Berkeley Lab scientists observed phosphorylation in living PC12 cells stimulated by nerve growth factor as they differentiated and sent out neuron-like neurites. The researchers imaged individual cells and simultaneously obtained absorption spectra using synchrotron radiation from the Advanced Light Source. Cells not stimulated with nerve growth factor did not differentiate and showed different infrared absorption spectra.
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.
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.
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