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Knowing
how a living cell works means knowing how the chemistry inside the cell
changes as the functions of the cell change. Protein phosphorylation,
for example, controls everything from cell proliferation to
differentiation to metabolism to signaling, and even programmed cell
death (apoptosis), in cells from bacteria to humans. It’s a chemical
process that has long been intensively studied, not least in hopes of
treating or eliminating a wide range of diseases. But until now the
close-up view—watching phosphorylation work at the molecular level as
individual cells change over time—has been impossible without damaging
the cells or interfering with the very processes that are being
examined.
“To
look into phosphorylation, researchers have labeled specific
phosphorylated proteins with antibodies that carry fluorescent dyes,”
says Hoi-Ying Holman of the U.S. Department of Energy’s Lawrence
Berkeley National Laboratory (Berkeley Lab). “That gives you a great
image, but you have to know exactly what to label before you can even
begin.”
Holman
and her coworkers worked with colleagues from the San Diego and
Berkeley campuses of the University of California to develop a new
technique for monitoring protein phosphorylation inside single living
cells, tracking them over a week’s time as they underwent a series of
major changes.
“Now
we can follow cellular chemical changes without preconceived notions of
what they might be,” says Holman, a pioneer in infrared (IR) studies of
living cells who is director of the Berkeley Synchrotron Infrared
Structural Biology program at Berkeley Lab’s Advanced Light Source (ALS)
and head of the Chemical Ecology Research group in the Earth Sciences
Division . “We’ve monitored unlabeled living cells by studying the
nonperturbing absorption of a wide spectrum of bright synchrotron
infrared radiation from the ALS.”
The researchers report their results in the American Chemical Society journal Analytical Chemistry.
Phosphorylation fundamentals
Phosphorylating
enzymes add one or more phosphate groups to three amino-acid residues
common in proteins—serine, threonine, or tyrosine—which activates the
proteins; removing the phosphate reverses the process. The research goal
is to learn exactly when proteins such as enzymes and receptors are
switched on and off by phosphorylation, and which cells within a
population are responding to cause specific changes—for example, during
differentiation of a progenitor cell into its functional form.
To
avoid killing cells or introducing modified proteins or foreign bodies
that may alter their behavior, scientists can use a method called
Fourier-transform infrared (FTIR) spectromicroscopy; because infrared
light has lower photon energy than x-rays, it can peer inside living
cells without damaging them. Different components and different states
of the cell absorb different wavelengths of the broad infrared spectrum;
applying the Fourier-transform algorithm allows signals of all
frequencies to be recorded simultaneously, pinpointing when, where, and
what chemical changes are occurring.
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Most
infrared sources are dim, however, so the information from typical IR
set-ups is limited in resolution and has a low signal-to-noise ratio.
Infrared from the ALS’s synchrotron light source is a hundred to a
thousand times brighter.
Previously
Holman and her colleagues have used IR beamline 1.4.3, managed by
Berkeley Lab’s Michael Martin and Hans Bechtel, to obtain spectra from
living organisms in rock, soil, and water. They have monitored ongoing
biochemistry within living bacteria adapting to stress, and more
recently within individual skin connective tissue cells (fibroblasts)
from patients with mitochondrial disorders. (Mitochondria are the
cellular organelles commonly known as the “power-plants” of the cell.)
The
present study was done with a line of cultured cells called PC12. When
nerve growth factor, a small protein, is introduced into a PC12 cell,
the cell begins to send out neurites resembling the projections from
nerve cell bodies. Although originally derived from a tumor of the rat’s
adrenal gland, PC12 has become, rather counterintuitively, a valuable
model of how nerve cells differentiate from their unspecialized
progenitors.
Berkeley
Lab postdoctoral fellow Liang Chen began the current experiments by
introducing nerve growth factor to groups of PC12 cells to induce them
to differentiate; one group of cells was left untreated as a control.
The cells were cultured on gold-coated slides in chambers maintained at
body temperature in a humidified environment and supplied with
nutrients. Individual cells of a group were positioned under the
infrared beam at the beamline 1.4.3 endstation.
FTIR
spectra were collected before and after the nerve growth factor was
introduced. After stimulation, the spectra were taken first at short
intervals, from two to sixty minutes apart. Additional spectra were
collected of cells in other groups on the third, fifth, and seventh day
of continued stimulation.
The
first day’s spectra revealed spikes in phosphorylation activity within
minutes after the addition of the nerve growth factor, in concert with
changes in the ratios of such important chemical contents of the cell as
proteins, carbohydrates, and lipids. Phosphorylation subsequently
waned, then picked up again in another burst of activity on Day 3, just
as the cells began to extend neurites.
By
comparing results with quantum chemistry simulations by Berkeley Lab’s
Zhao Hao—predicting what should be observed from first principles—as
well as with results from partial studies using other methods, the
researchers confirmed the monitoring of phosphorylation phases, their
timing, and their target proteins, along with associated changes in
other substances in the cell.
A new technique takes off
“This
experiment was a proof of the concept,” says Liang Chen. “We
demonstrated the dynamics of protein phosphorylation in controlling
differentiation in this biological system using synchrotron infrared
spectromicroscopy, and we pointed the way to answering the many
questions a biologist has to ask about measuring the coordination of
specific processes in real time.”
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Although
in this first experiment the team was not able to follow individual
cells continuously, they were able to monitor differentiation in groups
of cultured PC12 cells in real time, without labeling or any other
invasive procedure. It was the first step in an ambitious inquiry into
the real-time biochemistry of living mammalian cells over the long term.
At
beamline 1.4.3., with the help of new team members Kevin Loutherback
and Rafael Gomez-Sjoberg, the team is designing equipment to maintain
mammalian cells in a thin layer of culture media that will keep them
healthy yet not interfere with the infrared beam, while automatically
monitoring and adjusting temperature, humidity, and nutrient ratios, and
removing waste products. This will allow data on individual cells to be
gathered continuously throughout the entire phosphorylation process.
Meanwhile
the Berkeley Synchrotron Infrared Structural Biology program at ALS
beamline 5.4 is building multimodal facilities that will monitor cell
development in human cells, bacteria, and plants, within soils,
minerals, and other environments, via “hyperspectromicroscopy”—from the
ultraviolet through visible light and deep into the infrared.
Researchers will be able to choose the frequency window (or combination
of windows) best suited to the sample and the conditions – in Holman’s
words, “to watch almost everything at once.”
Says
Holman, “Many researchers from the medical communities are interested
in using the technology, and we are particularly interested in
collaborating with university centers and private firms that are seeking
a broad view of how promising drugs act within specific cells.”
Some
of the projects will target Alzheimer’s disease, macular degeneration
of the retina in diabetes, and mitochondrial diseases in children. In
addition, specific processes like protein glycation can also be
identified. Since different cells and different organisms respond
differently, the eventual goal is to develop specific ways to screen the
mechanisms of individual medicines.
