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
|Contact: Paul Preuss|
DOE/Lawrence Berkeley National Laboratory