based company founded by Quake, is developing a commercial version of the cell-culture plates along with a corresponding platform to run the plates. Other companies, such as CellASIC in Hayward, California, sell similar microfluidic cell culture devices. Their ONIX cell culture microfluidic plates range from $40–$80, and the corresponding platform, the ONIX Microfluidic Perfusion System, costs $10,000 to $15,000.

Detecting low levels of RNA

DEVELOPER

H. Kumar Wickramasinghe, Professor of Electrical Engineering and Computer Science, University of California, Irvine

PROJECT

When genes are expressed abnormally, a stem cell can become a cancer stem cell—or so one theory goes. Wickramasinghe wanted to test the theory, so he created a way to analyze slight differences in gene expression at different time points during a single cell’s life (Anal Biochem, doi:10.1016/j.ab.2010.08.014, 2010).

TECHNIQUE

Wickramasinghe sampled mRNA using extremely fine needles called dielectrophoretic nanoprobes, coated with primers for specific mRNA. When the 20-nm-wide needle tip pokes through the cell membrane, it generates an electrical field that attracts molecules from within the nucleus. Upon withdrawing the needle, the researchers counted the mRNAs selectively fished out by the primers—in this case, ones related to cancer-gene expression. “And since the cell doesn’t die, we could also inject something to silence a gene and sample the cell a week later to see what happens in real time,” says Wickramasinghe.

PROS

While many genomic analyses destroy cells, here they stay alive and ready for further perturbation and analysis. And, It’s fast—the process takes just a few minutes.

CONS

Cells must be stuck to a surface when they get their shot. If they aren’t naturally sticky, like fibroblasts, they must be treated to make them adherent before the experiment, adding an additional step. However, Wickramasinghe says it may be possible to inject cells in tissue culture that are embedded in an extracellular matrix.

CONSIDERATIONS

Wickramasinghe has proven that the probes work, but answering questions about cancer stem cells will be tricky for a different reason. Cancer biologist Edward Nelson at the University of California, Irvine, says the problem now is isolating the elusive cells. Yet the technique could be of use to researchers in other fields such as genetic imprinting, in which maternal and paternal alleles cause tiny fluctuations in gene expression.

SPECIAL EQUIPMENT

Collaborating with Wickramasinghe is presently the only way to use his dielectrophoretic nanoprobe needles. However, he says he is in the process of commercializing them.

Teeny tiny tension sensor

DEVELOPER

Martin Schwartz, Professor of Microbiology and Biomedical Engineering, University of Virginia and Taekjip Ha, Professor of Physics, University of Illinois at Urbana-Champaign

PROJECT

Cells use molecular forces when they protrude and retract during movement, and adhere to one another or to the extracellular matrix. Schwartz wanted to measure mechanical force in single cells, so he developed a tension sensor that directly measured tension across vinculin, a protein present at adhesive structures in migrating cells (Nature, 466:263-66, 2010).

TECHNIQUE

Schwartz’s sensor relies on a spring-like protein with fluorophores tethered to either end. When the “spring-protein” stretches, these fluorophores move apart and the light they emit decreases. Ha calibrated the tension sensor by applying a controlled amount of force on the “spring” and linking that to the resulting light intensity imaged through a microscope. Then the team inserted the sensor protein’s DNA sequence into a gene encoding vinculin and transfected the constructs into cells. Once expressed, the modified vinculin registered tension when it was stretched between the actin cytoskeleton and cell-surface adhesion receptors.

PROS

Mapping local forces with high accuracy; the sensor can detect changes with at least 100-fold greater sensitivity than simpler methods, which involve measuring the deformation of cell membrane surfaces.

CONS

Tweaking the protein carrying the tension sensor can change its function. “It took about 40 attempts before we got the construct right,” says Schwartz.

CONSIDERATIONS

Because they can measure local forces within cells, tension sensors inserted into other proteins might define forces that drive tumor cells to invade tissue or chromosomes to separate during cell division. However, researchers may need to alter the sensor design depending on the range of tension expected, Ha says. Their sensor only measures forces between 1–5 piconewtons.

SPECIAL EQUIPMENT

In addition to a confocal scanning microscope, you’ll need a high-power infrared laser to apply force to the tension sensor, as well as software to control it. Ha wrote the software and provides it freely to anyone who asks.

Mapping proteins

DEVELOPER

Hiromitsu Nakauchi, Professor of Stem Cell Biology and Regenerative Medicine, University of Tokyo

PROJECT

To elucidate molecular cascades involved in stem cell proliferation and renewal, Nakauchi perturbed hematopoietic stem cells and looked at the effects on protein phosphorylation (PNAS, 104:2349-54, 2007).

TECHNIQUE

Nakauchi used a simple immunostaining technique he calls the single-cell imaging of phosphorylation (SCIPhos) assay. In it, roughly 50 cells are placed in a drop on a glass slide, stained, and viewed through a confocal microscope. By staining phosphorylated proteins, he can infer which proteins are active or inactive, as well as determine their location within the cell. “Some of my students came up with this idea as an alternative to doing Western blotting, which uses up hundreds of cells,” he explains.

PROS

SCIPhos doesn’t just monitor phosporylated proteins, but also shows where proteins localize, which can reveal crucial biological information.

CONS

Each cell is imaged separately; analyzing thousands of cells is labor intensive.

CONSIDERATIONS

This technique is ideal for experiments involving a limited number of cells. The trick, says Nakauchi, was to figure out how to prevent evaporation. He simply draws a circle on the glass slide with a water-repellent pen before depositing the drop of cells on it, so that the liquid and cells don’t spread too thin. Nakauchi advises keeping the slide in a moisture chamber on ice and applying antibodies and reagents carefully.

SPECIAL EQUIPMENT

“Just a confocal microscope,” says Nakauchi. “It’s low tech and it works.”

Crosstown traffic monitor

DEVELOPERS

Robert Singer, Professor of Cell Biology, Albert Einstein College of Medicine, New York and David Grünwald, Professor of Bionanoscience, Delft University of Tech-nology, The Netherlands.

PROJECT

Messenger RNAs exit the nucleus through nuclear pores, but conventional microscopes can’t track the passage in real time. The authors developed an approach to visualize RNA movement in living cells (Nature, 467:604-07, 2010).

TECHNIQUE

Grünwald and Singer labeled RNAs and nuclear pores with fluorescent probes of two different colors. They then hooked two cameras up to a microscope in order to correct for optical aberrations. The pair of cameras each snapped 50 photos per second. In the ensuing analysis the images were aligned so that the distance between the probes could be precisely measured.

PROS

The aligned cameras can measure intermolecular distances with nanometer precision, unlike snapshots of two fluorescently labeled objects taken with a single camera.

CONS

The cameras and their snapshots must be perfectly aligned in order to accurately see where the two fluorescent spots are in relation to one another—no easy task, warns Singer.

CONSIDERATIONS

This imaging methodology enables researchers to see as yet unobserved interactions such as the dynamics between transcription factors and the genes they turn on. Researchers will have to be wary of background fluorescence, which can be overcome by exaggeratedly labeling the RNA. (Singer’s team labeled RNA with 24 fluorescent probes.) “It’s like the Milky Way on a dark night,” says Singer. “You see stars but they’re lost in a haze.”

SPECIAL EQUIPMENT

In addition to a confocal microscope, you need two highly sensitive cameras, a way to align those cameras at exactly the right angles, and filters that must be aligned as well. “The tools aren’t anything special,” Singer says, “but it takes a fair bit of expertise to set the system up.”

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